质谱法与直接测序法检测HBV P基因耐药突变的比较研究
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摘要
目的：用基质辅助激光解析电离飞行时间质谱法(MALDI-TOF MS)和PCR产物直接测序法两种方法检测乙型肝炎病毒P基因耐药位点的突变情况,并进行比较分析,进一步证实MALDI-TOF MS是一种快速、早期、准确发现核苷(酸)类药物治疗后耐药位点的检测方法。
方法：1、病例来源：(1)研究组：收集90例服用拉米夫定等核苷(酸)类药物治疗1年以上,HBV-DNA仍大于500copies/ml的慢性乙型病毒性肝炎患者血清,并根据核苷(酸)类药物用药情况分为6个组。(2)对照组：同时收集10名HBsAg阳性时间超过6个月,肝功能正常,HBV-DNA>1×105 copies/ml,始终未予抗病毒治疗的慢性乙型病毒性肝炎患者的血清作为对照组。2、检测方法：(1)采用MALDI-TOF MS检测HBV P基因已知11个变异位点的变异情况；基于SEQUENON公司质谱仪及配套芯片-试剂盒技术,通过TYPE4.0软件分析结果。(2)PCR产物直接测序法采用ABI3730XL测序仪,通过SequenceNavigator软件分析得出序列。
结果：1、两种方法检出结果比较：在服用拉米夫定等核苷(酸)类药物的90例病例血清中,MALDI-TOF MS测得53例耐药变异的病例,共86个耐药突变位点,PCR产物直接测序法仅检测出19例耐药变异的病例,共29个耐药突变位点,MALDI-TOF MS检出数明显高于直接测序法,两者间的差异有统计学意义(P<0.05)；在对照组中两种检测方法均没有发现已知的耐药变异位点。2、两种方法检测DNA的灵敏性比较：HBV-DNA水平在500-1000copies/ml的12例血清中,MALDI-TOF MS测得6例出现耐药变异位点,检出率50%,而PCR产物直接测序法在此病毒水平下未检出耐药变异位点；在相同的HBV-DNA水平下,MALDI-TOF MS变异检出数(率)均高于PCR产物直接测序法,两者间的差异有统计学意义(P<0.05)。3、MALDI-TOF MS检测准确性的结果分析：MALDI-TOF MS检测结果中,所用药物与拉米夫定耐药位点相关的5组中,检测到的变异位点与该药物已知的常见变异位点完全相符,相符率100%；单用阿德福韦酯组检出的变异位点与该药物已知的常见变异位点只部分相符,相符率70%。
结论：1、MALDI-TOF MS对已知耐药位点的检出数(率)高于PCR产物直接测序法；2、MALDI-TOF MS对已知耐药位点的检出的DNA灵敏性高于PCR产物直接测序法。3、MALDI-TOF MS准确性高,检测到已知耐药位点和实际用药耐药位点的相符率高。4、MALDI-TOF MS是为服用核苷(酸)类药物治疗效果不佳或者反弹的患者提供早期、灵敏、准确的发现已知南药位点的检测方法,可以为临床医生及时更改治疗方案提供帮助。
OBJECTIVE:To compare the differences between two methods of MALDI-TOF MS and PCR-direct DNA sequencing on hepatitis B viral P gene mutations detection.
METHODS:Study group consisted of 90 serum samples from 90 chronic hepatitis B patients received nucleoside analogues (NA) therapy for more than 1 year and HBV-DNA titer still higher than 500copies/ml; 10 serum samples from 10 chronic hepatitis B patients never treated with antiviral therapy and HBV-DNA titer higher than 1×105copies/mL comprised control group. The 90 samples included in study group then divided into 6 groups according to the different NA therapy. Next, the HBV P gene mutations were detected by MALDI-TOF MS and PCR-direct DNA sequencing at the same time, using TYPE4.0 software and Sequence Navigator software to analysis the two methods results separately.
RESULTS:
1. In study group, mutations were detected in 53 samples and total mutation sites were 86 by MALDI-TOF MS, however, by PCR-direct DNA sequencing, just 19 samples were found mutations and total 29 mutation sites were detected, the mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing and the difference was statistical significance (P<0.05). In control group, mutations were not detected whether by MALDI-TOF MS or by PCR-direct DNA sequencing.
2. For study group, in 12 samples with HBV-DNA titer between 500-1000 copies/ml,6 samples were detected mutations by MALDI-TOF MS, the detection rare was 50%. On the contrary, no mutation was detected by PCR-direct DNA sequencing. Similarly, at the other comparable lever of HBV-DNA titer, the mutations detection rate of MALDI-TOFMS also was higher than PCR-direct DNA sequencing.This result difference still was statistical significance (P<0.05)
3. Among the mutations detected samples by MALDI-TOF MS, sera from patients prescripted with Lamivudin or Telbivudine, the detected mutation sites were completely corresponded with the clinical known mutation sites special for Lamivudin, the consistent rate was 100%; however,sera from patients prescriped with Adefovir, the detected mutation sites were just partly corresponded with the clinical known mutation sites special for Adefovir, the consistent rate was 70%.
