Detection and Genotyping of Herps Simplex Virus Type 2 by DNA Sequencing / 解放军医学杂志

ABSTRACT

Objective Herpes simplex virus is the pathogenic agent of human herpes simplex. There are two genotypes of herpes simplex virus, HSV-1 and HSV-2. The clinical manifestations of HSV-1 and HSV-2 overlap each other and it is difficult to differentiate them clinically. Methods developed based on genome differences are efficient ones to differentiate the two genotypes of HSV. In this study, we attempted to develop a new method to detection and genotyping human HSV in clinical samples. Methods Swab samples were collected from genital lesions of patients and placed in transport media. Samples were used to inoculate Vero cells. After appearance of the cytotoxicity, the infection mixtures were collected, and subjected to genomic DNA extraction. Based on the conservation and variation of gD of HSV-1 and HSV-2, a pair of primers amplifying both of them were designed and synthesized. Sequence of the virus were amplified and cloned into pMD-18T, and then the sequence was determined by DNA sequencing. The sequence was compared to all the known sequences in Genebank by using BLAST. According to the BLAST results, the genus and genotype of the virus was determined. Results A DNA fragment of about 200 bp was successfully amplified. This DNA fragment was cloned and sequenced. The sequence was compared with other known sequences. the results showed that this sequence had the highest homology to gD of HSV, indicating that the virus in the sample was HSV-2. The BLAST results also showed that there were some differences in the sequence of gD to those previously isolated. Conclusion DNA sequencing of PCR amplification products is an efficient and definite method to detect and genotype HSV-1 and HSV-2 which otherwise are difficult to differentiate clinically.