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.

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.

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 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.

Direct detection of Actinobacillus pleuropneumoniae in swine lungs and tonsils by real-time recombinase polymerase amplification assay.

Actinobacillus pleuropneumoniae is the etiological agent of swine contagious pleuropneumoniae, which is distributed globally and associated with severe economic losses in the pig rearing industry. In this study, a real-time recombinase polymerase amplification assay (real-time RPA) based on the apxIVA gene was developed to rapid detect A. pleuropneumoniae. Real-time RPA was performed successfully in Genie III at the constant temperature of 39 °C for 20 min. The developed assay was highly specific for A. pleuropneumoniae, and the sensitivity at 95% probability was 536 fg of A. pleuropneumoniae genomic DNA. The real-time RPA for A. pleuropneumoniae was further evaluated on the 112 clinical swine lung and tonsil samples, and 25 (22.3%), 27 (24.1%), and 12 (10.7%) samples were positive for A. pleuropneumoniae by the real-time RPA, real-time PCR and bacterial isolation, respectively. With a real-time PCR as the reference method, the real-time RPA showed a diagnostic specificity of 98.8%, a diagnostic sensitivity of 88.9%, a positive predicative value of 96.0%, a negative predictive value of 96.5%, and a kappa value of 0.900. The above data demonstrated the well potentiality and usefulness of the developed real-time RPA assay in the reliable detection of A. pleuropneumoniae, especially in resource limited settings.

Iliac Aneurysms Treated with Endovascular Iliac Branch Device: A Systematic Review and Meta-analysis.

BACKGROUND: Iliac branch devices (IBDs) have been increasingly reported for treating aortoiliac aneurysms. However, there are still concerns regarding this device. The aim of this study was to evaluate the safety and outcomes of IBDs in treating aortoiliac aneurysms by performing a systematic review and meta-analysis. METHODS: The Medline, EMBASE, and Cochrane databases were systematically searched to identify studies on the management of aortoiliac aneurysms using IBDs. Studies were reviewed and selected using defined criteria by 2 independent investigators who abstracted data on the study characteristics, study quality, and outcomes. The extracted data were presented as a rate and converted through arcsine transformations. Individual studies were evaluated and analyzed for 7 outcomes, including technical success rate, 30-day mortality, 30-day patency, follow-up patency, endoleak rate, buttock claudication, and IBD-associated reintervention. The heterogeneity of the studies was determined using the chi-squared distribution-based Q test and quantified by I2 statistics. Meta-analyses were performed using both a random effects model and fixed effects model. RESULTS: Twenty-two studies with a total of 1064 patients met the inclusion criteria and were selected for analysis. The pooled technical success rate of IBD was 93% (95% confidence interval [CI]: 91-95%). After patients were treated with the IBD, the 30-day mortality rate was 2% (95% CI, 1-4%), 30-day patency rate was 93% (95% CI, 91-94%), follow-up patency was 86% (95% CI, 84-88%), endoleak rate was 12% (95% CI, 8-17%), buttock claudication rate was 6% (95% CI, 5-8%), and IBD-associated reintervention rate was 11% (95% CI, 8-14%). CONCLUSIONS: Our study demonstrates that treating aortoiliac aneurysm with IBD produces satisfactory outcomes in midterm follow-up.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 102 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas.

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2.

A visible and equipment-free recombinase polymerase amplification assay combined with a lateral flow strip (LFS RPA) was developed to detect canine parvovirus type 2 (CPV-2), which is the etiological agent of canine parvovirus disease. The CPV-2 LFS RPA assay was developed based on the VP2 gene and is performed in a closed fist using body heat for 15 min; the products are visible to the naked eye on the LFS within 5 min. The assay could detect CPV-2a, CPV-2b and CPV-2c, and there was no cross-reaction with the other viruses tested. Using the standard CPV-2 DNA as a template, the analytical sensitivity was 1.0 × 102 copies per reaction, which was the same result as that of a real-time PCR. The assay performance was further evaluated by testing 60 canine fecal samples, and CPV-2 DNA was detected in 46 samples (76.7%, 46/60) by LFS RPA, which was the same result as that of the real-time PCR assay and higher than that of the SNAP method (48.3%, 29/60). The novel CPV-2 LFS RPA assay is an attractive and promising tool for rapid and convenient diagnosis of CPV disease, especially cage side and in underequipped laboratories.

