Establishment of RT-qPCR detection method and molecular epidemiological investigation of Enterovirus G (EV-G) in Sichuan province

Abstract:

[Background] Enterovirus G (EV-G), commonly existing in pigs, can cause skin injury, muscle paralysis, pneumonia, fever, diarrhea and asymptomatic infection of pigs. [Objective] To establish a real-time quantitative polymerase chain reaction (RT-qPCR) for EV-G detection and carry out molecular epidemiological investigation in Sichuan province. [Methods] On the basis of primer design referring to the EV-G 5ʹUTR gene, SYBR Green I RT-qPCR (RT-qPCR for short) detecting all genotypes of EV-G was established and its sensitivity, specificity and repeatability were evaluated. Epidemic investigation of EV-G in Sichuan province was made with the established PCR method. [Results] The results showed that the C _t value of the RT-qPCR had a good linear relationship with the standard sample template in the concentration range of 1.89×10²-1.89×10⁸ copies/μL. In the specificity test, nine other swine viruses (PDCoV, PEDV, TGEV, JEV, PSV, PPV, PCV, PRV and PRRSV) could not be detected with the method. The minimum detection limitation was 1.89×10¹ copies/μL, and the intra-assay and inter-assay coefficients of variation were lower than 1% and 2%, respectively. A total of 431 samples with diarrhea in Sichuan province were detected by RT-qPCR, and the total positive rate of EV-G was 31.1%, indicating the virus was widespread in Sichuan province. Nine EV-G positive samples from Sichuan province were randomly selected to amplify VP1 gene for sequencing analysis. It was found that the prevalent genotypes of EV-G in Sichuan province were G1, G3, G4, and G9, but EV-G1 was the dominant genotype. [Conclusion] In this study, a RT-qPCR method for detecting EV-G genotypes is established, and the epidemic status of EV-G in Sichuan province is preliminarily mastered, which lays a foundation for further research on the virus.

Key words:Enterovirus G (EV-G);RT-qPCR;establishment;epidemiology;investigation