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Single nucleotide polymorphism (SNP), as one of the key components of the genetic factors, is important for disease detection and early screening of hereditary diseases. Current SNP genotyping methods require laboratory instruments or long operating times. To facilitate the diagnosis of hereditary diseases, we developed a new method referred to as the LwaCas13a-based SNP genotyping platform (Cas13a platform), which is useful for detecting disease-related SNPs. We report a CRISPR/Cas13a-based SNP genotyping platform that couples recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection for simple and fast genotyping of human disease-related SNPs. We used this Cas13a platform to identify 17 disease-related SNPs, demonstrating that position 2 in gRNA is suitable for the introduction of additional mismatches to achieve high discrimination in genotyping across a wide range of SNP targets. The discrimination specificity of 17 SNPs was improved 3.0–35.1-fold after introducing additional mismatches at position 2 from the 5′-end. We developed a method, which has a lower risk of cross-contamination and operational complexity, for genotyping SNPs using human saliva samples in an one-pot testing that delivers results within 60 min. Compared to TaqMan probe qPCR, RFLP, AS-PCR and other SNP genotyping methods, the Cas13a platform is simple, rapid and reliable, expanding the applications of the CRISPR/Cas system in nucleic acid detection and SNP genotyping. [ABSTRACT FROM AUTHOR]

Avian leukemia virus (ALV) is one of the main pathogens of poultry tumor diseases, and has caused significant economic losses to the poultry industry since its discovery. Therefore, establishing a rapid detection method is essential to effectively prevent and control the spread of ALV. In this study, specific CRISPR RNA (crRNA) and recombinase-aided amplification (RAA) primers with T7 promoter were designed based on the relatively conserved sequence of avian leukemia virus. When crRNA recognized the target sequence, Cas13a protein was activated to cut the reporting probes, and then the detection results were read by using lateral flow dipstick (LFD). The RAA-CRISPR/Cas13a-LFD reaction system was constructed. The RAA amplification time, Cas13a protein concentration, crRNA concentration and CRISPR reaction time were optimized to evaluate the specificity, sensitivity and reproducibility of the system. Finally, RAA-CRISPR/Cas13a-LFD method was compared with Polymerase chain reaction (PCR)-Agarose electrophoresis method and qPCR method in the detection of clinical samples, and the reliability of RAA-CRISPR/Cas13a-LFD method was evaluated. The results showed that the RAA-CRISPR/Cas13a-LFD method could effectively amplify the target gene at 37°C for 40 min, and the test results could be determined by LFD visual observation. The method had good specificity and no cross-reaction with Marek's disease virus (MDV), Fowl adenovirus (FAdV), Infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), and Infectious bronchitis virus (IBV). The minimum detection limit of the method was 100 copies/μL, and it had good repeatability and stability. The coincidence rate of clinical detection reached 97.69% and 99.23%. In summary, this study established a simple, efficient, accurate and visualized ALV detection method, which can be used for the prevention and rapid clinical diagnosis of avian leukosis (AL). [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I–V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I–V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of economic losses in sericulture. Thus, it is essential to establish rapid and effective method for BmNPV detection. In the present study, we have developed a recombinase-aided amplification (RAA) to amplify the BmNPV genomic DNA at 37 °C within 30 min, and achieved a rapid detection method by coupling with a lateral flow dipstick (LFD). The RAA-LFD method had a satisfactory detection limit of 6 copies/μL of recombinant plasmid pMD19-T-IE1, and BmNPV infection of silkworm can be detected 12 h post-infection. This method was highly specific for BmNPV, and without cross-reactivity to other silkworm pathogens. In contrast to conventional polymerase chain reaction (PCR), the RAA-LFD assay showed higher sensitivity, cost-saving, and especially is apt to on-site detection of BmNPV infection in the sericulture production. [ABSTRACT FROM AUTHOR]

BackgroundSenecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease.MethodsWe designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA.ResultsThe SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×101 copies/µL, 8.76×10-7 ng/µL, and 1×100.25 TCID50/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays.ConclusionThe SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 104 copies/μL and 103 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2–3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.

