基于受体捕获的人源诺如病毒检测方法的优化及应用

Optimization and Application of Receptors Capturing Based Human Noroviruses Assay

在原位捕获反转录实时定量PCR法(In situ capture real-time quantitative reverse transcription polymerase chain reaction,ISC-RT-qPCR)建立的基础上,本研究旨在优化该新型感染性HuNoVs的检测方法,并将其应用于市售贝类样品中HuNoVs的检测。本研究以2份HuNoVs临床腹泻样本3010(GI组)和3009(GII组)对ISC-RT-qPCR中猪胃粘膜提取物(porcine gastric mucin,PGM)包被浓度、待测样本孵育时间和孵育pH进行了优化;最后,对随机采集的40份市售贝类样品(文蛤、花蛤、海蛏、扇贝和鲍鱼)进行感染性HuNoVs的检测。结果表明,ISC-RT-qPCR最佳试验参数分别为:PGM包被浓度为1.0 mg/mL、待测样本孵育时间为30min、孵育pH(GI组,pH=3.0;GII组,pH=7.0);40份市售贝类样品中,贝类中HuNoVs总检出率为22.5%,多重检出率为5.0%,GI组和GII组HuNoVs的检出率分别为17.5%和10.0%;其中,市售文蛤的GI组和GII组HuNoVs检出率均为最高。因此,ISC-RT-qPCR是一种可用于市售贝类样品中感染性HuNoVs的检测方法,为食品中HuNoVs的检测与监测提供了新选择。

Based on the establishment of in situ capture real-time quantitative RT-PCR （ISC-RT-qPCR）, this study aimed at optimizing this new detection method to detect infectious HuNoVs, and applying on detections of HuNoVs in shellfish from retail markets. Stool samples, 3010 （GI） and 3009 （GII）,were used to optimize the conditions of ISC-RT-qPCR. The detection conditions of ISC-RT-qPCR, including coating concentration of porcine gastric mucin （PGM）, incubation time and incubation pH, were optimized. Forty shellfish samples （dam, astarte, razor clam, scallop, and abalone） were randomly collected from retail markets to detect infectious HuNoVs particles. The results indicated that the optimum conditions were. 1.0 mg/mL for PGM coating concentration,30 min for incubation time,and incubation pH 3.0 for GI and pH 7.0 for GII. In all 40 samples,the total and double positive rates of HuNoVs were 22. 50//00 and 5. 0%, respectively;detection rates of GI and GII HuNoVs were 17.5% and 10.0% , respectively. In all 5 kinds of shellfish,clam has the highest detection rates of both GI and GII HuNoVs. ISC-RT-qPCR is a good candidate method on detecting infectious HuNoVs in retail shellfish samples, thus provide another choice for detection and monitoring of HuNoVs in food samples.