CONCLUSIONS:
1. The mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing.
2. MALDI-TOF MS had higher sensitivity for known mutation sites detection compared with PCR-direct DNA sequencing.
3. MALDI-TOF MS had high consistent rate and was a accurate method for mutation detection.
4. MALDI-TOF MS was a rapid, sensitive and accurate method and could be used for monitoring mutations in chronic hepatitis B patients treated with NA therapy.
方法：1、病例来源：(1)研究组：收集90例服用拉米夫定等核苷(酸)类药物治疗1年以上,HBV-DNA仍大于500copies/ml的慢性乙型病毒性肝炎患者血清,并根据核苷(酸)类药物用药情况分为6个组。(2)对照组：同时收集10名HBsAg阳性时间超过6个月,肝功能正常,HBV-DNA>1×105 copies/ml,始终未予抗病毒治疗的慢性乙型病毒性肝炎患者的血清作为对照组。2、检测方法：(1)采用MALDI-TOF MS检测HBV P基因已知11个变异位点的变异情况；基于SEQUENON公司质谱仪及配套芯片-试剂盒技术,通过TYPE4.0软件分析结果。(2)PCR产物直接测序法采用ABI3730XL测序仪,通过SequenceNavigator软件分析得出序列。
结果：1、两种方法检出结果比较：在服用拉米夫定等核苷(酸)类药物的90例病例血清中,MALDI-TOF MS测得53例耐药变异的病例,共86个耐药突变位点,PCR产物直接测序法仅检测出19例耐药变异的病例,共29个耐药突变位点,MALDI-TOF MS检出数明显高于直接测序法,两者间的差异有统计学意义(P<0.05)；在对照组中两种检测方法均没有发现已知的耐药变异位点。2、两种方法检测DNA的灵敏性比较：HBV-DNA水平在500-1000copies/ml的12例血清中,MALDI-TOF MS测得6例出现耐药变异位点,检出率50%,而PCR产物直接测序法在此病毒水平下未检出耐药变异位点；在相同的HBV-DNA水平下,MALDI-TOF MS变异检出数(率)均高于PCR产物直接测序法,两者间的差异有统计学意义(P<0.05)。3、MALDI-TOF MS检测准确性的结果分析：MALDI-TOF MS检测结果中,所用药物与拉米夫定耐药位点相关的5组中,检测到的变异位点与该药物已知的常见变异位点完全相符,相符率100%；单用阿德福韦酯组检出的变异位点与该药物已知的常见变异位点只部分相符,相符率70%。
结论：1、MALDI-TOF MS对已知耐药位点的检出数(率)高于PCR产物直接测序法；2、MALDI-TOF MS对已知耐药位点的检出的DNA灵敏性高于PCR产物直接测序法。3、MALDI-TOF MS准确性高,检测到已知耐药位点和实际用药耐药位点的相符率高。4、MALDI-TOF MS是为服用核苷(酸)类药物治疗效果不佳或者反弹的患者提供早期、灵敏、准确的发现已知南药位点的检测方法,可以为临床医生及时更改治疗方案提供帮助。
OBJECTIVE:To compare the differences between two methods of MALDI-TOF MS and PCR-direct DNA sequencing on hepatitis B viral P gene mutations detection.
METHODS:Study group consisted of 90 serum samples from 90 chronic hepatitis B patients received nucleoside analogues (NA) therapy for more than 1 year and HBV-DNA titer still higher than 500copies/ml; 10 serum samples from 10 chronic hepatitis B patients never treated with antiviral therapy and HBV-DNA titer higher than 1×105copies/mL comprised control group. The 90 samples included in study group then divided into 6 groups according to the different NA therapy. Next, the HBV P gene mutations were detected by MALDI-TOF MS and PCR-direct DNA sequencing at the same time, using TYPE4.0 software and Sequence Navigator software to analysis the two methods results separately.
RESULTS:
1. In study group, mutations were detected in 53 samples and total mutation sites were 86 by MALDI-TOF MS, however, by PCR-direct DNA sequencing, just 19 samples were found mutations and total 29 mutation sites were detected, the mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing and the difference was statistical significance (P<0.05). In control group, mutations were not detected whether by MALDI-TOF MS or by PCR-direct DNA sequencing.
2. For study group, in 12 samples with HBV-DNA titer between 500-1000 copies/ml,6 samples were detected mutations by MALDI-TOF MS, the detection rare was 50%. On the contrary, no mutation was detected by PCR-direct DNA sequencing. Similarly, at the other comparable lever of HBV-DNA titer, the mutations detection rate of MALDI-TOFMS also was higher than PCR-direct DNA sequencing.This result difference still was statistical significance (P<0.05)
3. Among the mutations detected samples by MALDI-TOF MS, sera from patients prescripted with Lamivudin or Telbivudine, the detected mutation sites were completely corresponded with the clinical known mutation sites special for Lamivudin, the consistent rate was 100%; however,sera from patients prescriped with Adefovir, the detected mutation sites were just partly corresponded with the clinical known mutation sites special for Adefovir, the consistent rate was 70%.
CONCLUSIONS:
1. The mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing.
2. MALDI-TOF MS had higher sensitivity for known mutation sites detection compared with PCR-direct DNA sequencing.
3. MALDI-TOF MS had high consistent rate and was a accurate method for mutation detection.
4. MALDI-TOF MS was a rapid, sensitive and accurate method and could be used for monitoring mutations in chronic hepatitis B patients treated with NA therapy.
引文
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