HBV基因分型检测芯片的制备及临床应用
摘要
HBV基因分型检测芯片的制备及临床应用
前言
乙型肝炎病毒(HBV)的感染呈世界性分布,在我国尤其多见。HBV感染可导致急、慢性肝炎(CHB),甚至肝硬化,肝癌。由于病毒变异,HBV基因组分为多个型。根据其核苷酸序列差异,HBV可划分为(A-H)8种基因型。目前研究认为,HBV基因型是影响HBV感染者预后的重要因素之一,因此建立一套快速、准确的HBV分型检测技术十分必要。目前诊断HBV基因型的常用方法有DNA测序,聚合酶联反应-限制性片段长度多态性分析法(PCR-RFLP)等,在结果准确性、技术简单性方面各有优劣,且不能同时检测所有基因型。近几年发展起来的基因芯片技术弥补上述方法的缺陷。本课题的研究目的在于:建立DNA芯片检测HBV基因分型的研究方法;并对芯片检测的特异性、准确性、灵敏度和重复性进行测试、分析;通过调查杭州地区HBV的基因型分布对HBV基因分型检测芯片在临床上的应用进行初步探索,为病原体基因型检测芯片这种高科技方法在临床上的应用进行积极探索,积累有益经验。
方法
1.有关探针、引物设计
在GenBank数据库中查找HBV各型Pre-S基因组序列,并利用DNAStar软件进行MegAlign分析,找出其保守序列和特征序列。根据生物学软件Primer primer5.0设计出不同分型探针及其PCR引物。并对探针进行分析。
2.基因芯片的设计、制作
将设计好的多条寡核苷酸探针在醛基基片的特定位置上,用点样仪将探针固定,同一探针平行重复点样3次,以观察其重复性,为了能在每次实验时判定实验操作的正确性,以及是否存在污染,在芯片设计时增加了如下的质控点;标志列、阳性对照行、阴性对照行。
3.杂交、显色技术
提取血清中DNA,进行PCR扩增后滴于基因芯片上进行杂交,采用酶促显色技术检测杂交结果。
4.检测结果的准确性
随机抽取PCR阳性标本送交上海生工公司进行测序,测序结果与芯片杂交结果进行对比,观察芯片杂交反应的准确性。
5.检测结果的重复性分析
取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,观察结果是否一致。
结果
1.探针、引物设计
在Genbank中查到17个HBV Pre-S基因组序列,共为A、B、C、D、E、F、G7个基因型,对其进行了进化树分析和多序列比对。根据序列差异情况和保守序列分布情况设计出可以扩增出特异序列的PCR引物和7个探针,PCR产物长度为340bp左右。到NCBI上将设计好的探针进行BLAST,分析评分在前50个的序列及其完全匹配序列,与探针完全匹配的序列基因型一致的为97%。
2.临床标本检测
用制备的基因芯片检测HBV各基因型(A-G)标准病毒株DNA样本,检测结果与已知基因型(经核酸测序)完全一致;106例HBV-DNA阳性标本均测出其基因型,其中B型37例,占34.91%,C型69例,占65.09%。未发现其他基因型与混合型感染。
3.DNA序列分析
测序结果与芯片杂交结果完全一致,进一步证实了检测结果的特异性、准确性。经过测序分析,杂交特异性和探针匹配程度一致。
4.检测结果重复性测定和灵敏度分析
取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,结果一致,显示出良好的重复性。106例阳性血清,采用荧光定量PCR检测,HBV-DNA水平最低者为2~* 10~4 copies/ml,初步认定该芯片的检测灵敏度不低于此水平。
结论
1.通过对HBV基因组的生物信息学分析,可以设计出相应的探针和引物用于HBV基因分型芯片制作。
2.设计制备的HBV基因分型芯片能检测出HBV基因型,初步应用于临床后,显示出满意的准确性、特异性、稳定性和灵敏度。
3.杭州地区HBV感染者HBV基因型B、C两型均存在,但以C型为主,由于选择研究对象不同等因素的存在,其确切分布尚需大样本、多中心临床研究以进一步明确。
4.本研究结果表明,基因型检测芯片具有操作简单、自动化程度高、结果准确可靠等特点,有良好的临床应用前景。
Introduction
Hepatitis B virus(HBV)infection is a major cause of liver disease around the world,especially in China.HBV genome can be divided into several subtypes.According to the nucleotides sequence,HBV has been classified into 8 genotypes(A-H).The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy,so the genotyping of HBV had become more and more important.Currently there were some diagnostic methods of genotype,including the complete genome sequence analysis or S gene sequencing,PCR-RFLP and so on.But they have the disadvantages of a high cost and time-consuming to carry out or the result lack of reliability,and they haven't the ability to detect many.targets simultaneously.Recently,as the progress of functional genomic techniques,microarray has been developed into an ideal tool for genotyping.In this study we want to establish a integrated method genechip design for HBV genotyping,and do some test and analysis about the specificity and repetition of the chips and explore its clinical application,And,to investigate the distribution of hepatitis B virus genotypes in Hangzhou area.
