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The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%–25% tests positive and at a level +1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P  < .05 were found between the procedures using McNemar's χ 2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile. [ABSTRACT FROM AUTHOR]

The occurrence of Giardia and Cryptosporidium (oo)cysts in drinking source water poses a serious public health risk. Here, we established a method that combines membrane concentration and real-time polymerase chain reaction (PCR) to quantify Giardia and Cryptosporidium in drinking water. The water samples were filtered through a cellulose membrane to collect Giardia and Cryptosporidium, and then nucleic acids were extracted. Specific primers and probes were designed and synthesized according to the gph gene sequence of Giardia and 18S rRNA gene sequence of Cryptosporidium. The concentrations of the two targets were determined using real-time PCR technology. The sensitivity, specificity, and stability of the method were evaluated. Our findings revealed that the detection limits of real-time PCR method for detecting Giardia and Cryptosporidium were 0.926 and 0.65 copy/µL, respectively; the spiked recovery rates were above 60% and 38%, respectively, and relative standard deviations were under 0.95% and 2.26%, respectively. Therefore, this effective procedure based on the membrane concentration method and real-time PCR will be useful for detecting Giardia and Cryptosporidium in drinking water for purpose of continuous environmental monitoring. [ABSTRACT FROM AUTHOR]

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available. [ABSTRACT FROM AUTHOR]

Coxiella burnetii , the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii , based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible, and sensitive, allowing the detection of as few as 5 genome equivalents (GEs) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3 × 104 colony forming units per 0.5 g of soil (∼3 GEs per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples. [ABSTRACT FROM AUTHOR]

Salmonella is a crucial food‐borne pathogen causing food poisoning, leading to severe public health events. Here, we developed a technique by integrating recombinase polymerase amplification with CRISPR‐LbCas12a and employing two targets with engineered crRNA for detection of Salmonella (RPA‐LbCas12a‐TTECDS). Our findings revealed that this novel method rapidly detects trace Salmonella in food through fluorescence intensity and provides a template for other food‐borne pathogen detection methods. Further, crRNA was optimized to increase detection sensitivity. Double targets were used to enhance the detection accuracy, reaching the level of qPCR, which was superior to fluorescent RPA. The RPA‐LbCas12a‐TTECDS system specifically detected Salmonella levels as low as 50 CFU per ml at 37°C in 1 h. In summary, a simple, rapid, sensitive and high accuracy detection technique based on CRISPR‐Cas12a was created for Salmonella detection without complicated equipment. Significance and Impact of the Study: The emergence of CRISPR‐Cas12a provides a new technique for detection methods. In this study, we developed a rapid, sensitive, and high accuracy RPA‐LbCas12a‐TTECDS based toolbox for Salmonella detection. This method is easy to operate with a completion span of 50 min by a fluorescence detector. The simple and quick‐to‐read method is potentially useful as point‐of‐care testing for Salmonella. [ABSTRACT FROM AUTHOR]

We developed an assay comprising crude DNA lysis by simple heat treatment coupled loop-mediated isothermal amplification with hydroxynaphthol blue for Chlamydia trachomatis detection (petty patent pending), and evaluated the developed assay for its feasibility as a one-step point-of-care detection on 284 endocervical swab specimens from clinically symptomatic C. trachomatis and healthy subjects. This assay is sensitive to 0·04 pg of ompA, specific with six primers targeting C. trachomatis ompA region, rapid (45 min total assay time), inexpensive (approx. 3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay effectiveness (95% specificity, 90-100% sensitivity) to bacterial DNA isolation by a commercial kit coupled with polymerase chain reaction and gel electrophoresis (98-100% specificity, 87-100% sensitivity) based on the clinical samples test. The test result could be read by naked eye through the colour change from violet (negative) to sky blue (positive) for C. trachomatis-infected specimens. Further, this assay uses all safe chemical reagents and is hence safe to the users.Significance and impact of the studyChlamydia trachomatis is the major bacterial sexually transmitted disease worldwide. The clinical symptoms are broad, and chronic C. trachomatis infections could lead to blindness, ectopic pregnancy, sterility in males and females, and a higher risk of the development of cervical cancer. The result indicates the potential usefulness of our crude DNA lysis coupled loop-mediated isothermal amplification with hydroxynaphthol blue for a simple, rapid, specific, sensitive and cost-effective assay for C. trachomatis detection from suspected specimens. This assay offers an alternative in the clinical diagnosis of C. trachomatis in resource-limited health-care facilities and clinical laboratories in developing countries, and in field tests.

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic‐infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT‐PCR. Four primer‐pair‐produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one‐step m‐RT‐PCR assay. The m‐RT‐PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost‐effective and rapid multiplex RT‐RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India. [ABSTRACT FROM AUTHOR]

Oroxylum indicum is one of the valuable Dai pharmaceuticals; the dry seeds and bark of O. indicum were used to treat acute cough, sore throat and so on. Of the seven compounds from O. indicum were determined and obtained using the bioassay‐guided method. Among them, compound 7 was obtained from the plant for the first time. Eight bacterial strains and one yeast fungi were exposed to the compounds. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) or minimum fungicidal concentrations were determined according to the standard broth microdilution method. Baicalein (2) exhibited relative strong antibacterial activities with MIC of 8 μg ml−1 and MBC of 16 μg ml−1 against three MRSA strains of Staphylococcus aureus of SCCmec III type, whereas flavonoids 3, 5 and 7 showed some degree of activities against methicillin‐susceptible S. aureus (MSSA, ATCC 25923). The findings may offer new evidence that why O. indicum was used widely in Dai peoples' life. [ABSTRACT FROM AUTHOR]

