登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法，用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限；并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性，特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例；20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点，可用于登革热病毒的快速检测和基因分型鉴定。

A study of multiplex real-time PCR for detection and genotype of dengue virus

Objective To develop a multiplex real-time PCR of double target genes for laboratory diagnosis and genotype of dengue virus. Methods Specific and universal primers and TaqMan probes were designed according to the conserved sequence of dengue virus Ⅰ-Ⅳ types. The specificity, reproducibility and detection limit of the multiplex real-time PCR assay was evaluated and the assay was further validated with 20 positive samples. Results 20 dengue-positive RNA samples were found positive in the all general-purpose test. Results from the genotying study showed that there were 10 cases of dengue virus type-Ⅰ infection, 3 cases of dengue virus type-Ⅱ infection, 3 cases of dengue virus type-Ⅲ infection, and 4 cases of board dengue virus type-Ⅳ infection. Specimens from 20 normal asymptomatic subjects were found to be negative for HIV, HCV and HEV. Gradient detection of less than 5% coefficient of variation. I -IV of the dengue virus detection detection limit 103 copies/ml. Conclusions A multiplex real-time PCR of double target genes for laboratory diagnosis and genotype of dengue virus was established. This method is specific, reproducible, fast and easy in operation. It can be used for rapid detection and genotyping of dengue virus.