Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.     

The IS6110 repetitive DNA element of Mycobacterium tuberculosis is not detected in exhaled breath condensate of patients with active pulmonary tuberculosis

BACKGROUND: A large tertiary referral hospital in inner-city Chicago. OBJECTIVES: To determine whether the IS6110 repetitive DNA element of Mycobacterium tuberculosis is detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis. METHODS: Ten hospitalized patients with positive Ziehl-Neelson-stained sputum smears were studied. Concurrent sputum cultures for mycobacteria were performed as well. Exhaled breath condensate was collected from each patient within 6 days of initiating antituberculosis chemotherapy (median 1.5 days). These samples were analyzed by polymerase chain reaction (PCR) using primers designed to amplify the IS6110 DNA fragment of M. tuberculosis. Exogenous M. tuberculosis DNA was added to exhaled breath condensate samples to detect PCR inhibitors. Concurrent cultures of exhaled breath condensate for mycobacteria were performed. RESULTS:M. tuberculosis was identified in 9 of 10 sputum cultures. One isolate was identified as Mycobacterium kansasii. The IS6110 repetitive DNA element of M. tuberculosis was not detected in any of the 10 exhaled breath condensate samples. Exogenous M. tuberculosis DNA added to these samples elicited the characteristic band pattern of M. tuberculosis on agarose gel electrophoresis. No PCR inhibitors were detected. Cultures of exhaled breath condensate showed no growth of mycobacteria. CONCLUSIONS: The IS6110 repetitive DNA element of M. tuberculosis is not detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis.     

荧光定量PCR在肺结核临床诊断及疗效评估中的应用

目的 建立荧光定量PCR检测结核分枝杆菌方法,评估其在肺结核的临床诊断和疗效评估中的应用价值.方法 针对结核分枝杆菌保守序列IS6110基因设计引物和探针,建立荧光定量PCR方法.分别以荧光定量PCR、集菌培养和染色镜检法检测疑似肺结核和确诊肺结核患者随访的痰标本,比较各方法在结核病诊断以及治疗效果评估中的作用.结果 荧光定量PCR的检测灵敏度为4 Copies/反应,远高于抗酸染色法和集菌培养法.荧光定量PCR法与集菌培养法和抗酸染色法对临床样本的检出率分别为64.29％、35.12％和12.5％.患者治疗效果的随访检测结果显示,结核分枝杆菌的数量呈持续减少的趋势.结论 荧光定量PCR方法具有快速、敏感和特异性高的特点,可以快速、及时获取肺结核患者服药后的治疗效果,为医生的后续督导治疗提供依据,具有重要的临床意义.     

Comparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis

In developing countries the diagnosis of extrapulmonary tuberculosis (EPTB) is a major burning challenge. EPTB encounters many problems like pauci-bacillary nature, inadequate specimen volume. All the limitations reflect in the poor contribution of conventional bacteriological technique in the establishment of diagnosis of EPTB. Nucleic acid amplification methods are rapid and sensitive has modified strategies for the detection of mycobacterial DNA. A fragment of DNA of 123 bp belonging to insertion sequence IS6110 based on specific gene of Mycobacterium tuberculosis complex was amplified by polymerase chain reaction (PCR) for the rapid diagnosis of EPTB. The present study was to comparative evaluation of IS6110 PCR via conventional methods in the rapid diagnosis of new and Previously treated cases of extra pulmonary tuberculosis. Four hundred fifty specimens were collected from suspected cases of EPTB were processed for Mycobacteria by Zeihl Neelson (ZN) staining and BACTEC culture for M. tuberculosis. All the specimens were also processed for IS6110 based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. We found significant difference was seen in sensitivities of different tests. Of these 450 specimens, 60 (13.4%) were positive for AFB by ZN staining, 202 (45%) for BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 283 (63%) specimens (p< 0.05). However, there was no significant difference (p< 0.05) as far as specificity of different tests. We found that IS6110 PCR has higher sensitivity than smear microscopy and BACTEC culture in both cases of new cases as well as in previously treated cases. IS6110 PCR can be highly useful in diagnosis of new and treated cases of EPTB. It may facilitate therapeutic decisions for those with suspected of EPTB.     

Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis

PCR has emerged as a powerful technique for detection of various pathogens including Mycobacterium tuberculosis. In present study, eighty one samples of lymph node biopsies from clinically suspected cases of tuberculous lymphadenitis were examined for AFB, culture on L?wenstein Jensen medium and simultaneous use of two PCRs targeting IS6110 and MPB64. Positivity with M. tuberculosis culture and AFB was 13.6% and 28.4% respectively. All samples culture positive for nontuberculous mycobacteria were negative by both PCR systems. Higher proportion of positive results were observed with PCR targeting IS6110 by which 56 of 81 (69.1%) samples showed positive results as compared to PCR targeting MPB64 by which 39 of 81 (48.2 %) samples showed positive results. When combined, 63 out of 81 (77.8%) samples were detected positive for M. tuberculosis DNA. However, 7/81 (8.6 %) samples remained negative by IS6110 but positive by MPB64 method. Thus our data suggest that the use of one additional PCR (other than IS6110 system) can reduce false negativity of PCR results in the samples harboring zero copy of IS6110 element which is known to exist in Indian population.     

