建立等位基因特异性锁核酸实时荧光定量PCR检测HBV YIDD耐药突变及其临床应用评价
发布时间：2018-07-16 07:39
【摘要】：目的 1.建立等位基因特异性锁核酸实时荧光定量PCR（RT-AS-LNA-qPCR）检测HBVYIDD耐药突变，评价其性能特点和临床应用价值。 2.监测HBsAg浓度在恩替卡韦（ETV）治疗的HBeAg阳性的慢性乙型肝炎（CHB）患者血清中的动态变化，探讨其对治疗反应的预测价值。 方法 1.建立RT-AS-LNA-qPCR检测HBV YIDD耐药突变 1.1RT-AS-LNA-qPCR建立 包括反应体系组成、扩增条件、质粒标准品制备、标准曲线制备等。 1.2RT-AS-LNA-qPCR方法学评价 用RT-AS-LNA-qPCR检测重组野生、突变质粒标准品（1×1010copies/μl~1×101copies/μl），评价其线性范围、灵敏性、特异性、重复性、准确性及突变型HBV DNA检测灵敏度等。 1.3方法学比较 自建方法与测序法平行检测临床样本，通过Kappa一致性检验、Pearson相关分析等统计分析进行性能比较； 用克隆测序（金标准）法检测已用自建方法所检测出的含低水平突变DNA的临床样本，验证所建方法的准确性。 1.4RT-AS-LNA-qPCR临床应用价值评价 用自建方法检测含不同比例突变的临床混合模板，确定其能稳定、准确检测出的最低突变比例，评价用于临床样本检测的灵敏度； 用自建方法动态监测HBV野生株和突变株在患者体内的比例变化，评价其在少量突变检测中的临床应用价值。 2. HBsAg定量对HBeAg阳性的慢性乙型肝炎患者恩替卡韦治疗反应的预测价值 2.1临床病例和检测指标 选择接受ETV治疗（0.5mg/d）的HBeAg阳性CHB患者26例，并进行1年的随访研究； 分别于各CHB患者抗病毒治疗的0、3、6、9、12个月收集血清；化学发光法定量检测各时间点的HBsAg、HBeAg浓度；实时荧光PCR定量检测血清HBVDNA载量。 2.2ROC曲线分析 用ROC曲线分析HBsAg定量对HBeAg阳性的慢性乙型肝炎患者恩替卡韦治疗反应的预测价值，坐标点分析确定其最佳临界值。 结果 1.建立RT-AS-LNA-qPCR检测HBV YIDD耐药突变 1.1成功建立RT-AS-LNA-qPCR 成功制备了质粒标准品以及标准曲线；确定了反应体系的组成以及扩增条件。 1.2RT-AS-LNA-qPCR方法学评价 RT-AS-LNA-qPCR检测野生型、突变型标准质粒的线性范围均为1×109copies/μl~1×102copies/μl；检测下限均为1×101copies/μl；批内和批间变异系数（CV）在0.29%~2.72%之间；RT-AS-LNA-qPCR在1×109copies/μl、1×107copies/μl、1×105copies/μl野生背景下的突变检测灵敏度分别为10-6、10-4、10-2。 1.3方法学比较 RT-AS-LNA-qPCR和市售优秀HBV耐药突变测序检测试剂盒检测结果的完全一致率91.2%（93/102），部分一致率8.8%（9/102），未发现完全不一致（0/102），两法一致性好（Kappa=0.676, P=0.000）。克隆测序检结果与RT-AS-LNA-qPCR完全一致。 1.4RT-AS-LNA-qPCR临床应用价值评价 RT-AS-LNA-qPCR用于临床样本检测的灵敏度为0.03%；RT-AS-LNA-qPCR灵敏度高，可用于HBV耐药突变的早期检测。 2. HBsAg定量对HBeAg阳性的慢性乙型肝炎患者恩替卡韦治疗反应的预测价值 2.1ALT、HBV DNA、HBsAg检测结果 17例患者发生病毒学应答（VR+），9例未发生病毒学应答（VR-）；基线ALT水平VR+组[（141.82±77.29）IU/ml]与VR-组[（134.2±49.76）IU/ml]无明显差别（t=0.27, P=0.793）；HBV DNA VR+组[（6.76±1.00）lgIU/ml]明显低于VR-组[（7.65±0.87）lg IU/ml]（t=-2.27, P=0.033）。 HBsAg浓度VR+组[（3.79±0.61）lg IU/ml）]与VR-组[（4.19±0.43）lg IU/ml]无明显差别（t=-1.75, P=0.094）；HBsAg浓度与HBV DNA水平呈正相关（r=0.45,P=0.02）。HBsAg在治疗开始的前3个月下降较快，3个月后下降较缓慢。从基线到治疗3个月时，VR+组HBsAg平均下降（0.32±0.29）lg IU/ml，，VR-组下降（0.14±0.10）lg IU/ml，差异具有统计学意义（t=2.245, P=0.035）。 2.2ROC曲线分析 治疗3个月时lg HBsAg浓度的ROC曲线下面积最大（AUC=0.840, P=0.005），临界值3.8650lg IU/ml的Youden指数最大（0.602），其诊断敏感度为82.4%，特异度为77.8%。 结论 1.成功建立了RT-AS-LNA-qPCR检测HBV YIDD耐药突变。该技术线性范围宽、灵敏度高、特异性强、重复性好、检测下限低，优于传统的直接测序法，适合于临床实验室推广应用。 2. ETV治疗3个月时lg HBsAg≤3.8650IU/ml可作为预测ETV治疗1年病毒学应答的指标。
[Abstract]:Purpose
1 . To establish a real - time quantitative PCR ( RT - AS - LNA - qPCR ) for detecting HBV YIDD resistance mutation and evaluate its performance and clinical application value .
