PCR—膜芯片~(?)技术检测结核杆菌耐药基因突变的应用研究
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摘要
20世纪80年代后期以来,伴随人类免疫缺陷病毒(HIV)感染的流行、结核分枝杆菌(Mycobacterium tuberculosis,MTB)耐药菌株的出现以及流动人口的增加,全球结核病(tuberculosis,TB)疫情急剧恶化,严重威胁着人类健康。目前,全世界每年TB死亡人数高达300万。随着联合药物的使用,MTB的耐药菌株也在不断的出现和传播。据WHO 2008年1月的调查报告[1]显示,耐药结核病(drug-resistant tuberculosis,DR-TB )的发病率创历史最高纪录,中国结核病耐药的严重程度在全球范围内仅次于俄罗斯,DR-TB已成为我国严重的公共卫生和社会问题。
结核病实验室诊断和耐药性检测技术研究一直是国内外共同关注的焦点[2]。MTB培养加药敏试验仍是目前临床广泛采用鉴定MTB及其耐药性的“金标准”,但它对结核杆菌尤其是耐药菌株的检出率很低,所需时间较长,无法满足临床辅助诊断与指导用药的需要[3,4]。反向点膜杂交(reverse dot blot hybridization ,RDB)法应用DNA探针、核酸杂交、酶联显色3方面技术,同时检测MTB多个药物的耐药性[5],是目前国内外研究的热点。本课题组在前期研究中,以临床分离株为对象利用RDB法初步构建了检测耐药结核杆菌的膜芯片及反应体系。本研究以石蜡包埋组织标本为对象,从DNA的提取方法、多重PCR的反应体系及膜芯片的杂交条件三方面对整个检测体系进行优化,并将优化后的PCR-膜芯片体系直接用于检测石蜡包埋组织及痰液等临床标本的非培养结核杆菌及其耐药情况,结合药敏试验及测序结果对PCR-膜芯片检测结果进行比较分析。
目的:优化结核分枝杆菌耐药基因膜芯片检测体系,探讨PCR-膜芯片技术直接应用于临床标本中结核杆菌耐药性检测的方法。
方法:1、以石蜡包埋组织标本为对象,首先对提取DNA的方法进行优化。分别以氯化钠盐析法、酚-氯仿抽提法、一步法、试剂盒法提取石蜡包埋组织中结核杆菌的DNA,并通过琼脂糖凝胶电泳、PCR及TB膜芯片分析所提取DNA的质量;
2、根据课题组前期建立的结核杆菌多重PCR体系扩增石蜡包埋组织中结核杆菌的耐药基因,通过调整引物组合、模板DNA的加入量及PCR反应的循环次数进行优化;
3、根据课题组前期建立的结核杆菌耐药膜芯片体系检测石蜡包埋组织中结核杆菌耐药基因突变,通过调整膜芯片杂交时间及显色时间进行优化;
4、综合各项优化条件,构建优化后PCR-膜芯片检测体系。
5、采集100例石蜡包埋的肺或淋巴结组织标本(结核病人80例,非结核病人20例),采集82例痰标本(结核病人64例,非结核病人18例);
6、利用TB膜芯片检测石蜡包埋组织标本和痰标本中的结核杆菌IS6110基因,结果与抗酸染色镜检及痰涂片结果进行比较分析;
7、利用优化的PCR-膜芯片检测体系,直接检测TB膜芯片阳性的石蜡包埋组织及痰标本中的结核杆菌及其耐药基因突变,结果与药敏试验及DNA测序结果进行分析比较。
结果:1、氯化钠盐析法提取的DNA (19.338±6.270μg)和一步法提取的DNA(20.050±5.591μg)最多(P<0.001),PCR与TB膜芯片验证显示氯化钠盐析法与酚-氯仿法提取到质量较高的结核杆菌DNA;
2、扩增石蜡包埋组织中结核杆菌耐药基因的多重PCR的最适条件为:引物组合调整为RKE、IAG、PRP、RRI,模板DNA的加入量为5μl, PCR反应的循环次数为38;
3、检测石蜡包埋组织中结核杆菌耐药基因突变膜芯片杂交的最适条件为:杂交时间为6h及以上,显色时间为15min;
4、TB膜芯片检测石蜡包埋组织标本和痰标本中的结核杆菌IS6110基因,特异度分别为100%(20/20)和100%(18/18),灵敏度分别为52.5%(42/80)和81.3%(52/64)。
5、优化后PCR-膜芯片直接检测石蜡包埋组织结核杆菌耐药基因突变,在42例TB膜芯片确认结核杆菌阳性的标本中,检出5例存在耐药基因的突变,其突变位点与测序结果基本符合。
6、优化后PCR-膜芯片直接检测痰样结核杆菌耐药基因突变,在52例确认结核杆菌阳性的痰样中,检出5例存在耐药基因突变,其中4例临床药敏试验显示为耐药,该4例标本的PCR-膜芯片检测结果与药敏及测序结果基本符合;在痰培养阴性的样本中,PCR-膜芯片检测1例出现耐药基因突变,突变位点与测序结果一致,提示异烟肼耐药的可能。
结论:1、氯化钠盐析法具有高效简便的特点,是较为理想的石蜡包埋组织结核杆菌DNA的提取方法。
2、TB膜芯片检测石蜡包埋组织标本和痰标本中结核杆菌,检出率明显高于抗酸染色镜检法和痰涂片法。
3、优化后PCR-膜芯片体系可直接检测石蜡包埋组织及痰样等临床标本结核杆菌耐药基因突变;48小时内提供耐药信息,为临床耐药结核病的快速诊断提供有意义的辅助参考。
From later period of 1980s , tuberculosis (TB) global epidemic is fuelled by synergy with HIV, emergence of Mycobacterium tuberculosis (MTB) drug-resistant strains and the increase of floating population which seriously affects human’s health. 3 million people were killed by TB each year in the world.With the alliance use of drug,the emergence and transmission of MTB drug-resistant strains is continuous.The report in 2008 from WHO showed that the morbidity of drug-resistant tuberculosis (DR-TB) break through the records in history.The serious extent of DR-TB in china ranks only second to Russia , DR-TB has become serious public health and society problem in china .
