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The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing., (© 2021 John Wiley & Sons, Ltd.)

The method of direct quantitation of Lactobacillus spp. and L. acidophilus in intestinal contents based on real-time PCR was developed. It does not require culturing and allows estimating the number of living lactobacilli cells (measured in lg CFU) in absolute quantitative PCR format., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order to adopt this diagnostic method, the Italian National Reference Centre for Equine Diseases (NRC-ED) conducted a preliminary comparison between an end-point PCR, nested PCR, real-time PCR, and commercial real-time PCR, for the detection of T. equi and B. caballi, respectively. One hundred and three field samples, collected during spring-summer 2013 in Latium and Tuscany regions, were employed for the study, and results discordant between detection assays were confirmed by sequencing. The reference assay was defined as that showing the highest sensitivity, and the relative sensitivity (rSe) and specificity (rSp) of the other methods were estimated referring to this assay. Agreement between methods was estimated by calculating the concordance between each pair of methods. Although no statistical differences were detected among PCR-based methods, the non-commercial real-time PCR assays seemed to be the most suitable for detection of T. equi and B. caballi, respectively. An important advantage of direct PCR detection of the pathogen, in comparison to indirect detection using serological methods, is that it allows specific treatment against the causative pathogen species responsible of the infection as well as for the definition of the infectious status of an animal for international movement.

MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3'-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time., (© 2020 Her Majesty the Queen in Right of Canada Transboundary and Emerging Diseases © 2020 Wiley-VCH GmbH.)

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE , cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.

The unfolded protein response is a cellular adaptive mechanism localized in the endoplasmic reticulum. It involves three phases: the detection of increased presence of unfolded proteins as a result of cellular stressors; the execution of an adaptive cascade of events aimed at the enhancement of proper protein folding and degradation of improperly folded proteins; and finally, when stress is not alleviated, the execution of programmed cell death. The main effectors of the UPR are transcription factors involved in the upregulation of either chaperone proteins or proapoptotic proteins. Two of these transcription factors are CHOP and the spliced variant of XBP-1 (XBP1s). In this chapter, we describe a quantitative PCR method to detect the upregulation of CHOP and XBP1s mRNA during Tunicamycin-induced UPR.

Background: Droplet digital PCR (ddPCR) is a technology that has higher sensitivity than real-time PCR for the identification of trace DNA. However, the use of ddPCR for the detection of Mycobacterium tuberculosis DNA in pathological samples has not been fully studied., Methods: A total of 65 formalin-fixed and paraffin-embedded (FFPE) specimens were included in this study. Twenty samples with definite results for tuberculosis (TB) were used to establish the ddPCR system for TB detection. ddPCR was then conducted to detect TB DNA in the 45 patients who were classified as 'possible TB' (real-time PCR results in the gray area, Ziehl-Neelsen staining-negative, and hematoxylin and eosin staining showing morphology suspicious for TB). The clinical treatment and disease outcomes were followed to assess the accuracy of ddPCR in the detection of TB DNA., Results: Among the 45 possible TB samples, 26 were ddPCR-positive, 12 were ddPCR-negative, and seven were in the gray area. ddPCR improved the positive rate of 57.8% (26/45) for the samples that were in the gray area by real-time PCR. Moreover, several patients received anti-TB therapy, and the effective ratio of therapy for the ddPCR-positive, ddPCR-negative, and ddPCR-gray area cases was 61.9% (13/21), 50.0% (2/4), and 33.3% (1/3), respectively., Conclusions: ddPCR is more sensitive for detecting mild TB via FFPE samples than real-time PCR. The ddPCR method is of additional value in the diagnosis of TB from pathological samples., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The presence of Burkholderia cepacia complex (BCC) strains has resulted in recalls of pharmaceutical products, since these opportunistic pathogens can cause serious infections. Rapid and sensitive diagnostic methods to detect BCC are crucial to determine contamination levels. We evaluated bacterial cultures, real-time PCR (qPCR), droplet digital PCR (ddPCR), and flow cytometry to detect BCC in nuclease-free water, in chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions. Twenty BCC strains were each suspended (1, 10, 100, and 1000 CFU/ml) in autoclaved nuclease-free water, 10 μg/ml CHX, and 50 μg/ml BZK. Five replicates of each strain were tested at each concentration (20 strains × 4 concentrations × 5 replicates = 400 tests) to detect BCC using the aforementioned four methods. We demonstrated the potential of ddPCR and flow cytometry as more sensitive alternatives to culture-based methods to detect BCC in autoclaved nuclease-free water and antiseptics samples.

