极速热循环荧光定量PCR系统检测流感病毒的效果评价

An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus:evaluation on its efficacy

目的探讨极速热循环荧光定量PCR系统在甲型流感病毒(influenza A virus,IAV)和乙型流感病毒(influenza B virus,IBV)检测中的性能。方法构建IAV和IBV的质粒标准品,浓度梯度稀释,每个浓度重复检测20次后,制作浓度标准曲线,评价系统的最低检出限、线性范围和线性相关系数。收集来自南部战区疾病预防控制中心生物安全科2016年至2018年的临床样本278例(IAV阳性样本88例,IBV阳性样本87例,阴性样本103例),其中男性164人(59%),女性114人(41%),平均年龄24岁,通过重复检测相同IAV、IBV临床样本评价其分析重现性;将系统检测278例鼻/咽拭子临床样本与传统实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)结果比较评价其效果。结果极速热循环荧光定量PCR系统检测IAV和IBV质粒标准品的最低检出限为1×10^(0)copies/μL;线性检测范围是1×10^(0)~1×10^(5)copies/μL;此系统检测流感临床样本的敏感度为92.57%,特异度均为100%,阴、阳性预测值分别为88.79%和100%,与RT-qPCR结果的符合率为95.32%;IAV与IBV之间以及IAV和IBV与其他多种病原体之间无交叉反应;30 g/dL的血红蛋白、3.2 g/dL的甘油三酯和100μg/mL头孢曲松对检测结果无干扰。结论极速热循环荧光定量PCR系统体积小、重量轻、无需核酸提取步骤、耗时短、检测性能良好,适用于即时检测(point-of-care testing,POCT)。

Objective To evaluate the performance and application efficacies of a ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza A virus(IAV)and influenza B virus(IBV).Methods Our constructed plasmids of IAV and IBV was employed as standards,and then the concentration was diluted gradiently.After each concentration was repeatedly detected with the ultra-fast thermocycling fluorescence quantitative PCR system for 20 times,a standard curve was drawn to verify the minimum detection limit and liner range of the system and correlative coefficient.A total of 278 clinical samples(88 IAV-positive,87 IBV-positive,and 103 negative samples)were collected from the Biosafety Section of Center for Disease Control and Prevention,Southern Theater Command.They were isolated during 2016 and 2018,from the patients of 164 males(59%)and 114 females(41%),and at an average age of 24 years.The repeatability of the system was analyzed through the system repeatedly detect the identified 88 IAV-positive and 87 IBV-positive samples.The results of the system and traditional real-time q-PCR(RT q-PCR)were compared to evaluate the efficacy of the system.Results The results of ultra-fast thermocycling fluorescence quantitative PCR system showed that the minimum detection limit for detection of IAV and IBV plasmids was 1×10^(0)copies/μL,and the liner range ranged from 1×10^(0)to 1×10^(5)copies/μL.In detection of clinical samples,this system obtained a sensitivity of 92.57%,a specificity of 100%,and negative and positive predictive values of 88.79%and 100%,respectively,with a coincidence rate of 95.32%when compared with the results of traditional RT q-PCR.There were no cross-reactions between IAV and IBV or among them with other pathogens.No interference was observed in the detection results of influenza in presence of 30 g/dL hemoglobin,3.2 g/dL triglyceride and 100μg/mL ceftriaxone sodium.Conclusion The system shows good detection performance in detection of clinical samples,with advantages of small in size,light in weight,short in time,and no procedure of nucleic acid extraction,and is suitable for point-of-care testing(POCT).