Rapid authentication of characteristic milk powders by recombinase polymerase amplification assays.

The authentication of dairy species has great significance for food safety. This study focused on a more rapid method for identifying major dairy species, and specific recombinase polymerase amplification (RPA)-based assays for cattle, goat, sheep, camel and donkey were developed. Through the developed RPA-based assays, goats and sheep could be simultaneously identified and bovine families could be differentiated. The performances of the RPA assays were validated using 37 milk powder samples, of which 16.2% (6/37) were suspected of being adulterated and 24.3% (9/37) were potentially at risk of being wrongly identified as adulteration. The effectiveness of the developed assays for crude DNA detection was also validated by a rapid nucleic acid extraction kit, and results showed that the presence of large amounts of protein and fat did not affect the qualitative results. Therefore, these assays could combine with the rapid nucleic acids extraction methods for being used in field detection.

Rapid detection of Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays.

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.'s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Rapid detection of duck ingredient in adulterated foods by isothermal recombinase polymerase amplification assays.

Duck is often used in meat fraud as a substitute for more expensive meats. Rapid detection of duck ingredient in meat products is of great significance for combating meat fraud and safeguarding the interests of consumers. Therefore, we aim to develop duck-specific recombinase polymerase amplification (RPA)-based assays for the rapid detection of duck ingredient in animal-derived foods. Using Cytb gene as target, the real-time RPA and RPA combined with lateral flow strips (LFS RPA) were developed successfully for the rapid detection of ducks in 20 min at 39 °C and 40 °C, respectively. The assays did not show cross-reactions with 6 other livestock and poultry. The developed RPA assays could detect 10 pg duck genomic DNA per reaction and 0.1 % (w/w) duck ingredient in duck and mutton mixed powder within 30 min, including a rapid nucleic acid extraction. Furthermore, duck ingredient could be detected in 30 different actual foods including heat-processed meats and blood products. Therefore, duck-specific real-time RPA and LFS RPA assays were successfully developed with good specificity and sensitivity, which could enable rapid detection of duck ingredient in the field and provide technical support for combating the meat fraud.

Quantification of hepatitis E virus in raw pork livers using droplet digital RT-PCR.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. Some of the rise in hepatitis E infection in China may be linked to undercooked pork. In this study, we established a reverse transcription droplet digital PCR (RT-ddPCR) method to detect HEV in raw pork livers. The detection limit of the assay for HEV RNA was as low as 1.81 copies/µL. The suggested approach was validated on 14 samples, demonstrating greater sensitivity, specificity, and anti-interference performance features than RT-qPCR. Furthermore, we amplified the partial ORF2 gene by nested RT-PCR and sequenced for the HEV RNA positive samples. The prevalence of HEV in all collected samples was 2.24% (14/626), and the viral load was between 8.0 copies/µL and 8975 copies/µL. Specifically, the virus was detected in 10.62% (12/113) of the samples collected from the bio-safety disposal centers for dead livestock and poultry, in 0.67% (2/300) of the samples collected from the slaughterhouses, and none of the samples collected from the retail markets was HEV RNA positive. The subsequent phylogenetic analysis revealed that all HEV isolates belonged to the subtype 4d, which is one of the most common subtypes in northern China.

Rapid detection of bovine rotavirus a by isothermal reverse transcription recombinase polymerase amplification assays.

BACKGROUND: Bovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals. RESULTS: An RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively. CONCLUSIONS: In this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays.

Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/µl (95%CI: 126-333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/µl (95%CI: 20-57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

Development of a new TaqMan-based real-time RT-PCR assay for the specific detection of bovine kobuvirus.

Bovine kobuvirus (BKV) is a novel kobuvirus considered to be closely related to calf diarrhea and has become a worldwide epidemic. Currently, the BKV lacks an efficient and convenient detection method to assist the research on BKV prevalence. In this study, a new and specific TaqMan-based real-time RT-PCR for the detection of BKV was developed using the conserved region of the 3D gene. The assay was highly specific for BKV, without cross-amplification with other non-targeted pathogens. The limit of detection of this assay was 102 copies. Standard curves showed a strong linear correlation from 102 to 106 copies of BKV standard RNA per reaction, and the parameters revealed as a slope of -3.54, efficiency of 91.64%, and regression coefficients (R2) of 0.998. The assay was also reproducible, with the intra-assay and inter-assay coefficient of variation <1.0%. The newly developed real-time RT-PCR was validated using 243 fecal samples collected from diarrheic or non-diarrheic cattle from nine regions in Hebei province and revealed the positive detection of BKV at a ratio of 19.34% (47/243). Sequencing of partial 3D genes from 13 positive samples and the following phylogenetic analysis demonstrated the reliability of the assay. In conclusion, the newly developed TaqMan-based real-time RT-PCR could be used for the screening and epidemic monitoring of BKV.