Many studies continuely have foused on TB diagnosis in the laboratory and techniques of detecting DR-TB at home and abroad. MTB Culture and susceptibility test as the diagnostic gold standard is still widely used to identify MTB and its drug resistance in clinic. But it takes more time and it’s detection rate is too low to meet the requirement of supporting diagnosis and guiding the use of drug. Reverse dot blot hybridization (RDB), a hot research at prsent, that was used to detect the MTB multi-drug resistance with the combination of DNA probes, nucleic acid hybridization, enzyme-linked color. In our previous study, the gene membrane chip which detected MTB drug-resistant genes of Clinical isolates was initially constructed by using RDB. The detection system was optimized in our stduy by three aspects: the methods of extracting DNA, the multi-PCR reaction and the gene membrane chip conditions. Then the optimized PCR- membrane chip was applied to detect the MTB drug-resistant genes in clinical specimens such as the paraffin-embedded tissues and sputum, and the result was compared with susceptibility test and DNA sequencing.
Objective: To optimize the system of detecting MTB drug-resistant genes by gene membrane chip and study detection of MTB in clinical specimens by PCR-membrane chip directly.
Methods: 1. The DNA was extracted from paraffin-embedded tissues using salting-out procedure, phenol-chloroform method, one-step method and genomic DNA purification kit. The quality of extracted DNA was analyzed by agarose gel electrophoresis, PCR and TB- membrane chip.
2. According to multi-PCR system which was constructed in the earlier stage of our study, MTB drug-resistant genes in paraffin-embedded tissues were amplified and the multi-PCR system was optimized by adjusting the combination of primers, the quantity of DNA template and the cycling conditions in PCR reaction.
3. According to the system of gene membrane chip which was constructed in the earlier stage of our study, MTB drug-resistant genes in paraffin-embedded tissues were detected and the system of gene membrane chip was optimized by adjusting the time of hybridization and coloration.
4. The detection system of PCR-membrane chip was constructed by integrating all the optimized condictions.
5. 100 paraffin-embedded lung or lymph node tissue specimens were collected (80 cases of TB, 20 cases of non-TB), and 82 sputum specimens were collected (64 cases of TB, 18 cases of non-TB).
6. TB membrane chip was applied to detect the MTB IS6110 gene in the clinical specimens of paraffin-embedded tissues and sputum, and the result was compared with acid-fast staining and sputum smears.
