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(k>0.7)。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(k>0.6)。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。
Objective Respiratory viruses are the most common pathogens to cause respiratory tract infection in infants and children. The aim of the study was to establish a luminex-based molecular assay for rapid detection of four kinds of common respiratory viruses and provide measures for effective prevention and control. Methods 120 throat swab samples from patients with acute respiratory tract infection were collected in our hospital as disease group. 30 normal specimens were used as control group. Specific upstream and downstream primers, hybridization probes and super primers were designed on the basis of conserved sequences of Influenza A and B viruses （ FluA, FIuB）, respiratory syncytial virus types A and B （RSVA, RSVB） from available respiratory-virus sequence database. Recombinant plasmid and in vitro transcription RNA positive reference substances were established respectively. The testing sys- tem of Tem-PCR combined with luminex xMAP was built by amplification and optimization of hybridization. Comparative analysis were made between the detection results of the above method and those of single viral gene real-time PCR assay and luminex xTAG assay re- spectively. Results Rapid molecular assay was established to specifically detect the four kinds of respiratory viruses （FluA, FluB, RSVA and RSVB） with the sensitivity of 10 copies/ixL. Rapid molecular assay and single viral real-time PCR assay were utilized to de- tect the throat swabs （n = 120） from suspected patients, the positive result of the former was 31.7% （38/120） and the latter was 29.2% （35/120）. The consistency test result indicated the two methods were consistent without a significant difference （k 〉 0.7 ）. Several samples were detected by luminex xTAG assay simultaneously, in which good consistency and significant difference were found in two assays by statistical analysis （ k 〉 0. 6）. Conclusion Preliminary clinical application has confirmed the novel molecular assay is sensitive, specific and rapid in simuhaneous detection of FluA, FluB, RSVA and RSVB respiratory viruses, which provides experi- mental basis for accurate diagnosis of infected pathogens at early clinical stage.
出处
《医学研究生学报》
CAS
北大核心
2016年第9期958-963,共6页
Journal of Medical Postgraduates
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)
