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.

Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L.

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/µL, while the detection limit of RPA-LFD was 101 copies/µL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

Mucormycosis is a challenging disease to diagnose early. This study introduces a sensitive and rapid diagnostic approach using Loop-mediated isothermal amplification technology. Testing blood and urine samples from 30 patients revealed promising sensitivity and repeatability, indicating its potential for non-invasive diagnosis.

Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Clostridium piliforme.

BACKGROUND: Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme. OBJECTIVES: To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. METHODS: In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system. RESULTS: The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7  ng/µL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes. CONCLUSIONS: The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.

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.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. RESULTS: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. CONCLUSIONS: In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

A diagnostic approach to confirm Mycoplasma hyopneumoniae "Day zero" for pathogen eradication.

Breeding herds in the US are trending toward eradication of Mycoplasma hyopneumoniae (M. hyopneumoniae) due to the positive impact on welfare and downstream production. In an eradication program, "Day 0″ is the time point when the last replacement gilts to enter the herd were exposed to M. hyopneumoniae and marks the beginning of a herd closure. However, no specific diagnostic protocols are available for confirmation of successful exposure to define Day 0. Therefore, the objective of this study was to develop diagnostic guidelines, including sample collection approaches, for two common gilt exposure methods to confirm an entire population has been infected with M. hyopneumoniae following purposeful exposure. Forty gilts, age 21-56 days, were ear-tagged for longitudinal sample collection at five commercial gilt developer units (GDUs) and were exposed to M. hyopneumoniae by natural contact or aerosolization. Study gilts originated from sources known to be negative to major swine pathogens, including M. hyopneumoniae, and were sampled prior to exposure to confirm negative status, then every two weeks. Blood samples were collected for antibody detection, while laryngeal and deep tracheal secretions and pen based oral fluids were collected for PCR, and sampling continued until at least 85% of samples were positive by PCR. Detection of M. hyopneumoniae varied greatly based on sample type. Oral fluids showed the lowest detection in groups of gilts detected positive by other sample types. Detection by PCR in deep tracheal secretions was higher than in laryngeal secretions. Seroconversion to and PCR detection of M. hyopneumoniae on oral fluids were delayed compared to PCR detection at the individual level. By two weeks post-exposure, at least 85% of study gilts in three GDUs exposed by aerosolization were PCR positive in deep tracheal secretions. Natural contact exposure resulted in 22.5% of study gilts becoming PCR positive by two weeks post-initial exposure, 61.5% at four weeks, and 100% at six weeks on deep tracheal secretions. Deep tracheal secretions required the lowest number of gilts to sample for the earliest detection compared to all other samples evaluated. As observed in one of the GDUs using aerosolization, demonstration of failure to expose gilts to M. hyopneumoniae allowed for early intervention in the exposure protocol and delay of Day 0, for accurate timing of the eradication protocol. Sampling guidelines proposed in this study can be used for verification of M. hyopneumoniae infection of gilts following exposure to determine Day 0 of herd closure.

Rapid detection of goose astrovirus genotypes 2 using real-time reverse transcription recombinase polymerase amplification.

BACKGROUND: Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. RESULTS: The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/µL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. CONCLUSIONS: The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Multienzyme isothermal rapid amplification and lateral flow dipstick combination assay for visible detection of chicken chaphamaparvovirus.

Chicken chaphamaparvovirus (CkChpV) is a newly emerging pathogen that is currently prevalent in chickens with diarrhea symptoms. To diagnose CkChpV more conveniently and rapidly, this study established a multienzyme isothermal rapid amplification (MIRA) assay, with a reaction time of only 15 min and optimal reaction temperature of 38°C. In combination with the lateral flow dipstick assay, the CkChpV-MIRA assay can be completed within 20 min. We revealed that the detection limit of the MIRA assay using standard plasmids as templates was as low as 21.3 copies, and its sensitivity was 100 times higher than that of nested PCR. Moreover, the designed primer set and probe could only detect CkChpV specifically, and there was no cross reaction with avian nephritis virus, rotavirus, chicken parvovirus virus, Newcastle disease virus, and infectious bronchitis virus, which may cause diarrhea. These findings demonstrated that the CkChpV-MIRA assay established in this study is convenient, sensitive, and specific and does not require sophisticated equipment. It is more suitable for the detection of CkChpV in clinical samples.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Preliminary investigation of hepatitis E virus detection by a recombinase polymerase amplification assay combined with a lateral flow strip.

Hepatitis E virus (HEV) is a zoonotic pathogen that is a significant public health problem. Detecting HEV relies mainly on conventional PCR, which is time-consuming and requires sophisticated instruments and trained staff. We aimed to establish a reverse-transcription (RT)-recombinase polymerase amplification (RPA) assay (RT-RPA) combined with a lateral flow strip (LFS; RT-RPA-LFS) to rapidly detect HEV RNA in human and rabbit samples. With the optimal reaction conditions (37°C for 30 min), our assay detected as few as 1.0 × 102 copies/mL of HEV and showed no cross-reactivity with other hepatitis viruses. We tested 28 human samples (4 fecal and 24 serum samples) and 360 rabbit samples (180 fecal and 180 serum samples) with our RT-RPA-LFS assay and compared our assay to an RT-qPCR method. There was no significant difference (p > 0.05) in the test results between the 2 assays. Our RT-RPA-LFS assay detected both HEV3 and HEV4 genotypes. Our rapid, sensitive, and specific RT-RPA-LFS assay for the detection of HEV may provide a useful detection tool for limited-resource areas.

