贝类水产品星状病毒高效NanoPCR检测方法的建立及初步应用

Establishment and preliminary application of an efficient nanomaterial-assisted PCR assay for detection of astrovirus in shellfish aquatic products

人星状病毒(Human astrovirus,HAstV)是导致全球腹泻的主要病原体之一,主要存在于贝类水产品中.消费者通过加工、烹饪贝类水产品等过程感染HAstV,并伴有腹泻、呕吐等临床症状.因此,为及时发现贝类水产品中的HAstV和保障消费者食品安全,本研究将纳米材料融入到PCR检测方法中,建立一种操作简便、灵敏度高和特异性强的HAstV检测方法.针对具有保守HAstVORF2基因序列设计特异性引物,通过优化纳米金颗粒含量、引物浓度及退火温度,建立HAstV纳米PCR(Nanomaterial-assisted PCR,NanoPCR)检测方法.研究发现HAstVNanoPCR检测方法具有较高的敏感性和特异性,其最低病毒检测限为2.0×10^(0)copies/μL,灵敏度是传统PCR的1000倍,与贝类水产品中其他常见的病毒无交叉反应;HAstV NanoPCR方法检测贝类水产品HAstV检出率为7.5%,而传统PCR检出率为5.35%.研究表明建立的HAstV NanoPCR检测方法适用于筛选贝类水产品中的HAstV,后续可应用于检测水产品中HAstV流行情况,为保障消费者食品安全提供技术保障.

Human astrovirus(HAstV)is one of the main pathogens causing global diarrhea.HAstV mainly presents in shellfishes.Shellfishes are filter-feeding animals and their digestive tissues accumulated and enriched viruses in water.It can make them as the carriers of important transmission.Consumers were mainly infected with HAstV through processes of processing and cooking shellfishes.A tiny amount of virus can cause infection in young children and patients with acquired immune dysfunction.The patients were accompanied by clinical symptoms,such as diarrhea and vomiting.In order to timely detect HAstV in shellfishes and ensure safety of consumers,we established a simple,sensitive and specific method.The nanometer materials were integrated into PCR.We designed specific primers for the conserved HAstV ORF2 gene.By optimizing the content of AuNPs,the concentration of primers and the annealing temperature,we established an Nanomaterialassisted PCR(NanoPCR)method to detect HAstV.This study indicated that the HAstV NanoPCR assay has characteristics of high sensitivity and specificity.The minimum of viral detection line was 2.0×10^(0)copies/μL,the sensitivity of NanoPCR was 1000 times higher than traditional PCR.There was no cross reactions with other viruses in shellfishes.Using NanoPCR method to detect HAstV,the detection rate was 7.5%in shellfishes,while the detection rate of traditional PCR was 5.35%.Research has shown that the HAstV NanoPCR method is suitable for screening HAstV in shellfishes.It can be timely applied to detect the prevalence of HAstV in aquatic products and ensure to provide technical support for food safety of consumers.