Dataset Information

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

ABSTRACT: The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 10(0)?~?10(9) copies/?L. This method doesn't detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5%. The serum virus titers of P1 positive in this study were measured by this protocol to be 10(3) to 10(7) copies/mL.The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

SUBMITTER: He KW

PROVIDER: S-EPMC4583164 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

He Kong-wang KW Wen Li-bin LB Wang Yong-shan YS Lu Cheng-ping CP

BMC veterinary research 20150924

<h4>Background</h4>The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.<h4>Results</h4>A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as ...[more]

PMID: 26404908

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Project description:Porcine Circovirus-like (PCL) virus, a new emerging virus, has been widely detected in Guangdong, Guangxi, and Anhui provinces in China, which may be a novel agent causing severe diarrhea in newborn piglets and tending to spread widely. Evidence suggests that the virus is related to hemorrhagic enteritis and diarrhea, and many newborn piglets were emaciated to death after infection. Therefore, a sensitive, quick, and accurate detection system for virus detection and epidemiological investigation is necessary. In this study, we developed a real-time quantitative PCR assay based on SYBR green for the detection of PCL virus. The ORF4 conserved region of PCL virus was found by the alignment of the uploaded genome sequences to design specific primers, and the primers were tested and showed good specificity, sensitivity, and reproducibility. Approximately, 138 fecal samples were obtained from diarrheal pigs in South China from June to December 2021. Approximately, 22.46% (31/138) of the samples and 40% (8/20) of the pig farms were positive for PCL virus, respectively, by using this method. Moreover, it is worth noting that the virus was first detected in Hainan and Jiangxi Provinces of China, which means that the virus may spread widely in China. Through evolutionary tree analysis and partial sequence comparison, there are some differences of virus genes in each province, suggesting that there is a risk of variation, and the four PCL virus strains showed a sequence similarity of 86.7%-87.8% for the rep gene and 92.2%-92.9% for the Rep protein, respectively, with Bo-Circo-like virus that is detected in bovine, which further demonstrates a close relationship between the two viruses that originated from different animals. In conclusion, our study provides a useful diagnostic approach to PCL virus detection and epidemiological inquiry. Meanwhile, the epidemic data using this real-time qPCR assay provide evidence for the widespread variations and epidemic of the virus in South China, and warn the appropriate measures for prevention, and control of porcine circovirus-like virus infection should be under consideration in pig production.

Project description:BackgroundThe porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR.MethodsBased on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes.ResultsAll eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g.ConclusionGenotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Dataset Information

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

Publications

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

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