Edwardsiella piscicida, a significant intracellular pathogen, is widely distributed in aquatic environments and causes systemic infection in various species. Therefore, it's essential to develop a rapid, uncomplicated and sensitive method for detection of E. piscicida in order to control the transmission of this pathogen effectively. The recombinase-aided amplification (RAA) assay is a newly developed, rapid detection method that has been utilized for various pathogens. In the present study, a real-time RAA (RT-RAA) assay, targeting the conserved positions of the EvpP gene, was successfully established for the detection of E. piscicida. This assay can be performed in a one-step single tube reaction at a temperature of 39°C within 20 min. The RT-RAA assay exhibited a sensitivity of 42 copies per reaction at a 95% probability, which was comparable to the sensitivity of real-time quantitative PCR (qPCR) assay. The specificity assay confirmed that the RT-RAA assay specifically targeted E. piscicida without any cross-reactivity with other important marine bacterial pathogens. Moreover, when clinical specimens were utilized, a perfect agreement of 100% was achieved between the RT-RAA and qPCR assays, resulting a kappa value of 1. These findings indicated that the established RT-RAA assay provided a viable alternative for the rapid, sensitive, and specific detection of E. piscicida. [ABSTRACT FROM AUTHOR]

Eel (Anguilla sp.) is an important freshwater‐cultured species with high economic value in China. Anguillid herpesvirus 1 (AngHV‐1) has been proven to be the pathogen of "mucus sloughing and haemorrhagic septicaemia disease" in eels, resulting in significant mortality and substantial losses to the eel industry. Current diagnostic methods for detecting AngHV‐1 are limited to laboratory‐based tests, for example, conventional end‐point PCR and qPCR. Therefore, there is an urgent need to develop an accurate, rapid, and simple detection method for on‐site diagnosis of AngHV‐1. In this study, we developed a recombinase‐aided amplification combined lateral flow dipstick (RAA‐LFD) assay for the detection of AngHV‐1. The RAA‐LFD assay can be performed within a temperature range of 18–45°C, with a reaction time of just 10 min for amplification. Importantly, the established RAA‐LFD assay exhibited no reactivity with other common aquatic viral pathogens, indicating its high specificity. The limit of detection for this method is 102 copies of AngHV‐1, which is more sensitive than the established conventional end‐point PCR method similarly targeting ORF95. Clinical detection of the diseased samples demonstrated that the accuracy of RAA‐LFD was significantly higher than that of the conventional end‐point PCR. In conclusion, the developed RAA‐LFD assay has proven to be a convenient, rapid, sensitive, and reliable tool for on‐site diagnosis of AngHV‐1. This advancement will be invaluable for the prevention and control of AngHV‐1 in the eel farming industry. [ABSTRACT FROM AUTHOR]

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Casl3a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Casl3a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Casl3a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 10º copies/µL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection. [ABSTRACT FROM AUTHOR]

Background and Aim: Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. Materials and Methods: To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. Results: The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5’ end of the forward primer and with biotin at the 5’ end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/μL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen’s kappa coefficients showed a good agreement with the established techniques. Conclusion: The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

Chlamydia felis is an important zoonotic agent for humans and various animals. A recombinase-aided amplification (RAA) assay was developed for detecting C. felis. RAA can be performed in a closed tube at 39°C within 30 min. The detection limit was 10.6 copies of the C. felis plasmid DNA per reaction. No positive signals for other pathogens were detected. The coincidence rate of RAA and conventional PCR was 95.24% (20/21) and 100% (96/96) for positive and negative samples, respectively. The established RAA assay is a simple, rapid, highly sensitive, and specific method for detecting C. felis.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I–V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek’s disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I–V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Background & Aims Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR–Cas13a technology.Methods We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR–Cas13a combined with RT–PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR–Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT–qPCR and RT-ddPCR.Results For synthetic HDV RNA plasmids, the sensitivity of RT–PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT–qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT–qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT–qPCR, RT-ddPCR, RT–PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively.Conclusions We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Background and Aim: Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. Materials and Methods: To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. Results: The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5’ end of the forward primer and with biotin at the 5’ end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/µL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen’s kappa coefficients showed a good agreement with the established techniques. Conclusion: The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Modern Laboratory Medicine is the property of Journal of Modern Laboratory Medicine Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water. [ABSTRACT FROM AUTHOR]

With the improvement of people’s living standards, food safety has attracted more and more social attention. Organisms, especially microorganisms, are the most important factor that affects food safety. There are many methods currently available to detect biological (microbial) factors in the field of food safety, including isothermal amplification technology especially the new techniques of recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA) and enzymatic recombinase amplification (ERA), which is widely used for food safety detection because it can detect biological ingredients rapidly and accurately without relying on complicated instruments. The three isothermal amplification techniques require milder reaction conditions and simpler design of primers than others, despite having similar principles. In this article, we review the principles and concepts of these techniques and their recent application in the rapid detection of food pathogens, summarize their advantages and problems in food inspection, and present future prospects.