Methods
1.PCR primer and probe design of HBV genome.
HBV genotypes were found in NCBI's Genbank,and the results were alignmented with MegAlign and their conservative and specific sequences were determined.Primer Primer 5.0 was used to design genotype probes and PCR primers,the reliability of probes was analyzed.
2.Microarray fabrication
The oligonucleotide probes were stablized on the chip and each one spotted at a layout of 3×1 blocks to avoid possible hybridization failures.Six blocks of the HBV-Con probes targeting the conserved region of Pre-s monitor the PCR amplification,hybridization reactions and so on
3.Microarray hybridization and detection
After DNA samples were obtained and amplified with PCR,the HBV PCR products were hybridized with oligonucleotide probes.The hybridized results were colorized by BCIP/NBT.According to the hybridization signal and the corresponding probe sequence,HBV genotype was determined.
4.Veracity and specification of Hybridization
Random positive PCR products were sent for sequence analysis,and analysis results were compared with hybridization to test the veracity and specification of the genechips.
5.The repetition of genechips test.
20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples and the results were analyzed.
Results
1.PCR primer and chip probe design
We have found 17 HBV genotypes genome in Genbank,including7 genotypes:A,B,C,D,E,F and G.Phylogenetic tree and the evolutionary relationships were analyzed.PCR primers and 7 probes were designed according to the difference of the sequences and the conservative consequences at HBV pre-S gene region.The product of PCR was 340bp.Blasting the probes in NCBI'Genbank and analyzing the first 50 sequences of the results,we found the accuracy of completely matching with our designed probes is 97%.
2.Genotyping of clinical samples
We tested the clinical DNA samples,which genotypes were determined with genome sequence comparison method.The microarray hybridization assay displayed a complete match to the previously identified genotypes;106 clinical serum samples collected from HBV infection individuals in Hangzhou.Among them,37and69 samples were positive to genotypes B and C.
3.Validation of microarray genotyping
The results of hybrizations were identical with those of sequencing analysis.
4.The repetition and sensitiveness of genechips test.
20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples.The results are all positive.Of the 106 samples,,which HBV-DNA was positive,the lowest HBV-DNA title was 2~*10~4copies/ml,so we beliver the sensitiveness of the chips was not under the lever.
Conclusion
1.We have established a method of oligonucliotide probe designation according to the analysis of bioinformatics of HBV genome.
2.We have fabricated microarray and begun to apply the chips for clinical use.It is demonstrated that this assay is a promising method for virus genotyping.
3.HBV genotype B and C exsited in Hangzhou,and genotype C is a predominated genotype.However,To determine the distribution of HBV genotypes,we should take further investigation for HBV genotypes by enlarging study population.
4.The assay is a simple to perform and can be used as a rapid,accurate and high-throughput method.It has a high clinical value in detecting the HBV genotype.
