建立等位基因特異性鎖核酸實時熒光定量PCR檢測HBV YIDD耐藥突變及其臨床應(yīng)用評價
發(fā)布時間：2018-07-16 07:39
【摘要】：目的 1.建立等位基因特異性鎖核酸實時熒光定量PCR（RT-AS-LNA-qPCR）檢測HBVYIDD耐藥突變，評價其性能特點和臨床應(yīng)用價值。 2.監(jiān)測HBsAg濃度在恩替卡韋（ETV）治療的HBeAg陽性的慢性乙型肝炎（CHB）患者血清中的動態(tài)變化，探討其對治療反應(yīng)的預(yù)測價值。 方法 1.建立RT-AS-LNA-qPCR檢測HBV YIDD耐藥突變 1.1RT-AS-LNA-qPCR建立 包括反應(yīng)體系組成、擴(kuò)增條件、質(zhì)粒標(biāo)準(zhǔn)品制備、標(biāo)準(zhǔn)曲線制備等。 1.2RT-AS-LNA-qPCR方法學(xué)評價 用RT-AS-LNA-qPCR檢測重組野生、突變質(zhì)粒標(biāo)準(zhǔn)品（1×1010copies/μl~1×101copies/μl），評價其線性范圍、靈敏性、特異性、重復(fù)性、準(zhǔn)確性及突變型HBV DNA檢測靈敏度等。 1.3方法學(xué)比較 自建方法與測序法平行檢測臨床樣本，通過Kappa一致性檢驗、Pearson相關(guān)分析等統(tǒng)計分析進(jìn)行性能比較； 用克隆測序（金標(biāo)準(zhǔn)）法檢測已用自建方法所檢測出的含低水平突變DNA的臨床樣本，驗證所建方法的準(zhǔn)確性。 1.4RT-AS-LNA-qPCR臨床應(yīng)用價值評價 用自建方法檢測含不同比例突變的臨床混合模板，確定其能穩(wěn)定、準(zhǔn)確檢測出的最低突變比例，評價用于臨床樣本檢測的靈敏度； 用自建方法動態(tài)監(jiān)測HBV野生株和突變株在患者體內(nèi)的比例變化，評價其在少量突變檢測中的臨床應(yīng)用價值。 2. HBsAg定量對HBeAg陽性的慢性乙型肝炎患者恩替卡韋治療反應(yīng)的預(yù)測價值 2.1臨床病例和檢測指標(biāo) 選擇接受ETV治療（0.5mg/d）的HBeAg陽性CHB患者26例，并進(jìn)行1年的隨訪研究； 分別于各CHB患者抗病毒治療的0、3、6、9、12個月收集血清；化學(xué)發(fā)光法定量檢測各時間點的HBsAg、HBeAg濃度；實時熒光PCR定量檢測血清HBVDNA載量。 2.2ROC曲線分析 用ROC曲線分析HBsAg定量對HBeAg陽性的慢性乙型肝炎患者恩替卡韋治療反應(yīng)的預(yù)測價值，坐標(biāo)點分析確定其最佳臨界值。 結(jié)果 1.建立RT-AS-LNA-qPCR檢測HBV YIDD耐藥突變 1.1成功建立RT-AS-LNA-qPCR 成功制備了質(zhì)粒標(biāo)準(zhǔn)品以及標(biāo)準(zhǔn)曲線；確定了反應(yīng)體系的組成以及擴(kuò)增條件。 1.2RT-AS-LNA-qPCR方法學(xué)評價 RT-AS-LNA-qPCR檢測野生型、突變型標(biāo)準(zhǔn)質(zhì)粒的線性范圍均為1×109copies/μl~1×102copies/μl；檢測下限均為1×101copies/μl；批內(nèi)和批間變異系數(shù)（CV）在0.29%~2.72%之間；RT-AS-LNA-qPCR在1×109copies/μl、1×107copies/μl、1×105copies/μl野生背景下的突變檢測靈敏度分別為10-6、10-4、10-2。 1.3方法學(xué)比較 RT-AS-LNA-qPCR和市售優(yōu)秀HBV耐藥突變測序檢測試劑盒檢測結(jié)果的完全一致率91.2%（93/102），部分一致率8.8%（9/102），未發(fā)現(xiàn)完全不一致（0/102），兩法一致性好（Kappa=0.676, P=0.000）�？寺y序檢結(jié)果與RT-AS-LNA-qPCR完全一致。 1.4RT-AS-LNA-qPCR臨床應(yīng)用價值評價 RT-AS-LNA-qPCR用于臨床樣本檢測的靈敏度為0.03%；RT-AS-LNA-qPCR靈敏度高，可用于HBV耐藥突變的早期檢測。 