[A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique].
OBJECTIVE
To develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.


METHODS
The primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.


RESULTS
Of the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.


CONCLUSIONS
This method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.
Wei
Jun-feng
JF
Department of Infectious Diseases, Nanfang Hospital, School of Biotechnology, Southern Medical University, Guangzhou 510515, China. wjfhy@yeah.net
Zhang
Tai-song
TS
Huang
Hui-hong
HH
Zeng
Yan-li
YL
Zhang
Fan
F
Wang
Jun-jie
JJ
Zhou
Bin
B
Wu
Ying-song
YS
Liu
Shu-wen
SW
Hou
Jin-lin
JL
Guo
Ya-bing
YB
Zhou
Yuan-ping
YP
D005814:Genes, Viral; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D015151:Immunoblotting; D009693:Nucleic Acid Hybridization; D020133:Reverse Transcriptase Polymerase Chain Reaction