Detection of hepatitis A virus in strawberry by digital RT-PCR and fluorescence quantitative RT-PCR
10.3760/cma.j.cn112866-20230512-00051
- VernacularTitle:数字RT-PCR与荧光定量RT-PCR法检测草莓中甲型肝炎病毒
- Author:
Mengqi JIAO
1
;
Feng SHI
;
Wenjiao YIN
;
Jingyuan CAO
;
Shengli BI
Author Information
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
- Keywords:
Strawberry;
Hepatitis A virus;
Digital RT-PCR;
Fluorescence quantitative RT-PCR
- From:
Chinese Journal of Experimental and Clinical Virology
2023;37(4):443-448
- CountryChina
- Language:Chinese
-
Abstract:
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 0.4 μmol/L、0.4 μmol/L and 0.2 μmol/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.