Role of the major histocompatibility complex class II protein presentation pathway in bone immunity imbalance in postmenopausal osteoporosis.

Bone immunity regulates osteoclast differentiation and bone resorption and is a potential target for the treatment of postmenopausal osteoporosis (PMOP). The molecular network between bone metabolism and the immune system is complex. However, the molecular mechanism underlying the involvement of the major histocompatibility complex class II (MHC-II) molecule protein presentation pathway in PMOP remains to be elucidated. The MHC-II molecule is a core molecule of the protein presentation pathway. It is combined with the processed short peptide and presented to T lymphocytes, thereby activating them to become effector T cells. T-cell-derived inflammatory factors promote bone remodeling in PMOP. Moreover, the MHC-II molecule is highly expressed in osteoclast precursors. MHC-II transactivator (CIITA) is the main regulator of MHC-II gene expression and the switch for protein presentation. CIITA is also a major regulator of osteoclast differentiation and bone homeostasis. Therefore, we hypothesized that the MHC-II promotes osteoclast differentiation, providing a novel pathogenic mechanism and a potential target for the treatment of PMOP.

Emergence and genomic analysis of a novel ostrich-origin GPV-related parvovirus in China.

In recent years, ostrich disease characterized by paralysis and diarrhea has been circulating in some regions of China, causing huge economic losses to the ostrich breeding industry. In our study, clinical samples from diseased ostriches were collected, and only parvovirus was detected. The virus distribution analysis by histopathology and quantitative real-time PCR assays indicated that the virus had a wide range of tissue tropisms. The full-length genome of the ostrich parvovirus (OsPV) was sequenced and comprehensively analyzed. Interestingly, the phylogenetic and alignment results indicated that the OsPV and the goose parvovirus (GPV) form a separate branch. In contrast to GPV strains, OsPV showed 2 new 14 nucleotide deletions in the inverted terminal repeat (ITR) region. Furthermore, recombination analysis indicated that OsPV was a recombination strain between the vaccine strain SYG61v and the virulent strain B strain, with the major parent of OsPV as the SYG61v strain and the minor parent as the B strain. The 14 nucleotide deletions in the ITR region as well as recombination may be some of the reasons for the cross-species transmission of parvovirus from goose to ostrich. The above data will contribute to a better understanding of the molecular biology of the novel OsPV and help to develop the vaccine candidate strain.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays.

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.

Simultaneous quantification of hepatitis A virus and norovirus genogroup I and II by triplex droplet digital PCR.

The representative enteric viruses responsible for global foodborne outbreaks that have become an essential concern for health authorities are Norovirus (NoV) and Hepatitis A virus (HAV). Droplet digital PCR (ddPCR) has recently emerged as an alternative platform for virus quantification due to its high precision, ultra-sensitivity, and lack of a standard curve need. Using a ratio-based probe-mixing strategy, we established a triplex ddPCR method to detect norovirus genogroup I (GI), genogroup II (GII), and HAV in food, drinking water, and faecal samples. The probe concentration, annealing temperature, and annealing/extension time were all tuned in the PCR amplification program. The detection limit for NoV GI, NoV GII, and HAV was 7.5, 5.0, and 5.0 copies/reaction, respectively. Furthermore, the suggested approach was validated on 114 samples, demonstrating greater sensitivity, accuracy, and anti-interference performance features than RT-qPCR.

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples.

Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5-12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays' results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

Direct and Rapid Detection of Mycoplasma bovis in Bovine Milk Samples by Recombinase Polymerase Amplification Assays.

This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.

Carotid imaging changes and serum IL-1ß, sICAM-1, and sVAP-1 levels in benign paroxysmal positional vertigo.

Benign paroxysmal positional vertigo (BPPV) is the most common cause of vertigo. This study was performed to evaluate serum levels of inflammatory factors and changes in B-mode carotid ultrasound findings in patients with BPPV. The study population consisted of 90 BPPV patients and 90 age- and sex-matched controls. ELISA was used to compare the levels of inflammatory factors, such as interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), soluble intercellular adhesion molecule-1 (sICAM-1), prostaglandin-E2 (PG-E2), and soluble vascular adhesion protein-1 (sVAP-1), between BPPV patients and controls. In addition, the results of ultrasonographic imaging to determine carotid intima-media thickness (C-IMT), carotid atheromatous plaque, and vertebral artery stenosis were also compared between the BPPV and control groups. Serum levels of IL-1ß, sICAM-1, and sVAP-1 were significantly higher in BPPV patients than controls (P < 0.001, P < 0.05, and P < 0.001, respectively). C-IMT and vertebral artery stenosis were significantly different in BPPV patients compared to controls (both P < 0.05). There were no significant relations between other parameters and BPPV. IL-1ß, sICAM-1, and sVAP-1 are potentially associated with the pathogenesis of BPPV, and C-ITM and carotid vertebral stenosis may be useful reference imaging findings for the diagnosis of BPPV.