前言
乙型肝炎病毒(HBV)的感染呈世界性分布,在我国尤其多见。HBV感染可导致急、慢性肝炎(CHB),甚至肝硬化,肝癌。由于病毒变异,HBV基因组分为多个型。根据其核苷酸序列差异,HBV可划分为(A-H)8种基因型。目前研究认为,HBV基因型是影响HBV感染者预后的重要因素之一,因此建立一套快速、准确的HBV分型检测技术十分必要。目前诊断HBV基因型的常用方法有DNA测序,聚合酶联反应-限制性片段长度多态性分析法(PCR-RFLP)等,在结果准确性、技术简单性方面各有优劣,且不能同时检测所有基因型。近几年发展起来的基因芯片技术弥补上述方法的缺陷。本课题的研究目的在于:建立DNA芯片检测HBV基因分型的研究方法;并对芯片检测的特异性、准确性、灵敏度和重复性进行测试、分析;通过调查杭州地区HBV的基因型分布对HBV基因分型检测芯片在临床上的应用进行初步探索,为病原体基因型检测芯片这种高科技方法在临床上的应用进行积极探索,积累有益经验。
方法
1.有关探针、引物设计
在GenBank数据库中查找HBV各型Pre-S基因组序列,并利用DNAStar软件进行MegAlign分析,找出其保守序列和特征序列。根据生物学软件Primer primer5.0设计出不同分型探针及其PCR引物。并对探针进行分析。
2.基因芯片的设计、制作
将设计好的多条寡核苷酸探针在醛基基片的特定位置上,用点样仪将探针固定,同一探针平行重复点样3次,以观察其重复性,为了能在每次实验时判定实验操作的正确性,以及是否存在污染,在芯片设计时增加了如下的质控点;标志列、阳性对照行、阴性对照行。
3.杂交、显色技术
提取血清中DNA,进行PCR扩增后滴于基因芯片上进行杂交,采用酶促显色技术检测杂交结果。
4.检测结果的准确性
随机抽取PCR阳性标本送交上海生工公司进行测序,测序结果与芯片杂交结果进行对比,观察芯片杂交反应的准确性。
5.检测结果的重复性分析
取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,观察结果是否一致。
结果
1.探针、引物设计
在Genbank中查到17个HBV Pre-S基因组序列,共为A、B、C、D、E、F、G7个基因型,对其进行了进化树分析和多序列比对。根据序列差异情况和保守序列分布情况设计出可以扩增出特异序列的PCR引物和7个探针,PCR产物长度为340bp左右。到NCBI上将设计好的探针进行BLAST,分析评分在前50个的序列及其完全匹配序列,与探针完全匹配的序列基因型一致的为97%。
2.临床标本检测
用制备的基因芯片检测HBV各基因型(A-G)标准病毒株DNA样本,检测结果与已知基因型(经核酸测序)完全一致;106例HBV-DNA阳性标本均测出其基因型,其中B型37例,占34.91%,C型69例,占65.09%。未发现其他基因型与混合型感染。
3.DNA序列分析
测序结果与芯片杂交结果完全一致,进一步证实了检测结果的特异性、准确性。经过测序分析,杂交特异性和探针匹配程度一致。
4.检测结果重复性测定和灵敏度分析
取20份HBV-DNA阳性标本,每份标本用本法重复测定3次,结果一致,显示出良好的重复性。106例阳性血清,采用荧光定量PCR检测,HBV-DNA水平最低者为2~* 10~4 copies/ml,初步认定该芯片的检测灵敏度不低于此水平。
结论
1.通过对HBV基因组的生物信息学分析,可以设计出相应的探针和引物用于HBV基因分型芯片制作。
2.设计制备的HBV基因分型芯片能检测出HBV基因型,初步应用于临床后,显示出满意的准确性、特异性、稳定性和灵敏度。
3.杭州地区HBV感染者HBV基因型B、C两型均存在,但以C型为主,由于选择研究对象不同等因素的存在,其确切分布尚需大样本、多中心临床研究以进一步明确。
4.本研究结果表明,基因型检测芯片具有操作简单、自动化程度高、结果准确可靠等特点,有良好的临床应用前景。
Introduction
Hepatitis B virus(HBV)infection is a major cause of liver disease around the world,especially in China.HBV genome can be divided into several subtypes.According to the nucleotides sequence,HBV has been classified into 8 genotypes(A-H).The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy,so the genotyping of HBV had become more and more important.Currently there were some diagnostic methods of genotype,including the complete genome sequence analysis or S gene sequencing,PCR-RFLP and so on.But they have the disadvantages of a high cost and time-consuming to carry out or the result lack of reliability,and they haven't the ability to detect many.targets simultaneously.Recently,as the progress of functional genomic techniques,microarray has been developed into an ideal tool for genotyping.In this study we want to establish a integrated method genechip design for HBV genotyping,and do some test and analysis about the specificity and repetition of the chips and explore its clinical application,And,to investigate the distribution of hepatitis B virus genotypes in Hangzhou area.
Methods
1.PCR primer and probe design of HBV genome.
HBV genotypes were found in NCBI's Genbank,and the results were alignmented with MegAlign and their conservative and specific sequences were determined.Primer Primer 5.0 was used to design genotype probes and PCR primers,the reliability of probes was analyzed.
2.Microarray fabrication
The oligonucleotide probes were stablized on the chip and each one spotted at a layout of 3×1 blocks to avoid possible hybridization failures.Six blocks of the HBV-Con probes targeting the conserved region of Pre-s monitor the PCR amplification,hybridization reactions and so on
3.Microarray hybridization and detection
After DNA samples were obtained and amplified with PCR,the HBV PCR products were hybridized with oligonucleotide probes.The hybridized results were colorized by BCIP/NBT.According to the hybridization signal and the corresponding probe sequence,HBV genotype was determined.
