模拟水样中戊型肝炎病毒检测方法的比较

Comparison of methods for the detection of hepatitis E virus in simulated water samples

目的对模拟水样中戊型肝炎病毒(HEV)的检测方法进行比较, 为水中HEV的检测提供参考。方法自来水或蒸馏水模拟水样中人工污染HEV粪便悬液, 用正电荷滤膜-有机洗脱液洗脱法(方法1)进行前处理, 比较A、B和C三种核酸提取试剂盒提取效果;对模拟水样用方法1、方法2(正电荷滤膜-直接裂解法)、方法3(切向流超滤膜-无机洗脱液洗脱法)、方法4(切向流超滤膜-直接裂解法)进行前处理, A试剂盒进行核酸提取, Real time RT-PCR方法检测, 比较回收率;对模拟水样中不同浓度HEV回收率进行比较;比较自来水样中的抑制物对Real time RT-PCR的抑制作用;对不同批次自来水标本的HEV进行检测。结果试剂盒A核酸提取效果更好;方法1、2、3和4的回收率分别为7.31%、39.88%、6.85%和64.88%, 结果显示回收率具有统计学差异(F=114.069, P<0.001)。方法4添加高、中、低浓度HEV的回收率分别为65.26%、42.76%、32.79%。四种前处理方法抑制率均小于75%, 符合ISO(15216-2∶2019)要求。对20份自来水标本进行HEV检测, 结果均为阴性。结论本研究表明两种滤膜分别结合直接裂解法回收效果更好;方法4在小体积蒸馏水或自来水的HEV的检测中回收率较高, 但受水样体积、浊度等限制。对不同的水质及实验室条件可选择合适的方法进行检测。

Objective To compare the detection method of hepatitis E virus(HEV)in simulated water samples,and to provide a reference for the detection of HEV in water.Methods HEV fecal suspension was added to tap water or distilled water simulated water samples,and pretreatment was carried out by electropositive filter-organic eluent elution method(Method 1)to compare the extraction effect of the three nucleic acid extraction kits,A,B,and C.The simulated water samples were pre-treated by Method 1,2(electropositive filter-direct lysis method),3(tangential-flow ultrafiltration membrane-organic eluent elution method),and 4(tangential-flow ultrafiltration membrane-direct lysis method)for pretreatment,A kit for nucleic acid extraction,Real time RT-PCR method for detection and comparison of the recovery rate;comparison of the recovery rate of different concentrations of HEV in simulated water samples;comparing the inhibitory effects of inhibitors in tap water samples on real time RT-PCR;and detection of HEV in different batches of tap water specimens.Results Kit A nucleic acid extraction was better;the recoveries of method 1,2,3 and 4 were 7.31%,39.88%,6.85%and 64.88%,respectively,which showed a statistically significant difference in the recoveries(F=114.069,P<0.001).The recoveries of method 4 with the addition of high,medium and low concentrations of HEV were 65.26%,42.76%and 32.79%,respectively.The inhibition of all four pre-treatment method was less than 75%,which meets the requirements of ISO(15216-2∶2019).Twenty tap water specimens were tested for HEV and the result were negative.Conclusions This study showed that the two membranes better recovered in combination with direct lysis,respectively;Methods 4 had a higher recovery in the detection of HEV in small volumes of distilled or tap water,but it was limited by the volume of water samples,turbidity,and so on.Suitable method can be selected for different water quality and laboratory conditions.