Establishment and application of JEV,PRRSV and CSFV TaqMan triple RT-qPCR method
- VernacularTitle:JEV、PRRSV和CSFV TaqMan三重RT-qPCR方法的建立与应用
- Author:
Li ZHANG
1
;
Deyuan TANG
1
;
Zhiyong ZENG
1
;
Bin WANG
1
;
Shenglin YUAN
1
;
Xu CHEN
1
;
Zhengbo LIAO
1
;
Piao ZHOU
1
;
Song HE
1
;
Yinming MAO
1
;
Wenwen HU
1
;
Min ZHOU
1
;
Fangxin GAO
1
Author Information
- Publication Type:Journal Article
- Keywords: JEV; PRRSV; CSFV; triple RT-qPCR; clinical detection
- From: Chinese Journal of Veterinary Science 2025;45(9):1824-1833
- CountryChina
- Language:Chinese
- Abstract: To establish a TaqMan-based multiplex RT-qPCR method for the identification of Japa-nese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),and Classical swine fever virus(CSFV),this study designed and synthesized three pairs of specific primers and probes based on the conserved sequences of JEV E,PRRSV ORF6,and CSFV E2 a-vailable in the NCBI GenBank.By optimizing the reaction system and protocol,a multiplex RT-qPCR method for detecting these three viruses was developed and applied to the detection of clini-cal samples.The results showed that the established TaqMan multiplex RT-qPCR specifically am-plified the gene fragments of JEV,PRRSV,and CSFV,and did not amplify other non-target genes,indicating good specificity of the method.Intra-assay and inter-assay repeatability tests showed that the coefficient of variation(Cv)values were all below 3％,demonstrating that the method has ex-cellent repeatability.Sensitivity tests revealed that the minimum detectable amount for the recom-binant plasmids of the three viruses was 100 copies/pL.Using the established method,a total of 969 samples,including blood,aborted fetuses,semen,and deceased pigs,from 26 pig farms in Guizhou Province were tested.The detection rates were 34.3％(332/969)for JEV,28.3％(274/969)for PRRSV,and 19.8％(192/969)for CSFV.The co-infection rates were 10.1％(98/969)for JEV and PRRSV,12.1％(117/969)for JEV and CSFV,and 14.6％(141/969)for CSFV and PRRSV.Additionally,the triple co-infection rate of JEV,PRRSV,and CSFV was 7.9％(77/969).These results indicate that the TaqMan multiplex RT-qPCR method developed in this study is ef-fective for detecting these three viruses in pig farms,providing technical support for identifying vi-ral causes of reproductive disorders.