Molecular diagnostic assays for Cryptosporidium are usually based on PCR and may detect the entire genus or target specified species. Of the ~40 species, fewer than half have been reported from humans, and most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis. Here we describe a nested PCR for the detection of all Cryptosporidium spp. that can then be differentiated by sequencing the PCR amplicons, and a duplex, real-time PCR for the simultaneous detection and differentiation of C. parvum and C. hominis.

Adulteration of the high-value silver pomfret (Pampus argenteus) is a serious problem worldwide, necessitating accurate identification and quantification of the species. In this study, optimisation of the digital droplet PCR (ddPCR) assay for the identification and quantification of the silver pomfret was carried out. The primer and probe concentrations, melting temperature, and PCR cycle number were optimised by combining single-factor experiments with an orthogonal experimental design. The absolute limits of detection and quantification of the ddPCR were 2copies/μl and 21 copies/μl, respectively. Its sensitivity was 0.1% for meat mixtures and 0.5% for DNA mixtures. The ddPCR was 156 times more sensitive than the real-time PCR, although both methods had similar specificities. However, the overall time needed to complete the ddPCR method was twice that of the real-time PCR. Notwithstanding, the ddPCR methodology established in this study can be a valuable tool for addressing species adulteration issues., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales . Here, we developed a multiplex Taq Man-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and Taq Man fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 10 2 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Aggressive red imported fire ants (RIFAs) are expanding their habitat due to active international trade and global warming. To prevent infestation and settlement, RIFAs must be removed during the quarantine process. Because RIFAs are social insects and have different morphological characteristics depending on their castes, non-ant taxonomists have difficulty confirming RIFAs based on their morphological characteristics alone. The disadvantages of previously reported RIFA molecular diagnostics are that they require additional steps, such as restriction enzyme digestion followed by agarose gel electrophoresis separation or DNA sequence verification for polymerase chain reaction (PCR)-amplified products. To overcome these drawbacks, two RIFA-specific genes were selected and used to develop diverse PCR-based RIFA molecular diagnostic techniques. We found that RIFAs could be confirmed by conventional PCR targeting of two RIFA-specific genes followed by agarose electrophoresis separation. In addition, TaqMan probe real-time PCR methods had the advantage of confirming RIFAs immediately after the reactions were completed by observing fluorescence indexes. Finally, multiplex PCRs enhanced RIFA specificity and sensitivity. The new molecular diagnostic methods developed in this study had the advantages of reducing false positive and negative results together with high specificity and sensitivity for RIFAs., (© 2019 Wiley Periodicals, Inc.)

Brucellosis is primarily a worldwide zoonotic disease caused by Brucella species. The genus Brucella contains highly infectious species that are classified as biological threat agents. In this regard, the identification of Brucella can be a time-consuming and labor-intensive process posing a real risk of laboratory-acquired infection to the laboratory staff. This study aimed to present a novel conventional and real-time polymerase chain reaction (PCR) assay for the identification of Brucella abortus strains. Regarding this, two primers (bru ab2) were designed based on the unique loci encoding autotransporter-associated beta strand repeat-containing protein (ID:YP00113760). A total of 56 Brucella strains (e.g., reference, vaccinal, and field isolates) and Yersinia enterocolitica, as a non-Brucella isolate, were evaluated in conventional and real-time PCR systems. The results of the study indicated that 0.4 ng and 400 FG of genomic DNA of B. abortus strains can be detected by conventional and real-time PCR, respectively. The primers, bru ab2, were suitable for both PCR methods. Both methods were specific for the detection of all strains of the bacterium; however, real-time PCR assay was 1000-fold more sensitive than the conventional PCR method. Therefore, this new detection system could be a suitable selective modified method for the accurate identification of all B. abortus strains., (Copyright © 2019, Archives of Razi Institute. Published by Kowsar.)

