基于MassARRAY多重PCR-MS的登革病毒分型检测

Detection of Dengue virus typing based on MassARRAY multiplex PCR-MS spectrometry

目的建立一种基于MassARRAY技术的覆盖登革病毒4个血清型的多重PCR-MS检测方法,用于登革病毒分型及溯源。方法根据从NCBI网站获取的Ⅰ~Ⅳ型登革病毒全基因组序列设计9对引物,并引入伊蚊28S rRNA和人RNaseP基因的引物用于登革病毒溯源及质控。用该方法检测11份已知血清型的不同稀释度登革病毒阳性样本,评估方法的特异性、灵敏度。结果11份登革病毒样本经本方法检测全部为阳性,分型结果均与预期一致;各型引物组合未见交叉反应或交错检出。灵敏度应与荧光定量PCR方法相当。结论本研究建立的多重PCR-MS登革病毒分型检测方法具有高度特异性、灵敏度、高通量等优势,可用于登革病毒的快速检测、分型和样本溯源。

Objective To establish a MassARRAY based multiplex PCR-MS method for the detection of Dengue viruses with four serotypes,which can be used for typing and source tracing of Dengue virus.Methods Nine pairs of primers were designed according to the whole genome sequence of Dengue virus types I-IV obtained from the NCBI database.The primers of Aedes 28S rRNA and human RNaseP gene were introduced for sample tracing and quality control.Eleven Dengue virus positive samples with different dilutions of known serotypes were detected by using PCR-MS to evaluate the specificity and sensitivity of the method.Results All the 11 Dengue virus samples were positive by this method,and the typing results were consistent with expectations.No cross reaction or staggered detection was found in all primer combinations.The detection sensitivity was same as that of quantitative Real-time PCR.Conclusion The multiplex PCR-MS method for typing of Dengue virus established in this study displayed the advantages of high specificity,sensitivity,and throughput for detection of Dengue viruses.It should be of general uses for rapid detection,typing and sample tracing of Dengue virus.