2 . To monitor the dynamic changes of the serum levels of HBeAg - positive patients with HBeAg - positive HBeAg - positive patients treated with ETV , and to explore its predictive value for the therapeutic response .
method
1 . RT - AS - LNA - qPCR was established to detect HBV YIDD resistance mutation .
1.1RT-AS-LNA-qPCR寤虹珛
including reaction system composition , amplification conditions , plasmid standard preparation , standard curve preparation and the like .
1.2RT - AS - LNA - qPCR methodological evaluation
RT - AS - LNA - qPCR was used to detect the recombinant wild and mutant plasmid standard ( 1 脳 1010 copies / 渭l ~ 1 脳 101copies / 渭l ) . The linear range , sensitivity , specificity , repeatability , accuracy and mutation HBV DNA detection sensitivity were evaluated .
1.3 Comparison of methodology
The clinical samples were tested in parallel with the method of self - construction and sequencing , and the performance was compared by Kappa consistency test , Pearson correlation analysis and other statistical analysis .
The accuracy of the proposed method was verified by using clone sequencing ( gold standard ) method to detect the clinical samples containing low - level mutant DNA detected by the self - established method .
1.4RT - AS - LNA - qPCR Clinical Application Value Evaluation
a self - built method is used for detecting the clinical mixed template containing different proportion mutations , and determining the lowest mutation proportion which can be stably and accurately detected , and evaluating the sensitivity for clinical sample detection ;
The proportion of HBV wild strain and mutant strain in patients was dynamically monitored by means of self - construction method . The clinical application value of HBV wild strain and mutant strain in the detection of small number of mutations was evaluated .
2 . The predictive value of the quantitative analysis of HBsAg in patients with HBeAg - positive chronic hepatitis B patients
2.1 Clinical Case and Test Indicators
Twenty - six patients were selected to receive ETV therapy ( 0.5 mg / d ) , and a follow - up study was conducted for 1 year .
The serum was collected from 0 , 3 , 6 , 9 and 12 months of antiviral treatment respectively .
detecting the concentration of HBsAg and HBeAg at each time point by chemiluminescence ;
The serum HBV DNA content was quantitatively determined by real - time fluorescence PCR .
2.2 ROC Curve Analysis
ROC curves were used to analyze the predictive value of the clinical response of the patients with HBeAg - positive chronic hepatitis B patients , and the optimal critical value was determined by the coordinate point analysis .
Results
1 . RT - AS - LNA - qPCR was established to detect HBV YIDD resistance mutation .
1.1 Successful establishment of RT - AS - LNA - qPCR
The plasmid standard and standard curve were successfully prepared .
The composition of the reaction system and the amplification conditions were determined .
1.2RT - AS - LNA - qPCR methodological evaluation
RT - AS - LNA - qPCR was used to detect wild type , and the linear range of mutant standard plasmids was 1 脳 109 copies / 渭l ~ 1 脳 102copies / 渭l .
the detection limit is 1 * 101copies / 渭l ;
CV of intra - and inter - batch was 0.29 % ~ 2.72 % ;
The sensitivity of RT - AS - LNA - qPCR was 10 - 6 , 10 - 4 , 10 - 2 in 1 脳 109 copies / 渭l , 1 脳 107 copies / 渭l , 1 脳 105 copies / 渭l wild background .
