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6种不同实时荧光定量PCR仪对新型冠状病毒核酸检测性能的影响探讨

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文章导航 > 临床血液学杂志 > 2023 > 36(10): 697-701
陈家颖, 耿帜, 佘丹, 等. 6种不同实时荧光定量PCR仪对新型冠状病毒核酸检测性能的影响探讨[J]. 临床血液学杂志, 2023, 36(10): 697-701. doi: 10.13201/j.issn.1004-2806.2023.10.002
引用本文: 陈家颖, 耿帜, 佘丹, 等. 6种不同实时荧光定量PCR仪对新型冠状病毒核酸检测性能的影响探讨[J]. 临床血液学杂志, 2023, 36(10): 697-701. doi: 10.13201/j.issn.1004-2806.2023.10.002
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CHEN Jiaying, GENG Zhi, SHE Dan, et al. Effect of six different types of real-time fluorescent quantitative PCR instruments on analytic performance of 2019-nCoV nucleic acid detection[J]. J Clin Hematol, 2023, 36(10): 697-701. doi: 10.13201/j.issn.1004-2806.2023.10.002
Citation: CHEN Jiaying, GENG Zhi, SHE Dan, et al. Effect of six different types of real-time fluorescent quantitative PCR instruments on analytic performance of 2019-nCoV nucleic acid detection[J]. J Clin Hematol, 2023, 36(10): 697-701. doi: 10.13201/j.issn.1004-2806.2023.10.002
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6种不同实时荧光定量PCR仪对新型冠状病毒核酸检测性能的影响探讨

    • 华中科技大学同济医学院附属协和医院检验科(武汉，430022)

详细信息
    通讯作者: 陈凤花，E-mail：chfh100@126.com

  • 中图分类号: R446.1

Effect of six different types of real-time fluorescent quantitative PCR instruments on analytic performance of 2019-nCoV nucleic acid detection

    • Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

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    摘要
  • 目的 探讨6种不同型号的实时荧光定量PCR仪(分别为CFX96 DeepWell、GENTIER 96E、Cobas z480、LightCycler 480Ⅱ、ABI 7500、QuantStudio 5，依次标为A、B、C、D、E、F)对新型冠状病毒(2019-nCoV)核酸检测性能的影响，以便选择分析性能佳的检测系统用于临床检测。方法 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(假病毒)先10倍稀释接着2.5倍梯度稀释至1∶10、1∶25、1∶62.5、1∶156.25、1∶390.63、1∶976.56、1∶2 441.41和1∶6 103.52八个不同浓度，连续3 d内每天每个浓度做5个复孔，共计15个复孔，磁珠法提取核酸后应用同一种2019-nCoV扩增试剂在6种不同实时荧光定量PCR仪上进行扩增以评价其检出限；将1∶10和1∶25两个稀释度的质控品样本，连续5 d内每天每个浓度做5个复孔，共计25孔，磁珠法提取核酸后应用同一种2019-nCoV扩增试剂在6种不同实时荧光定量PCR仪上进行扩增以评价靶基因循环阈值(Ct)的重复性和实验室内精密度。结果 ① 6种实时荧光定量PCR仪对1∶10、1∶25和1∶62.5三个稀释度的质控品样本中ORF1ab和N基因的检出率均为100.0%；而对1∶156.25稀释样本中ORF1ab和N基因的检出率：仪器A为100.0%和100.0%，B为66.7%和86.7%，C为100.0%和100.0%，D为93.3%和93.3%，E为93.3%和93.3%，F为100.0%和100.0%；对1∶390.63稀释样本中ORF1ab和N基因的检出率：仪器A为60.0%和86.7%，B为60.0%和80.0%，C为66.7%和73.3%，D为73.3%和93.3%，E为66.7%和53.3%，F为73.3%和93.3%；1∶976.56、1∶2 441.41和1∶6 103.52三个稀释度的质控品样本中靶基因的检出率分别为4.00%~60.00%、6.67%~20.00%和0~20.00%。②6种不同实时荧光定量PCR仪对1∶10和1∶25两个稀释度的质控品样本中Ct的重复性和实验室内不精密度均 < 5.00%。结论 不同的实时荧光定量PCR仪对2019-nCoV核酸检测性能存在影响，该2019-nCoV扩增试剂在仪器C、F上的分析灵敏度优于另外4种实时荧光定量PCR仪。
    Objective To assess the effect of six different types of real-time fluorescent quantitative PCR instruments including Biorad CFX96 Deep Well, TianLong GENTIER 96E, Roche Cobas z480, Roche LightCycler 480Ⅱ, ABI 7500 and QuantStudio 5(labeled as A, B, C, D, E and F) on analytic performance of 2019-nCoV nucleic acid detection, and select the appropriate detection system with good analytic performance for clinical laboratory.Methods The commercial 2019-nCoV pseudovirus for internal quality control(IQC) was serially diluted with the clinical pooled 2019-nCoV negative nasopharyngeal swab specimens to eight dilutions including 1∶10, 1∶25, 1∶62.5, 1∶156.25, 1∶390.63, 1∶976.56, 1∶2 441.41 and 1∶6 103.52, which were extracted and tested five times per day in a single run for 3 consecutive days on six different types of real-time fluorescent quantitative PCR instruments using Sansure 2019-nCoV nucleic acid detection kit. The positive rate of each dilution was counted and the limit of detection(LoD) was evaluated by probit analysis. The two dilutions including 1∶10 and 1∶25 were extracted and tested five times per day in a single run for 5 consecutive days on six different types of real-time fluorescent quantitative PCR instruments using Sansure 2019-nCoV nucleic acid detection kit, and the repeatability and within-laboratory precision of the cycles threshold(Ct) of ORF1ab and Ngenes of 2019-nCoV were calculated following EP15-A3.Results ① The detection rates of three dilutions including 1∶10, 1∶25 and 1∶62.5 tested by six different types of real-time fluorescent quantitative PCR instruments were all 100%. The positive rates of ORF1ab and Ngenes of 2019-nCoV in the 1∶156.25 diluted sample tested by six different types of real-time fluorescent quantitative PCR instruments were 100.0% and 100.0% for A, 66.7% and 86.7% for B, 100.0% and 100.0% for C, 93.3% and 93.3% for D, 93.3% and 93.3% for E, and 100.0% and 100.0% for F, respectively. The positive rates of ORF1ab and Ngenes of 2019-nCoV in the 1∶390.63 diluted sample tested by six different types of real-time fluorescent quantitative PCR instruments were 60.0% and 86.7% for A, 60.0% and 80.0% for B, 66.7% and 73.3% for C, 73.3% and 93.3% for D, 66.7% and 53.3% for E, and 73.3% and 93.3% for F, respectively. The detection rates of target genes in three dilutions including 1∶976.56, 1∶2 441.41 and 1∶6 103.52 were 4.00%-60.00%, 6.67%-20.00% and 0-20.00%, respectively. ②The repeatability and within-laboratory precision(%CV) of the cycles threshold(Ct) of the target genes tested by six different types of real-time fluorescent quantitative PCR instruments were both less than 5%.Conclusion Different real-time fluorescent quantitative PCR instruments had effect on the analytic performance of 2019-nCoV nucleic acid detection, and the analytic sensitivity of the detection system C or F was better than that of the other four types of real-time fluorescent quantitative PCR instruments.
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  • 图 1  6种实时荧光定量PCR仪扩增1∶10、1∶25两个稀释度质控品中ORF1ab基因的Ct值

    下载: 全尺寸图片 幻灯片

    图 2  6种实时荧光定量PCR仪扩增1∶10、1∶25两个稀释度质控品中N基因的Ct值

    下载: 全尺寸图片 幻灯片

    表 1  实时荧光定量PCR循环参数设定

    步骤 名称 温度/℃ 时间 循环数
    1 逆转录 50 30 min 1
    2 cDNA预变性 95 1 min 1
    3 变性 95 15 s 45
    退火、延伸及荧光信号采集 60 30 s
    4 仪器冷却 25 10 s 1
    下载: 导出CSV

    表 2  6种不同实时荧光定量PCR仪对不同稀释度的质控品样本中2019-nCoV靶基因检出率的影响 例(%)

