摘要
目的建立甲型肝炎病毒(hepatitis A virus,HAV)数字芯片式RT-PCR(dRT-PCR)方法并与real time RT-PCR(RT-qPCR)方法进行比较,选出适用于检测草莓中HAV的最佳方法。方法提取HAV疫苗RNA,优化dRT-PCR的反应条件,评价其特异性;碱性洗脱-PEG浓缩法对草莓标本进行前处理后提取核酸,同时采用dRT-PCR与RT-qPCR方法,检测纯水或草莓基质中HAV疫苗RNA的敏感性、抑制率;比较人工污染草莓中HAV的回收率,并应用于市售标本中的检测。结果确立了dRT-PCR反应的最适退火温度为60℃,最佳引物、探针浓度为0.4μmol/L、0.4μmol/L、0.2μmol/L,特异度良好;两种检测方法检测HAV疫苗RNA在纯水或草莓基质中的敏感性没有明显差异,dRT-PCR的抑制率较低。dRT-PCR与RT-qRCR在检测较高浓度HAV污染草莓标本时的回收率分别为12.90±0.006%、30.12±0.02%;在检测较低浓度HAV污染草莓标本时的回收率分别为18.27±0.07%、10.85±0.03%,差异均具有统计学意义(P<0.05)。对市售标本进行检测,两种方法检测结果均为阴性。结论本研究建立的dRT-PCR法,与RT-qPCR检测不同基质中的HAV RNA敏感性没有明显差异,但dRT-PCR对PCR反应抑制物有较好的耐受能力,在检测低浓度HAV时回收率较高。两种检测方法均可用于草莓中甲型肝炎病毒的定量检测,可根据具体实际情况进行选择。
Objective To establish a digital droplet RT-PCR(dRT-PCR)method for Hepatitis A virus(HAV),and compare it with Real time RT-PCR(RT-qPCR)method,and select the best method for detecting hepatitis A virus in strawberry.Methods Extract HAV vaccine RNA,optimize the reaction conditions of dRT-PCR and evaluate its specificity;Alkaline elution-PEG concentration method was used to extract nucleic acid from strawberry samples.At the same time,dRT-PCR and RT-qPCR method were used to detect the sensitivity and inhibition rate of HAV vaccine RNA in pure water and strawberry matrix,and the recovery rate of HAV in artificially contaminated strawberry was compared,which was applied to the detection of commercially available samples.Results The optimal annealing temperature for dRT-PCR reaction was 60℃,and the optimal concentrations of primers and probes were 0.4μmol/L、0.4μmol/L and 0.2μmol/L,with good specificity.There is no significant difference in sensitivity between the two method in detecting HAV vaccine RNA in pure water and strawberry matrix.The inhibition rate of dRT-PCR is low.The recovery rates of dRT-PCR and RT-qRCR in the detection of strawberry samples contaminated with HAV at higher concentrations were 12.90±0.006%and 30.12±0.02%,respectively.The recovery rates of lower concentrations of HAV contaminated strawberry samples were 18.27±0.07%and 10.85±0.03%,respectively,and the difference was statistically significant(P<0.05).When strawberry samples on the market were tested,the result of both method were negative.Conclusions The sensitivity of dRT-PCR method established in this study is not significantly different from that of RT-qPCR in detecting HAV RNA in different substrates,but dRT-PCR has good tolerance to PCR reaction inhibitors and high recovery rate when detecting low concentration HAV.Both detection method can be used for quantitative detection of hepatitis A virus in strawberry,and can be selected according to the actual situation.
作者
焦梦琪
石峰
尹文娇
曹经瑗
毕胜利
Jiao Mengqi;Shi Feng;Yin Wenjiao;Cao Jingyuan;Bi Shengli(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)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2023年第4期443-448,共6页
Chinese Journal of Experimental and Clinical Virology
基金
国家卫生健康委疾控专项(131031103000200004)。