The protozoan parasite Tritrichomonas foetus may cause severe diarrhea in cats all over the world. In order to evaluate the methodology in coprological molecular diagnosis of feline tritrichomonosis, we compared previously published ("old") and newly developed ("novel") loop-mediated isothermal amplification (LAMP) (targeted to the T. foetus β-tubulin and the elf1α 1 gene, respectively) as well as an old conventional and an old and novel real-time PCR (all targeted to overlapping regions of T. foetus rDNA) assays regarding their diagnostic sensitivities and specificities. Here, the novel real-time PCR yielded the best methodical performance in that a sensitivity with a detection limit of <0.1 trophozoites (corresponding to ca.<0.13 trophozoites per mg feces) and a maximal specificity for diagnosis of Tritrichomonas spp. was achieved. The other test systems exhibited either an approximately 10-times lower sensitivity (<1 trophozoite corresponding to ca.<1.3 trophozoites per mg feces) (conventional PCR and both LAMP assays) or a lower specificity (old real-time PCR). Conversely, the diagnostic performance assessed with clinical fecal samples from cats demonstrated identical sensitivities (8 of 20 samples tested were positive) for the novel PCR and both LAMP assays. Diagnostic sensitivities were significantly higher than those found for the old real-time (5 positive samples) and conventional PCR (6 positive samples), respectively. Accordingly, our data suggested the novel PCR and both LAMP assays to be well suited molecular tools for direct (i.e. without including an in vitro cultivation step) coprological diagnosis of tritrichomonosis in cats. Interestingly, relative high (novel LAMP, 7 positive samples) to at least moderate (old LAMP, 6 positive samples and 1 sample with equivocal score) diagnostic sensitivities were also achieved by testing clinical samples upon simple visual inspection of colorimetric changes during the LAMP amplification reactions. Accordingly, both LAMP assays may serve as practical molecular tools to perform epidemiological studies on feline (and bovine as well as porcine) tritrichomonosis under simple laboratory conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a major human pathogen, causing a variety of nosocomial and community-acquired infections. While S. aureus usually grows well, there are situations where it cannot be isolated in culture, such as patients who have received prior antimicrobial therapy. There are commercially available tests for molecular identification of S. aureus and methicillin resistance; however, they often have limited utility due to restrictive specimen requirements, lack of data in pediatric populations and issues with specificity for methicillin resistance detections. Our objective was to evaluate the performance of laboratory-developed PCR assays that detect S. aureus and methicillin resistance directly from various specimen types. We developed two real-time PCR assays: 1) a singleplex assay targeting the nucA gene and 2) a multiplex PCR assay (mecA/SCC-orf PCRs) that detects the mecA gene and the conjunction region where SCCmec elements insert into the genome. A total of 538 pediatric specimens, including specimens from the lower respiratory tract (n = 149), abscess/wounds (n = 245), tissue and body fluids (n = 144), were tested and the results compared with culture and susceptibility testing. The nucA PCR is sensitive and specific for detection of S. aureus when compared with culture with an overall agreement of 93.1% and sensitivity and specificity of 93.5% and 93.0%, respectively. Among those culture-confirmed and nucA PCR positive specimens (n = 145), concordance between mecA/SCC-orf PCRs, using cycle threshold values for corroboration, and conventional methods was 98.6% and the sensitivity and specificity were 97.3% and 100%, respectively. The assays' performance suggests they are rapid, reliable tools to detect and differentiate between methicillin susceptible and methicillin resistant S. aureus in our pediatric patient population providing diagnostic impact when used in conjunction with culture., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD., Methods: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis., Results: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD., Conclusions: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis., Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 10 3  CFU/ml to 10 3 and 10 6  CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 10 2  CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories., Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

Chronic inflammatory diseases can be particularly challenging to diagnose and characterize, as inflammatory changes in tissue may not be present in blood. There is a crucial need to develop non-invasive biomarkers that would be useful in diagnosing disease and selecting medical therapies. For example, there are no blood tests to diagnose asthma, a common inflammatory lung disease. MicroRNA (miRNA) expression profiling in blood is emerging as a potentially sensitive and useful biomarker of many diseases. In particular, we have characterized a cost-effective PCR-based array technology to measure and profile circulating miRNAs in the plasma of patients with allergic rhinitis and asthma. Here, we describe the methods to isolate, quantify, and analyze miRNAs in the plasma of human subjects as well as ways to determine their diagnostic utility., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection., (Copyright © 2018. Published by Elsevier España, S.L.U.)

Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 108 copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.

Digital PCR (dPCR) is a 3rd generation technology that complements traditional end-point PCR and real-time PCR. It was developed to overcome certain limitations of conventional amplification techniques, in particular for the detection of small amounts of nucleic acids and/or rare variants. This technology is in a full swing because of its high sensitivity and major applications in various domains such as oncology, transplantation or non-invasive prenatal testing. Consequently, PCRd also has great interest in many areas of medical biology, particularly for clinical applications aiming at detecting and quantifying specific genetic or epigenetic alterations of nucleic acids, even with specimens containing very low concentration of the nucleic acids of interest (e.g. liquid biopsies). However, this technique requires a good training of users and compliance with certain precautions. A lack in such a knowledge can lead to many errors in the conduct of the experiment and the interpretation of the results. In this review, we present the context in which this technology has emerged by describing in particular its principle and the main factors that can influence the quality of the analysis. Then, we propose a number of practical recommendations for the implementation of a test based on dPCR in clinical laboratories with an eye on quality requirements.

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender-specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell-free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300-600 bp) in maternal circulation. The aim of this study was to assess this non-invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non-pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single-tube extraction without silicone membranes and phenol-chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real-time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single-tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real-time PCR test sensitivity in Group I was 90% and in Group II 91.6%., (© 2018 Blackwell Verlag GmbH.)

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

Objective: To compare two influenza polymerase chain reaction (PCR) methods., Methods: A total of 749 suspected MERS-CoV patients presenting at Johns Hopkins Aramco Healthcare, Saudi Arabia, each submitted a clinical sample for influenza A reflex testing using the on-site Cepheid® Xpert Flu assay and at the Ministry of Health laboratory by the Roche PCR assay., Results: There was 92.12% overall agreement between the two methods. Specificity of the Cepheid® Xpert Flu was 95.8% for H1N1 and 94.4% for total influenza A. Cepheid® Xpert Flu sensitivity for influenza A was 100% for younger patients (0-19-year age group) but significantly lower both for older patients (68.2% for 60-79-year and 50% for ≥80-year age groups) and overall for males compared to females (72.6% and 94.0%, respectively)., Conclusions: Specificity of the Cepheid® Xpert Flu test was high; however, sensitivity for total influenza A was lower particularly in males and older patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Weeds are serious problem in crop production and wild oat is a grass weed of economic and agronomic significance. We need to extend our basic knowledge of weeds especially in molecular genetics and gene expression. For study of gene expression by semi-quantitative and quantitative PCR, it is recommended that normalization of reference genes be carried out in order to select the most stable reference gene for a precise gene expression study. The purpose of this research was evaluation of four reference genes in response to treated and untreated (control) by herbicide in two tissues (stem and leaf) of non-target site resistance wild oat (A. ludoviciana). Four candidate reference genes including Actin, Ef1α (elongation factor 1 alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and TBP (TATA-box-binding protein) were used to determine stable reference gene exposed to the herbicide using the statistical methods of NormFinder, BestKeeper and delta-Ct. NormFinder indicated that TBP and Actin genes are the best combination of two genes for normalizing calculations (with a combined gene stability value of 0.012) for qPCR analysis under herbicide stress in different tissues of non-target site resistance wild oat. Based on the statistical results, the Ef1α gene was identified as the unstable reference gene. Totally, according to results of this study, TBP gene is the most stable reference gene and therefore, this gene can be used as a reference gene for future studies of quantitative PCR analysis of herbicide stress-responsive gene expression in wild oat and potentially in other grass weed species.

Wilms' tumor 1 (WT1) is overexpressed in various hematopoietic tumors and widely used as a marker of minimal residual disease. WT1 mRNA has been analyzed using quantitative real-time polymerase chain reaction (real-time PCR). In the present study, we analyzed 40 peripheral blood and bone marrow samples obtained from cases of acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome at Sapporo Medical University Hospital from April 2012 to January 2015. We performed quantification of WT1 was performed using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific‎) and compared the results between digital PCR and real-time PCR technology. The correlation between digital PCR and real-time PCR was very strong (R = 0.99), and the detection limits of the two methods were equivalent. Digital PCR was able to accurately detect lower WT levels compared with real-time PCR. Digital PCR technology can thus be utilized to predict WT1/ABL1 expression level accurately and should thus be useful for diagnosis or the evaluation of drug efficiency in patients with leukemia.