The genus Fusarium contains many fungal species known to be pathogenic to animals and plants alike. One species complex within this genus, the Fusarium solani species complex (FSSC), is of particular concern due to its high numbers of pathogenic members. FSSC members are known to contribute significantly to plant, human and other animal fungal disease. One member of the FSSC, Fusarium keratoplasticum, is of particular ecological concern and has been implicated in low hatching success of endangered sea turtle eggs, as well as contribute to human and other animal Fusarium pathogenesis. Species‐specific primers for molecular identification of F. keratoplasticum currently do not exist to our knowledge, making rapid identification, tracking and quantitation of this pathogenic fungus difficult. The objective of this study was to develop primers specific to F. keratoplasticum that could be applied to DNA from isolated cultures as well as total (mixed) DNA from environmental samples. RPB2 sequence from 109 Fusarium isolates was aligned and analysed to determine nucleotide polymorphisms specific to F. keratoplasticum useful for primer design. A set of primers were generated and found to be effective for identification of F. keratoplasticum from total DNA extracted from sand surrounding sea turtle nesting sites. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) and human astrovirus (HAstV) are important causative agents of acute gastroenteritis in children and adults. They are comprised of multiple genotypes and attention should be paid to genotype changes or emergence of new genetic variants. To study the prevalence and diversity of NoV GI, GII, and HAstV circulating in eastern China, we conducted a three‐year environmental surveillance in a coastal city of Yantai. Thirty‐six sewage samples were collected, processed, and examined for the presence of viral genomes by PCR. The results showed that NoV GI, GII, and HAstV were detected in all 36 samples. Six NoV GI genotypes, 11 NoV GII genotypes, and 5 HAstV serotypes were identified; GI.6, GII.17, and HAstV‐5 were the most prevalent types, respectively. Persistent existence of NoV GII.17 Kawasaki 308 variant was observed during whole study period. Phylogenetic analysis reflected multiple transmission lineages in local population for both viruses. Our results reflect continuous presence of enteric viruses in sewage, improve our understanding on their molecular epidemiology, and demonstrate surveillance on sewage is an effective approach in understanding the local circulation of enteric viruses. [ABSTRACT FROM AUTHOR]

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T‐2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 103 CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 104 CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin‐layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T‐2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 103 CFU per ml and for H7 positive E. coli is 1 × 104 CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 104 CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on‐site inspection tool for accurate and rapid detection of E. coli O157:H7 infection. [ABSTRACT FROM AUTHOR]

One of the major problems to be addressed in aquaculture is the prominence of antimicrobial resistance (AMR). The occurrence of bacterial infections in cultured fishes promotes the continuous use of antibiotics in aquaculture, which results in the selection of proliferated antibiotic‐resistant bacteria and increases the possibility of transfer to the whole environment through horizontal gene transfer. Hence, the accurate cultivation‐dependent and cultivation‐independent detection methods are very much crucial for the immediate and proper management of this menace. Antimicrobial resistance determinants carrying mobile genetic transfer elements such as transposons, plasmids, integrons and gene cassettes need to be specifically analysed through molecular detection techniques. The susceptibility of microbes to antibiotics should be tested at regular intervals along with various biochemical assays and conjugation studies so as to determine the extent of spread of AMR. Advanced omic‐based and bioinformatic tools can also be incorporated for understanding of genetic diversity. The present review focuses on different detection methods to unearth the complexity of AMR in aquaculture. This monitoring helps the authorities to curb the use of antibiotics, commencement of appropriate management measures and adequate substitute strategies in aquaculture. The long battle of AMR could be overcome by the sincere implementation of One Health approach. Significance and Impact of the Study: The use of antibiotics and increased antimicrobial resistance (AMR) are of major concerns in aquaculture industry. This could result in global health risks through direct consumption of cultured fishes and dissemination of AMR to natural environment through horizontal gene transfer. Hence, timely detection of the antimicrobial‐resistant pathogens and continuous monitoring programmes are inevitable. Advanced microbiological, molecular biological and omic‐based tools can unravel the menace to a great extent. This will help the authorities to curb the use of antibiotics and implement appropriate management measures to overcome the threat. [ABSTRACT FROM AUTHOR]

Saffold virus (SAFV) is an emerging human cardiovirus associated with respiratory and gastrointestinal infection, and, more recently, to symptoms related to the endocrine, cardiovascular, and neurological systems. Information about SAFV circulation in Italy is scarce. In order to provide insights into the epidemiology of SAFV in Italy, 141 raw sewage samples collected throughout Italy were tested using broad‐range nested RT‐PCR primers targeting the 5′‐NC region. Seven samples (5·0%) were confirmed as SAFV in samples collected in North, Centre and Southern Italy. Typing was attempted through amplification of the VP1 coding region, using both published and newly designed primers, and one sample was characterized as SAFV‐2. Significance and Impact of the Study: Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential. [ABSTRACT FROM AUTHOR]

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation‐based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real‐time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15–16·69. During cold‐starvation, PMA qPCR selectively excluded DNAs from heat‐killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). Significance and Impact of the Study: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold‐starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real‐time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold‐starvation conditions. [ABSTRACT FROM AUTHOR]

Shiga toxin‐producing Escherichia coli strains (STEC) are food‐borne pathogens. While E. coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non‐O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E. coli O‐serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture‐based methods. Fresh farm dairy effluent (FDE) (n = 36) and composite calf faeces (n = 12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx‐negative populations present in fresh dairy effluent and calf faeces. Significance and Impact of the Study: Shiga toxin‐producing Escherichia coli (STEC) are important food‐borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture‐based farms. Such information is required to develop risk‐assessment models aiming at limiting transmission of these STEC to human. Significance and Impact of the Study: Shiga toxin‐producing Escherichia coli (STEC) are important food‐borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture‐based farms. Such information is required to develop risk‐assessment models aiming at limiting transmission of these STEC to human. [ABSTRACT FROM AUTHOR]

