Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16 s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16 s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16 s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

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.

Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39°C. The RF-RAA outcomes are interpretable in 2 modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/µL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and real-time fluorescence quantitative polymerase chain reaction (qPCR) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.

Simultaneous Detection of Helicobacter pylori and Clarithromycin Resistance Mutations Using RAA-CRISPR/Cas13a Assay.

Background: Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance. Methods: We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency. Results: We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/µL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing. Conclusion: SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus.

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/µL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9.

BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device.

Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBRV) are the two most prevalent infectious diseases in cattle. They both can cause persistent infection and immunosuppression, resulting in significant economic losses in the livestock industry. Therefore, rapid detection of early BVDV and IBRV infections is crucial. In this study, a method for the rapid detection of BVDV and IBRV was established by using recombinase polymerase amplification (RPA) combined with lateral flow device (LFD). By optimizing the temperature and time conditions of the RPA reaction, the sensitivity, specificity, and clinical performance were evaluated. The results indicated that the RPA reaction could be completed at 40°C within 25 min. The LOD for BVDV and IBRV by RPA-LFD were 5.1 × 101 copies/µL and 6.65 × 101 copies/µL, respectively, with no cross-reactivity observed with other viruses such as CSFV, BRSV, BPIV3, BRV, and BCoV. Testing of 32 clinical samples showed consistent results between RPA-LFD and qPCR. The RPA-LFD method established in this study can be used for the rapid clinical detection of BVDV and IBRV, which providing a rapid and convenient molecular biology approach for on-site rapid detection and epidemiological investigations. Simultaneously, it offers technical support for the prevention and control of these viruses.

Development of a real-time recombinase-aided amplification assay for rapid and sensitive detection of Edwardsiella piscicida.

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.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus.

Among broilers, the main pathogen that leads to swollen head syndrome (SHS) is the subgroup C avian metapneumovirus (aMPV-C). The aMPV-C infection can lead to an upsurge in the rate of soft-shell eggs, resulting in reduced egg production and seriously affecting the economy of the livestock industry. Therefore, a rapid method for aMPV-C detection needs to be invented. According to the N gene of aMPV-C, we designed the specific probe and primer and created a reverse transcription recombinase-aided amplification assay (RT-RAA) for the detection of aMPV-C. aMPV-C could be detected quickly and specifically by this method at 41 °C for 30 min. The sensitivity assay inferred that the minimum detection threshold of RT-RAA was 3.38 × 101 copies/µL. A specificity assay showed that the RT-RAA method did not cross-react with other subgroups (aMPV-A, aMPV-B, aMPV-D) or other viruses (H9N2, NDV, IBV, IBDV). Forty samples of known clinical background were tested by RT-RAA and RT-qPCR. The two approaches had a 100% correlation rate. In conclusion, this research successfully created an RT-RAA assay for aMPV-C.

Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms.

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

Development and application of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid visual detection of anguillid herpesvirus 1.

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.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Development and performance of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium DNA in urine samples.

Rapid diagnosis of urogenital schistosomiasis caused by Schistosoma haematobium requires an accurate and timely assay, especially for low-intensity S. haematobium infection cases and in non-endemic areas. The mitochondrial cytochrome c oxidase 1 (cox1) gene fragment of S. haematobium was selected as detection target as this short fragment, which can be rapidly sequenced and yet possess good diagnostic resolution. A pair of primers and a fluorescent probe were designed according to the principle of recombinase-aided amplification (RAA), which was subsequently optimized and applied as an S. haematobium-specific RAA assay. Its diagnostic performance was validated for sensitivity and specificity in comparison to microscopy-based egg counting after urine filtration. The RAA assay could detect as little as 10 copies/µL of S. haematobium recombinant plasmid, and no cross-reactions were observed with S. mansoni, S. japonicum, Ancylostoma duodenale, Clonorchis sinensis, Echinococcus granulosus, or Ascaris lumbricoides. This test can be conducted at 39 °C and the whole RAA reaction can be completed within 20 min. The validation of the RAA assay showed that it had 100 % consistency with urine-egg microscopy, as it does not require an elaborate reading tool, is simple to use, and should be useful for field diagnostics and point-of-care applications.

[Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish].

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS: Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2.

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.

Development of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid detection of H7 subtype avian influenza virus.

Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 101 copies/µL, while RT-qPCR method was 1 × 101 copies/µL, and RT-PCR was 1 × 102 copies/µL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

Recombinase-aided amplification coupled with lateral flow dipstick for efficient and accurate detection of Bombyx mori nucleopolyhedrovirus.

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.

A Novel RAA Combined Test Strip Method Based on Dual Gene Targets for Pathogenic Vibrio vulnificus in Aquatic Products.

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.

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection.

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.

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips.

Meat adulteration is a major global concern that poses a threat to public health and consumer rights. However, current detection techniques, such as quantitative polymerase chain reaction (qPCR) and gas chromatography-mass spectrometry, are time-consuming and require sophisticated equipment. In this study, we developed a rapid onsite identification method for animal-derived ingredients by utilizing a fast nucleic acid lysis buffer to expedite the release of sample nucleic acids and combined it with dual-recombinase-aided amplification (dual-RAA) technology and visual multiplex lateral flow strips (MLFSs). Our method successfully detected duck- and bovine-derived, porcine- and bovine-derived, duck- and ovine-derived, and porcine- and ovine-derived meat in a rapid 20 min onsite detection assay, with a detection limit of 101 copies/50 µL reaction system for target genes. Moreover, our method accurately detected adulterated meat with proportions as low as 1:999. These findings have significant implications for food safety and the protection of consumer rights.