猪戊型肝炎病毒(HEV)RT-qRPA检测方法的建立及应用评价

Evaluation of RT-qRPA method for the detection of swine hepatitis E virus

为了建立一种快速、高效检验猪肝样品中猪戊型肝炎病毒(HEV)污染情况的新方法,即HEV实时荧光逆转录-重组酶聚合酶扩增(RT-qRPA)检测方法。参考TwistDx公司RPA技术说明,以基因4型猪HEV分离株的保守基因序列为模板设计合成特异性引物和EXO探针建立HEV RT-qRPA检测方法。以HEV国际检测金标准RT-qPCR方法为参照,对新建立的RT-qRPA检测方法的特异性、敏感性及可重复性进行对比验证评价,进一步通过对锦州市猪肝样品中HEV的污染情况进行对比检测分析,评估RT-qRPA检测方法的临床可靠性。结果显示,本研究所建立的RT-qRPA检测方法在39℃恒温条件下20 min即可检出阳性值,且特异性强、敏感性高,HEV RNA的最低检测限约17.7拷贝,与RT-qPCR标准参考方法相同。148份猪肝脏样品中HEV的阳性检出率为0.8%(1/148),与RT-qPCR方法的检出结果一致。本研究成功建立了HEV RT-qRPA检测方法,该方法敏感性高、特异性好、操作简单、反应快速,为下一步开发POCT便携检测设备应用于我国畜牧兽医基层工作的HEV检测提供了技术参考。

The purpose of this study was to establish a simple,rapid and efficient real-time reverse transcription-recombinase polymerase amplification(RT-qRPA)assay for HEV contaminated in raw pig liver.The specific primers and EXO probe for RT-qRPA assay was designed based on the conserved region in genotype 4 swine HEV isolate(DQ279091)according to TwistDx description.The specificity,sensitivity and reproducibility of the RT-qRPA assay were analyzed by comparing with the gold standard RT-qPCR assay.Further,the clinical reliability of the RT-qRPA assay was evaluated by detecting the HEV contamination in the pork liver from Jinzhou markets.The results showed that the established RT-qRPA assay in this study could specifically performed under the constant temperature of 39℃for 20 min,without cross reaction to other internal control viruses.The minimum detection limit of RT-qRPA assay for HEV RNA was 17.7 copies/reaction,which was the same as the reference standard method.HEV RNA positive detection rate was 0.8%(1/148)in 148 pig liver samples by both methods.These dates indicate that the HEV RT-qRPA assay established in this study was sensitivity,specificity,and rapidly.It provides a technical reference for the developing of POCT portable testing equipment for HEV testing in the grassroots work of husbandry and veterinary in China.