Abstract: Fresh‐cut produce is at greater risk of Salmonella contamination. Detection and early warning systems play an important role in reducing the dissemination of contaminated products. One‐step Reverse Transcription Polymerase Chain Reaction (RT‐qPCR) targeting Salmonella tmRNA with or without a 6‐h enrichment was evaluated for the detection of Salmonella in fresh‐cut vegetables after 6‐h storage. LOD of one‐step RT‐qPCR was 1·0 CFU per ml (about 100 copies tmRNA per ml) by assessed 10‐fold serially diluted RNA from 106 CFU per ml bacteria culture. Then, one‐step RT‐qPCR assay was applied to detect viable Salmonella cells in 14 fresh‐cut vegetables after 6‐h storage. Without enrichment, this assay could detect 10 CFU per g for fresh‐cut lettuce, cilantro, spinach, cabbage, Chinese cabbage and bell pepper, and 102 CFU per g for other vegetables. With a 6‐h enrichment, this assay could detect 10 CFU per g for all fresh‐cut vegetables used in this study. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh‐cut vegetable processing to strengthen food safety assurance. Significance and Impact of the Study: Significance and Impact of the Study: Fresh‐cut produce is at greater risk of Salmonella contamination. Rapid detection methods play an important role in reducing the dissemination of contaminated products. One‐step RT‐qPCR assay used in this study could detect 10 CFU per g Salmonella for 14 fresh‐cut vegetables with a 6‐h short enrichment. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh‐cut vegetable processing to strengthen food safety assurance. [ABSTRACT FROM AUTHOR]

Abstract: Salmonid alphaviruses (SAV) severely infect farmed salmonids and rainbow trout. Owing to the recent increase in fish import trade, several SAV unreported countries, such as China, may face serious threat of this infection. Thus, it is necessary to develop efficient detection methods for the prevention and diagnosis of SAV infection. In this study, we selected a conserved segment of the SAV E1 protein as a target immunogen for the preparation of monoclonal antibodies (mAbs) specific to SAV. A sensitive indirect immunofluorescence (IF) method was developed using 26E9C2 mAb that detected E1 protein and identified subtypes 1, 2 and 5 of SAV. Thus, this assay provides a sensitive and specific detection method, and an improved technical support for the clinical diagnosis and epidemiological study of SAV. Significance and Impact of the Study: In this study, we successfully prepared two monoclonal antibodies (mAbs) against three subtypes of salmonid alphavirus (SAV), and established an indirect immunofluorescence assay that can be used to diagnose and prevent SAV from entering SAV unreported countries, such as China, through international trade. The application of this method will contribute to controlling SAV infection and reducing economic losses. [ABSTRACT FROM AUTHOR]

Abstract: The total direct count (TDC) microbial enumeration method is rapid and suitable for analysing environmental samples containing numerous un‐culturable micro‐organisms. Conventional TDC methods require the addition of a fluorescent stain and are thus unsuitable for automatic monitoring. We unexpectedly found that heated micro‐organisms emit strong autofluorescence. This study was conducted to determine how heating enhances the autofluorescence of bacteria and fungi and to evaluate whether the phenomenon could be exploited to develop a new TDC method. Bacterial autofluorescence was augmented by heating cells at 200°C. ELISA results indicated that levels of advanced glycation end products (AGEs) increased in heated microbes. Catechin, an inhibitor of the Maillard reaction, disrupted the intensification of autofluorescence. These results suggest that the enhanced autofluorescence is associated with the formation of AGEs and that the reaction could be utilized as alternative probe in TDC methods. Significance and Impact of the Study: Autofluorescence of bacteria and fungi was prominently intensified by heat treatment at 200°C. This phenomenon was associated with advanced glycation end products formed in micro‐organisms via the Maillard reaction. The fluorescence signal was strong enough to be utilized as an alternative probe for fluorescent dye in the total direct count method. This phenomenon could be incorporated in an automatic apparatus for microbial enumeration, as it does not require staining. [ABSTRACT FROM AUTHOR]

The aim of this research was to optimize two multiplex polymerase chain reaction ( PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli ( STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Significance and Impact of the Study: Two multiplex polymerase chain reaction ( PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli ( STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in food and environmental samples. [ABSTRACT FROM AUTHOR]

The objective of the present study was to assess the occurrence of major waterborne enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E virus) along the Tiber River in Italy, in areas affected by different kinds of anthropogenic pressure (agricultural, urban, industrial and pristine). Moreover, in light of the recent abundant detection of human bocavirus in urban wastewater samples in Italy, the occurrence of this virus was also assessed. Virus detection was based on nested PCR followed by sequencing, and on real-time PCR. A correlation with anthropogenic pressure was observed. The urban and industrial areas were the most contaminated (100 and 75% of samples were positive for at least one virus respectively). The agricultural area was less contaminated, with 50% of samples positive. None of the samples collected in a pristine area were positive for viruses. The most frequently detected virus was human bocavirus, identified in 37·5% of samples, followed by norovirus and enterovirus (28% each) and adenovirus (21·6%). Rotavirus, and hepatitis A and E viruses were less common (<9%). Although Human Bocavirus is not considered a waterborne pathogen, the widespread contamination of river waters suggests that virus transmission via the water route should not be neglected. Significance and Impact of the Study To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded. [ABSTRACT FROM AUTHOR]

