多重RT-PCR同步检测A组轮状病毒P基因型

唐少文; 叶临湘; 王斌; 周敦金; 杨继红

多重RT-PCR同步检测A组轮状病毒P基因型

Simultaneous detection of genotype P of human group a rotaviruses by multiplex RT-PCR

摘要

摘要:
目的建立A组轮状病毒VP4基因型的多重PCR分型方法, 并用此方法对武汉地区婴幼儿腹泻样本进行检测。
方法在分析A组轮状病毒VP4基因的基础上, 选取引物con2、con3进行第一轮RT-PCR, 然后以A组轮状病毒5个P基因型P6、P8、P9、P4和P10的特异性引物, 进行第二轮巢式PCR, 建立多重巢式RT-PCR同步检测A组轮状病毒P基因型技术。
结果利用该方法可以同时检出5种不同基因型标准株混合物中的各个型别。将纯化的A组轮状病毒标准株RNA系列稀释后, 用本方法进行检测, 其灵敏度为0.1ng/μl。同时还对40份从武汉市儿童医院采集的A组轮状病毒阳性的腹泻儿童大便样本进行检测, 其中P8有29例、P4有5例, 另外发现有P8与P4混合感染4例。说明该方法对实际样本中的混合感染也能很好地分型。
结论应用多重巢式RT-PCR对A组轮状病毒基因型研究将为临床诊断和实验研究提供一种快速、灵敏和特异的检测手段。

Abstract:
Objective To establish a convenient method for simultaneously determining P-genotype of human group A rotavirus using a multiplex polymerase chain reaction.
Methods 6 oligonucleotide primers which were type-specific respectively for P8, P4, P6, P9and P10 P-genotype of human group A rotavirus were used.The amplified product of 346 bp, 484 bp, 268 bp, 392 bp and 584 bp represented genotype P8, P4, P6, P9 and P10 respectively.The concentration of primers for multiplex PCR was optimized.
Results All five P-genotypes of human group A rotavirus could be typed simultaneously from a mixture of 5 standard strains and the sensitivity of the assay is 0.1 ng/μl.40 clinical samples were tested.P8 was 72.5%, P4 was 12.5%.
Conclusion The multiplex PCR appears to be rapid, sensitive and specific method for detecting human group a rotavirus' genotype P and may be applied to clinical diagnosis and laboratory work.

HTML全文

参考文献(8)

施引文献

资源附件(0)