[应用肝炎基因诊断芯片对丙型肝炎病毒进行基因分型]
[Genotyping of hepatitis C virus by hepatitis gene diagnosis microarray].
作者信息
Zhao Wei, Liu Wei, Liu Quanjun, Zhang Lin, Zhou Zhenxian, Liu Xinjue, Zhang Hanrong
机构信息
Department of Pathology, Nanjing Second Hospital, Medical College of Southeast University, Nanjing 210003, China.
出版信息
Zhonghua Yi Xue Za Zhi. 2002 Sep 25;82(18):1249-53.
OBJECTIVE
To study the preparation of hepatitis C virus (HCV) diagnosis microarray and its accuracy in diagnosis of gene type of hepatitis C virus.
METHODS
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.
RESULTS
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.
CONCLUSION
Hepatitis gene microarray can be used in detection of serum HCV RNA and in diagnostic genotyping with great accuracy.
目的
研究丙型肝炎病毒（HCV）诊断芯片的制备及其对丙型肝炎病毒基因分型诊断的准确性。
方法
在丙型肝炎病毒基因的5′非编码区和C区设计探针及引物。探针由DNA合成仪合成。将探针溶于碳酸钠缓冲液制成终浓度为50 μmol/L的探针溶液。采用基于点样针的PixSys5500点样机器人及CMP3点样针进行丙型肝炎病毒基因分型芯片点样。将探针点样于经特殊处理的载玻片上制备基因芯片。从南京二院住院患者中获取60份HCV RNA阳性血清样本（实验组），从健康体检者中获取60份HCV RNA阴性血清样本（对照组）。采用荧光定量PCR对血清HCV RNA进行定量检测。对实验组（HCV RNA浓度大于500拷贝/ml）和对照组（HCV RNA浓度小于500拷贝/ml）血清标本中的HCV RNA进行分离纯化，逆转录及巢式PCR扩增，然后采用基因芯片及HCV RNA测序进行基因分型。实验采用双盲法。
结果
基因芯片检测显示，实验组血清标本均为HCV RNA阳性，其中46例为1b型、3例为3a型、3例为3b型、2例为2a型、2例为2b型、2例为1b + 2a型、2例为3a型。核苷酸测序检测显示，50例为1b型、3例为3a型、3例为3b型、2例为2a型、2例为2b型。双盲检测结果显示，两种方法对HCV基因分型的符合率为93.3%。
结论
肝炎基因芯片可用于血清HCV RNA检测及诊断基因分型，准确性高。
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