Immunomagnetic separation used with culture based methods has been a useful technique in the detection of pathogens. However, previous studies have not answered many of the necessary questions for real world applications. The objective of this study was to assess the efficacy of different immunomagnetic separation ( IMS) bead types in recovery of the correct serogroup from a mixture of big six non-O157 Shiga toxin-producing Escherichia coli strains. To determine the impact of different matrices on recovery, samples of sterile phosphate buffered saline ( PBS), sterile and non-sterile cattle faeces, ground beef and lettuce were inoculated with 10 CFU per ml mixture of isolates representing the six serogroups. After a 6 h incubation at 37°C, samples were mixed with IMS beads from three different commercial sources and plated on eosin methylene blue agar ( EMB). Three suspect E. coli colonies were selected from each EMB plate and multiplex polymerase chain reaction was used to determine the serogroup. The rate of correct identification varied with the serogroup, IMS bead manufacturer and matrix. Overall, recovery of the correct serogroup became less likely with increase in matrix complexity, with enrichments containing lettuce having the greatest number of bead types with significantly lower likelihood of correct recovery compared to recovery in PBS. Significance and Impact of the Study The need to accurately and efficiently detect Shiga toxin-producing Escherichia coli ( STEC) O26, O45, O103, O111, O121 and O145, which have caused outbreaks on numerous occasions, is a major public health and food safety concern in the United States. Detecting these STEC serogroups can be challenging because methods to detect non-O157 serogroups have not been refined as compared to those for O157. Immunomagnetic separation ( IMS) has the potential to isolate STEC from a mixture in complex matrices. Our results highlight the need for optimization of IMS-based detection of STEC to effectively recover the targeted serogroup from a variety of sample matrices. [ABSTRACT FROM AUTHOR]

Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10−5 ng DNA (SV3) and 0.4 × 10−6 ng DNA ( U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. Significance and Impact of the Study Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a fastidious organism that requires a lengthy incubation time in liquid growth media for detection. The objective of this study was to develop a medium formulation using readily available supplements that enhanced early growth of F. tularensis. Francisella tularensis live vaccine strain was used to evaluate the growth responses for each of the media formulations tested. Growth in brain heart infusion broth supplemented with 2% Vitox, 10% Fildes and 1% histidine ( BVFH) resulted in a significant increase in growth after 8 h incubation compared to other media formulations tested ( P < 0·001). Virulent strains of F. tularensis grown in BVFH medium demonstrated similar enhanced early growth. Cell densities of 3·9-5·2 log10 CFU per ml were obtained after 24 h of growth in BVFH from a 1-2 cell ml−1 starting inoculum of the virulent Type A Schu4 strain, indicating the suitability of this medium in rapidly amplifying low starting titres of F. tularensis. Collectively, these results indicate that the novel formulation of the BVFH medium was capable of producing enhanced growth response for F. tularensis. Significance and Impact of the Study The need for rapid cultivation of Francisella tularensis is essential for detection and monitoring during natural outbreak events or intentional bioterrorism attacks. The addition of selected supplements into the base medium BHI (BVFH) developed in this study enhanced growth of F. tularensis Type A1, A2 and B strains compared to BHI alone. Growth of these organisms in BVFH will allow for improved response time should a natural or intentional contamination event occur. [ABSTRACT FROM AUTHOR]

Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridization ( FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between 2 and 3 h), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH. Significance and Impact of the Study Xanthomonas citri subsp. citri (Xcc) is an aggressive and hardy pathogen of citrus plants worldwide. Outbreaks are difficult and costly to contain and the establishment of citrus canker results in restricted trade. In order to extend the existing toolkit for identification of Xcc we developed a novel diagnostic approach based on fluorescence in situ hybridization. Our approach is of comparable specificity and sensitivity to existing methods but can be performed directly on infected tissue making it significantly faster than existing PCRs, and requiring fewer laboratory resources. [ABSTRACT FROM AUTHOR]

Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4 527 samples of DNA from ticks in Japan, which were then analysed by real-time PCR and nested PCR. Francisella DNA was detected by real-time PCR in 2·15% (45/2 093) of Ixodes ovatus, 0·66% (14/2 107) of I. persulcatus, 8·22% (6/73) of I. monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I. ovatus and I. persulcatus, respectively, which were positive according to real-time PCR. Phylogenetic analysis showed that the sequence from I. ovatus and I. persulcatus were clustered with F. tularensis type B strains distributed in Eurasia. Microinjected live F. tularensis persisted in ticks, whereas heat-killed F. tularensis decreased. Microinjected F. tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F. tularensis contributes to the adaptation or survive of bacterial infection in ticks. Significance and Impacts of the Study Francisella tularensis has been detected in ticks, suggesting that it is a tick-borne pathogen. However, F. tularensis has not been detected in ticks in Japan since 1991. In this study, we performed a large-scale analysis of DNA isolated from ticks in Japan and detected F. tularensis by real-time polymerase chain reaction (PCR) and nested PCR. We found that F. tularensis could survive in ticks based on an experimental tick-infection model. We also identified a bacterial factor that contributes to survival in ticks. Our results suggest that ticks are candidate vectors that mediate F. tularensis infection in Japan. [ABSTRACT FROM AUTHOR]

Cronobacter is an emerging food pathogen, especially in infants and neonates, often associated with the ingestion of contaminated Powdered Infant Formula (PIF). Therefore, regulations require the control of the absence of Cronobacter and of Salmonella, another important food pathogen, in these food products. So far, reference and alternative methods take up to several days, and no validated method exists for the simultaneous detection of these two pathogens. In this work, we propose to address this issue by an innovative and easy-to-operate assay, named Plasmonic Immuno-Assay (PlasmIA), and by producing dedicated polyclonal antibodies. Our approach is based on Surface Plasmon Resonance imaging of antibody-arrays and bacterial growth during a standardized enrichment. Such a single-step assay enables the multiplex detection of both Cronobacter and Salmonella, with concentrations smaller than 30 CFU cells in 25 g PIF samples, in less than 1 day. Significance and Impact of the Study Among bacterial pathogens involved in food contamination, Cronobacter and Salmonella are of particular interest. Nevertheless, all detection methods used so far require several days to assess food safety. In the present paper, we describe the first multiplex immuno-assay ever described for fast and specific detection of these two pathogens in food samples. Such advances were made possible by combining the advantages of protein microarrays with on-biochip culture of contaminated food samples and an easy-to-operate optical detection. By doing so, we managed to detect both viable Cronobacter and Salmonella occurring during the enrichment phase. [ABSTRACT FROM AUTHOR]