7. The detection system of PCR-membrane chip was applied to detect the MTB drug-resistant genes in the TB-positive specimens of paraffin-embedded tissues and sputum, and the result was compared with susceptibility test and DNA sequencing.
Results: 1. The quantity of DNA extracted by salting-out procedure (19.338±6.270μg) and one-step method (20.050±5.591μg) was more than that of the other methods (P<0.001); PCR and membrane chip show that the quality of TB-DNA extracted by salting-out procedure and phenol-chloroform method was better than that of the other methods.
2. The most optimum condition of multi-PCR for amplifying MTB drug-resistant genes in paraffin-embedded tissues: the combination of primers were adjusted to RKE、IAG、PRP、RRI; the quantity of DNA template was 5μl; the cycling conditions in PCR reaction was 38.
3. The most optimum condition of gene gmembrane chip for detecting MTB drug-resistant genes in paraffin-embedded tissues: the time for hybridization was 6 hours or more; and the time for coloration was 15min.
4. MTB-IS6110 gene in paraffin-embedded tissue and sputum specimens were detected by TB membrane chip: The specificity was 100% (20/20) and 100% (18/18) respectively; and the sensitivity was 52.5% (42/80) and 81.3% (52/64), respectively.
5. MTB drug-resistant genes in paraffin-embedded tissue specimens were detected by PCR-membrane chip : In 42 TB-positive cases, there are 5 cases of drug-resistant gene mutations and the mutation sites were consistent with the sequencing.
6. MTB drug-resistant genes in sputum specimens were detected by PCR-membrane chip : In 52 TB-positive samples, there were 5 cases of drug-resistant gene mutations and 4 of them were consistent with the susceptibility test and the sequencing results. In sputum culture-negative samples, 1 case of gene mutation was detected by PCR-membrane chip and the mutation which prompted the possibility of INH resistance was consistent with the sequencing.
Conclusion: 1. The salting-out procedure for DNA extraction of mycobacterium tuberculosis from paraffin-embedded tissues is more efficient and simple.
2. The detection rate of TB membrane chip which was applied to detect the MTB IS6110 gene in the clinical specimens of paraffin-embedded tissues and sputum is significantly higher than that of acid-fast staining method and sputum smears.
3. MTB drug-resistant genes in clinical specimens including paraffin-embedded tissues and sputum can be detected derectly by using PCR-membrane chip which can provide resistant informations within 48 hours and provide a meaningful reference for the rapid diagnosis of clinical DR-TB.
结核病实验室诊断和耐药性检测技术研究一直是国内外共同关注的焦点[2]。MTB培养加药敏试验仍是目前临床广泛采用鉴定MTB及其耐药性的“金标准”,但它对结核杆菌尤其是耐药菌株的检出率很低,所需时间较长,无法满足临床辅助诊断与指导用药的需要[3,4]。反向点膜杂交(reverse dot blot hybridization ,RDB)法应用DNA探针、核酸杂交、酶联显色3方面技术,同时检测MTB多个药物的耐药性[5],是目前国内外研究的热点。本课题组在前期研究中,以临床分离株为对象利用RDB法初步构建了检测耐药结核杆菌的膜芯片及反应体系。本研究以石蜡包埋组织标本为对象,从DNA的提取方法、多重PCR的反应体系及膜芯片的杂交条件三方面对整个检测体系进行优化,并将优化后的PCR-膜芯片体系直接用于检测石蜡包埋组织及痰液等临床标本的非培养结核杆菌及其耐药情况,结合药敏试验及测序结果对PCR-膜芯片检测结果进行比较分析。
目的:优化结核分枝杆菌耐药基因膜芯片检测体系,探讨PCR-膜芯片技术直接应用于临床标本中结核杆菌耐药性检测的方法。
