海南省2019年9例登革病毒的鉴定及E基因序列分析

目的 对2019年海南省暴发的16例疑似登革热（dengue fever, DF）患者血清标本进行调查研究,从分子水平对登革病毒（Dengue virus, DENV）的E基因进行分析,对此次登革病毒进行鉴定,并对登革热的防控给出可行性意见。方法 对患者血清进行核酸检测、分型,将登革病毒阳性标本进行分离及核酸检测,利用实时荧光定量PCR技术对病毒进行分型,通过RT-PCR扩增E基因片段RT-PCR产物经过琼脂凝胶电泳成像系统观察条带结果,根据条带的有无判断阴性或阳性,根据条带的大小判断登革病毒的型别,得到9条DENV-1型E基因序列,基因测序之后与2019年中国其他省、国外选取的共11条DENV-1型E基因序列进行比较中,通过MEGA7.0.26软件从分子水平进行同源性比较以及系统进化树构建。结果 通过血清学鉴定结果以及实时荧光定量PCR技术检测得出的Ct值,可知研究的16份患者血清中,有9份经过鉴定为DENV-1型,7例为阴性结果。系统进化树表明,2019年海南9例DENV-1病例与参考序列MK517727 Guangzhou 2019、MK517719 Guangzhou 2019、MK517720 Guangzhou 2019同源性最高为99.73%~99.80%,与3株作为参考序列的外源株,序列相似度都相对较低,结果可靠。结论 得到的E基因长度为1 485 bp,2019年海南省9株DENV-1型为输入型流行株,根据进化树亲缘性分析,此次海南DENV-1病例与广州省的登革病例同源性最高。

Identification of 9 cases of Dengue virus and E gene sequence analysis in Hainan, 2019

Objective Serum samples of 16 suspected dengue fever patients in Hainan Province in 2019 were investigated. The E gene of dengue virus was analyzed at the molecular level, and the secondary dengue virus was identified, and feasible suggestions for the prevention and control of dengue fever were given. Methods Nucleic acid detection and typing were performed on patients' sera. Positive samples of dengue virus were isolated and nucleic acid detection was performed. Real-time quantitative PCR was used to type the virus, and E gene fragments were amplified by RT-PCR. According to the size of the bands, the genotypes of dengue virus were determined, and 9 E gene sequences of DENV-1 were obtained. After gene sequencing, a total of 11 E gene sequences of DENV-1 selected from other provinces in China and foreign countries in 2019 were compared. Homology comparison was conducted at the molecular level by using Mega 7.0.26 software and phylogenetic tree was constructed. Results According to the results of serological identification and Ct values obtained by real-time quantitative PCR, 9 of the 16 sera in the study were identified as DENV-1, and 7 cases were negative. Phylogenetic tree showed that 9 DENV-1 cases in Hainan in 2019 had the highest homology of 99.73%-99.80% with reference sequences MK517727 Guangzhou 2019, MK517719 Guangzhou 2019 and MK517720 Guangzhou 2019. The sequence similarity was relatively low with the three foreign strains as reference sequences, and the results were reliable. Conclusion s The total length of E gene is 1 485 bp. Nine DENV-1 strains in Hainan Province are imported epidemic strains in 2019. According to phylogenetic analysis, the DENV-1 cases in Hainan Province has the highest homology with the dengue cases in Guangzhou Province.