Duck tembusu virus (DTMUV) has caused significant economic losses to the poultry industry in China since the spring of 2010. In this study, a nano-PCR assay targeting E gene of DTMUV was developed and their sensitivities and specificities were investigated. Under the optimized conditions of nano-PCR assay for detection of DTMUV, the nano-PCR assay was 10-fold more sensitive than a conventional PCR assay. The lower detection limit of the nano-PCR assay was 1.8 × 102 copies μl -1 of DTMUV RNA, as no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nano-PCR assay for the detection of DTMUV. The sensitive, and specific nano-PCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of DTMUV-infection. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila has been increasingly implicated as the aetiologic agent of various human diseases. Therefore, reliable laboratory detection and identification of this bacterium has become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method for rapid detection of Aer. hydrophila using a cross-priming amplification ( CPA) assay with the desA gene as the target. The desA gene is crucial for the survival and growth of Aer. hydrophila under iron starvation. The results can be visualized as colour changes without opening the reaction tubes. No false-positive results were observed for the 33 non- Aer. hydrophila strains tested to evaluate assay specificity. The limit of detection for Aer. hydrophila was approximately 200 copies of desA per reaction (on reference plasmids) and 5 × 103 CFU g−1 Aer. hydrophila in simulated human stool, which is the same sensitivity as a qPCR assay. The performance of the CPA assay was also evaluated with 100 stool specimens from diarrhoea patients and 40 environmental water samples. In conclusion, the simplicity, cost-effectiveness and nearly instrument-free platform of the CPA assay make it practical for use in primary care facilities and smaller clinical laboratories. Significance and Impact of the Study Aeromonas hydrophila is a human pathogen that infects via exposed wounds or ingestion of contaminated water and food. In this study, a CPA-based PCR method was developed for specific, rapid, cost-effective detection of Aer. hydrophila, and the test results could be visualized without opening the reaction tubes. This is the first report on the application of the CPA method for the detection of Aer. hydrophila. This novel method could be practical for use in primary care facilities and smaller clinical laboratories. [ABSTRACT FROM AUTHOR]

Embellisia astragali is an important seedborne pathogenic fungus responsible for yellow stunt and root rot of standing milk vetch ( Astragalus adsurgens). The current detection methods that are based on culture and morphological identification are time-consuming, laborious and not always reliable. A PCR-based diagnostic method was developed with the species-specific primer pairs AatpF and AatpR designed from the sequence of the plasma membrane ATPase gene of E. astragali. The specificity of the primers was verified by PCR analysis of DNA from three strains of E. astragali and 19 isolates of nontarget fungi from other genera. A single 135-bp amplicon was detected only from E. astragali isolates, and no cross-reactions were observed with any other tested isolates. The detection limit of this new method was 5 pg of template DNA. The specific primers enabled the detection of E. astragali from the seed of standing milk vetch. This species-specific PCR method provides quick, simple, powerful and reliable detection of E. astragali. Significance and Impact of the Study PCR-based detection is fast, convenient, precise and sensitive compared to the traditional methods of pathogen detection. This study develops the first PCR method for the detection of Embellisia astragali in standing milk vetch seeds. The species-specific primer set designed from the plasma membrane ATPase gene of E. astragali can detect the pathogen. This assay could be applied in the standing milk vetch seed industry. [ABSTRACT FROM AUTHOR]

The viable plate count ( VPC) is considered as the reference method for bacterial enumeration in periodontal microbiology but shows some important limitations for anaerobic bacteria. As anaerobes such as Porphyromonas gingivalis are difficult to culture, VPC becomes time-consuming and less sensitive. Hence, efficient normalization of experimental data to bacterial cell count requires alternative rapid and reliable quantification methods. This study compared the performance of VPC with that of turbidity measurement and real-time PCR (q PCR) in an experimental context using highly concentrated bacterial suspensions. Our TaqMan-based q PCR assay for P. gingivalis 16S r RNA proved to be sensitive and specific. Turbidity measurements offer a fast method to assess P. gingivalis growth, but suffer from high variability and a limited dynamic range. VPC was very time-consuming and less repeatable than q PCR. Our study concludes that q PCR provides the most rapid and precise approach for P. gingivalis quantification. Although our data were gathered in a specific research context, we believe that our conclusions on the inferior performance of VPC and turbidity measurements in comparison to q PCR can be extended to other research and clinical settings and even to other difficult-to-culture micro-organisms. Significance and Impact of the Study Various clinical and research settings require fast and reliable quantification of bacterial suspensions. The viable plate count method (VPC) is generally seen as 'the gold standard' for bacterial enumeration. However, VPC-based quantification of anaerobes such as Porphyromonas gingivalis is time-consuming due to their stringent growth requirements and shows poor repeatability. Comparison of VPC, turbidity measurement and TaqMan-based q PCR demonstrated that q PCR possesses important advantages regarding speed, accuracy and repeatability. [ABSTRACT FROM AUTHOR]