Precise diagnosis is a key measurement for malaria control and elimination, traditional microscopy and rapid diagnostic tests cannot satisfy the requirements especially in the low transmission endemic areas or in the malaria elimination phase. Polymerase chain reaction (PCR) with high sensitivity and specificity can be considered as a diagnostic standard while no uniform PCR assay was established due to variations in their performance and lack of formal external quality assurance programs for validation for PCR assays in use. Here, 24 articles including 43 paired comparative evaluations limited to paired comparison of diagnostic performance between real-time PCR and conventional PCR to detect plasmodium in blood samples of human subjects from clinics or the field are systematically summarized. And according to the Landis and Koch classification, nineteen pairs showed almost perfect agreement, followed by 8 pairs of moderate agreement and 4 pairs of good agreement, while the kappa values of 12 pairs couldn't be examined. Moreover, the performance of 14 pairs were completely the same and 8 pairs had no differences, but 14 pairs were significant different including 8 pairs of real-time PCR with better performance than conventional PCR. Therefore, it is still an outstanding issue to choose PCR methods, and more work such as the standardization of materials and methods in use and their availability are needed to settle priority to better promote the role of malaria diagnosis reference laboratories.

After several decades of relatively modest use, in the last several years digital PCR (dPCR) has grown to become the new gold standard for nucleic acid quantification. This coincides with the commercial availability of scalable, affordable, and reproducible droplet-based dPCR platforms in the past five years and has led to its rapid dissemination into diverse research fields and testing applications. Among these, it has been adopted most vigorously into clinical oncology where it is beginning to be used for plasma genotyping in cancer patients undergoing treatment. Additionally, innovation across the scientific community has extended the benefits of reaction partitioning beyond DNA and RNA quantification alone, and demonstrated its usefulness in evaluating DNA size and integrity, the physical linkage of colocalized markers, levels of enzyme activity and specific cation concentrations in a sample, and more. As dPCR technology gains in popularity and breadth, its power and simplicity can often be taken for granted; thus, the reader is reminded that due diligence must be exercised in order to make claims not only of precision but also of accuracy in their measurements.

Purpose: Bartonella is an increasingly isolated emerging pathogen that can cause severe illness in humans, including cat scratch disease (CSD). The bacteria are difficult to grow and thus many detection methods have been developed, especially molecular. We previously developed a PCR method targeting ribC to identify Bartonella sp. A manufactured kit (RealCycler BART, Progenie Molecular) was commercialised shortly thereafter for the detection of Bartonella infection, including Bartonella henselae., Methodology: We performed a comparison between this test and our in-house PCR assay on 73 lymphadenopathy samples sent to the laboratory for suspicion of CSD.Results/Key findings. Among the 28 positive samples for Bartonella, 21 were identified by the two PCR assays, and seven by the commercial kit only., Conclusion: The performance of this commercial kit suggests that it could be a suitable alternative to our in-house PCR assay, highlighting the importance of the molecular methods used to diagnose CSD.

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species., (Copyright © 2017 Kordalewska et al.)

DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to develop a molecular technique to determine the level of human originated DNA contamination in unhygienic food products. In the study, four model foods were prepared under both hygienic (H) and non-hygienic (NH) conditions and the human originated microbial loads of these products were determined. DNA was extracted from the model foods and human buccal samples by GIDAGEN Multi-fast DNA isolation kit. A primer specific region of human mitochondrial D-Loop was designed. The level of human DNA contamination in the model foods was determined by real-time PCR. The sensitivity of the technique developed here was 0.00001ng DNA/PCR. In addition, the applicability of the traceable molecular hygiene control method (TMHCM) was tested in 60 food samples from the market. The results of this study demonstrate that DNA based TMHCM can be used to predict to what extent foods meet the human oriented hygienic conditions., (Copyright © 2016. Published by Elsevier Ltd.)

