[Genotyping of hepatitis C virus by hepatitis gene diagnosis microarray]

To study the preparation of hepatitis C virus (HCV) diagnosis microarray and its accuracy in diagnosis of gene type of hepatitis C virus.

Probe and primer and primers were designed in 5'-untranslated region and C region of hepatitis C virus gene. The probes were synthesized by DNA synthesizer. Solutions of probe of the final concentration of 50 micromol/L were made by dissolving the probes into sodium carbonate buffer. Hepatitis C virus genotype array spotting was performed by pin-based spotting robot PixSys5500 with CMP3 pin. The gene chips were prepared by spotting the probes onto the specially treated glass sliders. Sixty HCV RNA positive serum samples were obtained from the in-patients of the Nanjing Second Hospital (experimental group), and 60 HCV RNA negative serum samples were obtained from the healthy people undergoing physical examination (control group). Quantitative examination of serum HCV RNA was made by fluorescent quantitation PCR. The HCV RNA in the serum specimens of the experimental group (with the HCV RNA concentration of more than 500 copies/ml) and of the control group (with the HCV RNA concentration of less than 500 copies/ml) was isolated and purified, underwent reversed transcription and nested PCR to be amplified, and then genotyped by gene microarray and HCV RNA sequencing. During the experiment, double blind method was used.

Tested by the gene microarray, the serum specimens in the experimental group were all HCV RNA positive, out of which 46 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, 2 cases were 2b type, 2 cases were 1b + 2a type, and 2 cases were 3a type. Tested by nucleotide sequencing assay, 50 cases were 1b type, 3 cases were 3a type, 3 cases were 3b type, 2 cases were 2a type, and 2 cases were 2b type. The double-blind test results showed a coincidence rate of 93.3% in genotyping HCV by these two methods.

Hepatitis gene microarray can be used in detection of serum HCV RNA and in diagnostic genotyping with great accuracy.