Pantoea ananatis is a major pathogen that causes the new bacterial blight in rice, and its symptoms very similar to rice bacterial blight. Therefore, there is a dire need for an accurate and rapid method for detecting P. ananatis. In this study, an early and rapid visual detection method for P. ananatis was established. Using GyrB gene as the target sequence, an innovative recombinase-aided amplification detection system integrated with a lateral flow dipstick (RAA-LFD) was constructed. The optimized RAA-LFD detection method can be initiated at body temperature and does not rely on precise instruments. It does not require DNA extraction and can be used directly with plant tissue fluids. The results can be visualized after 10 minutes of amplification. The specificity and sensitivity tests showed that the RAA-LFD method could detect P. ananatis, whereas other common plant pathogens were not detected, and its detection sensitivity for P. ananatis DNA reached 100 copies/µL. The detection of diseased tissues indicated that this method could accurately detect P. ananatis in artificially inoculated rice tissues in the early stages of infection before symptoms. The RAA-LFD detection system established in this study is simple and fast, with visual results, excellent specificity, and high sensitivity. It is semi-quantitative and should be used for the early detection and rapid field diagnosis of new leaf blight, which provides technical support for the early warning and real-time detection of field samples. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hookworm infection is one of the most common neglected tropical diseases and is mainly found in tropical and subtropical areas. Two species of human hookworm are distributed in China, i.e., Ancylostoma duodenale (AD) and Necator americanus (NA). Background: Traditional microscopic technology such as the Kato-Katz method is not suitable for hookworm diagnosis due to the rapid degeneration of fragile hookworm eggs or for species identification of hookworm infection. The aim of the present study was to establish and evaluate a novel nucleic acid detection method based on recombinase-aided isothermal amplification (RAA) for the detection of hookworm infections and species identification. Methods: Based on the specific target gene sequences of hookworms (5.8S rRNA for AD and ITS2 for NA, respectively), we designed and synthesized amplification primers and fluorescence probes referring to the principle of the fluorescence recombinase-aided amplification (RAA) technique. Results: Each assay provided specific amplification of larval DNA from AD and NA by fluorescence RAA, and the detection limits in plasmids reached 102 copies and 10 copies, respectively. Genomic DNA of two hookworm species was successfully detected at a concentration of 0.1 pg/μL, revealing a high detection sensitivity. No positive amplification occurred for genomic DNA from crossed hookworm species and genomic DNA from Cryptosporidium, Giardia lamblia, Strongyloides stercoralis, Schistosoma japonicum, Ascaris lumbricoides, and Clonorchis sinensis, revealing a satisfactory specificity. Fecal sample detection results demonstrated a similar efficacy to the Kato-Katz method; however, it had a greater sensitivity than the larvae culture method. Conclusion: A simple and rapid nucleic acid method was successfully established based on RAA, which improved the detection efficacy and species identification for human hookworm infections. [ABSTRACT FROM AUTHOR]

The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.

ABSTRACT: In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA–RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA–RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA–RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF.

Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water.

ABSTRACT: Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

A recent, unprecedented outbreak of human mpox virus infection has led to cases in non-African nations, and the number of confirmed or suspected cases outside of Africa has exceeded 1,000 within 5 weeks. Mpox may pose a double threat to public health in the context of the ongoing COVID-19 pandemic. It is difficult to distinguish mpox virus infection from other diseases in the early stages, and patients are contagious from the onset of nonspecific symptoms; therefore, it is crucial to develop rapid and specific diagnostic methods. The diagnosis of mpox relies on real-time polymerase chain reaction, a time-consuming method that requires a highly sophisticated thermal cycler, which makes it unsuitable for widespread use in underdeveloped areas, where the outbreak is still severe. In this study, we developed a recombinase-aided amplification (RAA) assay that can detect mpox virus within 5–10 minutes. The conserved regions of the A27L gene and F3L gene were selected as targets, as they amplify well from different mpox virus clades with no cross-reaction from other pathogens. The sensitivity of this RAA assay is 10 copies/reaction for the A27L gene and 102 copies/reaction for the F3L gene. When applied to simulated clinical samples, both targets showed 100% specificity, and the detection limits were consistent with the sensitivity results. Moreover, through clinical blinded sample detection, RAA exhibits the same detection power as RT-PCR. In summary, the RAA mpox assay described here exhibits rapid detection, high sensitivity and specificity, and low operational difficulty, making it suitable for mpox virus detection in less developed countries and regions.

