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摘要: 目的 评估A、B、C、D 4种新型冠状病毒(2019-nCoV)核酸检测试剂盒的分析性能，以便选择性能佳、性价比高的试剂用于临床检测。方法 ① 将国家卫生健康委临床检验中心和湖北省临床检验中心2021年2019-nCoV(含变异株)核酸检测室间质量评价样本36例(27例阳性和9例阴性)，磁珠法提取核酸后分别应用4种试剂扩增以评价其符合率; ②应用2019-nCoV阴性混合鼻咽拭子样本将商品化2019-nCoV RNA液体室内质控品(以ORF1ab浓度赋值17 300 copies/mL)稀释12倍到1 441.7 copies/mL，连续5 d每天做5个复孔，共计25孔，磁珠法提取核酸后分别应用4种试剂扩增以评价其重复性和实验室内精密度; ③应用2019-nCoV阴性混合鼻咽拭子样本将2019-nCoV RNA液体室内质控品(以ORF1ab浓度赋值17 300 copies/mL)梯度稀释成567.7、216.3和108.2 copies/mL 3种浓度，连续3 d每种浓度每天做7~8个复孔，共计23个复孔，磁珠法提取核酸后分别应用4种试剂扩增以评价其分析灵敏度。结果 ① 对36例2019-nCoV核酸检测室间质量评价(EQA)样本，试剂A、B和D的符合率均为100%(36/36)，试剂C的符合率为97.2%(35/36)，但试剂A、B和C分别有2、6和10个样本需要复查后才能判定结果。②4种试剂检测1 441.7 copies/mL 2019-nCoV RNA质控品稀释液的靶基因循环阈值(Ct)重复性和实验室内精密度均小于5%。③4种试剂A、B、C、D对576.7 copies/mL 2019-nCoV RNA质控品稀释液的检出率分别为95.7%、34.8%、8.7%和100%，对216.3 copies/mL稀释液的检出率则分别为95.7%、13.0%、17.4%和91.3%，A和D的分析灵敏度明显优于B和C。结论 4种新型冠状病毒核酸检测试剂的分析性能存在差异，D最优，A次之，明显优于B和C。Abstract: Objective To assess the analytic performance of four different 2019-nCoV nucleic acid detection kits(labeled A, B, C and D respectively), and select the kits with good analytic performance and high cost performance for clinical laboratory.Methods ① These 36 samples(27 positive and 9 negative for 2019-nCoV RNA) for external quality assessment(EQA) from National Center For Clinical Laboratories(NCCL) and Hubei Center For Clinical Laboratory(HBCCL) were extracted and tested using these four kits to evaluate overall agreement. ②The 2019-nCoV pseudovirus with 17 300 copies/mL of ORF1ab measured by droplet digital PCR was diluted with clinical pooled 2019-nCoV negative nasopharyngeal swab specimens to 1 441.7 copies/mL. The 1 441.7 copies/mL of 2019-nCoV pseudovirus was extracted and tested five times per day in a single run for 5 consecutive days using these four kits, and the repeatability and within-laboratory precision were calculated following EP15-A3. ③The 567.7, 216.3 and 108.2 copies/mL of 2019-nCoV pseudovirus were extracted and tested seven or eight times per day in a single run for 3 consecutive days using these four kits. The positive rate of each dilution was counted and the analytic sensitivity was evaluated.Results ① For these 36 EQA samples, the concordance rates were 100%(36/36) for A, B and D, and 97.2%(35/36) for C, respectively. But there were 2, 6, and 10 EQA specimens which needed to be tested again by kit A, B and C respectively. ②The repeatability and within-laboratory precision(%CV) of the cycles threshold(Ct) of the target genes tested by these four kits at 1 441.7 copies/mL were less than 5%. ③The detection rates of 576.7 copies/mL of 2019-nCoV pseudovirus were 95.7% for A, 34.8% for B, 8.7% for C and 100% for D, respectively. The detection rates of 216.3 copies/mL of 2019-nCoV pseudovirus were 95.7% for A, 13.0% for B, 17.4% for C and 91.3% for D, respectively.Conclusion The analytic performance of these four 2019-nCoV RNA detection kits was different. The analytic performance of D was the best, followed by A, and the analytic performance of A and D was better than that of B and C.