Rapid detection of norovirus genogroup II in clinical and environmental samples using recombinase polymerase amplification.

Norovirus is the leading cause of acute gastroenteritis all over the world, and the most genotype that causes its epidemic is norovirus genogroup II (NoVs GII). Rapid detection of NoVs is important because it can facilitate timely diagnosis. In this study, we designed universal specific primers and an Exo probe to hybridize to all genetic clusters of NoVs GII based on the conserved region at the ORF1-ORF2 junction of the genome. For the first time, we established a rapid and reliable reverse transcription recombinase polymerase amplification (RT-RPA) method for the detection of NoVs GII within 20 min. This method can specifically amplify NoVs GII, and the detection limit was as low as 1.66 × 102 copies/µL. The method was validated in terms of LOD, accuracy, and specificity. We tested 55 real samples including foods, water, and feces. The results showed a sensitivity of 96% and specificity of 100% to NoVs GII. The whole procedure can be operated by a mobile suitcase laboratory, which is useful for resource-limited diagnostic laboratories. This novel real-time RT-RPA assay is an accurate tool for point-of-care testing of NoVs, providing practical support for norovirus-caused disease diagnosis and prevention.

Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae in sheep.

BACKGROUND: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. RESULTS: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae, as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminants. The limit of detection of LFS RPA assay was 1.0 × 101 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100 and 98.73%, diagnostic sensitivity of 90.63 and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. CONCLUSIONS: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae in sheep, especially in resource-limited settings. However, the effectiveness of the developed RPA assays in the detection of M. ovipneumoniae in goats needs to be further validated.

Development of a recombinase polymerase amplification assay for rapid detection of Haemophilus parasuis in tissue samples.

Haemophilus parasuis is the etiological agent of Glässer's disease in swine, which associates with severe economic losses in the swine industry worldwide. A real-time recombinase polymerase amplification assay (real-time RPA) was developed for direct and rapid detection of H. parasuis basing on the translation-initiation factor IF2 (infB) gene. The assay was performed successfully at 39°C for 20 min in Genie III, which is portable and chargeable by battery. The developed assay was highly specific for H. parasuis, and the limit of detection of the assay was 6.0 × 103  fg of H. parasuis genomic DNA, which was the same as that of a real-time PCR developed previously. The assay was further evaluated on 68 pig tissue samples, and 18 (26.5%), 20 (29.4%), and 8 (11.8%) samples were positive for H. parasuis by the real-time RPA, real-time PCR and bacterial isolation, respectively. With the bacteria isolation as the reference method, the real-time RPA showed a diagnostic specificity of 83.33% and a diagnostic sensitivity of 100%. The above data demonstrated the well-potentiality and usefulness of the developed real-time RPA assay in reliable diagnosis of swine Glässer's disease, especially in resource limited settings.

Development and evaluation of a rapid and sensitive RPA assay for specific detection of Vibrio parahaemolyticus in seafood.

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a leading cause of food poisoning and is of great importance to public health due to the frequency and seriousness of the diseases. The simple, timely and efficient detection of this pathogen is a major concern worldwide. In this study, we established a simple and rapid method based on recombinase polymerase amplification (RPA) for the determination of V. parahaemolyticus. According to the gyrB gene sequences of V. parahaemolyticus available in GenBank, specific primers and an exo probe were designed for establishing real-time recombinase polymerase amplification (real-time RPA). RESULTS: The real-time RPA reaction was performed successfully at 38 °C, and results were obtained within 20 min. The method only detected V. parahaemolyticus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. The study showed that the detection limit (LOD) of real-time RPA was 1.02 × 102 copies/reaction. For artificially contaminated samples with different bacteria concentrations, V. parahaemolyticus could be detected within 5-12 min by real-time RPA in oyster sauce, codfish and sleeve-fish at concentrations as low as 4 CFU/25 g, 1 CFU/25 g and 7 CFU/25 g, respectively, after enrichment for 6 h, but were detected in a minimum of 35 min by real-time PCR (Ct values between 27 and 32). CONCLUSION: This study describes a simple, rapid, and reliable method for the detection of V. parahaemolyticus, which could potentially be applied in the research laboratory and disease diagnosis.