荧光标记杂交双探针PCR融解曲线法在临床的应用评价  

Evaluation of fluorescent hybridization biprobe PCR and melting curve assay

作　　者：张淑云[1] 刘伟[1] 李迪[2] 谷鸿喜[2] 仰曙芬[1] 周志红[1] 杜博[1] 金茜[1] 常曼丽[1] 

机构地区：[1]哈尔滨医科大学附属第二医院科研实验中心,黑龙江省哈尔滨市150086 [2]哈尔滨医科大学微生物学教研室,黑龙江省哈尔滨市150086

出　　处：《世界华人消化杂志》2005年第11期1291-1294,共4页World Chinese Journal of Digestology

基　　金：黑龙江省自然科学基金资助项目;No.D0307~~

摘　　要：目的:对国产荧光标记杂交双探针PCR融解曲线法(FH—PCR—MC)检测乙型肝炎病毒聚合酶酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸基序(HBVYMDD)变异的试剂进行临床应用评价.方法:慢性乙型肝炎患者外周血浆标本217份,分别采用荧光标记Taqman探针定量PCR法(FT—PCR)和荧光标记杂交双探针PCR融解曲线法(FH—PCR—MC)检测HBVDNA含量和YMDD及其变异;其中78份HBVDNA阳性标本采用基因型特异性多引物对巢式PCR法进行基因分型(A—F),30份标本用PCR产物克隆测序法检测HBVYMDD及其变异.结果:血浆标本HBVDNA阳性率(≥1.0×106copies/L)为75.6%(164/217).YMDD及其变异检出率为67.7%(147/217).其中YMDD44.9%(66/147),YIDD22.5%(33/147).YVDD17.7%(26/147),YIDD/YVDD混合株10.9%(16/147),其他混合株4.0%(6/147);结合HBVDNA含量,HBVYMDD及其变异的检出下限为HBVDNA=5.0×106copies/L,但在106copies/L时检出率较低,仅为36.4%,随着HBVDNA含量增高,检出率显著增高(>80%),且在107copies/L时即有显著增高(χ2=7.177,P<0.01).在基因分型的78份标本中,C基因型占89.8%,B和D基因型分别占8.9%和1.3%,YMDD及其变异总检出率为100%,变异总检出率为59.0%;1份D基因型YMDD及其变异检测为YIDD/YVDD混合变异;B,C两种基因型变异检出率分别为71.4%和55.7%,但二者无统计学差异.以测序法的检测结果为相对标准,则FH—PCR—MC法检测HBVYMDD及其变异的相对敏感性、特异性和符合率分别为96.3%(26/27),100%(3/3)和96.7%(29/30);对于HBVYMDD及其变异的类型,两种方法检测结果一致.结论:FH—PCR—MC法是一种快速、特异的HBVYMDD及其变异检测方法,具有较高的检出率,且能区分YMDD及其变异的类型.AIM: To evaluate a fluorescent hybridization biprobe PCR and melting curve assay for detection of (hepatitis B virus) YMDD mutation associated with lamivudine therapy. METHODS: HBV DNA and YMDD mutations in the 217 clinical serum specimens from patients with chronic HBV infection who were treated with lamivudine (100 mg/day) were detected by the fluorescence quantitative polymerase chain reaction (PCR) using TaqMan probe (FT-PCR) and the fluorescent hybridization biprobe PCR and melting curve assay (FH-PCR-MC), respectively. Seventy-eight positive sera were then genotyped by nested PCR with six pairs of HBV genotype-specific primes (A to F), and the cloned DNA fragments derived from conventional PCR of HBV YMDD of 30 positive sera were sequenced. RESULTS: Among 217 samples, 75.6%(164/217) were HBV DNA positive, and 67.7% (147/217) were HBV YMDD positive, including YMDD 44.9%(66/147),YIDD22.5%(33/147),YVDD 17.7%(26/147),YI/VDD 10.9%(16/147), and others 4.0% (6/147). In HBV DNA ≥ 107copies/L, all the positive and mutant rates of YMDD have no significant difference in different HBV DNA levels. Among 78 genotype samples (genotype C 89.8%, B 8.9% and D 1.3%), the positive and mutant rates of YMDD were 100%(78/78) and 58.97%(46/78) respectively. One genotype D was YIDD/YVDD. The mutant rates of YMDD in genotype B and C were 71.4% and 55.7% respectively, but have no marked difference (P>0.5). Using the results by DNA sequencing as reference standard, the relative specificity, sensitivity and over-all accordance of FH-PCR-MC were 96.3% (26/27), 100% (3/3) and 96.7% (29/30) respectively. The results of YMDD typing by FH-PCR-MC were confirmed by the sequencing of clones. CONCLUSION: The fluorescent hybridization biprobe PCR and melting curve assay kit in detection of HBV YMDD mutation has high sensitivity and specificity. It is a convenient and rapid test kit, and may be used in YMDD genotyping.

关 键 词：荧光标记 曲线法 双探针 融解 杂交 乙型肝炎病毒聚合酶 慢性乙型肝炎患者 DNA含量 YMDD 天门冬氨酸 临床应用评价 定量PCR法 Taqman 巢式PCR法 HBV 血浆标本 基因分型 D基因型 总检出率 检测结果 克隆测序法 PCR产物 基因型变异 

分 类 号：R446.5[医药卫生—诊断学]