修回日期: 2002-08-01
接受日期: 2002-08-09
在线出版日期: 2003-02-15
目的: 探讨基因芯片技术在乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)及HBV YMDD变异株检测中的应用.
方法: 采用HBV、HCV联合基因芯片检测40份血清标本, 并与HBV, HCV基因定量检测方法比较; 采用HBV YMDD变异株芯片检测20份血清标本, 并与错配PCR及DNA序列测定方法比较.
结果: HBV, HCV联合基因芯片与HBV DNA定量检测的符合率为85%(34/40), HBV检出率为83%(19/23), 假阳性率为12%(2/17); 与HCV RNA定量检测的符合率为85%(34/40), HCV检出率为58%(7/12), 假阳性率为4%(1/28). HBV YMDD变异株芯片与本室设计的错配PCR符合率为70%(14/20), 5份芯片检测的标本与DNA序列测定结果-致, 该芯片尚可检测出不同病毒体或变异体的混合感染.
结论: HBV, HCV联合基因芯片对HBV的检测有较高的敏感性, 但存在-定程度的非特异性; 对HCV的检测敏感性偏低, 但特异性较高. HBV YMDD变异株芯片敏感性和特异性均较高, 同时能检测出病毒变异体的共生现象.
引文著录: 李刚, 舒欣, 马会慧, 陈伟, 陈文思, 陈青, 江元森, 姚集鲁. 基因芯片技术检测HBV HCV及HBV YMDD变异株. 世界华人消化杂志 2003; 11(2): 178-181
Revised: August 1, 2002
Accepted: August 9, 2002
Published online: February 15, 2003
AIM: To investigate the effect of DNA microarray in detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and HBV YMDD mutants.
METHODS: HBV and HCV in 40 serum samples were detected by mixed microarray and quantitative determination method as well; 20 serum samples from patients with hepatitis B treated with lamivudine were detected by microarray loaded HBV YMDD mutants gene, and were simultaneously tested with mismatched PCR and DNA sequencing for comparison.
RESULTS: The coincident rate of mixed microarray and quantitative determination of HBV DNA was 85% (34/40). The detectable rate of HBV by mixed microarray was 83% (19/23); 2 of 17 samples showed false positive reaction. The coincident rate of mixed microarray and HCV RNA quantitative determination was 85% (34/40). The detectable rate of HCV by mixed microarray was 58% (7/12). One of 28 samples showed false positive reaction. The coincident rate of HBV YMDD mutants microarray and mismatched PCR was 70% (14/20). Mixed infection of wild and mutant HBV or different mutants were detected by microarray.
CONCLUSION: Mixed microarray has high sensitivity and low non-specificity in detection of HBV, but has lower sensitivity and higher specificity in detection of HCV. Detection of HBV YMDD mutants and mixed infection with microarray had higher sensitivity and specificity.
- Citation: Li G, Shu X, Ma HH, Chen W, Chen WS, Chen Q, Jiang YS, Yao JL. Detection of HBV, HCV and HBV YMDD mutants by DNA microarray. Shijie Huaren Xiaohua Zazhi 2003; 11(2): 178-181
- URL: https://www.wjgnet.com/1009-3079/full/v11/i2/178.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v11.i2.178
微阵列(microarray)亦称为生物芯片(biochip), 其中用于检测基因的生物芯片称为基因芯片(genechip, Affymetrix公司注册商标). 芯片是一种融电子学, 生物学, 分子生物学, 物理学, 化学, 计算机科学为-体的高度交叉的新技术. 具有高通量, 自动化程度高, 系统化等特点[1-6]. 目前, 该技术已应用于药物筛选, 基因表达谱测定, 多态性分析, 新基因鉴定, 测序, 临床检测等领域的研究[7-23]. HBV和HCV是引起慢性肝炎的最主要原因, 这两种病毒基因及其变异株的检测对于临床诊断, 治疗方案选择及药物疗效评估有重要价值[24-28]. 本文采用基因芯片技术检测HBV, HCV基因及HBV YMDD变异株, 并与其他检测方法进行比较, 以探讨该法临床应用的可行性.
血清标本60例采自广州中山大学附属第三医院传染病科1999-10/2001-09门诊和住院患者, -30℃保存备用. 患者年龄14-72(平均35.1)岁. 其中40份为乙、丙型肝炎基因芯片检测所用标本, 男25例, 女15例. 20份为HBV YMDD变异株检测所用标本, 男16例, 女4例, 均为拉米夫定(贺普丁)治疗(100 mg/d)超过1年的病例.
