多重RT-PCR MassARRAY技术检测27种呼吸道病原体方法的建立和临床应用评价

Establishment and clinical application of multiple RT-PCR MassARRAY for the determinations of 27 respiratory pathogens

目的建立一种可同时检测27种呼吸道病原体的多重逆转录聚合酶链反应(RT-PCR)MassARRAY技术,并探讨其临床应用价值。方法针对呼吸道病原体设计扩增引物和延伸引物,并构建病原体标准质粒;按梯度稀释标准质粒,用于考察多重RT-PCR MassARRAY方法的灵敏度、特异性和重复性。分别采用建立的多重RT-PCR MassARRAY方法和实时RT-PCR方法检测207份呼吸道感染患者鼻咽拭子样本,比较2种方法的检测性能。结果建立的多重RT-PCR MassARRAY方法对29种病原体的最低检出限为1×10^(1)~1×10^(3)拷贝/μL,特异性高。207例鼻咽拭子样本中共检出57份阳性,阳性检出率为27.54%。细菌性病原体中检出率较高的是肺炎链球菌、卡他莫拉菌、肺炎支原体和流感嗜血杆菌;病毒性病原体阳性检出率较高的为副流感病毒、人偏肺病毒和甲型流感病毒。2种方法检测结果总符合率为99.03%,具有高度的一致性(kappa=0.98)。结论基于RT-PCR MassARRAY技术的27种呼吸道病原体检测方法具有较高的灵敏度和特异性,且操作简单,通量高,可以为临床呼吸道病原体感染的诊断提供实验室依据。

Objective To establish a method based on multiple reverse transcription polymerase chain reaction(RT-PCR)MassARRAY for the determinations of 27 respiratory pathogens,and to investigate its clinical application value.Methods Multiple polymerase chain reaction amplification and extension primers were designed for pathogens,and the sensitivity,specificity and repeatability of multiple RT-PCR MassARRAY were evaluated with the standard plasmid of pathogens established.Totally,207 clinical specimens were determined by multiple RT-PCR MassARRAY,and the results were compared with real-time RT-PCR.Results The minimum detection limit of the established multiple RT-PCR MassARRAY for 27 respiratory pathogens was 1×10^(1)~1×10^(3) copies/μL,with a high specificity.The 57 of 207 clinical specimens were positive,and the positive rate was 27.54%.Streptococcus pneumoniae,Moraxella catarrhalis,Mycoplasma pneumoniae and Haemophilus influenzae were the most common bacterial pathogens,and parainfluenza virus,human metapneumovirus and influenza A virus had high detection rates.”Compared with real-time RT-PCR,the total consistency rate was 99.03%,with high consistency and a kappa value of 0.98.Conclusions The detection system of 27 respiratory pathogens based on multiple RT-PCR MassARRAY has high sensitivity and specificity,simple operation and high throughput,which can provide a reference for the clinical diagnosis of respiratory pathogens.