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The Burkholderia cepacia complex (BCC) is a group of opportunistic pathogens, including Burkholderia cepacia, Burkholderia multivorans, Burkholderia vietnamiensis and Burkholderia ambifaria, which can cause severe respiratory tract infections and lead to high mortality rates among humans. The early diagnosis and effective treatment of BCC infection are therefore crucial. In this study, a novel and rapid recombinase-aided amplification (RAA) assay targeting the 16S rRNA gene was developed for BCC detection. The protocol for this RAA assay could be completed in 10 min at 39°C, with a sensitivity of 10 copies per reaction and no cross-reactivity with other pathogens. To characterize the effectiveness of the RAA assay, we further collected 269 clinical samples from patients with bacterial pneumonia. The sensitivity and specificity of the RAA assay were 100% and 98.5%, respectively. Seven BCC-infected patients were detected using the RAA assay, and three BCC strains were isolated from the 269 clinical samples. Our data showed that the prevalence of BCC infection was 2.60%, which is higher than the 1.40% reported in previous studies, suggesting that high sensitivity is vital to BCC detection. We also screened a patient with B. vietnamiensis infection using the RAA assay in clinic, allowing for appropriate treatment to be initiated rapidly. Together, these data indicate that the RAA assay targeting the 16S rRNA gene can be applied for the early and rapid detection of BCC pathogens in patients with an uncharacterized infection who are immunocompromised or have underlying diseases, thereby providing guidance for effective treatment. [ABSTRACT FROM AUTHOR]

Foodborne pathogenic bacteria have become a worldwide threat to human health, and rapid and sensitive bacterial detection methods are urgently needed. In this study, a facile microfluidic chip was developed and combined with recombinase-aided amplification (RAA) for rapid and sensitive detection of Salmonella typhimurium using a non-contact eddy heater for dynamic lysis of bacterial cells and a 3D-printed fan-shaped active mixer for continuous-flow mixing. First, the bacterial sample was injected into the chip to flow through the spiral channel coiling around an iron rod under an alternating electromagnetic field, resulting in the dynamic lysis of bacterial cells by this non-contact eddy heater to release their nucleic acids. After cooling to ~75 °C, these nucleic acids were continuous-flow mixed with magnetic silica beads using the fan-shaped mixer and captured in the separation chamber using a magnet. Finally, the captured nucleic acids were eluted by the eluent from the beads to flow into the detection chamber, followed by RAA detection of nucleic acids to determine the bacterial amount. Under the optimal conditions, this microfluidic chip was able to quantitatively detect Salmonella typhimurium from 1.1 × 102 to 1.1 × 105 CFU/mL in 40 min with a detection limit of 89 CFU/mL and might be prospective to offer a simple, low-cost, fast and specific bacterial detection technique for ensuring food safety. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 10 4 copies/μL and 10 3 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2-3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa . The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr , in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr , the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhou, Shi, Mu, Tian, Zhou, Lyu and Chen.)