In this study, we developed and validated a loop-mediated isothermal amplification ( LAMP) assay for Salmonella detection targeting bcfD gene, a conserved fimbrial operon gene existing in Salmonella. The Salmonella LAMP assay we developed successfully amplified 44 Salmonella strains (14 standard strains and 30 clinical isolates), but none of 9 non- Salmonella standard strains ( Proteus mirabilis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Campylobacter jejuni and Vibrio parahemolyticus). The detection limit was 5 CFU of Salmonella pure culture or 200 CFU of artificially spiked faeces per reaction system (equivalent to 5000 CFU g−1 of faeces), and this method could directly detect Salmonella in chicken faeces free of pre-enrichment in a reaction time of 25 min. Our experiments show that the LAMP method we developed is a rapid, sensitive, specific and practical method for Salmonella detection. The Salmonella LAMP assay can potentially serve as new on-site diagnostics in the food and agricultural industries. Significance and Impact of the Study A loop-mediated isothermal amplification (LAMP) assay was established to detect Salmonella targeting bcfD gene, a conserved fimbrial operon gene. The detection limit was 5 CFU of Salmonella pure culture or 200 CFU of artificially spiked faeces per reaction system (equivalent to 5000 CFU g−1 of faeces), and this method could directly detect Salmonella in chicken faeces free of pre-enrichment in a reaction time of 25 min. The Salmonella LAMP assay is a rapid, sensitive, specific and practical method for Salmonella detection and can potentially serve as new on-site diagnostics in the food and agricultural industries. [ABSTRACT FROM AUTHOR]

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (m PCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current m PCR covered much more target bacterial species compared with previously reported species-specific m PCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (m PCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The m PCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The m PCR detection limits for each target bacterial genera were 50 colony-forming units ( CFU) in pure culture and 100 CFU in fish tissue samples. In conclusion, the m PCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. Significance and Impact of the Study The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The m PCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This m PCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice. [ABSTRACT FROM AUTHOR]

Salmonella-contaminated poultry house dust plus 10 g chicken faeces inoculated with Salmonella Enteritidis and then frozen for storage and transport were used as candidate external quality assurance test samples. Variations in faeces sample preparation, storage and culture were examined initially. This indicated that, within modest limits, the age of the inoculating culture and of the faeces did not affect detection, nor did swirling the pre-enrichment culture or extending its duration. Under optimal conditions of preparation and storage, Salmonella numbers of 70 colony-forming units (CFU) and above were reliably detected at the originating laboratory. A ring trial was performed, involving 13 external UK laboratories plus the originating laboratory. Faeces samples inoculated with Salmonella Enteritidis were frozen, transported on dry ice and tested by the ISO 6579:2002 (Annex D) method. Detection by the originating laboratory was consistent with the previously established lower limit for reliability of 70 CFU. However, the sensitivity of detection by the external laboratories was apparently poorer in several cases, with significant interlaboratory variation seen at the lowest inoculum level, using Fisher's exact test. Detection of Salmonella in poultry house dust appeared to be more sensitive and uniform among laboratories. Significance and Impact of the Study Salmonella surveillance and control regimes in the European poultry industry and elsewhere require sensitive culture detection of Salmonella in environmental samples, including poultry faeces. A ring trial was conducted, and the results highlighted that some of the participating laboratories failed to identify Salmonella. This suggests that contaminated frozen faeces cubes could be beneficial to assess proficiency, according to the results of this preliminary study. The data obtained in this study can be used as an indication for the design of realistic external quality assurance for laboratories involved in official testing of Salmonella in poultry flocks. [ABSTRACT FROM AUTHOR]

Accurate and rapid analytical methods are essential for the detection and identification of biological warfare agents ( BWA). Although various studies have investigated the use of MALDI- TOF MS for bacterial classification, only a few studies have examined the applicability of method for the identification of BWAs. This study aimed to generate, collect and analyse Bacillus spore aerosol particles of 2-10 μm, the optimal size of a BWA. Bacillus spore aerosol particles of 2-10 μm were rapidly analysed using direct in situ MALDI- TOF MS without pretreatment processes. A mass spectrum of Bacillus spore aerosol particles was successfully detected. For real-time detection and identification, a mass spectral database of Bacillus spores was constructed, and an algorithm was developed and applied. Bacillus spore particles were rapidly detected and identified by MS, which can be used for the detection and inspection of BWAs. Significance and Impact of the Study Methods that permit accurate and fast detection and identification are essential in the case of pathogens such as Bacillus anthracis, which can be used as biological warfare agents or bioterrorism materials. Here, direct in situ MALDI- TOF MS allowed the high-throughput detection and identification of aerosolized Bacillus spore particles, and this method could be effectively applied for the detection and inspection of BWAs. In this context, we believe that this method may contribute to the realization of real-time and on-site identification of BWAs. [ABSTRACT FROM AUTHOR]

F + RNA phages are commonly used as indicators of faecal contamination. This study evaluated a fluorescent method for the detection of F + RNA phages based on testing the phage-mediated release of β-galactosidase. Factors that may potentially interfere with phage detection were investigated, and the assay was optimized. Low numbers of F + RNA phages were detected by the fluorescent method coupled with a concentration step using a Disruptor filter. The fluorescent method, when used alone, detected 1 log PFU ml−1 of F+RNA phages within 3 h, while 0·01 PFU ml−1 was detected within 5 h when the method was combined with the concentration step. This is the first time to combine a fluorescent method with a filtration step by the use of Disruptor filter for rapid detection of low numbers of F + RNA phages, and this method can be adapted to detect other lytic phages infecting host cells that produce measurable enzyme activity. Significance and Impact of the Study A fluorescent method coupled with Disruptor filtration was evaluated for the first time to rapidly detect low numbers of F + RNA phages. Rapid detection of F + RNA phages provides an effective way to monitor faecal contamination of environmental water and thus helps prevent contamination of fresh produce via irrigation. [ABSTRACT FROM AUTHOR]

