双重荧光数字PCR方法定量检测贝类中诺如病毒

Quantitative detection of Noroviruses in shellfish by dual fluorescence digital PCR

目的建立双重荧光微滴式数字反转录聚合酶链式反应(microdrop digital reverse transcription polymerase chain reaction,RT-ddPCR)法定量检测贝类样品中诺如病毒。方法确定RT-ddPCR方法的最佳退火温度和探针浓度,建立贝类中诺如病毒双重荧光数字PCR检测方法,并定量检测秦皇岛2015~2017年7月采集的贝类样品中的诺如病毒。结果贝类中诺如病毒RT-ddPCR检测方法线性范围为5×10^4~5×10^0 copy/µL,检出限为1.0 copy/20µL的反应体系。用数字PCR方法检出秦皇岛贝类样品中诺如病毒总阳性率为67%,GⅠ型病毒平均浓度为8.70×10^2 copy/g消化腺,GⅡ型病毒平均浓度为1.04×10^3 copy/g消化腺。结论与荧光定量反转录聚合酶链式反应(fluorescence quantitative reverse transcription polymerase chain reaction,RT-qPCR)方法相比,数字PCR方法在检测贝类诺如病毒时有明显优势,能提高病毒检出率,不依赖标准品进行绝对定量。

Objective To establish a method for the quantitative determination of Norovirus in shellfish samples by a dual fluorescent digital reverse transcription polymerase chain reaction(RT-ddPCR). Methods The annealing temperature and probe concentrations of RT-ddPCR experiment were optimized to establish a dual fluorescence digital PCR method for detection of Norovirus in shellfish,and the Norovirus in shellfish samples collected from Qinhuangdao from July of 2015 to 2017 were quantitatively detected. Results The linear range of RT-ddPCR for detection of Norovirus in shellfish was 5×10^4-5×10^0 copy/μL,and the limit of detection was 1.0 copy/20 μL reaction system. The total positive rate of Norovirus in shellfish samples from Qinhuangdao was 67%. The average concentration of GI virus was 8.70×10^2 copy/g digestive gland,and 1.04×10^3 copy/g digestive gland for GII type. Conclusion Compared with fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR),digital PCR has obvious advantages in detecting shellfish Norovirus,which can improve the detection rate,and does not rely on standard materials for absolute quantification.