方法:1、以石蜡包埋组织标本为对象,首先对提取DNA的方法进行优化。分别以氯化钠盐析法、酚-氯仿抽提法、一步法、试剂盒法提取石蜡包埋组织中结核杆菌的DNA,并通过琼脂糖凝胶电泳、PCR及TB膜芯片分析所提取DNA的质量;
2、根据课题组前期建立的结核杆菌多重PCR体系扩增石蜡包埋组织中结核杆菌的耐药基因,通过调整引物组合、模板DNA的加入量及PCR反应的循环次数进行优化;
3、根据课题组前期建立的结核杆菌耐药膜芯片体系检测石蜡包埋组织中结核杆菌耐药基因突变,通过调整膜芯片杂交时间及显色时间进行优化;
4、综合各项优化条件,构建优化后PCR-膜芯片检测体系。
5、采集100例石蜡包埋的肺或淋巴结组织标本(结核病人80例,非结核病人20例),采集82例痰标本(结核病人64例,非结核病人18例);
6、利用TB膜芯片检测石蜡包埋组织标本和痰标本中的结核杆菌IS6110基因,结果与抗酸染色镜检及痰涂片结果进行比较分析;
7、利用优化的PCR-膜芯片检测体系,直接检测TB膜芯片阳性的石蜡包埋组织及痰标本中的结核杆菌及其耐药基因突变,结果与药敏试验及DNA测序结果进行分析比较。
结果:1、氯化钠盐析法提取的DNA (19.338±6.270μg)和一步法提取的DNA(20.050±5.591μg)最多(P<0.001),PCR与TB膜芯片验证显示氯化钠盐析法与酚-氯仿法提取到质量较高的结核杆菌DNA;
2、扩增石蜡包埋组织中结核杆菌耐药基因的多重PCR的最适条件为:引物组合调整为RKE、IAG、PRP、RRI,模板DNA的加入量为5μl, PCR反应的循环次数为38;
3、检测石蜡包埋组织中结核杆菌耐药基因突变膜芯片杂交的最适条件为:杂交时间为6h及以上,显色时间为15min;
4、TB膜芯片检测石蜡包埋组织标本和痰标本中的结核杆菌IS6110基因,特异度分别为100%(20/20)和100%(18/18),灵敏度分别为52.5%(42/80)和81.3%(52/64)。
5、优化后PCR-膜芯片直接检测石蜡包埋组织结核杆菌耐药基因突变,在42例TB膜芯片确认结核杆菌阳性的标本中,检出5例存在耐药基因的突变,其突变位点与测序结果基本符合。
6、优化后PCR-膜芯片直接检测痰样结核杆菌耐药基因突变,在52例确认结核杆菌阳性的痰样中,检出5例存在耐药基因突变,其中4例临床药敏试验显示为耐药,该4例标本的PCR-膜芯片检测结果与药敏及测序结果基本符合;在痰培养阴性的样本中,PCR-膜芯片检测1例出现耐药基因突变,突变位点与测序结果一致,提示异烟肼耐药的可能。
结论:1、氯化钠盐析法具有高效简便的特点,是较为理想的石蜡包埋组织结核杆菌DNA的提取方法。
2、TB膜芯片检测石蜡包埋组织标本和痰标本中结核杆菌,检出率明显高于抗酸染色镜检法和痰涂片法。
3、优化后PCR-膜芯片体系可直接检测石蜡包埋组织及痰样等临床标本结核杆菌耐药基因突变;48小时内提供耐药信息,为临床耐药结核病的快速诊断提供有意义的辅助参考。
From later period of 1980s , tuberculosis (TB) global epidemic is fuelled by synergy with HIV, emergence of Mycobacterium tuberculosis (MTB) drug-resistant strains and the increase of floating population which seriously affects human’s health. 3 million people were killed by TB each year in the world.With the alliance use of drug,the emergence and transmission of MTB drug-resistant strains is continuous.The report in 2008 from WHO showed that the morbidity of drug-resistant tuberculosis (DR-TB) break through the records in history.The serious extent of DR-TB in china ranks only second to Russia , DR-TB has become serious public health and society problem in china .
Many studies continuely have foused on TB diagnosis in the laboratory and techniques of detecting DR-TB at home and abroad. MTB Culture and susceptibility test as the diagnostic gold standard is still widely used to identify MTB and its drug resistance in clinic. But it takes more time and it’s detection rate is too low to meet the requirement of supporting diagnosis and guiding the use of drug. Reverse dot blot hybridization (RDB), a hot research at prsent, that was used to detect the MTB multi-drug resistance with the combination of DNA probes, nucleic acid hybridization, enzyme-linked color. In our previous study, the gene membrane chip which detected MTB drug-resistant genes of Clinical isolates was initially constructed by using RDB. The detection system was optimized in our stduy by three aspects: the methods of extracting DNA, the multi-PCR reaction and the gene membrane chip conditions. Then the optimized PCR- membrane chip was applied to detect the MTB drug-resistant genes in clinical specimens such as the paraffin-embedded tissues and sputum, and the result was compared with susceptibility test and DNA sequencing.
