Establishment and application of quadruplex RT-qPCR for differentiation of viral pathogens associated with diarrhea in pig herds
10.16303/j.cnki.1005-4545.2025.11.04
- VernacularTitle:鉴别猪病毒性腹泻病原的四重荧光定量RT-PCR方法的建立及应用
- Author:
Chunlin LI
1
;
Zhou SHA
;
Jin CUI
;
Hui ZHENG
;
Fulong NAN
;
Yaqin DONG
;
Rong WEI
;
Rui WU
;
Bo NI
Author Information
1. 黑龙江八一农垦大学,动物科技学院,黑龙江大庆 163319;佳木斯大学,黑龙江佳木斯 154000
- Publication Type:Journal Article
- Keywords:
TGEV;
PEDV;
PDCoV;
PoRVA;
quadruplex RT-qPCR
- From:
Chinese Journal of Veterinary Science
2025;45(11):2325-2333,2342
- CountryChina
- Language:Chinese
-
Abstract:
A quadruplex RT-qPCR method was developed for rapid identification and diagnosis of transmissible gastroenteritis virus(TGEV)of swine,porcine epidemic diarrhea virus(PEDV),porcine deltacorona virus(PDCoV),and porcine rotavirus type A(PoRVA).The full-length sequences of PEDV(77 strains),TGEV(63 strains),PDCoV(17 strains)that are prevalent in China,as well as the 85 VP6 gene sequences of PoRVA,were downloaded from the NCBI database for homology analysis.Based on the relatively conserved sequences,the corresponding primers and probes for each virus were designed and used to establish the quadruplex RT-qPCR method.After optimization of the probes and the reaction conditions,the specificity,sensitivity,and repeatability were determined.Using the established method,109 clinical samples of diarrhea were tested and further compared with the results by standard method.The results showed that the quadruplex RT-qPCR method established in this experiment has good amplification effect,with the C,value linearly correlated with the copies of templates(R2＞0.99).Specificity assay demonstrated that the quadruplex RT-qPCR method can identify TGEV,PEDV,PDCoV,PRoVA strains,and do not de-tect African swine fever virus(ASFV),porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV)and other epidemic viruses.Sensitivity assay showed that the detection limits for TGEV,PEDV,PDCoV and PoRVA were 10,20,20 and 50 copies/μL,respectively.The method exhibits excellent reproducibility,with coefficients of variation(Cv)for both intra-and inter-assay repli-cates being less than 1％.Detection of 109 samples of diarrhea by this method yielded the coinci-dence rate of 100％with the industry standard,indicating high practical applicability.The devel-oped method possesses the advantages of strong strain compatibility,high sensitivity,strong speci-ficity,good repeatability and stability.It is suitable for virus diagnosis and large-scale clinical sam-ple testing,providing technical support for disease prevention and control as well as epidemiologi-cal investigation.
