Hepatitis gene chip in detecting HBV DNA, HCV RNA in serum and liver tissue samples of hepatitis patients.

OBJECTIVE: To study the preparation of diagnostic gene chip for detecting hepatitis B virus (HBV) and hepatitis C virus (HCV) and its accuracy in detecting HBV DNA and HCV RNA in serum and liver tissues. METHODS: The probes, which depend on the conservative gene fragment of hepatitis virus, was designed, synthesized and spotted on the modified glass. The probes and some other control probes were assembled on the diagnostic microarray of hepatitis virus. The gene of hepatitis virus, purified from blood or tissue, was labeled with fluorescence and hybridized to the microarray. The hybridized microarry was scanned with microarray scanner and the diagnostic result was analyzed from the scanning data. Fourty patients with hepatitis B virus and 40 healthy people or 40 patients with hepatitis C virus were subjected to detection of HBV DNA and HCV RNA with the hepatitis virus gene chip by the double-blind method. Paraffin liver specimens obtained from 99 cases of posthepatitic cirrhosis were used to detect HBV DNA. The liver tissues and serum from 15 cases of chronic hepatitis B were used to detect HBV DNA. Simultaneously, HBsAg and HBcAg were detected in the serum by fluorescence microparticle quantitation, HBV DNA and HCV RNA in the serum by PCR, and HBcAg in liver tissues by immunocytochemistry or HBV DNA by in situ molecular hybridization. RESULTS: Chip detection of serum specimens showed that 30 patients were HBV DNA positive and 10 HBV DNA negative in the 40 patients with HBV positive, 25 patients were HCV RNA positive and 15 patients were HCV RNA negative in the 40 patients with HCV positive, and all were HBV and HCV negative in the 40 healthy people. In 15 patients with HBV marker positive who were subjected to liver biopsy, 15 patients were detected HBV DNA positive in serum by gene chip, 15 patients HBcAg positive in liver tissues by immunocytochemistry, 14 patients HBV DNA positive in liver tissues by in situ molecular hybridization, and 14 patients HBV DNA positive in liver tissues by gene chip. Paraffin liver tissues specimens from the 99 patients with posthepatitis B cirrhosis showed that 67 patients were detected HBcAg positive by immunocytochemistry, 53 patients HBV DNA positive by in situ molecular hybridization, and 46 patients HBV DNA positive by gene chip. In the 46 patients, 40 patients were detected HBV DNA and HBcAg positive by in situ molecular hybridization and immunocytochemistry, 6 patients only HBcAg positive, and 33 patients HBcAg negative. CONCLUSIONS: The designed diagnostic gene chip can be used to simultaneously detect serum HBV DNA and HCV RNA, but the positive rate of HCV RNA diagnosed by this chip is lower. The gene chip can detect HBV DNA in serum and in liver tissue.

[Detection and identification of emergence of HBV YMDD motif mutation during lamivudine treatment by hybridization to an oligonucleotide array].

OBJECTIVE: Gene chip technology was set up to quickly and accurately detect and identify the HBV P gene YMDD motif mutation during the chronic hepatitis treated with lamivudine. METHODS: DNA microarrays were prepared by spotting fluorescence labeled probes of target genes onto specially lattice glass slides with robotics. The serum samples from 30 patients with hepatitis B after 68-week treatment with lamivudine were detected double-blind by gene chips and by nucleotide sequences assay technique to identify the rate of emergence of HBV P gene YMDD motif mutation. RESULTS: Twenty-one patients were found HBV P gene YMDD motif mutation by the gene chips including 11 cases with YVDD and 10 cases with YIDD motif mutation. By direct sequencing of the PCR products, 11 cases were found to have YVDD with adenine741 changed into cytidine resulted in methionine552 changed into valine in which 6 cases with adenine669 changed into cytidine and leucine changed into methionine, 10 cases had YIDD motif mutation with guanosine743 altered thymidine methionine552 changed into isoleucine, including 3 cases with thymidine281 changed into cytidine and leucine565 altered proline. CONCLUSION: The gene chip can be used to test HBV YVDD,YIDD motif mutation compared with nucleotide sequences assay technique, the accuracy rate was 100%.