2. HBsAg定量對HBeAg陽性的慢性乙型肝炎患者恩替卡韋治療反應(yīng)的預(yù)測價值 2.1ALT、HBV DNA、HBsAg檢測結(jié)果 17例患者發(fā)生病毒學(xué)應(yīng)答（VR+），9例未發(fā)生病毒學(xué)應(yīng)答（VR-）；基線ALT水平VR+組[（141.82±77.29）IU/ml]與VR-組[（134.2±49.76）IU/ml]無明顯差別（t=0.27, P=0.793）；HBV DNA VR+組[（6.76±1.00）lgIU/ml]明顯低于VR-組[（7.65±0.87）lg IU/ml]（t=-2.27, P=0.033）。 HBsAg濃度VR+組[（3.79±0.61）lg IU/ml）]與VR-組[（4.19±0.43）lg IU/ml]無明顯差別（t=-1.75, P=0.094）；HBsAg濃度與HBV DNA水平呈正相關(guān)（r=0.45,P=0.02）。HBsAg在治療開始的前3個月下降較快，3個月后下降較緩慢。從基線到治療3個月時，VR+組HBsAg平均下降（0.32±0.29）lg IU/ml，，VR-組下降（0.14±0.10）lg IU/ml，差異具有統(tǒng)計學(xué)意義（t=2.245, P=0.035）。 2.2ROC曲線分析 治療3個月時lg HBsAg濃度的ROC曲線下面積最大（AUC=0.840, P=0.005），臨界值3.8650lg IU/ml的Youden指數(shù)最大（0.602），其診斷敏感度為82.4%，特異度為77.8%。 結(jié)論 1.成功建立了RT-AS-LNA-qPCR檢測HBV YIDD耐藥突變。該技術(shù)線性范圍寬、靈敏度高、特異性強(qiáng)、重復(fù)性好、檢測下限低，優(yōu)于傳統(tǒng)的直接測序法，適合于臨床實驗室推廣應(yīng)用。 2. ETV治療3個月時lg HBsAg≤3.8650IU/ml可作為預(yù)測ETV治療1年病毒學(xué)應(yīng)答的指標(biāo)。
[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 鹵 77.29 ) IU / ml and VR - treated group ( 134.2 鹵 49.76 ) IU / ml ( t = 0.27 , P = 0.793 ) ;
HBV DNA VR + ( 6.76 鹵 1.00 ) lgIU / ml ( t = - 2.27 , P = 0.033 ) was significantly lower than that in VR - group ( 7.65 鹵 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 鹵 0.61 ) lg IU / ml ) and VR - group ( 4.19 鹵 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 鹵 0.29 ) lg IU / ml , VR - group decreased ( 0.14 鹵 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
本文編號：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 鹵 77.29 ) IU / ml and VR - treated group ( 134.2 鹵 49.76 ) IU / ml ( t = 0.27 , P = 0.793 ) ;
HBV DNA VR + ( 6.76 鹵 1.00 ) lgIU / ml ( t = - 2.27 , P = 0.033 ) was significantly lower than that in VR - group ( 7.65 鹵 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 鹵 0.61 ) lg IU / ml ) and VR - group ( 4.19 鹵 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 鹵 0.29 ) lg IU / ml , VR - group decreased ( 0.14 鹵 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
本文編號：2125702
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