靶序列富集多重PCR-液相芯片联合检测4种常见呼吸道病毒的初步应用

发布时间：2018-12-25 19:19

【摘要】：目的呼吸道病毒是引起婴幼儿呼吸道感染的最主要病原体,为快速准确诊断其感染,文中建立一种基于液相芯片系统的4种常见呼吸道病毒快速分子检测方法,为有效防控其发生和流行提供有力手段。方法采集南京总医院儿科就诊的有急性呼吸道感染症状的患儿咽拭子标本120例及30例正常对照标本,分别纳入患儿组和对照组。针对A型流感病毒(Flu A)、B型流感病毒(Flu B)、呼吸道合胞病毒A型(RSVA)和B型(RSVB)的保守区序列分别设计并合成了特异性引物、探针和通用引物,构建4种病原体阳性参比品,通过对扩增和杂交条件的优化,建立了靶序列富集多重PCR(Tem-PCR)和Luminex液相芯片(x MAP)技术相结合的检测系统,并对该检测系统进行了特异性、灵敏度评价。收集以急性上呼吸道感染为主要表现的患儿咽拭子标本,用上述建立的方法进行检测分析,分别与单病毒基因荧光定量PCR检测方法及x TAG液相芯片法进行比对检测。结果建立了Flu A、Flu B、RSVA和RSVB4种呼吸道病毒的特异性快速分子检测方法且灵敏度可达10拷贝/μL。用快速分子检测方法和单病毒基因荧光定量PCR法对120例疑似患儿咽拭子标本进行检测,快速法阳性检出率为31.7%(38/120),荧光定量法阳性检出率为29.2%(35/120)。经一致性检验分析提示2种方法一致性较强(k0.7)。部分标本同时采用x TAG液相芯片试剂盒进行检测,经一致性检验分析提示2种方法一致性较好(k0.6)。结论临床的初步应用证实了4种常见呼吸道病毒快速分子检测方法具备灵敏、特异、快速等优点,为临床早期、准确判断呼吸道感染的病原体提供实验依据。
[Abstract]:Objective Respiratory tract virus (RTV) is the most important pathogen causing respiratory tract infection in infants. In order to diagnose respiratory tract infection quickly and accurately, a rapid molecular detection method of four common respiratory viruses based on liquid chip system was developed in this paper. For effective prevention and control of its occurrence and prevalence to provide effective means. Methods the throat swabs of 120 children with symptoms of acute respiratory tract infection and 30 normal controls were collected from pediatrics department of Nanjing General Hospital. Specific primers, probes and universal primers were designed and synthesized for the conserved regions of (Flu B), respiratory syncytial virus A (RSVA) and B type (RSVB) (RSVB) of influenza A virus (Flu A), B). Four kinds of pathogen positive reference products were constructed. By optimizing the conditions of amplification and hybridization, a detection system combining target sequence enrichment multiple PCR (Tem-PCR) and Luminex liquid chip (x MAP) technique was established. The specificity and sensitivity of the detection system were evaluated. The pharyngeal swabs of children with acute upper respiratory tract infection were collected and analyzed by the established method. The results were compared with single virus gene fluorescence quantitative PCR assay and x TAG liquid chip assay. Results A rapid and specific molecular method for the detection of respiratory viruses from Flu Agna Flu BRSVA and RSVB4 was established with a sensitivity of up to 10 copies / 渭 L. The rapid molecular detection method and single virus gene fluorescence quantitative PCR method were used to detect the throat swabs of 120 suspected children. The positive rate of rapid detection was 31.7% (38 / 120). The positive rate of fluorescence quantitative method was 29.2% (35 / 120). The results of consistency test showed that the two methods had strong consistency (k 0.7). Some samples were detected by X TAG liquid chip kit at the same time. The results of consistency analysis showed that the two methods had good consistency (K0.6). Conclusion the preliminary clinical application has proved that the four rapid molecular detection methods of respiratory tract virus have the advantages of sensitivity specificity and rapidity which provides experimental basis for the early clinical diagnosis of respiratory tract infection pathogens.
【作者单位】：南京军区南京总医院中心实验科;南京军区南京总医院儿科;
【基金】：全军医学科技“十二五”科研项目(CWS129811298) 南京军区医学科技重大专项(14ZX17)
【分类号】：R725.6;R440

【相似文献】