以可乐为代表的饮品中戊型肝炎病毒富集及检测方法比较  

Comparison of enrichment and detection methods for hepatitis E virus in beverages represented by cola

作　　者：张瑞婷 王秋媛 尹文娇[1] 曹经瑗[1] Zhang Ruiting;Wang Qiuyuan;Yin Wenjiao;Cao Jingyuan(National Health Commission Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2025年第1期122-127,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家科技重大专项(2018ZX10201002-009-002)。

摘　　要：目的对模拟可乐样本中戊型肝炎病毒(Hepatitis E Virus,HEV)的富集及核酸检测方法进行比较。方法使用正电荷滤膜-直接裂解法(方法1)、切向流超滤膜-直接裂解法(方法2)以及方法3、4(在方法1、2基础上分别增加PCR抑制剂去除步骤)对人工污染HEV的可乐样本进行富集,实时荧光定量RT-PCR(RT-qPCR)检测,比较回收率及抑制率。应用数字RT-PCR(RT-dPCR)及RT-qPCR检测中、低浓度人工污染可乐样本中HEV的回收率;以及在不同基质中HEV RNA检测的抑制率及敏感性。选用方法3对8份市售可乐样本进行病毒富集,RT-qPCR及RT-dPCR进行HEV RNA检测。结果RT-qPCR检测方法3、方法4的HEV回收率(10.44%、10.16%)均高于方法1、方法2的回收率(4.89%、0.32%),差异有统计学意义(P<0.05)。方法3、4抑制率均小于方法1、2。RT-qPCR及RT-dPCR检测中浓度人工污染可乐样本中HEV的回收率分别为17.04%和16.28%,对低浓度人工污染可乐样本中HEV的回收率分别为6.91%和4.65%,两种检测方法在同一浓度的回收率差异均无统计学意义(P=0.260,P=0.107);可乐基质对RT-qPCR和RT-dPCR检测均有一定抑制作用。8份市售可乐样本HEV RNA检测均为阴性。结论可乐饮品中HEV的检测可选用正电荷滤膜-直接裂解法+抑制剂去除(方法3)或切向流超滤膜-直接裂解法+抑制剂去除(方法4)富集,再进行RT-dPCR或RT-qPCR检测,病毒检测回收率较高。ObjectiveTo compare enrichment and nucleic acid detection method for hepatitis E virus(HEV)in simulated cola samples.MethodsCola samples experimentally contaminated with HEV were enriched using positively charged filter membrane-direct lysis(Method 1),tangential flow ultrafiltration membrane-direct lysis(Method 2),and Method 3 and 4(with the addition of a PCR inhibitor removal step on the basis of Method 1 and 2,respectively),and were assayed by real-time fluorescence quantitative RT-PCR(RT-qPCR),and the recoveries and inhibition rates were compared.Digital RT-PCR(RT-dPCR)and RT-qPCR were applied to detect the recovery of HEV in different medium and low concentrations of experimentally contaminated cola samples;and the inhibition rate and sensitivity of HEV RNA detection in different matrices.Methods 3 was selected for virus enrichment of 8 commercially available cola specimens,RT-qPCR and RT-dPCR for HEV RNA detection.ResultsThe HEV recoveries of method 3 and 4(10.44%and 10.16%)were higher than those of method 1 and 2(4.89%and 0.32%),and the differences were statistically significant(P<0.05).The inhibition rates of method 3 and 4 were smaller than the inhibition rates of method 1 and 2.The recoveries of HEV in medium concentration artificially contaminated cola samples by RT-qPCR and RT-dPCR were 17.04%and 16.28%,respectively,and for low concentration artificially contaminated cola samples were 6.91%and 4.65%,respectively,and the differences in recoveries between the two assays at the same concentration were not statistically significant(P=0.260,P=0.107);Cola matrix inhibits the detection of both RT-qPCR and RT-dPCR assays.Eight commercially available cola specimens were negative for HEV.ConclusionsDetection of HEV in cola beverages can be done by positively charged filter membrane-direct lysis+inhibitor removal(method 3)or tangential flow ultrafiltration membrane-direct lysis+inhibitor removal(method 4)enrichment,followed by RT-dPCR or RT-qPCR,with a high recovery of virus detection.

关 键 词：戊型肝炎病毒 可乐 富集 RT-QPCR RT-dPCR 

分 类 号：R51[医药卫生—内科学]