4.Veracity and specification of Hybridization
Random positive PCR products were sent for sequence analysis,and analysis results were compared with hybridization to test the veracity and specification of the genechips.
5.The repetition of genechips test.
20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples and the results were analyzed.
Results
1.PCR primer and chip probe design
We have found 17 HBV genotypes genome in Genbank,including7 genotypes:A,B,C,D,E,F and G.Phylogenetic tree and the evolutionary relationships were analyzed.PCR primers and 7 probes were designed according to the difference of the sequences and the conservative consequences at HBV pre-S gene region.The product of PCR was 340bp.Blasting the probes in NCBI'Genbank and analyzing the first 50 sequences of the results,we found the accuracy of completely matching with our designed probes is 97%.
2.Genotyping of clinical samples
We tested the clinical DNA samples,which genotypes were determined with genome sequence comparison method.The microarray hybridization assay displayed a complete match to the previously identified genotypes;106 clinical serum samples collected from HBV infection individuals in Hangzhou.Among them,37and69 samples were positive to genotypes B and C.
3.Validation of microarray genotyping
The results of hybrizations were identical with those of sequencing analysis.
4.The repetition and sensitiveness of genechips test.
20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples.The results are all positive.Of the 106 samples,,which HBV-DNA was positive,the lowest HBV-DNA title was 2~*10~4copies/ml,so we beliver the sensitiveness of the chips was not under the lever.
Conclusion
1.We have established a method of oligonucliotide probe designation according to the analysis of bioinformatics of HBV genome.
2.We have fabricated microarray and begun to apply the chips for clinical use.It is demonstrated that this assay is a promising method for virus genotyping.
3.HBV genotype B and C exsited in Hangzhou,and genotype C is a predominated genotype.However,To determine the distribution of HBV genotypes,we should take further investigation for HBV genotypes by enlarging study population.
4.The assay is a simple to perform and can be used as a rapid,accurate and high-throughput method.It has a high clinical value in detecting the HBV genotype.
引文
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8. Kurbanov F,Tanaka Y,Fujiwara K,et al. A new subtype Ac of hepatitis B virus and recombination between genotypes A and E in Cameroon. J Gen Virol,2005 Jul;86(Pt7):2047-56.
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3. Okamoto H,Tsuda F,Sakugawa H,et al. Typing hepatitis B virus by homology in nucleotide sequence:comparision of surface antigen subtypes.J Gen Virol.1998,69:2575-2583.
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11. Lindh M,Hannoun C,Dhillon AP,et al. Core promoter mutations and genotypes in relation to virus replication and liver damage in East Asian hepatitis B virus carriers.J infect Dis, 1999,179(4):775-782.
12. Kao JH.,Chen PJ, Lai MY,et al. Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol,2002,40(4): 1207-1209.
13. Kao JH,Chen PJ,Lai MY,et al. Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers. J Med Virol,2004,72(3):363-369.
14. Tsubota A,Arase Y,Ren F,et al. Genotype may correlate with liver carcinogenesis and tumor characteristics in cirrhotic patients infected with hepatitisB virus subtype adw [J]. J Med Virol,2001 ,.65(2):257-265.
15. Kao JH,Chen PJ,Lai MY,et al.Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitisB[J].Gastroenterology,2001,120(6):1564-1565.
16.Sugauchi F,Orito E,Ichida T,et al.Epidemiologic and virologic characteristics of hepatitis B virus genotype B having the recombination with genotype C.Gastroenterology,2003,124(4):925-932.
17.Sanchez-Tapias JM,CostaJ,Mas A,et al.Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitisB in western patients.Gastroenterology 2002;123:1848-1856
18.Enomoto M,Tamori A,Nishiguchi S.Hepatitis B virus genotypes and response to antiviral therapy.Clin Lab.2006;52(1-2):43-7.
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39.Sumi H,Yokosuka O,Seki N,et al.Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease.Hepatology,2003,37:19-26.
1.Kao JH, Chen PJ,Lai MY,et al.Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B.GastroenteroIogy 2000;118:554-559.
2.Kao JH. Hepatitis B viral genotypes:clinical relevance and molecular characteristics. J Gastroenterol Hepatol 2002; 17:643-650.
3. Wright TL.Introduction to chronic hepatitis B infection.Am J Gastroenterol, 2006, 101 Suppl 1:S1-6.
4. Okamoto H,Tsuda F,Sakugawa H,et al. Typing hepatitis B virus by homology in nucleotide sequence:comparision of surface antigen subtypes.J Gen Virol.1998,69:2575-2583.
