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A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets

Author:
Wang, Zhao, Liu, Yebing, Lin, Wencheng, Cui, Shangjin
Source:
Molecular biology reports 2012 v.39 no.12 pp. 10013-10017
ISSN:
0301-4851
Subject:
Porcine reproductive and respiratory syndrome virus, Classical swine fever virus, genes, lungs, heart, Suid alphaherpesvirus 1, detection limit, endometrium, spleen, viruses, pathogenesis, testes, virulence, pathogens, Porcine teschovirus, fetus, sows, kidneys, Porcine circovirus, quantitative polymerase chain reaction, Encephalomyocarditis virus, piglets
Abstract:
A real-time polymerase chain reaction with SYBR Green was developed for the detection and quantification of encephalomyocarditis virus (EMCV) in porcine tissues; the method uses two primers specific for the 3D gene. The detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001 TCID₅₀/ml. The assay was linear over a 10⁷dilution range of template concentrations and was specific for EMCV; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductive and respiratory virus, classical swine fever virus, pseudorabies virus, or porcine teschovirus). This assay detected EMCV titers at least 10⁴smaller than the routine PCR assay. To increase our understand of EMCV pathogenesis, the new method was used to quantify levels of EMCV genome in various tissues of artificially challenged sows and piglets. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. The real-time PCR method described here should be useful for the study of EMCV infection and distribution in pigs.
Agid:
1206844