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Duck is commonly used for premium meat adulteration. A simple, rapid, accurate, and affordable method is urgently needed for duck ingredient identification. Therefore, we developed two recombinase polymerase amplification (RPA) assays based on the duck-specific Cytb gene for rapid detection of duck ingredient in this study, and there were less than 30 min from sample to answer. Two RPA assays were validated to demonstrate good inter- and intra-species specificity. The real-time RPA could detect as low as 10 pg of duck DNA while the RPA combined with lateral flow test strips (LFS RPA) could detect down to 1 pg, and both could detect 0.1% duck meat adulteration. Then the two RPA assays applicability was confirmed using 113 different foods. The effects of background DNA and whole blood on RPA amplification were also analyzed, which did not affect the feasibility of the developed two RPA assays. The developed real-time RPA and LFS RPA would be promising user-friendly on-site detection methods for rapid detecting duck ingredient in different meat products.

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., 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 infB gene.

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.

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.

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.

Background: Aircrew fatigue is a major contributor of operational errors in civil and military aviation, which translates a comprehensive performance associated with both neuromuscular and cognitive states. Here we have used untargeted and non-invasive urinary metabolomics to explore flight fatigue-related aberrations. In this sense, we aimed to identify biomarkers that could better monitor pilot fatigue and also assess its severity to prevent ‘nonfunctional over-reached’ state, thus promoting flight safety. Methods: In this study, 22 active-duty male pilots, who conducted different flight hour duties, were recruited to mimic different levels of fatigue. For this, respective urine samples were collected, before and after flight, and analyzed by liquid chromatography/mass spectrometry (LC/MS). Results: Except for the fatty acids and some amino acids, significant changes on metabolite levels were observed during the progression of flight fatigue. Most of these metabolites corresponded to acyl carnitines, carbohydrates, purines and indoles. The majority of amino acids were downregulated after the flight mission. A total of 61 metabolites were found to be significantly changed along with the extent of flight fatigue. To efficiently discriminate the occurrence of flight fatigue, three candidate biomarkers (beta-guanidinopropionic acid, 3-dehydro-L-gulonate and 2-propylpent-3-enoic acid) were further characterized. Lastly, Bayes discriminant function models were established to stratify pilots with severity of fatigue and, therefore, to aid in flight risk management.Conclusion: To our knowledge, this study inaugurally provides a metabolic profiling in response to flight fatigue, thus offering a novel and effective way to monitor and manage this physiological condition.

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.

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.

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 ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 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 , especially in resource-limited settings.

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-1β (IL-1β), 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-1β, sICAM-1, and sVAP-1 were significantly higher in BPPV patients than controls (P P P P

Porcine circovirus type 3 (PCV3) is a novel pathogen first identified in the United States in 2016. As there is a high possibility that no clinical signs of infection are observed in the host, an accurate and sensitive method is needed for quarantine on numerous live pigs especially for international pig trade. In this study, a TaqMan‐based real‐time PCR assay specifically for PCV3 was established without cross‐reactions with other non‐targeted pig viruses. The sensitivity of the current approach is about 1.5 × 101 copies μL−1 plasmid DNA while the sensitivity of the conventional PCR is about 1.5 × 102 copies μL−1 plasmid DNA. Further, this assay was applied in the retrospective quarantine on serum samples of 601 commercial live boars imported to China from the United States, France and the United Kingdom from 2011 to 2017. The results revealed that PCV3 could be detected positive in the commercial boars imported from the United States and the above‐mentioned western European countries and phylogenetic study also revealed that viral isolates were grouped with some isolates from Korea and the United States. Our study suggested that PCV3 may be prevalent globally since 2011.

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.

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.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene . The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis , as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10 −1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

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.

A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R2 value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.

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.

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 102 copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.

Porcine epidemic diarrhea (PED), which is caused by porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious enteric disease characterized by severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets. A real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed based on the nucleocapsid gene of PEDV. RT-RPA assay was performed at 40 °C for 20 min. The assay could detect both the classical and variant PEDV strains, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed PEDV RNA as template, the analytical sensitivity was 23 copies per reaction. The assay performance was evaluated by testing 76 clinical samples. PEDV RNA positive rate was 55.3% (42/76) by RT-RPA and 59.2% (45/76) by real-time RT-PCR. The diagnostic agreement between the two assays was 96.1% (73/76), and the R2 value of the two assays was 0.903 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for simple, rapid and reliable detection of PEDV in resource-limited diagnostic laboratories and on-site facilities.

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 I 2 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.

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. Electronic supplementary material The online version of this article (10.1186/s12917-017-1232-z) contains supplementary material, which is available to authorized users.

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.

Introduction It remains unclear whether exposure to repeated positive acceleration (+Gz) can exacerbate endothelial dysfunction on the basis of hyperlipidemia. The aim of this study was to investigate the effect of repeated +Gz exposure on endothelial function in high-fat-diet-induced hyperlipidemic rats. Material and methods Male Sprague-Dawley rats were randomly divided into control, repeat +Gz exposure, high-fat diet (HFD), and +Gz + HFD groups. The rats in the +Gz group were exposed to +Gz and the rats in the HFD group were fed a diet with 2% cholesterol. The rats in the +Gz + HFD group received both the +Gz exposure and HFD. Eight weeks later, the endothelium-dependent relaxation of the aorta was tested and the ultrastructure of the endothelial cells was observed using transmission electron microscopy. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression of endothelial function-associated proteins. Results Repeated +Gz exposure elevated the serum level of LDL-C in HFD rats. In the +Gz + HFD rats, the ACh-induced relaxation in the aorta rings was significantly attenuated and the endothelial cells of the aorta were dramatically damaged compared with HFD rats. Nitric oxide content and eNOS expression in the aortic tissue were markedly decreased and the oxidative stress was more serious in the +Gz + HFD rats compared with HFD rats. In addition, repeated +Gz exposure significantly increased serum ox-LDL level and LOX-1 expression in the aorta of HFD rats, thereby activating NF-κB p65 and upregulating the expression of interleukin 6, ICAM-1 and VAP-1. Conclusions Repeated +Gz exposure promotes endothelial dysfunction in HFD-induced hyperlipidemic rats.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.

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.