• Tuberculosis and Respiratory Diseases
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• v.50 no.2
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• pp.213-221
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• 2001

Background : There is a well recognized interlaboratory variation in the results using the polymerase chain reaction(PCR) to detect the IS6110 sequence. The clinical utility of a commercially developed PCR test(Amplicor) in bronchial washings for detecting pulmonary tuberculosis in smear negative patients was evaluated. The sensitivity and specificity of Amplicor was compared with that of an in-house PCR test used for detecting the IS6110 sequence of Mycobacterium tuberculosis(M.tbc) in the bronchial washing fluid. Methods : 66 patients whose sputum smear for M. tbc were negative or who could not produce any sputum were recruited from January 1999 to July 1999. They all had a bronchoscopy performed to determine if there were signs of hemoptysis, patients who could not cough up sputum, lung lesion that exclude pulmonary tuberculosis. Pulmonary tuberculosis was diagnosed on the basis of a positive culture or a response to anti-tuberculosis therapy. Results : 19 patients with tuberculosis were identified and samples from 16 patients were later confirmed by culture. Bronchial washing for Amplicor PCR revealed a sensitivity, specificity, positive and negative predictive values of 94.7%, 97.9%, 94.7%, 97.9%, respectively. Using IS6110 based PCR, the sensitivity, specificity, positive and negative predictive values were of 73.7%, 87.2%, 70%, 89.1% respectively. Conclusion : Bronchial washing for Amplicor PCR proved to be more useful than IS6110 based PCR in rapidly diagnosing smear negative pulmonary pulmoary tuberculosis in patients where tuberculosis was likely to be differential and rapid diagnosis was essential for optimal treatment.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.745-752
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• 2015

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from non-tuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/discrimination of this bacterium from clinical samples.

• Biomedical Science Letters
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• v.10 no.2
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• pp.163-169
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• 2004

Worldwide, tuberculosis remains one of the leading infectious diseases, accounting for nearly 3 million deaths and more than 8 million new cases annually. DNA typing of Mycobacterium tuberculosis is important for the control of tuberculosis, since it can be used to track transmission route of tuberculosis, source of internal laboratory contaminations, and to answer questions on the nature of tuberculosis infections such as reactivation or exogenous reinfection of disease. At present, IS6110-based RFLP is the choice of method for typing large numbers of clinical isolates of M. tuberculosis, since it has the highest resolution power. However, RFLP requires long time, high cost and qualified experts, so only reference level laboratories can use the RFLP technique. In order to have an optional molecular typing method suitable for the clinical settings, this study evaluated the use of one of PCR-based typing methods, IS6110-based outward PCR for typing clinical isolates of M. tuberculosis. In brief, the results from this study showed that IS6110-based RFLP is useful to discriminate diverse clinical isolates of M. tuberculosis as well as to identify clinical isolates that belong to the same family or cluster groups that have been previously classified by RFLP analysis. In addition, the banding profiles resulted from IS6110-based outward PCR seemed to represent genomic characteristics of M. tuberculosis, since strains belong to the K-family generated unique band that is not present in any other strains but present only in the genome of K-family strains. The IS6110-based outward PCR was also shown to be useful with DNAs isolated directly from liquid cultures indicating this method can be suitable for typing M. tuberculosis in clinical settings.

• Tuberculosis and Respiratory Diseases
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• v.44 no.6
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• pp.1245-1255
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• 1997

Background : Rifampicin(RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant(MDR) tuberculosis. RPoB gene encodes the $\beta$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And rpoB gene mutations are the cause of RFP resistance of M. tuberculosis. Although several reports showed that PCR-SSCP would be a rapid diagnostic method for identifying the RFP resistance, there were few reports Performed using direct, clinical specimens. So we Performed PCR-SSCP analysis of rpoB gene of M. tuberculosis in direct, clinical specimens. Methods : 75 clinical specimens were collected from patients at Asan Medical Center from June to August 1996. After PCR of IS 6110 fragments, 43 both AFB smear-positive and IS6110 fragment PCR-positive specimens were evaluated. The RFP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. DNA was extracted by bead beater method. And heminested PCR was done using 0.1ul(1uCi) [$\alpha-^{32}P$]-dCTP. SSCP analysis was done using non-denaturating MDE gel electrophoresis. Results : The results of PCR of IS6110 fragments of M. tuberculosis were positive in 55(73%) cases of 75 AFB smear-positive clinical specimens. Of the 55 specimens, RFP susceptibility was confirmed in only 43 specimens. Of the 43 AFB smear-positive and IS6110 fragment-positive specimens, 29 were RFP susceptible and 14 were RFP resistant. All the RFP susceptible 29 strains showed the same mobility compared with that of RFP sensitive H37Rv in SSCP analysis of ropB gene. And all the other RFP resistant 13 strains showed the different mobility. In other words they showed 100% identical results between PCR-SSCP analysis and traditional susceptibility test. Conclusion : The PCR-sseP analysis of rpoB gene in direct clinical specimens could be used as a rapid diagnostic method for detecting RFP resistant M. tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.42 no.5
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• pp.695-702
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• 1995

