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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100~200 μL待测样本, 加入112~224 μL样本处理液Ⅰ, 5~10 μL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20 μL复溶缓冲液重悬沉淀; 取10 μL重悬液, 加入50 μL 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101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-Na2(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。 Abstract: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. Results The lower limit of detection(LOD) of the ZIKV assay for MR766 and ZKC2/2016 strains was 101 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-Na2(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 false negatives. Conclusions The detection method for ZIKV has good specificity and repeatability. The detection results are not affected by interfering substances. The LOD meets clinical requirements, and the operation is simple. It can be used as a backup diagnostic tool for ZIKV and it needs to be further verified in real clinical samples. -
Key words:
- Zika virus /
- rapid detection /
- virus enrichment /
- performance evaluation
作者贡献：李阿茜、孔令君负责实验设计，数据分析，论文撰写和修改；刘洋、李川参与实验设计，负责实验执行和实验结果整理；伊洁负责模拟临床样品评价部分的实验执行和数据分析；梁米芳、窦亚玲参与研究设计，并对论文进行审阅。利益冲突：所有作者均声明不存在利益冲突 -
表 1 病毒富集法对不同滴度寨卡病毒样本的检测结果
毒株 病毒滴度(TCID50/mL) 检测阳性率[%, (n/N)] MR766 106 100(9/9) (非洲型) 105 100(9/9) 104 100(9/9) 103 100(9/9) 102 100(9/9) 101 100(9/9) 100 66.7(6/9) 10-1 11.1(1/9) ZKC2/2016 106 100(9/9) (亚洲型) 105 100(9/9) 104 100(9/9) 103 100(9/9) 102 100(9/9) 101 100(9/9) 100 88.9(8/9) 10-1 0(0/9) TCID50：半数组织培养感染剂量 
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表 2 病毒富集法检测寨卡病毒的抗干扰性评价结果
干扰物质 干扰物质浓度 检测阳性率[%, (n/N)] 胆红素 15 mg/L 100(3/3) 血红蛋白 100 mg/L 100(3/3) 甘油三酯 2000 mg/L 100(3/3) IgG 37.5 g/L 100(3/3) EDTA-Na2 2.5 g/L 100(3/3)
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表 3 病毒富集法精密度评价结果
毒株 病毒浓度 批次 批次内CV(%) 批次间CV(%) MR766 高浓度(106 TCID50/mL) 批次1 6.35 4.99 (非洲型) 批次2 2.26 批次3 4.13 中浓度(104 TCID50/mL) 批次1 2.17 2.25 批次2 2.63 批次3 1.34 低浓度(102 TCID50/mL) 批次1 1.94 2.15 批次2 2.58 批次3 1.83 ZKC2/2016 高浓度(106 TCID50/mL) 批次1 6.31 8.31 (亚洲型) 批次2 6.81 批次3 7.40 中浓度(104 TCID50/mL) 批次1 3.10 3.17 批次2 2.99 批次3 2.16 低浓度(102 TCID50/mL) 批次1 3.58 3.35 批次2 2.66 批次3 1.06 CV：变异系数；TCID50：同表 1
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表 4 病毒富集法对模拟临床样本寨卡病毒的检测结果(n)
检测结果 实际结果 总计 阳性(+) 阴性(-) 阳性(+) 73 0 73 阴性(-) 0 271 271 总计 73 271 344
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