依赖核酸序列扩增技术检测呼吸道合胞病毒方法的建立

doi:10.3969/j.issn.1673-8640.2016.03.004

摘要/Abstract

摘要：

目的建立依赖核酸序列扩增技术（NASBA）检测呼吸道合胞病毒（RSV）的方法。方法依据美国国家生物技术信息中心（NCBI）基因数据库,利用Primer 5软件进行RSV特异性引物设计。收集经免疫荧光法检测确认RSV阳性的咽拭子样本,以同批检测RSV阴性样本作为阴性对照。优化样本处理条件和扩增反应体系,建立NASBA检测RSV的方法,并与逆转录聚合酶链反应（RT-PCR）进行比较。结果采用直接冻融法处理咽拭子样本,取上清检测;引物设计原则为引物加T7启动子序列。NASBA在RSV RNA标准品浓度为2.81×10² 拷贝/µL时仍能检测出目的片段,而RT-PCR在2.81×10⁴ 拷贝/µL时目的条带隐约可见,提示NASBA的灵敏度比RT-PCR高出至少100倍。NASBA在7例RSV阳性咽拭子样本中特异性扩增出RSV RNA,而在其他病毒阳性（包括流感病毒A阳性2例、副流感病毒3阳性2例、腺病毒阳性1例）及RSV阴性（3例）样本中未见目的条带,提示NASBA的特异性较高。结论建立的NASBA特异性强、灵敏度高、耗时相对较短,克服了目前实验室病毒检测方法的局限性,为RSV的检测提供了有效的实验室检测手段。

关键词: 呼吸道合胞病毒, 依赖核酸序列扩增技术, 方法学评价

Abstract:

Objective To establish a nucleic acid sequence-based amplification （NASBA） to detect respiratory syncytial virus（RSV）. Methods According to the alignment results from the National Center for Biotechnology Information （NCBI） gene databank,the specific primers for RSV were designed by Primer 5 software. Throat swab samples being positive for RSV by immuno-fluorescence assay were collected,and the corresponding negative samples were as controls. Through optimizing the sample process and amplification system, NASBA for detecting RSV was established and compared to reverse transcription polymerase chain reaction （RT-PCR）. Results Direct freeze-thawing throat swab samples were optimal for the sample process,and the supernatant was as the target for detecting. The specific primers were added with T7 promoter sequence. NASBA could detect the standard RSV RNA at 2.81×10² copies/µL,but RT-PCR could only show blurry products even up to 2.81×10⁴ copies/µL,which indicated that the sensitivity of NASBA assay was 100 times at least higher than that of RT-PCR. NASBA could detect RSV RNA in all of the 7 throat swab samples,while they showed negative in samples,which were positive for other viruses（2 cases of influenza A virus,3 cases of parainfluenza virus and 1 case of adenovirus）and negative RSV samples （3 cases）. These results indicated that NASBA had high specificity. Conclusions The established NASBA is a highly specific,sensitive and time-saving method,overcoming the limitations of current detections for RSV. It is an effective method to detect RSV.

Key words: Respiratory syncytial virus, Nucleic acid sequence-based amplification, Methodology evaluation

中图分类号:

R446.1

杨海鸥, 叶星晨, 傅启华. 依赖核酸序列扩增技术检测呼吸道合胞病毒方法的建立[J]. 检验医学, 2016, 31(3): 168-172.

YANG Haiou, YE Xingchen, FU Qihua. Establishment of nucleic acid sequence-based amplification for detection of respiratory syncytial virus[J]. Laboratory Medicine, 2016, 31(3): 168-172.

图/表 7

图1 RSV N基因序列保守性分析

表1 NASBA-RSV引物序列

名称	序列（5′~3′）
F端	AATTCTAATACGACTCACTATAGGGAGAAGGAAAGTCCTACAAAAAAATGC
R端	AATTCTAATACGACTCACTATAGGGAGAAGGATCTATCTCCTGCTGCTAAT

图2 患者咽拭子样本不同处理方法的比较

图3 患者咽拭子样本直接进行扩增曲线

图4 NASBA扩增条件的优化

图5 NASBA与一步法RT-PCR的灵敏度比较

图 6 NASBA检测RSV的特异性

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