病例初筛: HBsAg, HBeAg, 抗HBs, 抗HBe, 及抗HBc采用酶免疫试验(EIA)法(美国Abbott诊断试剂), 抗HCV IgM, 抗HCV IgG采用ELISA法(沈阳惠民试剂公司), 实验操作按试剂盒说明. HBV DNA, HCV RNA定量检测: 采用荧光定量PCR方法 设备为GeneAmp 5 700 Sequence Detection System (applied biosystems公司), 试剂购自广州中山大学达安基因诊断中心. 按说明书进行操作. 错配PCR检测HBV YMDD变异株: 所用引物由课题组设计, 序列见表1.取待检血清50 mL, 加入裂解液5 mL(含EDTA和25 mmol/L MgCl2), 在98℃变性15 min, 离心取上清5 mL进行PCR扩增, 第1次扩增用外引物P24, P26, 第2次扩增正链内引物为P25, 负链内引物分别用YMDD, YVDD, YIDD特异的引物P27, P28, P30.反应总体积为50 mL, 含dNTP(2.5 mmol/L), Taq DNA聚合酶(2ü), 在94℃ 30 s, 57℃ 30 s, 72℃ 1 min, 30个循环. 第2次扩增时退火温度根据引物TM值进行调整, 扩增产物166 bp, 取10 mL在20g/L琼脂糖凝胶中电泳, 紫外灯下观察结果. DNA序列测定: 用引物P25, P26对5份标本进行扩增, 反应条件同上, 获得扩增产物249 bp, 电泳后采用胶纯化DNA方法, 用QIA Quick Gel Extraction Kit纯化, 按说明书操作. 将纯化PCR产物直接测序, 经双脱氧链末端终止法进行正反方向测序, 在ABI373全自动测序仪上进行, 测序工作由北京赛百盛公司完成. 基因芯片检测: 芯片制备 由上海博华基因芯片技术有限公司制备, 包括迈科锐HBV、HCV联合基因检测芯片和HBV YMDD(拉米夫定耐药基因)检测芯片. 血清核酸扩增 取待检测血清10 mL, 采用常规酚、氯仿、异戊醇方法抽取核酸, 异丙醇沉淀. 反转录及PCR扩增 反转录反应体积15 mL, 含抽提核酸, dNTP, 反转录酶, 42℃ 30 min, 取5 mL用于PCR扩增, 共35个循环, 再吸取PCR产物2 mL, 加入CY5标记的dUTP, 进行PCR标记, 共35个循环. 杂交 标记的产物沉淀后加入杂交试剂, 并与芯片分别于95℃变性5 min, 冷却后将杂交液10 mL滴于芯片的上样区, 封闭后在湿盒内42℃杂交1.5 h, 然后用0.1×SSC洗涤10 min, 双蒸水冲洗并避光晾干. 扫描及信号分析 将杂交后的芯片插入ScanArray 300扫描仪(美国)中进行扫描, 扫描强度55-60%, 获得的图片用GenArray-Check软件分析各点的信号状况, 结果根据信号强度及比值作出判断(图1, 表2).
| 编号 | 极性 | 位置 | 序列 |
| p24 | + | 457-476 | AGGAAGCTTGCCCGTTTGTC(外正) |
| p25 | + | 594-613 | CTAAGCTTGTATTCCCATCC(内正) |
| p26 | - | 843-824 | TAGAATTCAAATGTATACCC(外负) |
| p27 | - | 760-741 | CCCAATACCACATCATCCAT(YMDD) |
| p28 | - | 760-741 | CCCAATACCACATCATCCAC(YVDD) |
| p30 | - | 760-743 | CCCAATACCACATCATCA(YIDD) |
| 平均值 | 比值 | 结果 | |
| YVDD突变点 | 13 | 795.16 | 1.07 共生 |
| 单点差异对照 | 2782.76 | ||
| 单点差异对照 | 5 002.95 | ||
| YMDD野生点 | 12 886.75 | ||
| A548V突变点 | 629.18 | 0.64 | 野生 |
| 单点差异对照 | 580.87 | ||
| 单点差异对照 | 546.46 | ||
| A548V野生点 | 981.00 | ||
| L528M突变点 | 5 383.92 | 0.99 | 共生 |
| 单点差异对照 | 729.66 | ||
| L528M野生点 | 1 319.74 | ||
| L528M野生点 | 5 440.26 | ||
| YIDD突变点 | 2 264.35 | 0.15 | 野生 |
| 单点差异对照 | 870.77 | ||
| 单点差异对照 | 1 313.16 | ||
| YMDD野生点 | 14 675.54 |
在40份血清标本中, 通过检测HBsAg及HBV DNA定量证实23份为HBV感染者, 通过检测抗HCV及HCV RNA定量证实12份为HCV感染者, 其余5份为阴性对照. 基因芯片检测结果与上述标本-致的有34份, 符合率为85%(34/40). 在23份HBV感染者中, 芯片检测19份为HBV阳性, 阳性率为83%(19/23); 在其余17份HBV阴性标本中, 有2例为阳性(12%). 在12例HCV感染者中, 芯片检测7例为HCV阳性, 阳性率58%(7/12); 28份HCV阴性标本中, 1份为阴性(1/28).