Background: Ionising radiation induces molecular alterations, such as formation of ions, free radicals, and new stable molecules, and cleavage of the chemical bonds of the molecules present in food. Irradiation-treated meat should be labelled to control the process and to ensure free consumer choice. Therefore, sensitive analytical methods are required to detect the irradiation dose., Results: Meat samples were exposed to radiation doses of 0, 0.272, 0.497, 1.063, 3.64, 8.82 and 17.42 kGy in an industrial 60 Co gamma cell. Primers were designed to amplify 998, 498 and 250-base pair (bp) regions of the 18S rRNA gene of nuclear DNA from the irradiated samples. A new DNA-based method was developed to quantify the radiation exposed to the unstored meat and the meat stored at -20 °C for 3 and 6 months. The method was able to detect meat samples stored and unstored with dose limits of 1.063 and 3.64 kGy, respectively., Conclusion: The level of irradiation can be detected using primer pairs that target particularly different-sized sequences for DNA amplification by PCR. This method can be widely used for the analysis of not only meat samples, but also all biological materials containing DNA. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Shortage of human organs forced the development of xenotransplantation using cells, tissues, and organs from pigs. Xenotransplantation may be associated with the transmission of porcine zoonotic microorganisms, among them the porcine cytomegalovirus (PCMV). To prevent virus transmission, pigs have to be screened using sensitive methods. In order to perform regular follow-ups and further breeding of the animals, samples for testing should be collected by low-invasive or non-invasive methods. Sera, ear biopsies, as well as oral and anal swabs were collected from ten 10-day-old Aachen minipigs (AaMP) and tested for PCMV using sensitive nested polymerase chain reaction (PCR) as well as uniplex and duplex real-time PCR. Porcine cytomegalovirus DNA was detected most frequently in oral and anal swabs. Comparison of duplex and uniplex real-time PCR systems for PCMV detection demonstrated a lower sensitivity of duplex real-time PCR when the copy numbers of the target genes were low (less 200). Therefore, to increase the efficacy of PCMV detection in piglets, early testing of oral and anal swabs by uniplex real-time PCR is recommended., Competing Interests: G.H. produces and supplies the Aachen Minipigs. The other authors report no conflict of interests.

Uveitis is a sight-threatening intraocular inflammatory disorder which may occur from both infectious and non-infectious or autoimmune causes. The frequency of infectious uveitis and autoimmune uveitis varies depending on countries and regions. According to a nationwide survey conducted by the Japanese Ocular Inflammation Society, infectious and non-infectious uveitis accounted for 16.4 and 50.1% of new patients, respectively while the remaining 33.5% of new uveitis cases were not classified or were idiopathic uveitis. Infectious uveitis is particularly important because it causes tissue damage to the eye and may result in blindness unless treated. However, it can be treated if the pathogenic microorganisms are identified promptly and accurately. Remarkable advancements in molecular and immunological technologies have been made in the last decade, and the diagnosis of infectious uveitis has been greatly improved by the application of molecular and immunological investigations, particularly polymerase chain reaction (PCR). PCR performed on a small amount of ocular samples provides a prompt, sensitive, and specific molecular diagnosis of pathogenic microorganisms in the eye. This technology has opened a new era in the diagnosis and treatment of uveitis, enabling physicians to establish new clinical entities of uveitis caused by infectious microorganisms, identify pathogens in the eyes of many patients with uveitis, and determine prompt diagnosis and appropriate therapy. Here we review the PCR process, new PCR tests specialized for ocular diseases, microorganisms detected by the PCR tests, diseases in the eye caused by these microorganisms, and the clinical characteristics, diagnosis, and therapy of uveitis.

Acanthamoeba species are free-living amebae that can be found in almost every range of environments. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease that is predominantly associated with contact lens use, the epidemiology of which is related to the specific genotype of Acanthamoeba. This study reports seven (7/16; 43.75%) positive cases. Detection of Acanthamoeba in corneal scrapings is based on cultivation and polymerase chain reaction (PCR) combined with the molecular taxonomic identification method. By PCR, seven samples were positive; cultivation was successful for five samples, probably because of the low quantity of samples. Genotype identification was carried out with a real-time fluorescence resonance energy transfer PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. All seven detected Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK from human samples. Genotyping allowed the identification of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in Hungary.