    仪器 不同稀释度的质控品样本
    1∶10 1∶25 1∶62.5 1∶156.25 1∶390.63 1∶976.56 1∶2 441.41 1∶6 103.52
    A
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 15(100.0) 9(60.0) 4(26.7) 3(20.0) 3(20.0)
      N 15(100.0) 15(100.0) 15(100.0) 15(100.0) 13(86.7) 10(66.7) 5(33.3) 0
    B
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 10(66.7) 9(60.0) 2(13.3) 3(20.0) 1(6.67)
      N 15(100.0) 15(100.0) 15(100.0) 13(86.7) 12(80.0) 10(66.7) 6(40.0) 2(13.3)
    C
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 15(100.0) 10(66.7) 9(60.0) 2(13.3) 0
      N 15(100.0) 15(100.0) 15(100.0) 15(100.0) 11(73.3) 10(66.7) 2(13.3) 1(6.67)
    D
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 14(93.3) 11(73.3) 6(40.0) 1(6.67) 1(6.67)
      N 15(100.0) 15(100.0) 15(100.0) 14(93.3) 14(93.3) 11(73.3) 6(40.0) 1(6.67)
    E
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 14(93.3) 10(66.7) 5(33.3) 2(13.3) 0
      N 15(100.0) 15(100.0) 15(100.0) 14(93.3) 8(53.3) 5(33.3) 3(20.0) 1(6.67)
    F
      ORF1ab 15(100.0) 15(100.0) 15(100.0) 15(100.0) 11(73.3) 7(46.7) 1(6.67) 0
      N 15(100.0) 15(100.0) 15(100.0) 15(100.0) 14(93.3) 9(60.0) 4(26.7) 0
    下载: 导出CSV

    表 3  6种不同实时荧光定量PCR仪检测2019-nCoV核酸的相对LoD 95%CI

    仪器 Probit分析算得的相对LoD
    ORF1ab基因 N基因
    A 1∶90.79(1∶170.87~1∶30.05) 1∶312.36(1∶505.26~1∶122.91)
    B 1∶48.36(1∶91.56~1∶16.52) 1∶121.05(1∶239.07~1∶34.14)
    C 1∶193.64(1∶317.23~1∶78.05) 1∶202.57(1∶343.28~1∶76.49)
    D 1∶133.50(1∶228.08~1∶51.45) 1∶256.45(1∶447.14~1∶89.92)
    E 1∶132.34(1∶220.50~1∶397.36) 1∶88.53(1∶162.59~1∶30.53)
    F 1∶217.13(1∶339.08~1∶92.45) 1∶360.52(1∶562.02~1∶165.95)
    下载: 导出CSV

    表 4  6种不同实时荧光定量PCR仪对2019-nCoV检测精密度的影响

    仪器 重复性精密度 实验室内精密度
    1∶10 1∶25 1∶10 1∶25
    SD CV/% SD CV/% SD CV/% SD CV/%
    A
      ORF1ab 0.26 0.81 0.55 1.64 0.26 0.81 0.55 1.64
      N 0.24 0.75 0.43 1.28 0.27 0.82 0.50 1.48
    B
      ORF1ab 0.29 0.91 0.33 0.98 0.29 0.91 0.33 0.98
      N 0.21 0.64 0.40 1.19 0.33 1.01 0.48 1.42
    C
      ORF1ab 0.30 0.94 0.36 1.07 0.64 1.96 0.88 2.56
      N 0.31 0.92 0.31 0.89 0.34 1.01 0.42 1.19
    D
      ORF1ab 0.28 0.85 0.31 0.91 0.37 1.13 0.75 2.21
      N 0.28 0.82 0.38 1.09 0.33 0.98 0.38 1.09
    E
      ORF1ab 0.45 1.38 0.35 1.02 0.47 1.43 0.40 1.18
      N 0.27 0.84 0.34 0.99 0.27 0.84 0.34 0.99
    F
      ORF1ab 0.28 0.85 0.49 1.41 0.37 1.13 0.59 1.70
      N 0.26 0.79 0.27 0.78 0.40 1.21 0.35 1.01
    下载: 导出CSV
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收稿日期:  2023-04-13
刊出日期:  2023-10-01

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