ABSTRACT As a notifiable terrestrial and aquatic animal disease listed by World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]), classical swine fever (CSF) has caused great economic losses to the swine industry worldwide during recent decades. Differentiation of infected and vaccinated animals (DIVA) is urgent for eradication of CSF. In this study, a diagnostic platform based on CRISPR/Cas13a was established with the ability to differentiate between classical swine fever virus (CSFV) virulent and vaccine strains. In combination with reverse transcription recombinase-aided amplification (RT-RAA), the detection limit for CSFV synthetic RNA templates reached 3.0 × 102 copies/μL. In addition, with boiling and chemical reduction, heating unextracted diagnostic samples to obliterate nucleases (HUDSON) treatment was introduced to inactivate nucleases and release viral genome, achieving robust pretreatment of tested sample before CRISPR/Cas13a detection without the need to extract viral nucleic acids. HUDSON-RT-RAA-CRISPR/Cas13a can directly detect cell cultures of virulent Shimen strain and vaccine hog cholera lapinized virus (HCLV) strain, with the detection limit of 3.5 × 102 copies/μL and 1.8 × 102 copies/μL, respectively, which was equally sensitive to nested PCR (nPCR) and 100 times more sensitive than antigen enzyme-linked immunosorbent assay (ELISA). Meanwhile, HUDSON-RT-RAA-CRISPR/Cas13a showed no cross-reactivity with bovine viral diarrhea virus (BVDV), atypical porcine pestivirus (APPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2), exhibiting good specificity. At last, a total of 50 pig spleen samples with suspected clinical signs were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a, nPCR, and antigen ELISA in parallel. HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0% with nPCR and 82.0% coincident rate with antigen ELISA, respectively. IMPORTANCE Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. Methods with the ability to precisely differentiate classical swine fever virus (CSFV) virulent strains from vaccine strain hog cholera lapinized virus (HCLV) are urgently needed. Combining the high sensitivity of isothermal recombinase-aided amplification (RAA) with the accurate molecular sensing ability of Cas13a, we presented a novel method for CSFV detection without the need to extract viral nucleic acids, which showed great advantage to traditional detection methods for precise differentiation of CSFV virulent strains and vaccine strain, providing a novel powerful tool for CSF eradication.

Salmonella, as an important foodborne pathogen, can cause various diseases, such as severe enteritis. In recent years, various types of nucleicacid-intercalating dyes have been utilized to detect viable Salmonella. However, in principle, the performance of existing nucleic acid dyes is limited because they depend on the integrity of cell membrane. Herein, based on the metabolic activity of bacteria, a novel DNA dye called thiazole orange monoazide (TOMA) was introduced to block the DNA from dead bacteria. Recombinase-aided amplification (RAA) was then performed to detect viable Salmonella in samples. In this study, the permeability of TOMA to the cell membrane of Salmonella was evaluated via confocal laser scanning microscopy and fluorescence emission spectrometry. The limit of detection (LOD) of the TOMA–RAA method was 2.0 × 104 CFU/mL in pure culture. The feasibility of the TOMA–RAA method in detecting Salmonella was assessed in spiked milk. The LOD for Salmonella was 3.5 × 102 CFU/mL after 3 h of enrichment and 3.5 × 100 CFU/mL after 5 h of enrichment. The proposed TOMA–RAA assay has great potential to be applied to accurately detect and monitor foodborne pathogens in milk and its byproducts. [ABSTRACT FROM AUTHOR]

BACKGROUND: Klebsiella pneumoniae is a zoonotic opportunistic pathogen, leading to severe infections in dairy cows and humans. Efficient, on‐site and accurate detection of K. pneumoniae is necessary to reduce the harm of cow mastitis and human infections. The objective of this study was to establish a recombinase‐aided amplification (RAA) method combined with lateral flow dipstick (LFD) for rapid detection of K. pneumoniae. RESULTS: The primer concentration, incubation temperature and incubation time of the RAA reaction were optimized. When the primer concentration was 100 nmol L–1, the strongest band could be obtained by incubation at 37 °C for 20 min. The RAA‐LFD method had high specificity to K. pneumoniae and showed no cross‐reaction with other pathogens. In addition, the detection limit of RAA‐LFD for K. pneumoniae was 20 fg genomic DNA and 2.5 × 102 CFU mL−1 of bacteria in pure culture, which is 100 times higher than that of polymerase chain reaction (PCR) detection. Moreover, the RAA‐LFD method can detect K. pneumoniae at initial concentrations as low as 2.5 CFU per 25 mL in artificially spiked milk samples after at least incubation for 6 h. Importantly, RAA‐LFD had a high agreement with a test accuracy of 96.9%, compared with the biochemical identification method. Also, the detection accuracy of RAA‐LFD was higher than that of the PCR assay (95.3%). CONCLUSIONS: The results demonstrated that the RAA‐LFD assay is an accurate, sensitive, simple and point‐of‐use detection method for K. pneumoniae, which could be used as a potential application in the research laboratory and for disease diagnosis. © 2021 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Malaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion transmitted-malaria (TTM). Therefore, blood banks need a rapid screening tool to detect Plasmodium species. Methods: We designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of Plasmodium species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated. Results: The RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/μL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with P. falciparum , P. vivax , P. ovale , and P. malariae , the sensitivity was 0.1 pg/μL, 10 pg/μL, 10-100 pg/μL, and 100pg/μL, respectively. The sensitivity of this assay was 100pg/μL. No cross-reaction with other transfusion-transmissible parasites was detected. Conclusions: The results demonstrated that this RAA-LFD assay was suitable for reliable field detection of Plasmodium species in low-resource settings with limited laboratory capabilities. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is one of the major pathogenic Vibrio species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE–RAA–CRISPR) for detecting V. parahaemolyticus in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for V. parahaemolyticus, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 101 CFU/mL in pure cultures and 7.3 × 101 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE–RAA–CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement. [ABSTRACT FROM AUTHOR]