Pathogenicity and molecular characterization of a fowl adenovirus 4 isolated from chicken associated with IBH and HPS in China.

BACKGROUND: Since July in 2015, an emerging infectious disease, Fowl adenovirus (FAdV) species C infection with Hepatitis-Hydropericardium syndrome was prevalent in chicken flocks in China. In our study, one FAdV strain was isolated from commercial broiler chickens and was designated as SDSX1.The phylogenetic information, genetic mutations and pathogenicity of SDSX1 were evaluated. RESULTS: The phylogenetic analysis indicated that SDSX1 is a strain of serotype 4, FAdV-C. The amino acid analysis of fiber-2 showed that there were more than 20 mutations compared with the non-virulent FAdV-C strains. The pathogenic evaluation of SDSX1 showed that the mortality of one-day-old chickens inoculated SDSX1 was 100%. The typical histopathological changes of SDSX1 were characterized by the presence of basophilic intranuclear inclusion bodies in hepatocytes. The virus copies in different tissues varied from107 to 1011 per 100 mg tissue and liver had the highest virus genome copies. CONCLUSION: In conclusion, the isolate SDSX1, identified as FAdV-4, could cause one-day-old chicks' typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were the highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1.

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. RESULTS: The FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively. CONCLUSIONS: The performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

Endovascular Chimney Technique for Aortic Arch Pathologies Treatment: A Systematic Review and Meta-Analysis.

BACKGROUND: Endovascular chimney technique has been increasingly reported in treating aortic arch pathologies. However, there are still concerns about this technique. The aim of this study is to evaluate the safety and outcome of this technique by performing a systematic review and meta-analysis. METHODS: Medline, EMBASE, and Cochrane databases were systematically searched to identify studies on the management of aortic arch pathologies using chimney technique. Studies were reviewed and selected using a priori defined criteria by 2 independent observers who abstracted data on study characteristics, study quality, and outcomes. The extracted data were presented as rate and were converted through arcsine transformations. Individual studies were evaluated and analyzed for 5 ending factors such as technical success, 30-day mortality, patency, perioperative endoleak, and stroke. Heterogeneity of the studies was determined using the chi-square distribution-based Q test and quantified by I2 statistics. The meta-analyses were performed using both random effects model and fixed-effect model. RESULTS: Twelve studies with a total of 379 patients met the inclusion criteria. The pooled technical success rate of chimney technique was 91% (95% confidence interval [CI]: 87-94%). After patients were treated with chimney technique, the rate of 30-day mortality was 4% (95% CI: 2-7%), the rate of patency was 93% (95% CI: 89-96%), the rate of perioperative endoleak was 21% (95% CI: 17-26%), and the rate of stroke was 5% (95% CI: 3-8%). CONCLUSIONS: This study indicates that using chimney technique for treating aortic arch pathologies produced satisfied midterm outcomes in both elective and emergent situations.

Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp.

Cronobacter spp. is an opportunistic pathogen that is associated with rare but life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (PIF). In the present study, we developed recombinase polymerase amplification (RPA) and real-time RPA for the detection of Cronobacter spp. in PIF for the first time by targeting the ompA gene. The specificity and sensitivity of the RPA and real-time RPA were validated and the practical applicability of these methods for the detection of Cronobacter spp. in artificially contaminated PIF samples was proved by comparing their reaction time, sensitivity, and efficacy with those of real-time PCR and the Chinese traditional method. The RPA and real-time RPA assays reduced the analysis time to less than 15 min and the results were as reliable as those of real-time PCR. Taken together, the RPA and real-time RPA assays served as fast, reliable, and sensitive techniques for the detection of Cronobacter spp.

Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus.

A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 102 copies. The assay was useful in the prevention and control of BADS.

Recombinase polymerase amplification assay for rapid detection of porcine circovirus 3.

The objective of this study was to develop a real-time recombinase polymerase amplification (rt-RPA) assay for the rapid detection of porcine circovirus 3 (PCV3). Specific RPA primers and exo probes were designed for the cap gene of PCV3 within the conserved region of viral genome. The amplification was performed at 38 °C for 20 min. The rt-RPA was specific for PCV3, as there was no cross-reaction with other pathogens tested. Using the recombinant plasmid pUC57-PCV3 as template, the analytical sensitivity was 23 copies. Of the 186 clinical samples, PCV3 DNA was identified in the 51 samples by the rt-RPA, and the positive rate was 27.4% (51/186). The diagnostic agreement between the rt-RPA and real-time PCR was 96.2%. The R2 value of rt-RPA and real-time PCR was 0.919 by linear regression analysis. The developed rt-RPA assay shows promise for rapid and sensitive detection of PCV3 in diagnostic laboratories and at point-of-need, thus facilitating the prevention and control of PCV3.

Development of a real-time recombinase polymerase amplification assay for rapid and sensitive detection of porcine circovirus 2.

Porcine diseases associated with porcine circovirus 2 (PCV-2) infection have resulted in significant economic losses worldwide. A real-time recombinase polymerase amplification (RPA) assay was developed to detect PCV-2 using primers and an exo probe specific for the ORF2 gene. The reaction process can be completed in 20 min at 38 °C. The assay only detects PCV-2, as there was no cross-reaction with other pathogens important in pigs. Using the PCV-2 genomic DNA as template, the analytical sensitivity of the real-time RPA was 103 copies. The assay performance was evaluated by testing 38 field samples and compared with real-time PCR. The two assays demonstrated a 100% diagnostic agreement, and PCV-2 DNA was detected in 26 samples. The R2 value of real-time RPA and real-time PCR was 0.954 by linear regression analysis. The real-time RPA assay provides an alternative tool for rapid, simple, and reliable detection of PCV-2, especially in remote and rural areas.

Development of a TaqMan-based real-time PCR assay for rapid and specific detection of fowl aviadenovirus serotype 4.

Twelve serotypes of fowl aviadenovirus, namely, FAdV-(1-8a and 8b-11), have been identified, among which FAdV-4 is the aetiologic agent of hepatitis hydropericardium syndrome (HHS) in chickens. Outbreaks of HHS have been documented in many countries, causing significant economic losses. Real-time PCR methods described so far in the literature cross-detect different serotypes of FAdVs. In this study, we aimed to develop a TaqMan-based real-time PCR assay for the specific detection of FAdV-4. A pair of primers targeting the hexon gene and a TaqMan probe were designed. Using different copy numbers of plasmid DNA carrying the hexon gene as template, we showed the detection limit of this assay was 101 copies/reaction, which was 10 times higher than conventional PCR. The assay was highly specific for FAdV-4 and did not cross-detect 11 other serotypes of FAdVs, avian influenza virus, Newcastle disease virus, infectious bronchitis virus or subgroup J of the avian leukosis virus. The reproducibility of the assay was assessed by five independent reactions using different copy numbers of plasmid DNA (103 and 105) as template, and the results showed 0.56-1.15% coefficient of variation for inter-assay variability. Furthermore, the assay was validated with 80 clinical samples. Real-time PCR showed that 76 out of 80 samples were positive for FAdV-4 (95.0% positivity) while 68 out of 80 were tested positive by conventional PCR (85.0% positivity). Our data suggest this real-time PCR assay could be an attractive tool for screening, confirmatory diagnosis and specific differentiation of FAdV-4 infection.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

BACKGROUND: Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. METHODS: A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. RESULTS: The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R2 value of the positive results was 0.947. CONCLUSIONS: The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2.

BACKGROUND: Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene. RESULTS: The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 105-101 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 101 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results. CONCLUSION: The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.