Propidium monoazide is a DNA-intercalating dye. PMA- qPCR has been reported as a novel method to detect live bacteria in complex samples. In this study, this method was used to monitor the sterilization effects of UHP, ultrasound and high PEF on Escherichia coli O157: H7. Our results showed that all three sterilization techniques are successful to kill viable E. coli O157: H7 cells under their appropriate conditions. PMA- qPCR can effectively monitor the amount of DNA released from viable E. coli O157: H7 cells, and the results from PMA- qPCR were highly consistent with those from plate counting after treatment with UHP, ultrasound and high PEF. The maximal Δ Ct between PMA- qPCR and qPCR obtained in this study was 10·39 for UHP, 5·76 for ultrasound and 2·30 for high PEF. The maximal sterilization rates monitored by PMA- qPCR were 99·92% for UHP, 99·99% for ultrasound and 100% for high PEF. Thus, PMA- qPCR can be used to detect the sterilization effect on food and water supplies after treatment with UHP, ultrasound and high PEF. [ABSTRACT FROM AUTHOR]

Curvularia lunata ( Wakker) Boed, the causative agent of Curvularia leaf spot in maize, was determined according to conidiophore and conidium morphology in a previous study. In the current study, a sensitive polymerase chain reaction assay was developed for the detection of C. lunata. Two specific forward ( Clg D1/ Clg D2) and one reverse primers ( Clg D3) were designed based on a Ras-related ( Clg2p) gene. Eight C. lunata isolates that represent different virulent strains in maize, six other Curvularia spp., and 22 fungal plant pathogens were used to test the specificity of the primers. PCR amplification using Clg D1/ Clg D3 as the first-round primers resulted in an 870-bp band from the C. lunata isolates. The detection sensitivity using Clg D1/ Clg D3 was 100 pg of genomic DNA. In the second round of PCR, a 1 : 50 dilution of the first-round PCR products was used as a template with the Clg D2/ Clg D3 primer pair, which increased the detection sensitivity to 1 fg. This semi-nested PCR procedure could also be used to detect C. lunata from infected maize leaves. The proposed PCR-based assay may be used for diagnosing and monitoring maize Curvularia leaf spot. Significance and Impact of the Study The semi-nested PCR assay may provide researchers and laboratory technologists a tool to rapidly detect C. lunata, which causes maize Curvularia leaf spot, compared with histological examination. [ABSTRACT FROM AUTHOR]

The object of this study was to develop a reverse transcription-loop-mediated isothermal amplification ( RT-LAMP) assay for detecting hepatitis D virus ( HDV) genotype 1. With an alignment analysis, a highly conserved sequence (nt 820-1020) was chosen as a suitable target to design LAMP primers. The optimal condition of RT-LAMP was a 25-μl reaction volume, which consists of the following components: 1·6 μmol l−1 each of FIP and BIP, 0·2 μmol l−1 each of F3 and B3, 1·5 μmol l−1 dNTPs, 4 mmol l−1 MgSO4, 8 U Bst DNA polymerase, 2U M-MLV and 2 μl extracted RNA sample. The amplification reaction was carried out at 65°C for 50 min. Compared with conventional qualitative or quantitative real-time reverse transcription polymerase chain reaction, the results of RT-LAMP indicated a 1000-fold increase in sensitivity for detecting HDV. There was no cross-reaction for the RT-LAMP method between HDV 1 and HIV, HAV, HBV, HCV and HEV. [ABSTRACT FROM AUTHOR]

Aims: To design a primer set enabling the identification through PCR of highquality DNA for routine and high-throughput genomic screening of a diverse range of cyanobacteria. Methods and Results: A codon-equivalent multiple alignment of the phycocyanin alpha-subunit coding sequence (cpcA) of 22 cyanobacteria was generated and analysed to produce a single degeneracy primer set with virtually uniform product size. Also, an 18S ribosomal RNA detection set is proposed for rejecting false positives. The primer sets were tested against five diverse cyanobacteria, Chlorella vulgaris, Saccharomyces cerevisiae, and Escherichia coli. All five cyanobacteria showed positive amplification of cpcA product with homogeneous fragment length, and no products were observed for any other organism. Additionally, the only product formation observed for the 18S rRNA set was in C. vulgaris and S. cerevisiae. Conclusions: The newly proposed primer set served as effective check primers for cyanobacteria. Cyanobacteria gDNA had a positive, homogenous result, while other bacteria, eukaryotes and alga tested were negative. Significance and Impact of the Study: These novel, broad-spectrum primers will greatly increase the utility of PCR on newly discovered cyanobacterial species. [ABSTRACT FROM AUTHOR]

Aims: This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. Methods and Results: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. Conclusions: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. Significance and Impact of the Study: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction. [ABSTRACT FROM AUTHOR]

Aims: A rapid real-time PCR-based method for the detection of Listeria monocytogenes was applied to the examination of 44 Quargel cheese samples from a recent outbreak in Austria and compared to the standard method according to ISO-16140. Methods and Results: The combined enrichment/real-time PCR method amplifying the prfA locus was performed according to [Rossmanith et al. (2006) Res Microbiol, 157, 763-771]. Qualitative and quantitative examination of the samples was performed according to the standard method ISO-11290. Comparison of the combined enrichment/real-time PCR method with ISO-11290 resulted in 100% relative accuracy, 100% relative sensitivity and 100% relative specificity. Conclusions: A previously published study describing the validation of the method, including samples after storage at −80°C, resulted in lower performance values. In contrast, the samples were stored at +4°C in this study. The results of this study indicate an effect of storage, thus masking the true performance of the method. Significance and Impact of the Study: The results of this study are discussed together with the previously published data to demonstrate the excellent qualities of this rapid (≤30 h) method when applied to fresh specimens stored at +4°C. [ABSTRACT FROM AUTHOR]