Multi-detection for paramphistomes using novel manually designed PAR-LAMP primers and its application in a lateral flow dipstick (LAMP-LFD) system.

Loop-mediated isothermal amplification (LAMP) has been applied for the detection of various parasites, and its application in lateral flow dipstick (LFD) can improve the convenience of point-of-care diagnosis. A novel PAR-LAMP probe and primers were designed by manual selection from a region of low variation in the ITS-2 DNA sequence. Up to six species of rumen fluke were detected by LAMP and LAMP-LFD in this study. Target specificity and sensitivity were tested, revealing a high target specificity (accuracy) and a low limit of detection (sensitivity). Different target sensitivities of paramphistome were presented, including 5 pg for Gastrothylax crumenifer and Carmyerius sp.; 1 pg for Fischoederius elongatus, Orthocoelium parvipapillatum, and O. dicranocoelium; and 0.1 pg for Paramphistomum epiclitum. LAMP-LFD can detect a paramphistome egg even in contaminated in feces that was spiked with the egg under laboratory conditions. In addition, natural paramphistome infection in cattle from Surat Thani and Khon Kaen provinces, Thailand, was evaluated by detection of egg contamination in fecal specimens using PAR-LAMP primers. The PAR-LAMP detection result was also statistically evaluated by microscopic examination of feces. This study presents the application of novel manually designed primers in a LAMP-LFD system for improving performance in detection and diagnosis assays for paramphistomosis.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Development of a direct duplex real-time PCR assay for rapid detection of domestic cat hepadnavirus.

Domestic cat hepadnavirus (DCH) is a novel hepadnavirus, first identified in 2018. DCH is generally detected using conventional PCR assays, which include time-consuming agarose gel electrophoresis. We developed a rapid, sensitive, and specific real-time PCR (rtPCR) assay for the detection of the DCH genome. To streamline the procedure, our rtPCR assay was carried out using blood samples, without DNA extraction. A consensus primers/probe set was designed based on the nucleotide sequences of the surface/polymerase gene of all DCH strains available in GenBank. To exclude the possibility that the PCR reaction was blocked by anticoagulants, we also used a primers/probe set for amplifying the cat beta-actin gene as a reference gene. Our direct duplex rtPCR assay had high sensitivity, with a limit of detection of 10 copies/µL of blood for DCH. Our direct duplex rtPCR assay should be a useful tool for DCH detection and surveillance.

Simple and rapid detection of severe fever with thrombocytopenia syndrome virus in cats by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay using a dried reagent.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes lethal hemorrhagic diseases in human, cats, and dogs. Several human cases involving direct transmission of SFTSV from diseased animals have been reported. Therefore, rapid diagnosis in veterinary clinics is important for preventing animal-to-human transmission. Previously, we developed a simplified reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for human that does not require RNA extraction for detecting the SFTSV genome. In this study, we improved the simplified RT-LAMP assay for cats by introducing a dried reaction reagent and investigated the applicability of this method for diagnosing SFTS in cats. SFTSV RNA was detected in 11 of 12 cats naturally infected with SFTSV by RT-LAMP assay using both liquid and dried reagents. The RT-LAMP assay using liquid and dried reagents was also applicable to the detection of SFTSV genes 3-4 days after challenge in cats experimentally infected with SFTSV. The minimum copy number of SFTSV genes for 100% detection using the RT-LAMP assay with liquid and dried reagents was 4.3 × 104 and 9.6 × 104 copies/mL, respectively. Although the RT-LAMP assay using the dried reagent was less sensitive than that using the liquid reagent, it was sufficiently sensitive to detect SFTSV genes in cats with acute-phase SFTS. As the simplified RT-LAMP assay using a dried reagent enables detection of SFTSV genes more readily than the assay using a liquid reagent, it is applicable for use in veterinary clinics.

Research Note: Application of reverse-transcription recombinase-aided amplification-lateral flow dipstick method in the detection of infectious bursal disease virus.

Infectious bursal disease (IBD) is a highly contagious viral disease caused by infectious bursal disease virus (IBDV) in chickens. The consequent immunosuppression and secondary infection affect the healthy development of chicken industry. In this study, specific primers and probes were screened in the conserved region of IBDV VP2 gene sequence, and reverse transcription-recombinase-aided amplification (RT-RAA) was combined with lateral flow dipstick (LFD) for establishing RT-RAA-LFD method for detection of IBDV in chickens. The reaction conditions of RT-RAA-LFD assay were optimized, and the specificity, sensitivity, and repeatability were verified. The results showed that the RT-RAA-LFD method could amplify the IBDV target fragment at 37°C for 15 min, and the required primer and probe concentration was 1,250 nmol/L. The detection results were directly observed by the dipstick, the lowest detectable limit (LDL) for IBDV was 10 copies/µL, and there was no cross reaction with several common immunosuppressive pathogens in poultry. The total coincidence rate of sample test results between RT-RAA-LFD and reverse transcription-polymerase chain reaction (RT-PCR) was 95.83%. Due to advantages of high sensitivity, strong specificity, easy operation, fast detection, the established RT-RAA-LFD method can provide some technical support and new solutions for local laboratory to detect IBDV.