Chlamydia psittaci is the causative agent of psittacosis, a worldwide zoonotic disease. A rapid, specific, and sensitive diagnostic assay would be benefit for C. psittaci infection control. In this study, an assay combining recombinase-aided amplification and a lateral flow strip (RAA-LF) for the detection of active C. psittaci infection was developed. The RAA-LF assay targeted the CPSIT_RS02830 gene of C. psittaci and could be accomplished in 15 min at a single temperature (39°C). The analytical sensitivity of the assay was as low as 1 × 100 copies/μl and no cross-reaction with some other intracellular pathogens was observed. Moreover, all feces samples from mice infected with C. psittaci at day-1 post-infection were positive in the RAA-LF assay. In conclusion, the RAA-LF assay provides a convenient, rapid, specific and sensitive method for detection of active C. psittaci infection and it is also suitable for C. psittaci detection in field. [ABSTRACT FROM AUTHOR]

In addition to Cronobacter spp., Klebsiella pneumoniae is another opportunistic bacterial pathogen present in powdered infant formula (PIF) that can cause pneumonia, septicemia, and other diseases. In this study, a rapid and specific method based on a fluorescence probe was developed for detecting viable K. pneumoniae in PIF samples via the combination of recombinase-aided amplification (RAA) with thiazole orange monoazide (TOMA) dye (the TOMA–RAA assay hereafter). As a novel photosensitive DNA-intercalating dye, TOMA was used to penetrate bacterial cells, including both dead and viable cells, as verified by confocal laser scanning microscopy and fluorescent emission spectrometry. Importantly, the RAA assay exhibited good performance in detecting K. pneumoniae within 40 min at 39°C. Under optimal conditions, the TOMA–RAA assay can detect as low as 2.6 × 103 cfu/mL of K. pneumoniae in pure culture and 2.3 × 104 cfu/g of K. pneumoniae in spiked PIF sample. After 3 h of pre-enrichment, 3 × 100 cfu/g of K. pneumoniae can be detected. Furthermore, the TOMA–RAA assay displayed an excellent anti-interference ability to nontarget bacteria. In short, the proposed method has great potential application for the rapid and accurate detection of viable K. pneumoniae in PIF. [ABSTRACT FROM AUTHOR]

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/µl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti. [ABSTRACT FROM AUTHOR]

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

BackgroundMalaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion transmitted-malaria (TTM). Therefore, blood banks need a rapid screening tool to detect Plasmodium species.MethodsWe designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of Plasmodium species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated.ResultsThe RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/μL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with P. falciparum, P. vivax, P. ovale, and P. malariae, the sensitivity was 0.1 pg/μL, 10 pg/μL, 10-100 pg/μL, and 100pg/μL, respectively. The sensitivity of this assay was 100pg/μL. No cross-reaction with other transfusion-transmissible parasites was detected.ConclusionsThe results demonstrated that this RAA-LFD assay was suitable for reliable field detection of Plasmodium species in low-resource settings with limited laboratory capabilities.

Chlamydia psittaci is the causative agent of psittacosis, a worldwide zoonotic disease. A rapid, specific, and sensitive diagnostic assay would be benefit for C. psittaci infection control. In this study, an assay combining recombinase-aided amplification and a lateral flow strip (RAA-LF) for the detection of active C. psittaci infection was developed. The RAA-LF assay targeted the CPSIT_RS02830 gene of C. psittaci and could be accomplished in 15 min at a single temperature (39°C). The analytical sensitivity of the assay was as low as 1 × 100 copies/μl and no cross-reaction with some other intracellular pathogens was observed. Moreover, all feces samples from mice infected with C. psittaci at day-1 post-infection were positive in the RAA-LF assay. In conclusion, the RAA-LF assay provides a convenient, rapid, specific and sensitive method for detection of active C. psittaci infection and it is also suitable for C. psittaci detection in field.