一种直接检测结核病痰标本的PCR技术的建立与评价

目的建立并评价聚合酶链式反应(polymerase chain reaction,PCR)在结核病痰标本检测中的应用价值。方法根据结核分枝杆菌复合体IS6110序列设计引物INS1和INS2,并建立PCR反应体系和反应条件。运用PCR方法分别检测标准菌株、结核分枝杆菌PCR检测标准品和拟诊结核病患者痰标本,采用痰涂片和细菌培养为对照。结果比较的统计学分析采用卡方检验。结果PCR方法对结核分枝杆菌、牛分枝杆菌、卡介苗标准株的最小检出浓度分别达到102,103,103个细菌/毫升,能够特异地检出结核分枝杆菌复合体。在PCR检测的574例拟诊病例中,PCR检测阳性病例241例,42%;痰涂片和细菌培养的阳性率分别为19.69%和26.31%,PCR检测阳性率高于传统细菌学检验方法,经χ2检验,差异具有显著统计学意义(χ2=103.67,P<0.01)。以痰培养结果为标准,计算PCR检测方法的敏感度为67.53%。结论PCR检测方法与传统的细菌学检测方法相比可以提高阳性标本检出率,而且具有快速、特异、简便的特点,有望成为结核病大规模筛查和临床快速检测方法。     

Rapid and efficient detection of extra-pulmonary Mycobacterium tuberculosis by PCR analysis.

SETTING: The diagnosis of extra-pulmonary tuberculosis (EPTB) remains an important clinical problem, primarily because of the inadequate sensitivity of conventional bacteriologic methods for detecting Mycobacterium tuberculosis in extra-pulmonary specimens. OBJECTIVE: To evaluate whether a IS6110-based polymerase chain reaction (PCR) method can be utilized to detect M. tuberculosis in non-pulmonary specimens. DESIGN: Specimens from 286 Mexican patients with a presumptive clinical diagnosis of EPTB were prospectively examined by Ziehl-Neelsen staining, mycobacterial culture on L?wenstein-Jensen slants, and by PCR. The DNA for PCR was extracted by the buffer lysis method and phenol-guanidine thiocyanate-chloroform. Primers that amplify a 200 bp fragment from the insertion-like M. tuberculosis sequence element IS6110 were utilized. RESULTS: Our results demonstrate that this PCR method is highly specific (100%) for identifying M. tuberculosis from a variety of specimens including cerebrospinal fluid (CSF), pleural fluid, ascitic fluid, pericardial fluid, urine, and lymph node exudate. Moreover, the sensitivity of PCR for detecting M. tuberculosis in CSF (94%), pleural fluid (94%), ascitic fluid and other extrapulmonary specimens (93%) greatly exceeds the sensitivity of conventional smear and culture methods. CONCLUSION: These results demonstrate that PCR can be a highly specific and sensitive aid in the detection of M. tuberculosis from extra-pulmonary specimens.     

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

SETTING: The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. OBJECTIVE: To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. DESIGN: A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. RESULTS: We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. CONCLUSION: The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.     

The relative frequency of Mycobacterium tuberculosis and Mycobacterium avium infections in HIV positive patients, Ahvaz, Iran

ObjectiveTo estimate the prevalence of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium avium (M. avium) infections in HIV-positive patients suspected to have pulmonary and extrapulmonary mycobacterial co-infection using PCR technique.MethodsTotally 50 samples comprising sputum, pleural fluid and CSF taken from HIV positive patients suspected to have mycobacterial infection, were processed. The demographic information and results of acid fast staining and culture were recorded for each patient. The PCR for detecting of M. tuberculosis comprised of specific primers targeting IS6110 gene sequence. For detecting of M. avium, PCR with primers that amplifies the mig gene were used.ResultsFrom 50 samples processed, 45 were sputum (90%), 3 pleural fluid (6%) and 2 CSF (4%). In total, 8 (16%) were culture positive, 7 had positive acid fast staining (14 %) and 13 samples (26%) were positive using PCR technique. All the positive samples were sputum and belonged to patients with pulmonary infection. Of these, 9 were positive for M. tuberculosis (69.2%) and 4 were identified as M. avium (30.8%), which 2 out of 13 positive samples showed mixed infections by both mycobacteria.ConclusionsThe PCR shows the highest detection rate (26%) of mycobacteria compared with culture and acid fast staining. The majority of infections were with M. tuberculosis (18%) and this shows the importance of this mycobacterial co-infection in HIV positive patients in the region of study.     

Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.     

Evaluation of polymerase chain reaction, tuberculostearic acid analysis, and direct microscopy for the detection of Mycobacterium tuberculosis in sputum.

Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.     

结核分枝杆菌IS6110序列在石蜡组织中的检测、克隆与测序

目的：探讨套式-聚合酶链反应（Nested-PCR)检测石蜡组织中结核分枝杆菌的特异性和敏感性。方法：采用套式-PCR检测石蜡包埋组织中结核菌复合体特异插入序列IS6110，并对部分标本的PCR产物进行克隆和测序。结果：31例结核标本石蜡组织检出结核菌DNA共28例，套式-PCR的敏感度为90.3%,特异度为100%。阳性预测值为100%。随机选取两例PCR产物没是序结果与结核菌标准株H37Rv同源性分别为97%和95.3%。结论：套式-PCR检测常规石蜡包埋组织中结核菌IS6110序列具有特异性强和敏感性高的特点，可应用于临床诊断，尤其是对那些常规苏木精-伊红染色和抗酸染色无法确诊的病例更具意义。     

利用PCR技术检测537份新疆结核病人痰标本中的结核分枝杆菌

目的 提高聚合酶链反应在检测人结核分枝杆菌中的特异性和敏感性。方法 在聚合酶链反应中对4种DNA片段即结核杆菌特异插入列IS6110,IS1081,和16SrRNA,65kDa摸板进行了比较;为获取较多的细菌DNA,临床痰标本处理采用了国际标准化方法主要包括用一定量的N－乙酰－L半胱胺酸和氢氧化钠处理痰标本;改进了RCR混和液的成份即添加了甘油,dUTP－尿嘧啶糖基化酶。结果 选择IS6110作为结核杆菌特异性摸板用于检测细菌DNA,制备出了6批人结核杆菌PCR检测试剂盒,在对来自新疆结核病研究所的537份痰标本的检测中发现,谝试剂盒检测的特异性为65.97%,敏感性为93.53%。结论 改良TB－PCR试剂盒显示有较高的敏感性,特异性还有待于进一步完善,该试剂盒在临床诊断中有一定的价值。     

Detection of M. tuberculosis in clinical samples of diversified nature by IS6110 based PCR

Performance of the polymerase chain reaction technique based on IS6110 sequence was evaluated in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. One hundred and seventy two samples were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests amplifying 123bp region of IS6110 sequence. A significant difference was seen in the sensitivities of different tests, the figures being 83% for PCR test, 35.2% for smear examination, 47.16% for LJ culture and 53.45% for BACTEC culture (p < 0.05). However, no significant difference was found as far as specificity was concerned. PCR test sensitivity in. pulmonary and extrapulmonary clinical samples were 90.14% and 77.27% respectively and found to be significantly higher (p < 0.05) when compared with those of other tests. The mean detection time for M. tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. PCR based on IS6100 sequence is highly sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.     

Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.     

Establishment and effect evaluation of polymerase spiral reaction for detection of Mycobacterium tuberculosis

目的 建立聚合酶螺旋反应(polymerase spiral reaction,PSR)检测结核分枝杆菌的方法并评价效果。方法 针对结核分枝杆菌插入序列IS6110设计引物进行PSR检测。提取结核分枝杆菌基因组DNA,加入Bst DNA聚合酶的RM2×反应液中,在荧光定量PCR仪上于63℃反应45min,通过荧光信号进行检测。通过对引物序列进行优化,筛选出最佳引物序列,并对其检测特异性和最低检出限进行评估。于2019年5—8月间从郑州市第六人民医院连续收集200例肺结核患者痰液样本(同一患者收集3份),对痰标本进行痰涂片、固体痰培养和PSR检测。以痰培养结果为参照,评价PSR对结核病患者的检测效能。结果 通过引物序列优化,筛选出一套对结核分枝杆菌检测的PSR引物,使用该引物进行PSR的最低检出限可达到10³菌落形成单位(CFU)/ml;检测特异性实验表明该方法与15株非结核分枝杆菌无交叉反应。200例肺结核患者中,痰涂片检测阳性48例,阳性检出率为24.0%(48/200);痰培养检测阳性83例,阳性检出率为41.5%(83/200);PSR方法检测阳性87例,阳性检出率为43.5%(87/200)。以痰培养结果为标准,PSR检测结核病的敏感度和特异度分别为96.4%(80/83)、94.0%(110/117),阳性预测值为92.0%(80/87),阴性预测值为97.3%(110/113),与痰培养检测方法基本一致(Kappa值为0.898)。结论 PSR检测适用于结核分枝杆菌的快速筛查。     

