One-step multiple TaqMan real-time RT-PCR for simultaneous detection of swine diarrhea viruses

Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.

Analysis of Common Respiratory Infected Pathogens in 3100 Children after the Coronavirus Disease 2019 Pandemic.

OBJECTIVE: To investigate the epidemiological features in children after the coronavirus disease 2019 (COVID-19) pandemic. METHODS: This study collected throat swabs and serum samples from hospitalized pediatric patients of Renmin Hospital of Wuhan University, Wuhan, Hubei province, China before and after the COVID-19 pandemic. Respiratory infected pathogens [adenovirus (ADV), influenza virus A/B (Flu A/B), parainfluenza virus 1/2/3 (PIV1/2/3), respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP), and Chlamydia pneumoniae (CP)] were detected. The pathogens, age, and gender were used to analyze the epidemiological features in children after the COVID-19 pandemic. RESULTS: The pathogen detection rate was significantly higher in females than in males (P<0.05), and the infection of PIV1 and MP was mainly manifested. After the COVID-19 pandemic, PIV1, PIV3, RSV, and MP had statistically different detection rates among the age groups (P<0.05), and was mainly detected in patients aged 0-6 years, 0-3 years, 0-3 years, and 1-6 years, respectively. When comparing before the COVID-19 pandemic, the total detection rate of common respiratory pathogens was lower (P<0.05). Except for the increase in the detection rate of PIV1 and CP, the infection rate of other pathogens had almost decreased. CONCLUSION: The prevention and control measures for the COVID-19 pandemic effectively changed the epidemiological features of common respiratory tract infectious diseases in pediatric children.

Identification of coronaviruses in farmed wild animals reveals their evolutionary origins in Guangdong, southern China.

Coronavirus infections cause diseases that range from mild to severe in mammals and birds. In this study, we detected coronavirus infections in 748 farmed wild animals of 23 species in Guangdong, southern China, by RT-PCR and metagenomic analysis. We identified four coronaviruses in these wild animals and analysed their evolutionary origins. Coronaviruses detected in Rhizomys sinensis were genetically grouped into canine and rodent coronaviruses, which were likely recombinants of canine and rodent coronaviruses. The coronavirus found in Phasianus colchicus was a recombinant pheasant coronavirus of turkey coronavirus and infectious bronchitis virus. The coronavirus in Paguma larvata had a high nucleotide identity (94.6-98.5 per cent) with a coronavirus of bottlenose dolphin (Tursiops truncates). These findings suggested that the wildlife coronaviruses may have experienced homologous recombination and/or crossed the species barrier, likely resulting in the emergence of new coronaviruses. It is necessary to reduce human-animal interactions by prohibiting the eating and raising of wild animals, which may contribute to preventing the emergence of the next coronavirus pandemic.

Public health risks of regional assessment of the coronavirus diseases 2019 in Wenzhou, Zhejiang

Objective To establish an index system for comprehensive evaluation of public health risks of the coronavirus diseases 2019(COVID-19),and to evaluate the COVID-19 in different counties(districts) of Wenzhou, so as to provide scientific evidence for the implementation of targeted prevention and control measures. Methods Rank-sum ratio(RSR) was used to evaluate 12 quality indicators of 5 categories by brain storming method with data including the incidence of COVID-19 and public health risks of COVID-19 epidemic in Wenzhou during January and February, 2020.The regional public health risks of COVID-19 were ranked according to rank-sum ratio size. Results The top three counties(districts) with most reported cases were Yueqing,Rui′an and Lucheng,accounting for 61.90%(312/504) of the total number in Wenzhou.The ratio of import cases to indigenous cases was 1∶1.39.The top three counties(districts) reported most clustering epidemics were Yueqing,Rui′an and Lucheng,accounting for 62.03%(49/79) of the total number in Wenzhou.About 70.63%(356/504)of the cases were found before the medical visit.The time from onset to first medical visit was 2.39 d, and the time from medical visit to diagnosis was 4.49 d.The average number of close contacts for confirmed cases was 29,and the top three counties(districts)with most reported average number of close contacts were Jingkai(135 cases),Dongtou(59 cases)and Cangnan(50 cases).Among all13 counties(districts),Longgang,Wencheng and Jingkai were at lower level,Yueqing and Lucheng were at higher level,and the other counties(districts)were at the middle level of public health risks of COVID-19. Conclusions RSR method is flexible and simple.It has no specific requirements on samples,and can eliminate the effects of comprehensive assessment indexes of different dimension.It has high value in evaluating public health risks of the COVID-19.The evaluating of public health risks of COVID-19 should be strengthened.Targeted prevention and control measures should be taken to positively prevent the outbreak of COVID-19.

Chinese Society of Interventional Radiology Expert Consensus on the prevention and control of COVID-19 in interventional radiology procedures (first edition).

The COVID-19 pandemic seriously threatens the lives of the general public and poses momentous challenges to all medical workers, including those engaged in interventional radiology, who play an important role in the diagnosis and treatment of various diseases. To further standardize the prevention and control of nosocomial infections and ensure the safety of doctors and patients, the Chinese Society of Interventional Radiology (CSIR) organized multidisciplinary experts in the field of interventional radiology in China to prepare an "Expert Consensus" elaborating and summarizing the protective strategies and suggestions for medical workers in the field of interventional radiology when they engage in interventional diagnosis and treatment activities against the background of novel coronavirus infection control. The aim is to provide a reference for interventional procedures in hospitals and other medical institutions at all levels in China and worldwide. The key points include the following: (I) non-emergency interventional diagnosis and treatment should be suspended while work is ongoing to prevent and control the spread of COVID-19; (II) protective measures should be taken according to the appropriate level designated for COVID-19 infection prevention and control; (III) patients should take measures to protect themselves when they want to see a doctor, including accessing outpatient services online and other relevant channels of consultation.