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| 实时荧光RT-PCR法和NASBA方法在呼吸道多病原检测中的应用评价 | |
| 引用本文： | 李爱华,王雪,龚成,罗琴,罗明,李茂中,董梅,黄芳.实时荧光RT-PCR法和NASBA方法在呼吸道多病原检测中的应用评价[J].现代预防医学,2022,0(7):1279-1283. |
| 作者姓名： | 李爱华 王雪 龚成 罗琴 罗明 李茂中 董梅 黄芳 |
| 作者单位： | 1.北京市疾病预防控制中心/北京市预防医学研究中心免疫预防所，北京 100013；2.首都医科大学公共卫生学院 |
| 摘 要： | 目的 评价实时荧光RT-PCR和核酸依赖性序列扩增法（NASBA）两种方法在呼吸道多病原检测中的应用价值。方法 收集北京地区急性呼吸道感染病例标本140份，分别采用实时荧光RT-PCR方法和NASBA方法对样本进行平行检测并对两种方法的病原检出限、重复性、检测一致性及检测时间等指标进行比较。结果 两种检测方法批内重复CV值均<5%，重复性较好；实时荧光RT-PCR方法对脊灰毒株核酸和H1N1病毒核酸最低检出限分别为5.62 CCID50 /0.1 ml（107 倍稀释）和103倍稀释度，NASBA方法最低检出限分别为56.2 CCID50 /0.1 ml（106 倍稀释）和102倍稀释。实时荧光RT-PCR灵敏度略优于NASBA方法。针对两种方法共同检测呼吸道病原体种类，实时荧光RT-PCR和NASBA方法检测阳性率分别为89.28%（125/140）、70%（98/140）。两种方法对甲型H1N1病毒、肠道病毒及新型冠状病毒的检测结果一致性达100%，对甲型流感病毒、甲型H3N2、副流感病毒1-4型的检测结果基本一致， Kappa 值范围为0.81~1；对冠状病毒229E/HKU1/OC43/NL63、鼻病毒、呼吸道合胞病毒和乙型流感病毒的检测结果一致性较好， Kappa 值范围为0.61~0.80；对人偏肺病毒的检测结果一致性差， Kappa 值仅为0.55。实时荧光RT-PCR方法检测时间为90 min，NASBA方法仅需40 min即可完成。 结论 实时荧光RT-PCR方法灵敏度较高，适合对大批量临床样本进行筛查；NASBA方法检测时间短，不易污染，适用于时限性强但对灵敏度要求不太高的应用场景，如临床上重症呼吸道感染者的快速检测。建议根据不同需求和应用场景，选择合适的检测方法和产品。 |
| 关 键 词： | 呼吸道多病原 实时荧光RT-PCR方法 NASBA方法 |
Comparative evaluation of Real-Time RT-PCR and NASBA for respiratory pathogens detection |
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| LI Ai-hua,WANG Xue,GONG Cheng,LUO Qin,LUO Ming,LI Mao-zhong,DONG Mei,HUANG Fang.Comparative evaluation of Real-Time RT-PCR and NASBA for respiratory pathogens detection[J].Modern Preventive Medicine,2022,0(7):1279-1283. | |
| Authors: | LI Ai-hua WANG Xue GONG Cheng LUO Qin LUO Ming LI Mao-zhong DONG Mei HUANG Fang |
| Affiliation: | *Beijing Center for Disease Control and Prevention, Institute of Immunoprophylaxis, Beijing Preventive Medicine Research Center, Beijing 100013, China |
| Abstract: | Objective To evaluate the application of Real-Time RT-PCR and NASBA assays in the detection of respiratory pathogens. Methods A total of 130 respiratory infectious samples and 10 SARS-CoV-2 samples were collected from Beijing from 2019 to 2020. The samples were tested in parallel by Real-Time PCR and NASBA assays. The consistency rates, detection capability and intra-batch repeatability were compared. Results The two methods showed excellent repeatability in test with CV <5%. The detection limits of Real-Time RT-PCR method for poliovirus nucleic acid and H1N1 virus nucleic acid (5.62 CCID 50 /0.1 ml, 103fold dilution) was lower than that of NASBA (56.2 CCID 50 /0.1 ml, 103fold dilution), which indicated that Real-Time RT-PCR was more sensitive. For the pathogens detected by both assays, the positive rates were 89.28%（125/140）and 70%（98/140） for Real-Time RT-PCR and NASBA, respectively. The results of H1N1, EV and SARS-CoV-2 by the two methods were 100% identical. The Kappa values of FluA, H3N2, coronavirus, PIV1, PIV2, PIV3 and PIV4 were between 0.81-1. The results of detection of coronaviruses 229E/HKU1/OC43/NL63, RHV, RSV and FluB were similar, the Kappa value ranged from 0.61 to 0.80. The consistency of detection for MPV by the two assays was rather poor, with the kappa value only 0.55. Conclusion Real-Time RT-PCR has high sensitivity, which is suitable for the detection of large quantities of clinical samples. The NASBA method has the advantages of short detection time, less contamination of RNA product. It is suitable for the time-limited but less sensitive application, such as rapid detection of patients with severe respiratory infection. It is recommended to select appropriate detection methods and products according to different needs and application scenarios. |
| Keywords: | Respiratory pathogens Real-Time RT-PCR NASBA |
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