结核分枝杆菌聚合酶螺旋反应检测方法的建立及效果评价

目的 建立聚合酶螺旋反应(polymerase spiral reaction,PSR)检测结核分枝杆菌的方法并评价效果。方法 针对结核分枝杆菌插入序列IS6110设计引物进行PSR检测。提取结核分枝杆菌基因组DNA,加入Bst DNA聚合酶的RM2×反应液中,在荧光定量PCR仪上于63℃反应45min,通过荧光信号进行检测。通过对引物序列进行优化,筛选出最佳引物序列,并对其检测特异性和最低检出限进行评估。于2019年5—8月间从郑州市第六人民医院连续收集200例肺结核患者痰液样本(同一患者收集3份),对痰标本进行痰涂片、固体痰培养和PSR检测。以痰培养结果为参照,评价PSR对结核病患者的检测效能。结果 通过引物序列优化,筛选出一套对结核分枝杆菌检测的PSR引物,使用该引物进行PSR的最低检出限可达到10³菌落形成单位(CFU)/ml;检测特异性实验表明该方法与15株非结核分枝杆菌无交叉反应。200例肺结核患者中,痰涂片检测阳性48例,阳性检出率为24.0%(48/200);痰培养检测阳性83例,阳性检出率为41.5%(83/200);PSR方法检测阳性87例,阳性检出率为43.5%(87/200)。以痰培养结果为标准,PSR检测结核病的敏感度和特异度分别为96.4%(80/83)、94.0%(110/117),阳性预测值为92.0%(80/87),阴性预测值为97.3%(110/113),与痰培养检测方法基本一致(Kappa值为0.898)。结论 PSR检测适用于结核分枝杆菌的快速筛查。     

The usefulness of PCR amplification of the IS6110 insertion element of M. tuberculosis complex in ascitic fluid of patients with peritoneal tuberculosis

Background: The diagnosis of tuberculous peritonitis (TP) may be difficult and elusive. The present study was designed to demonstrate the diagnostic usefulness of a nested polymerase chain reaction (PCR) assay, specific for the IS6110 insertion element of M. tuberculosis complex, in patients with ascites who were suspected of having TP in order to achieve a more timely diagnosis and treatment. Methods: Three HIV-negative patients suffering from fever and ascites were evaluated for suspected TP. Specimens were obtained from ascitic fluid, bone marrow, and peripheral blood and analyzed by both conventional methods and nested PCR for the presence of bacilli. Response to antituberculous treatment was considered as the final criterion for diagnosis of peritoneal tuberculosis. Results: All three patients had an excellent response to antituberculous therapy. Our PCR-based protocol detected M. tuberculosis complex DNA in the ascitic fluid of all patients, whereas conventional methods failed to establish the disease. Furthermore, in one patient, M. tuberculosis was also detected in both bone marrow and peripheral blood. Conclusions: PCR amplification of the IS6110 sequence of M. tuberculosis complex in ascitic fluid is a useful tool when peritoneal tuberculosis is suspected. However, its validity still needs to be established.     

PCR指印技术对新疆结核病病原Bacter460液体培养标本进行菌株分型

以结核分枝杆菌特异插入序列IS6 110两端序列为模板 ,设计一对外向PCR引物进行PCR扩增 ,从而建立一种结核分枝杆菌的快速分子生物学分型技术 ,该试验的基础是利用IS6 110在结核分枝杆菌染色体DNA中反复重复且IS6 110序列之间相距较近 ,经PCR扩增呈现多条带型构成DNA指印。在对新疆结核病人的 31份液体培养标本的PCR检测呈现 6种指印 ,该PCR分型技术对结核分枝杆菌的分型所需时间短 ,不需细菌再培养 ,DNA纯化和酶切、萨瑟恩转印或核酸杂交等繁琐步骤。证明该法是一种快速、准确的鉴定与分型方法并可直接进行分子流行病学研究     

Usefulness of self ligation mediated polymerase chain reaction: a rapid method for fingerprinting in molecular epidemiology of tuberculosis

Restriction fragment length polymorphism (RFLP) analysis based on the insertion sequence IS 6110 has been used as one of the powerful tools for epidemiological study of tuberculosis. However this technique requires more than 1 micro-gram of DNA and two days for completion. To overcome these inconvenience, we have modified a PCR-based method, self ligation mediated PCR (SL-PCR) on the molecular epidemiological study. This method uses a pair of primers whose orientations are from inside to outside of IS 6110. The DNA fragments flanking IS 6110 are amplified by the PCR by using the Sau 3A I digested and ligated chromosomal DNA of Mycobacterium tuberculosis strains. By using this method, M. tuberculosis strains can be differentiated within 8 hours.