1.3 Comparison of methodology
The complete agreement rate of RT - AS - LNA - qPCR and commercial excellent HBV - resistant mutation sequencing detection kit was 91.2 % ( 93 / 102 ) . The partial coincidence rate was 8.8 % ( 9 / 102 ) . There was no complete inconsistency ( 0 / 102 ) . There was good agreement between the two methods ( Kappa = 0.676 , P = 0.000 ) . The results of clone sequencing were completely consistent with RT - AS - LNA - qPCR .
1.4RT - AS - LNA - qPCR Clinical Application Value Evaluation
The sensitivity of RT - AS - LNA - qPCR was 0.03 % .
RT - AS - LNA - qPCR has high sensitivity and can be used for early detection of HBV - resistant mutation .
2 . The predictive value of the quantitative analysis of HBsAg in patients with HBeAg - positive chronic hepatitis B patients
2.1 ALT , HBV DNA , HBsAg Test Results
virological response ( VR + ) in 17 patients and no virological response ( VR - ) in 9 cases ;
There was no significant difference ( t = 0.27 , P = 0.793 ) between the baseline ALT level VR + arm group ( 142.82 卤 77.29 ) IU / ml and VR - treated group ( 134.2 卤 49.76 ) IU / ml ( t = 0.27 , P = 0.793 ) ;
HBV DNA VR + ( 6.76 卤 1.00 ) lgIU / ml ( t = - 2.27 , P = 0.033 ) was significantly lower than that in VR - group ( 7.65 卤 0.87 ) lg IU / ml ( t = - 2.27 , P = 0.033 ) .
There was no significant difference ( t = - 1.75 , P = 0.094 ) between the HBsAg concentration VR + group ( 3.79 卤 0.61 ) lg IU / ml ) and VR - group ( 4.19 卤 0.43 ) lg IU / ml ( t = - 1.75 , P = 0.094 ) ;
The concentration of HBsAg was positively correlated with HBV DNA level ( r = 0.45 , P = 0.02 ) . The average decrease of HBsAg in VR + group ( 0.32 卤 0.29 ) lg IU / ml , VR - group decreased ( 0.14 卤 0.10 ) lg IU / ml and the difference was statistically significant ( t = 2.245 , P = 0.035 ) .
2.2 ROC Curve Analysis
The maximum area under ROC curve ( AUC = 0.840 , P = 0.005 ) , the critical value 3.8650lg IU / ml Youden index was the largest ( 0.602 ) . The diagnostic sensitivity was 82.4 % and the specificity was 77.8 % .
Conclusion
1 . RT - AS - LNA - qPCR was successfully established to detect HBV YIDD resistance mutation . The technique has wide linear range , high sensitivity , strong specificity , good repeatability and low detection limit , which is superior to the traditional direct sequencing method and is suitable for clinical laboratory application .
2 . When ETV was treated for 3 months , lg HBsAg 鈮
本文编号：2125702
[Abstract]:Purpose
1 . To establish a real - time quantitative PCR ( RT - AS - LNA - qPCR ) for detecting HBV YIDD resistance mutation and evaluate its performance and clinical application value .
2 . To monitor the dynamic changes of the serum levels of HBeAg - positive patients with HBeAg - positive HBeAg - positive patients treated with ETV , and to explore its predictive value for the therapeutic response .
method
1 . RT - AS - LNA - qPCR was established to detect HBV YIDD resistance mutation .
1.1RT-AS-LNA-qPCR寤虹珛
including reaction system composition , amplification conditions , plasmid standard preparation , standard curve preparation and the like .
1.2RT - AS - LNA - qPCR methodological evaluation
RT - AS - LNA - qPCR was used to detect the recombinant wild and mutant plasmid standard ( 1 脳 1010 copies / 渭l ~ 1 脳 101copies / 渭l ) . The linear range , sensitivity , specificity , repeatability , accuracy and mutation HBV DNA detection sensitivity were evaluated .
1.3 Comparison of methodology
The clinical samples were tested in parallel with the method of self - construction and sequencing , and the performance was compared by Kappa consistency test , Pearson correlation analysis and other statistical analysis .
The accuracy of the proposed method was verified by using clone sequencing ( gold standard ) method to detect the clinical samples containing low - level mutant DNA detected by the self - established method .
1.4RT - AS - LNA - qPCR Clinical Application Value Evaluation
a self - built method is used for detecting the clinical mixed template containing different proportion mutations , and determining the lowest mutation proportion which can be stably and accurately detected , and evaluating the sensitivity for clinical sample detection ;
The proportion of HBV wild strain and mutant strain in patients was dynamically monitored by means of self - construction method . The clinical application value of HBV wild strain and mutant strain in the detection of small number of mutations was evaluated .