Abstract Background Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment. Method The rapid reverse transcription recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39 °C in 30 min. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial reverse transcription real-time fluorescence quantitative PCR (RT-qPCR). Results At 95% probability, the detection sensitivity of RT-RAA was 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared of RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p

Foodborne diseases caused by foodborne pathogenic microorganisms have long been a severe public health problem. Timely and accurate detection of food-borne microorganisms can minimize their harm and enable the development of efficient control strategies. In this study, we establish a multiplex nucleic acid detection method based on recombinase-aided amplification reaction on a suspension microarray (referred to as SC-RAA) to identify and quantify microorganisms. The experimental results confirmed that this approach has an outstanding sensitivity, with a limit of detection (LOD) of 0.8 fM and a linear range of 0.8 fM–80 pM, spanning five orders of magnitude. Moreover, the prepared method also showed exquisite specificity, which the amplified signal can be detected only when the target nucleic acid is present. The recovery rate of the SC-RAA assay was 82.2%–113.4%, suggesting that the SC-RAA assay is performing well and accurately measuring the concentration of the substance in the sample. In addition, the consistency analysis showed that the results of the SC-RAA detection were also in good agreement with those of traditional qPCR detection (R2 > 0.95). These results highlight the potential of the SC-RAA assay to be applied as a multiplex and sensitive DNA detection platform for identifying and quantifying foodborne pathogenic microorganisms. [ABSTRACT FROM AUTHOR]

Hookworm infection is one of the most common neglected tropical diseases and is mainly found in tropical and subtropical areas. Two species of human hookworm are distributed in China, i.e., Ancylostoma duodenale (AD) and Necator americanus (NA). Background: Traditional microscopic technology such as the Kato-Katz method is not suitable for hookworm diagnosis due to the rapid degeneration of fragile hookworm eggs or for species identification of hookworm infection. The aim of the present study was to establish and evaluate a novel nucleic acid detection method based on recombinase-aided isothermal amplification (RAA) for the detection of hookworm infections and species identification. Methods: Based on the specific target gene sequences of hookworms (5.8S rRNA for AD and ITS2 for NA, respectively), we designed and synthesized amplification primers and fluorescence probes referring to the principle of the fluorescence recombinase-aided amplification (RAA) technique. Results: Each assay provided specific amplification of larval DNA from AD and NA by fluorescence RAA, and the detection limits in plasmids reached 102 copies and 10 copies, respectively. Genomic DNA of two hookworm species was successfully detected at a concentration of 0.1 pg/μL, revealing a high detection sensitivity. No positive amplification occurred for genomic DNA from crossed hookworm species and genomic DNA from Cryptosporidium, Giardia lamblia, Strongyloides stercoralis, Schistosoma japonicum, Ascaris lumbricoides, and Clonorchis sinensis, revealing a satisfactory specificity. Fecal sample detection results demonstrated a similar efficacy to the Kato-Katz method; however, it had a greater sensitivity than the larvae culture method. Conclusion: A simple and rapid nucleic acid method was successfully established based on RAA, which improved the detection efficacy and species identification for human hookworm infections.

ABSTRACT: Duck circovirus disease (DuCVD), as an immunosuppressive disease, is a threat to the poultry industry. In order to diagnose this disease quickly and accurately, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established to detect duck circovirus (DuCV). The results showed that the quantity of amplification products was positively correlated with the value of fluorescence signal. Obvious detection results can be observed at 41°C after 15 min reaction. This method has good specificity and has no cross reaction with Muscovy duck parvovirus (MDPV), duck enteritis virus (DEV), fowl adenovirus (FAdV), porcine circovirus (PCV), and duck hepatitis A virus (DHAV). The sensitivity test showed that the minimum concentration of template detected by RF-RAA for DuCV was 10° copies/μL, and its sensitivity was 10 times higher than that of real-time fluorescence-based quantitative PCR (RFQ-PCR) and 10,000 times higher than that of polymerase chain reaction (PCR). Fifty-two clinical samples were detected by RF-RAA and RFQ-PCR, and the coincidence rate of the two methods was 98.08%. This method has the advantages of simple operation, good specificity and high sensitivity, and can be used for laboratory detection and clinical diagnosis of DuCV.