Preparation and clinical application of cDNA microarray for combined detection of hepatitis virus
- VernacularTitle:病毒性肝炎联合诊断cDNA芯片的研制与临床应用性研究
- Author:
Zhaohui SUN
;
Shuyan WANG
;
Min WEI
- Publication Type:Journal Article
- Keywords:
hepatitis, viral, human;
oligonucleotide array sequence analysis;
nucleic acid hybridization
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop the cDNA microarray for the combined detection of hepatitis virus, and to study the feasibility of applying the microarray in clinical setting. Methods For the small and simple genome of HBV and HDV, the specific primers of PCR were designed with Primer Premier 5.0 program according to the conserved region of HBV and HDV, and 10 and 4 gene fragments were obtained respectively, which could be used as the probes of gene chip. As for the complex genome of HCV, the technique of restriction display PCR (RD-PCR) was employed. Some of gene fragments were selected which were comparatively more specific and sensitive as microarray probes. In order to explore the experimental conditions of microarray in vitro detection, three types of gene chip were prepared successively including HBV and HDV simultaneous detection, HCV detection and modified HCV detection. Results The hybridized signals on the gene chip showed that the effect in detection was satisfactory. Through the prepared gene chips mentioned above, some probes with good quality were selected and the microarray was prepared for HBV, HCV and HDV simultaneous detection. The diagnostic capability of the microarray was evaluated following the washing and scanning steps. Linearity: Serial dilutions of the target DNA or cDNA showed that a strong linear relationship existed between the various concentrations of target DNA or cDNA and the fluorescence intensities obtained from microarray assay (r=0.990 2, r=0.992 1, r=0.981 9), and that the detection range for the microarray was from 104 to 1011 copies/ml. Specificity: Samples from other viruses such as YFV, JET and DV were also subjected to the test and the results were all negative. Reproducibility: The reproducibility of this assay system was evaluated by repeated measurements, and the within-run coefficient of validation of HBV, HCV and HDV were 7.1%, 7.2% and 6.6%, respectively, while the between-run coefficient of validation was 7.9%, 8.2% and 7.6%, respectively. Accuracy: By using the BLAST and the GenBank database, the identity of the obtained sequences including 16 fragments of PCR, 24 RD fragments of HCV and some positive serum samples were verified, each sequenced product was confirmed to be a genome fragment of expected size. In order to fulfill the request of clinical diagnosis, a modified protocol for microarray detection was established. This new protocol consisted of two hours of hybridization, omitting the steps of prehybridization and purification of samples, and the hybridization temperature was elevated from 42℃ to 52℃, et al. The whole protocol could be completed in less than 8 hours. 98, 42 and 5 serum samples from hepatitis B, C and D patients and 130 samples from healthy people were analyzed, respectively, by microarray assay and real-time PCR (Taqmam method). There was a significant correlation between the results of these assays (HBV, r=0.985 4 and HCV, r=0.958 2, P