2 . The predictive value of the quantitative analysis of HBsAg in patients with HBeAg - positive chronic hepatitis B patients
2.1 Clinical Case and Test Indicators
Twenty - six patients were selected to receive ETV therapy ( 0.5 mg / d ) , and a follow - up study was conducted for 1 year .
The serum was collected from 0 , 3 , 6 , 9 and 12 months of antiviral treatment respectively .
detecting the concentration of HBsAg and HBeAg at each time point by chemiluminescence ;
The serum HBV DNA content was quantitatively determined by real - time fluorescence PCR .
2.2 ROC Curve Analysis
ROC curves were used to analyze the predictive value of the clinical response of the patients with HBeAg - positive chronic hepatitis B patients , and the optimal critical value was determined by the coordinate point analysis .
Results
1 . RT - AS - LNA - qPCR was established to detect HBV YIDD resistance mutation .
1.1 Successful establishment of RT - AS - LNA - qPCR
The plasmid standard and standard curve were successfully prepared .
The composition of the reaction system and the amplification conditions were determined .
1.2RT - AS - LNA - qPCR methodological evaluation
RT - AS - LNA - qPCR was used to detect wild type , and the linear range of mutant standard plasmids was 1 脳 109 copies / 渭l ~ 1 脳 102copies / 渭l .
the detection limit is 1 * 101copies / 渭l ;
CV of intra - and inter - batch was 0.29 % ~ 2.72 % ;
The sensitivity of RT - AS - LNA - qPCR was 10 - 6 , 10 - 4 , 10 - 2 in 1 脳 109 copies / 渭l , 1 脳 107 copies / 渭l , 1 脳 105 copies / 渭l wild background .
1.3 Comparison of methodology
The complete agreement rate of RT - AS - LNA - qPCR and commercial excellent HBV - resistant mutation sequencing detection kit was 91.2 % ( 93 / 102 ) . The partial coincidence rate was 8.8 % ( 9 / 102 ) . There was no complete inconsistency ( 0 / 102 ) . There was good agreement between the two methods ( Kappa = 0.676 , P = 0.000 ) . The results of clone sequencing were completely consistent with RT - AS - LNA - qPCR .
1.4RT - AS - LNA - qPCR Clinical Application Value Evaluation
The sensitivity of RT - AS - LNA - qPCR was 0.03 % .
RT - AS - LNA - qPCR has high sensitivity and can be used for early detection of HBV - resistant mutation .
2 . The predictive value of the quantitative analysis of HBsAg in patients with HBeAg - positive chronic hepatitis B patients
2.1 ALT , HBV DNA , HBsAg Test Results
virological response ( VR + ) in 17 patients and no virological response ( VR - ) in 9 cases ;
There was no significant difference ( t = 0.27 , P = 0.793 ) between the baseline ALT level VR + arm group ( 142.82 卤 77.29 ) IU / ml and VR - treated group ( 134.2 卤 49.76 ) IU / ml ( t = 0.27 , P = 0.793 ) ;
HBV DNA VR + ( 6.76 卤 1.00 ) lgIU / ml ( t = - 2.27 , P = 0.033 ) was significantly lower than that in VR - group ( 7.65 卤 0.87 ) lg IU / ml ( t = - 2.27 , P = 0.033 ) .
There was no significant difference ( t = - 1.75 , P = 0.094 ) between the HBsAg concentration VR + group ( 3.79 卤 0.61 ) lg IU / ml ) and VR - group ( 4.19 卤 0.43 ) lg IU / ml ( t = - 1.75 , P = 0.094 ) ;
The concentration of HBsAg was positively correlated with HBV DNA level ( r = 0.45 , P = 0.02 ) . The average decrease of HBsAg in VR + group ( 0.32 卤 0.29 ) lg IU / ml , VR - group decreased ( 0.14 卤 0.10 ) lg IU / ml and the difference was statistically significant ( t = 2.245 , P = 0.035 ) .
2.2 ROC Curve Analysis
The maximum area under ROC curve ( AUC = 0.840 , P = 0.005 ) , the critical value 3.8650lg IU / ml Youden index was the largest ( 0.602 ) . The diagnostic sensitivity was 82.4 % and the specificity was 77.8 % .
Conclusion
1 . RT - AS - LNA - qPCR was successfully established to detect HBV YIDD resistance mutation . The technique has wide linear range , high sensitivity , strong specificity , good repeatability and low detection limit , which is superior to the traditional direct sequencing method and is suitable for clinical laboratory application .
2 . When ETV was treated for 3 months , lg HBsAg 鈮
本文编号：2125702
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