猪肠病毒G (EV-G)荧光定量PCR检测方法的建立与四川地区分子流行病学调查

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

摘要:

【背景】猪肠病毒G (Enterovirus G，EV-G)在猪群中普遍存在，可引起猪的皮肤损伤、肌肉麻痹、肺炎、发热、腹泻等，也存在无症状感染。【目的】建立检测EV-G的荧光定量PCR (real-time quantitative PCR，RT-qPCR)方法并开展四川地区分子流行病学调查。【方法】根据EV-G的5ʹUTR基因保守序列设计特异性引物，建立可检测EV-G各基因型的RT-qPCR，并评估其灵敏性、特异性和重复性，开展四川地区EV-G的流行调查。【结果】标准品在1.89×10²-1.89×10⁸ copies/μL浓度范围内线性关系良好；在特异性试验中，其他9种猪病毒(猪德尔塔冠状病毒、猪流行性腹泻病毒、传染性胃肠炎病毒、日本乙型脑炎病毒、猪萨佩罗病毒、猪细小病毒、猪圆环病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒)检测均为阴性；最低检测浓度为1.89×10¹ copies/μL；组内和组间变异系数分别小于1%和2%。检测2013-2021年四川省431份猪腹泻样品，EV-G总体阳性率为31.1%，表明该病毒在四川地区的感染较普遍。随机选取9份四川的EV-G阳性样品扩增VP1基因并测序分析，发现四川EV-G流行的基因型包括G1、G3、G4和G9，但以EV-G1为优势基因型。【结论】本研究建立了一种检测EV-G各基因型的RT-qPCR方法，并初步掌握了四川EV-G的流行现状，为后续深入开展该病毒的研究奠定了基础。

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.