PCR-Membrane Chip Technology For The Detection Of Resistant Gene Mutations In Mycobacterium Tuerculosis

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.