Aims: A diagnostic protocol was developed for rapid detection of Vibrio corallilyticus by method of loop-mediated isothermal amplification (LAMP). Methods and Results: For cloning and sequencing of rpo A gene of V. corallilyticus, a set of four LAMP primers were designed by targeting the rpoA gene. With Bst DNA polymerase, the reaction time and temperature were optimized for 70 min at 65°C, respectively. The amplification products were detected by electrophoresis. The detection limit of V. corallilyticus by LAMP was 3·6×103 CFU ml−1 (8 CFU per reaction), but PCR could detect up to 3·6×104 CFU ml−1 (72 CFU per reaction). The LAMP method was ninefold more sensitive than conventional PCR. The results also indicated that the LAMP reaction was highly specific to V. corallilyticus. Conclusions: The LAMP assay was a sensitive, specific and cost-effective method for the rapid detection of V. corallilyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. corallilyticus infection. It can replace laborious biochemical tests for the identification of V. corallilyticus. [ABSTRACT FROM AUTHOR]

Aims:  Vibrio vulnificus is a major cause of seafood-related deaths in the United States. Several biomarkers, e.g. the virulence-correlated gene ( vcg), 16S rRNA, and the capsular polysaccharide operon (CPS) have been used to differentiate virulent- from nonvirulent-type V. vulnificus strains. In this study, we combined the use of these biomarkers with a species-specific V. vulnificus cytolysin/haemolysin gene ( vvhA) to develop two pairs of multiplex PCR assays that simultaneously detect and characterize V. vulnificus strains. Methods and Results:  The first multiplex PCR pair amplified four genes ( vvhA, vcg, 16S rRNA, and CPS), with one for virulent-type and the other one for nonvirulent-type V. vulnificus strains, while the second pair targeted three of those genes excluding CPS. Primer concentration and annealing temperature were optimized for the four multiplex PCR assays. When testing ten V. vulnificus reference strains and 80 field oyster isolates, results from each multiplex PCR matched 100% with known strain characteristics for these target genes. Conclusions:  The optimized multiplex PCR assays were capable of simultaneously detecting and characterizing V. vulnificus with high specificity and speed. Significance and Impact of the Study:  Multiplex PCR assays designed in this study are valuable tools for microbial ecology and epidemiology studies. They may facilitate better control of V. vulnificus risks in oysters, thereby reducing the number of illnesses and deaths because of V. vulnificus in the long run. [ABSTRACT FROM AUTHOR]

Aims: To develop fast and reliable sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4-mm-diameter, 43-cm-long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1–4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56·6% for the scheme based on a single drill to 96·8% for the scheme based on four drillings analysed separately. Both schemes significantly differ ( P < 0·05) from those based on two and three drillings, whose efficiency was 72·6% and 83·9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of the Study: Fast and reliable sampling procedures make DNA-based diagnosis more suitable for tree inspection procedures. [ABSTRACT FROM AUTHOR]

Aims: The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. Methods and Results: A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65°C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10−5 dilution level (equivalent to 17·2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. Conclusions: The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. Significance and Impact of the Study: This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to develop a real-time quantitative PCR test to recognize and quantify the DNA levels of the increasingly important barley pathogen Ramularia collo-cygni. Methods and Results: The method described uses specifically designed primers and a molecular beacon probe based on an internal transcribed spacer (ITS) sequence. Pathogen extracted from barley leaves could be quantified to the picogram level in both leaves showing symptoms of infection and symptomless barley leaves. Conclusions: A relationship between R. collo-cygni DNA levels and disease symptoms was established in spring barley under natural infection conditions. Significance and Impact of the Study: To our knowledge, this is the first report of a test of this type and makes an important contribution to studies into the life cycle of this pathogen. [ABSTRACT FROM AUTHOR]

Aims: In this study, we report a new, simple methodology for the monitoring of Cryptosporidium oocysts and Giardia cysts in drinking water samples, ranging from 10- to 1000-l, which combines a new ARAD microfibre filtration of the (oo)cysts from drinking water and loop-mediated isothermal amplification (LAMP) of a human pathogenic Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium meleagridis and Giardia duodenalis Assemblage A and B specific DNA sequence. Methods and Results: During the evaluation of the new concentration and detection technique, spiked reagent and matrix water samples plus blank samples were filtered and tested. In total, 27 samples have been investigated. The results clearly demonstrate that the methodology of using a new ARAD filter, which passed through 1000 l of drinking water with high turbidity (2 NTU), and followed by the LAMP assay was able to detect at least one (oo)cyst in 10 l of drinking water based on a 1000-l sample, taken over a 24-h period. Conclusions: The described protozoa detection methodology is sensitive, rapid and cost-effective. Significance and Impact of the Study: This effective procedure will be useful for small waterworks to achieve continuous monitoring and is also of value for screening catchments to identify those that require further treatment and more detailed microscopic counts. [ABSTRACT FROM AUTHOR]

Aims: To develop a scanning electron microscopic approach using in situ hybridization (SEM–ISH) for gaining both genetic and morphological information about target bacteria. Methods and Results: Target cells were hybridized with DNA-targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM–ISH enabled to detect low copy number target DNA sequences in individual cells. Conclusions: SEM–ISH allowed the in situ detection of bacteria carrying a specific gene. Significance and Impact of the Study: Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials. [ABSTRACT FROM AUTHOR]

Aims: To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV). Methods and Results: Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption–elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6·9 × 104 and 1·5 × 104 copies l−1, respectively. Conclusions: Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks. Significance and Impact of the Study: This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period. [ABSTRACT FROM AUTHOR]