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表 1 4种试剂的概况
试剂 靶基因 内参 PCR反应液体积/μL 核酸模板量/μL 荧光PCR仪 扩增时间/min 靶基因阳性判断Ct参考值 分析灵敏度参考值/(copies·mL-1) 省集采每人份价格/元 A ORF1ab，N，E 外源性 20 5 Lightcycler 480 Ⅱ 80 ≤43 200 7.20 B ORF1ab，N 内源性 20 5 ABI 7500 90 ≤40 500 8.00 C ORF1ab，N 外源性 10 10 ABI 7500 76 < 40 200 5.38 D ORF1ab，N 内源性 30 20 Cobas z480 105 ≤40 200 9.10 
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表 2 4种试剂检测NCCL 2019-nCoV核酸EQA样本结果
EQA样本号 浓度/(copies·mL-1) A B C D 202111 7.5×102 三阳 双阳 N单阳1) 双阳 202112 3.0×103 三阳 双阳 N单阳1) 双阳 202113 1.5×103 三阳 双阳1) N单阳1) 双阳 202114 6.0×103 三阳 双阳 N单阳1) 双阳 202115 - - - - - F202111 1.5×103 三阳 双阳1) N单阳1) 双阳 F202112 - - - - - F202113 7.5×102 三阳 双阳1) -1) 双阳 F202114 6.0×103 三阳 双阳 N单阳1) 双阳 F202115 3.0×103 三阳 双阳 N单阳1) 双阳 核酸快速检测EQA样本号前加F，表中浓度来自NCCL室间质量评价结果报告，采用数字PCR定量; 1)第一次检测结果为单阳，双孔复查核酸后严格按试剂说明书判定结果。
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表 3 4种试剂检测NCCL 2019-nCoV变异株核酸EQA样本结果
EQA样本号 A B C D 2021A01 - - - - 2021A02 三阳1) 双阳 双阳2) 双阳 2021A03 双阳(ORF1ab与N) 双阳1) 双阳 双阳 2021A04 三阳 双阳 双阳 双阳 2021A05 - - - - 2021A06 三阳 双阳 双阳 双阳 2021A07 双阳(ORF1ab与N)1) ORF1ab单阳1) 双阳 双阳 2021A08 三阳 双阳 双阳 双阳 2021A09 三阳 双阳 双阳 双阳 2021A10 三阳 双阳 双阳 双阳 2021A11 三阳 双阳 双阳 双阳 2021A12 - - - - 2021A13 三阳 双阳 双阳2) 双阳 2021A14 双阳(ORF1ab与N) 双阳1) 双阳 双阳 2021A15 三阳 双阳 双阳 双阳 2021A16 三阳 双阳 双阳 双阳 1)第一次检测结果为单阳，双孔复查核酸后严格按相应试剂说明书判定; 2)第一次检测结果为双靶基因S型扩增曲线，但其中N Ct位于40~45，双孔复查核酸后严格按试剂说明书判定结果。
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表 4 4种试剂的精密度评价结果
试剂 靶基因 重复性 实验室内精密度 SD CV/% SD CV/% A ORF1ab 0.78 2.29 1.28 3.77 N 0.78 2.44 0.85 2.65 E 1.24 3.68 1.29 3.83 B ORF1ab 1.04 3.08 1.07 3.15 N 0.54 1.71 0.56 1.77 C ORF1ab 1.19 3.47 1.27 3.70 N 0.91 2.68 1.19 3.48 D ORF1ab 0.65 1.94 0.89 2.68 N 0.51 1.65 0.68 2.22
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表 5 4种试剂的分析灵敏度评价结果
例(%) 试剂 不同浓度质控品稀释液中检测到ORF1ab阳性情况(23例) 576.7 copies/mL 216.3 copies/mL 108.2 copies/mL A 22(95.7) 22(95.7) 14(60.9) B 8(34.8) 3(13.0) 3(13.0) C 2(8.7) 4(17.4) 3(13.0) D 23(100) 21(91.3) 16(69.6)
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| 引用本文: |
陈家颖, 黄达, 耿帜, 等. 4种新型冠状病毒核酸检测试剂的分析性能评价[J]. 临床血液学杂志, 2022, 35(8): 547-551. doi: 10.13201/j.issn.1004-2806.2022.08.003
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| Citation: |
CHEN Jiaying, HUANG Da, GENG Zhi, et al. Performance evaluation of four different 2019-nCoV nucleic acid detection kits[J]. J Clin Hematol, 2022, 35(8): 547-551. doi: 10.13201/j.issn.1004-2806.2022.08.003
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