Purpose: Fungi of Scopulariopsis and Microascus genera cause a wide range of infections, with S. brevicaulis being the most prevalent aetiological agent of mould onychomycosis. Proper identification of these pathogens requires sporulating culture, which considerably delays the diagnosis. So far, sequencing of rDNA regions of clinical isolates has produced ambiguous results due to the lack of reference sequences in publicly available databases. Thus, there is a clear need for the development of new molecular methods that would provide simple, rapid and highly specific identification of Scopulariopsis and Microascus species. The objective of this study was to develop simple and fast assays based on PCR and real-time PCR for specific detection of fungi from Scopulariopsis and Microascus genera, and separately, S. brevicaulis species., Methods: On the basis of alignment of β-tubulin gene sequences, Microascus/Scopulariopsis-specific primers were designed and S. brevicaulis-specific primers were reevaluated. DNA from cultured fungal isolates, extracted in a two-step procedure, was used in Microascus/Scopulariopsis-specific and S. brevicaulis-specific PCR and real-time PCR followed by electrophoresis or melting temperature analysis, respectively., Results: The specificity of the assays was confirmed, as positive results were obtained only for Scopulariopsis spp. and Microascus spp. isolates tested in Microascus/Scopulariopsis-specific assay, and only for S. brevicaulis and S. koningii (syn. S. brevicaulis) isolates in a S. brevicaulis-specific assay, respectively, and no positive results were obtained neither for other moulds, dermatophytes, yeast-like fungi, nor for human DNA., Conclusions: The developed assays enable fast and unambiguous identification of Microascus spp. and Scopulariopsis spp. pathogens.

This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We assess the concordance between low level HCV values obtained using the VERSANT HCV RNA 1.0 Assay (kPCR) and COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test v2.0. The correlation between the values obtained by the two RT-PCR assays for samples with quantifiable HCV RNA levels revealed that viral load measured by kPCR significantly correlated with that of the CAP/CTM (R=0.644, P<0.0001). The results show a good concordance (n=126/144, 87%); discordant results were mainly observed in the assessment of values below the lower limit of detection of the assays. These variations may have an impact on clinical decisions for patients on HCV triple therapy or interferon- free regimens. It is therefore recommended to monitor individual patients with the same test throughout treatment.

Background: In non-small cell lung cancer (NSCLC) the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene mutations have been reported in fewer than 5% of primary tumors., Materials and Methods: We assessed PIK3CA gene mutations in 145 tissue samples from central nervous system (CNS) metastases of NSCLC using three polymerase chain reaction (PCR) techniques: high resolution melting-PCR (HRM-PCR), allele-specific-quantitative PCR (ASP-qPCR) and TaqMan PCR., Results: HRM analysis allowed us to select three PIK3CA-positive specimens (2.1% of the studied group) and ASP-qPCR techniques identified them as one E542K and two H1047R substitutions, which were confirmed by TaqMan probes. The PIK3CA mutations were indicated only in males (3% of all males). One of the patients was reported to be a non-smoker with adenocarcinoma (AC; 2.5% of the AC group), however, the other two patients were smokers with squamous cell carcinoma (SCC; 3.4% of SCC group)., Conclusion: This is the first report of the presence of PIK3CA gene mutation in CNS-metastatic lesions of NSCLC worldwide that could broaden therapeutic choices in such patients., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CYP2A6 genotyping is of clinical importance--CYP2A6 gene variants influence nicotine metabolism and are associated with nicotine dependence, cigarettes per day, smoking cessation and the risk for tobacco-associated cancers. CYP2A6 gene variants also influence the metabolism of therapeutic drugs, such as the anticancer agents, tegafur and letrozole. Over the years, CYP2A6 genotyping methods have evolved to incorporate novel gene variants and to circumvent genotyping errors resulting from the high degree of homology between CYP2A6 and neighboring CYP2A genes. Herein, CYP2A6 genotyping strategies are described for commonly genotyped functionally significant alleles including SNPs, small insertions/deletions and more complex structural variants. The methods presented utilize higher throughput SYBR green real-time PCR technology in addition to standard thermocycling.

The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.