在20份拉米夫定治疗1年后的血清标本中, 有3份为HBV DNA定量检测阴性, 用错配PCR和芯片检测亦为阴性. 另17份HBV DNA定量检测阳性标本中, 错配PCR检测发现8份为YMDD野生株, 8份YVDD变异株, 1份YIDD变异株; 芯片检测发现5例为YMDD野生株, 6例为YVDD变异株, 1例YIDD变异株, 2例为YVDD变异株与野生株共生, 1例为YVDD变异株与YIDD变异株共生, 其余2例为阴性. 其中5份标本用DMA序列测定法检测, 结果与芯片检测基本-致, 仅1例DNA序列测定为YIDD变异株, 而芯片检测为YIDD变异株与YVDD变异株共生. 5例序列测定标本中, 1例与错配PCR不符. 芯片检测与错配PCR结果完全-致的有8例(8/17); 部分-致的有3例(3/17), 表现为错配PCR检测为单-病毒株, 芯片检测为两种病毒株共生.
目前, 临床上常用的病毒性肝炎特异性病原检测方法主要有ELISA法检测病毒抗原或抗体, 以及PCR法定性或定量检测病毒基因. 就确定病毒感染而言, 抗原抗体检测仍然是首选方法, 而病毒基因的检测对于了解患者的病毒载量, 病毒复制状况, 传染性大小, 抗病毒药物疗效等有较大意义[29-33]. 上述两种方法每次只能检测一种病原体或一种基因, 也难以检测基因的变异, 特别是多种变异体存在的情况下. 基因芯片则可弥补上述不足, 他具有高通量的特点, 一个芯片可同时检测多个病原体或变异体[34], 而病毒性肝炎是由多种肝炎病毒引起的常见病, HBV和HCV是慢性肝炎的主要病原体, 临床上很多情况下对两种病毒的基因都要进行检测. 另外, 乙型肝炎病毒基因容易发生变异, 特别是拉米夫定治疗后有部分患者出现病毒变异, 而且变异株可以不止一种, 变异株的出现表明病毒出现了耐药. 在上述情况下, 基因芯片可同时检测两种以上病原体或变异体的优势可凸现出来. 我们用HBV、HCV联合基因芯片对40份标本进行检测, 并与HBV DNA定量检测作了比较, 二者的符合率为85%(34/40), 在23份HBsAg阳性及HBV DNA 定量阳性者中, 芯片检测出率为83(19/23). 在17份HBsAg阴性及HBV DNA定量阴性标本中, 有2例芯片检测阳性, 假阳性率12%. 在HCV检测方面, 芯片与抗HCV及HCV RNA定量检测的符合率亦为85%(34/40), 但检出率偏低, 为58%(7/12), 同时假阳性率也较低, 仅为4%(1/28). 从上述数据看出, HBV、HCV联合基因芯片对HBV检测有较高的敏感性, 但存在-定的非特异性; 对HCV的敏感性偏低, 但特异性较高. 芯片对HCV检测敏感性偏低可能与血标本中病毒量低有关. 在HBV YMDD变异株检测方面, 芯片检测结果与DNA序列测定-致. 我们仅对5例进行了序列测定, 由于标本数偏少, 得出的结论须待更大量的标本来证实. 芯片检测与本室设计的错配PCR法符合率为70%(14/20), 而芯片可检测出3例不同病毒株混合感染的共生现象. 由此可见, 芯片检测HBV YMDD变异株的敏感性和特异性均较高, 且能检出病毒变异体的共生状况.
基因芯片检测的生物学基础是核酸杂交[35,36], 但在实际操作中, 单纯采用核酸杂交进行检测, 其信号强度不足, 导致敏感性低. 在目前阶段, 样品扩增, 如通过PCR, 仍是其必不可少的一个步骤, 是提高敏感性的措施, 但亦导致其步骤的复杂化及PCR带来的假阴性或假阳性. 因此, 从多个方面进行优化, 包括标记方式的改进, 增强信号强度, 提高信号检测仪器的灵敏度, 杂交温度的控制等是今后基因芯片能否在临床广泛应用的关键[37-39]. 本结果显示, 基因芯片在HBV、HCV基因检测中是一种较为有效的手段, 在同时检测HBV、HCV两种病毒及病毒变异株中有其独到的优势, 临床应用中可作为其他检测方法的补充, 在分析结果时须参考其他临床资料再作出判断.
编辑: N/A
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