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一种寨卡病毒富集检测方法的性能评价

Evaluation of an Assay of Zika Virus Detection Without RNA Extraction
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摘要 目的 对一种无需核酸提取纯化的寨卡病毒实时荧光定量反转录PCR检测方法的性能进行评价。方法 该检测方法基于实时荧光定量反转录PCR技术,样本处理采用病毒富集法:取100~200μL待测样本,加入112~224μL样本处理液Ⅰ,5~10μL样本处理液Ⅱ,混匀后进行离心富集(12 000 r/min,离心半径8.6 cm,离心5 min)。离心后弃上清液,加入20μL复溶缓冲液重悬沉淀;取10μL重悬液,加入50μL PCR反应液上机检测。从定量检测下限、交叉反应、抗干扰性、精密度方面评价该检测方法的性能。收集北京协和医院检验科临床样本(血清、尿液、唾液样本),随机选取约1/5构建寨卡病毒感染模拟临床样本,进一步评定该检测方法的性能。结果 该检测方法对MR766、ZKC2/2016毒株的检测下限均为10;半数组织培养感染剂量(50%tissue culture infective dose, TCID50)/mL;对17种常见虫媒病原体检测结果均为阴性,无交叉反应发生;潜在干扰物质胆红素(15 mg/L)、血红蛋白(100 mg/L)、甘油三酯(2000 mg/L)、IgG(37.5 g/L)和EDTA-Na;(2.5 g/L)对其检测结果无干扰;检测低、中、高浓度寨卡病毒的批次内和批次间Ct值变异系数均<10%,精密度良好。共收集344份北京协和医院检验科临床样本,其中构建寨卡病毒感染模拟临床样本73份。采用该检测方法对344份样本进行检测,结果均与实际相符,无假阳性和假阴性。结论 寨卡病毒简单富集后再检测的方法特异性高、重复性好,检测结果不受干扰物质的影响,检测下限满足临床需求,且操作简便,但应用效果仍需在真实临床样本中进一步验证。 Objective To evaluate a Zika virus(ZIKV) test based on real-time reverse-transcriptase-polymerase chain reaction(rRT-PCR) that can be performed without RNA isolation. Methods The detection method is based on rRT-PCR technology, and a virus-enrichment method is used for sample processing. The sample processing procedure is: Add 112-224 μL sample treatment solution Ⅰ and 5-10 μL sample treatment solutionⅡ to 100-200 μL clinical samples, vortex and then centrifuge at a speed of 12 000 r/min and a centrifugation radius of 8.6 cm for 5 minutes. After centrifugation, discard the supernatant, add 20 μL of solution to resuspend the precipitate, and then add 10 μL of the resuspended precipitate into the PCR reaction solution(50 μL) for detection. The performance of the assay was evaluated from the aspects of the lower limit of detection, cross reactions, interfering substances and precision. Clinical samples(serum, urine and saliva samples) were collected from the Clinical Laboratory of Peking Union Medical College Hospital, and 1/5 of these samples were randomly selected to simulate clinical samples of ZIKV infection to further evaluate the performance of the method.ResultsThe lower limit of detection(LOD) of the ZIKV assay for MR766 and ZKC2/2016 strains was 10~1 50% tissue culture infective dose(TCID50)/mL. A panel of 17 potential cross substances was tested and no cross-reactivity was detected. Potential interfering substances including bilirubin(15 mg/L), hemoglobin(100 mg/L), triglycerides(2000 mg/L), IgG(37.5 g/L) and EDTA-Na;(2.5 g/L) were evaluated and no obvious interference was observed. The intra-lots and inter-lots variations that were measured by coefficient of variation(CV), were lower than 10%. A total of 344 clinical samples from the Clinical Laboratory of Peking Union Medical College Hospital were collected;73 simulated clinical samples of ZIKV infection were constructed. The detection results of 344 samples by this method were consistent with the expected results without false positives and fa
作者 李阿茜 孔令君 刘洋 伊洁 李川 梁米芳 窦亚玲 LI Aqian;KONG Lingjun;LIU Yang;YI Jie;LI Chuan;LIANG Mifang;DOU Yaling(Department of Viral Hemorrhagic Fever,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Department of Clinical Laboratory,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100730,China)
出处 《协和医学杂志》 CSCD 2022年第3期455-460,共6页 Medical Journal of Peking Union Medical College Hospital
基金 北京市临床重点专科医学检验科卓越项目(ZK201000)。
关键词 寨卡病毒 快速检测 病毒富集法 性能评价 Zika virus rapid detection virus enrichment performance evaluation
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