Objective: To optimize the system of detecting MTB drug-resistant genes by gene membrane chip and study detection of MTB in clinical specimens by PCR-membrane chip directly.
Methods: 1. The DNA was extracted from paraffin-embedded tissues using salting-out procedure, phenol-chloroform method, one-step method and genomic DNA purification kit. The quality of extracted DNA was analyzed by agarose gel electrophoresis, PCR and TB- membrane chip.
2. According to multi-PCR system which was constructed in the earlier stage of our study, MTB drug-resistant genes in paraffin-embedded tissues were amplified and the multi-PCR system was optimized by adjusting the combination of primers, the quantity of DNA template and the cycling conditions in PCR reaction.
3. According to the system of gene membrane chip which was constructed in the earlier stage of our study, MTB drug-resistant genes in paraffin-embedded tissues were detected and the system of gene membrane chip was optimized by adjusting the time of hybridization and coloration.
4. The detection system of PCR-membrane chip was constructed by integrating all the optimized condictions.
5. 100 paraffin-embedded lung or lymph node tissue specimens were collected (80 cases of TB, 20 cases of non-TB), and 82 sputum specimens were collected (64 cases of TB, 18 cases of non-TB).
6. TB membrane chip was applied to detect the MTB IS6110 gene in the clinical specimens of paraffin-embedded tissues and sputum, and the result was compared with acid-fast staining and sputum smears.
7. The detection system of PCR-membrane chip was applied to detect the MTB drug-resistant genes in the TB-positive specimens of paraffin-embedded tissues and sputum, and the result was compared with susceptibility test and DNA sequencing.
Results: 1. The quantity of DNA extracted by salting-out procedure (19.338±6.270μg) and one-step method (20.050±5.591μg) was more than that of the other methods (P<0.001); PCR and membrane chip show that the quality of TB-DNA extracted by salting-out procedure and phenol-chloroform method was better than that of the other methods.
2. The most optimum condition of multi-PCR for amplifying MTB drug-resistant genes in paraffin-embedded tissues: the combination of primers were adjusted to RKE、IAG、PRP、RRI; the quantity of DNA template was 5μl; the cycling conditions in PCR reaction was 38.
3. The most optimum condition of gene gmembrane chip for detecting MTB drug-resistant genes in paraffin-embedded tissues: the time for hybridization was 6 hours or more; and the time for coloration was 15min.
4. MTB-IS6110 gene in paraffin-embedded tissue and sputum specimens were detected by TB membrane chip: The specificity was 100% (20/20) and 100% (18/18) respectively; and the sensitivity was 52.5% (42/80) and 81.3% (52/64), respectively.
5. MTB drug-resistant genes in paraffin-embedded tissue specimens were detected by PCR-membrane chip : In 42 TB-positive cases, there are 5 cases of drug-resistant gene mutations and the mutation sites were consistent with the sequencing.
6. MTB drug-resistant genes in sputum specimens were detected by PCR-membrane chip : In 52 TB-positive samples, there were 5 cases of drug-resistant gene mutations and 4 of them were consistent with the susceptibility test and the sequencing results. In sputum culture-negative samples, 1 case of gene mutation was detected by PCR-membrane chip and the mutation which prompted the possibility of INH resistance was consistent with the sequencing.
Conclusion: 1. The salting-out procedure for DNA extraction of mycobacterium tuberculosis from paraffin-embedded tissues is more efficient and simple.
2. The detection rate of TB membrane chip which was applied to detect the MTB IS6110 gene in the clinical specimens of paraffin-embedded tissues and sputum is significantly higher than that of acid-fast staining method and sputum smears.
3. MTB drug-resistant genes in clinical specimens including paraffin-embedded tissues and sputum can be detected derectly by using PCR-membrane chip which can provide resistant informations within 48 hours and provide a meaningful reference for the rapid diagnosis of clinical DR-TB.
引文
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