5.Norder H,Courouce AM,Magnius LO.Complete genomes,phylogenotic relatedness,and strutuctural proteins of six strains of hepatitis B virus,four of which represent two new genotypes.Virology 1994;198:489-503.
6. Okamoto H,Imail M,Shimozaki M,et al.Nucleotide sequence of a cloned hepatitis B virus genome,subtype ayr:comparison with genomes of the other three subtypes[J].J Gen Virol,1986,67(Ptl1):2305-2314.
7. Norder H,Hammas B,Lofdahl ,et al.Comparision of the aminoacid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains[J].J Gen Virol, 992,73 (Pt5): 1201 -1208.
8. Kurbanov F,Tanaka Y,Fujiwara K,et al. A new subtype Ac of hepatitis B virus and recombination between genotypes A and E in Cameroon. J Gen Virol,2005 Jul;86(Pt7):2047-56.
9. Kobayashi M,Suzuki F,Akuta N,et al. Virological differences between patients infected with subtypes Ba and Bj of hepatitis B virus genotype B.J Gastroenterol Hepatol, 2005 Apr,20:570-6.
10.Kao JH,Chen PJ,Lai MY,et al.Clinical and virological aspects of blood donors infected with hepatitis B virus genotypes B and C[J].J Clin Microbiol,2002,40(1):22-25.
11. Xia G,Omana V N,Jia Z,et al.Characterization and distribution of hepatitis B virus genotypes and subtypes in 4 provinces of China[J]. Chin J Epidemiol, 2001,22(5):348-351.
12. Miyakawa Y,Mizokami M.Classifying hepatitis B Virus genotypes.Intervirology 2003;46:329-38.
13. Lindh M,Hannoun C,Dhillon AP,et al. Core promoter mutations and genotypes in relation to virus replication and liver damage in East Asian hepatitis B virus carriers.J infect Dis,1999,179(4):775-782.
14. Kao JH.,Chen PJ, Lai MY,et al. Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol,2002,40(4): 1207-1209.
15. Kao JH,Chen PJ,Lai MY,et al. Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers. J Med Virol,2004,72(3):363-369.
16. Tsubota A,Arase Y,Ren F,et al. Genotype may correlate with liver carcinogenesis and tumor characteristics in cirrhotic patients infected with hepatitisB virus subtype adw [J]. J Med Virol,2001,65(2):257-265.
17. Kao JH,Chen PJ,Lai MY,et al.Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitisB[J].Gastroenterology,2001,120.(6): 1564-1565.
18. Sugauchi F,Orito E,Ichida T,et al. Epidemiologic and virologic characteristics of hepatitis B virus genotype B having the recombination with genotype C.Gastroenterology,2003,124(4):925-932.
19. Sanchez-Tapias JM,CostaJ,Mas A,et al. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitisB in western patients.Gastroenterology 2002;123:1848-1856
20. Enomoto M,Tamori A,Nishiguchi S.Hepatitis B virus genotypes and response to antiviral therapy.Clin Lab.2006;52(1-2):43-7.
21. Flink HJ,Van Zonneveld M,Hansen BE,et al.Treatment with Peg-interferon alpha-2b for HbeAg-positive chronic hepatitis B:HBsAg loss is associated with HBV genotype.Am J Gastroenterol.2006 Feb;101(2):297-303.
22.Orito E,Fujiwara K,Tanaka Y,et al.A case-control study of response to lamivudine therapy for 2years in Japanese and Chinese patients chronically infected with hepatitis B virus of genotypes Bj,Ba and C.Hepatol Res.2006Jun;35(2):127-34.
23.Enomoto M,Tamori A,Nishiguchi S.Hepatitis B virus genotypes and response toantiviral therapy.Clin Lab.2006;52(1-2):43-7.
24.Idrees M,Khan S,Riazuddin S.Common genotypes of hepatitis B virus.J Coll Physicians Surg Pak.2004 Jun;14(6):344-7.
25.任来峰.乙型肝炎病毒基因分型方法研究进展[J].国际病毒学杂志,2006,13(1):26-29.
26.Sun ZH,Zheng WL,Zhang B,et al.Detection of hepatitis D virus by cDNA microarray method.Hepatobiliary Pancreat Dis Int,2004,3(3):423-427.
27.Chen LY,Huang J,Zhang XP,et al.Clinical evaluation of oligonucleotide microarrays for the detection of HBV mutants associated with lamivudine resistance.Pharmacogenomics,2005,6(7):721-730.
28.李凌,马文丽.DNA芯片技术研究进展.中国生物化学与分子生物学报,2000,16(2):151.