Background: Since polymerase chain reaction(PCR) was devised by Saiki in 1985, it has been used extensively in various fields of molecular biology. Clinically, PCR is especially useful in situation when microbiological or serological diagnosis is limited by scanty amount of causative agents. Thus, PCR can provide rapid and sensitive way of detecting M. tuberculosis in tuberculosis pleurisy which is diagnosed in only about 60 % of cases by conventional method. Method: To evaluate the diagnostic usefulness of PCR in tuberculosis pleurisy, The results of PCR was compared with those of conventional method, including pleural biopsy. The pleural effusion fluid was collected from 7 proven patients, 7 clinically suspected patients and control group(7 patients with malignant effusion). We extracted DNA from pleural fluid by modified method of Eisennach method(1991). The amplification target for PCR was 123 base pair DNA, a part of IS6110. Result: 1) Sensitivity of PCR: We detected upto 50fg DNA. 2) In patients with pleural effusion of proven tuberculosis, the positive rate of PCR was 85.7%(6/7). In patients with pleural effusion of clinically suspected tuberculosis, the positive rate was 71.5%(5/7). In control group, positive rate was 0%(0/7). Conclusion: We concluded that PCR method could be a very rapid, sensitive and specific one for diagnosis of M tuberculosis in pleural effusion. Further studies should be followed for the development of easier method.

• 보건세계
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• v.40 no.12 s.448
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• pp.26-28
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• 1993

세포나 미생물의 특정 DNA 부위를 증폭하고자 할때 이용되는 PCR(polymerase chain reaction, 핵산중합효소 연쇄반응)은 1983년 미국 Cetus사의 Mullos에 의해 고안되었고, 그후 PCR에 의한 마이코 박테리아의 동정 및 검출이 특정 염기서열의 연구가 이루어지면서 활발히 시도되었으며, 아이젠하크 등은 IS6110을 이용하여 본격적으로 PCR이 결핵균 검출에 유용한 도구로 사용될 수 있다는 가능성을 제시하였다. PCR에 의한 DNA증폭은 단시간 내에 이루어진다는 장점 때문에 유전병 및 미생물의 진단에 이용될 뿐 아니라 법의학 분야 및 장기이식에 따른 조직접합 시험에도 이용될 것으로 예상된다. 이에 따라 국내에서도 결핵연구원에서 최초로 일반 임상검사에 적용함으로써 현재 많은 호평을 받고 있는 PCR에 대하여 알아본다

• Korean Journal of Veterinary Service
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• v.30 no.3
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• pp.393-406
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• 2007

Bovine tuberculosis is an important zoonosis worldwide. Mycobacterium bovis, the causative agent of this disease in cattle, is also a pathogen for humans and several economically important animals. The cases of tuberculosis are reported in two cow found at slaughter house located in Gwangju city. Histopathologically, in the lymph nodes, granulomas consisted of large areas of necrosis surrounded by variable thick bands of cellular infiltrate containing macrophages, Langhans-type multinucleated giant cells and lymphocytes. Lesions in the lung followed the same developmental pattern as did lesions in the lymph nodes with some exceptions. With the acid-fast staining, numerous mycobacteria were revealed in the lung and lymph nodes. M bovis was confirmed as a causative agent in these cattle using bacterial isolation and PCR and restriction fragment length polymorphism method based on a unique 12.7 kb fragment insertion sequence from the Mycobacterium tuberculosis genome and the pncA polymorphism, The insertion element IS6110 and IS1081 were present M bovis isolated from lungs and lymph nodes of cattle using PCR assay. These cases are interesting and important in public health aspect that M bovis-infected cattle were found during a routine post-mortem inspection at slaughter house.

• Korean Journal of Veterinary Service
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• v.25 no.2
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• pp.135-140
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• 2002

The purpose of this study was to detect Mycobacterium bovis in cattle(serum, milk, lung, lymph node) by PCR. Nineteen samples from 7 skin test-positive cattle were analyzed. The amplified band of IS6110 by PCR was detected from 2 samples in lung and Iymph node. But the sensitivities of the present methods for detecting M bovis in milk and serum are deficient. Because the PCR sensitivity has been shown to be hindered by the method used to isolate the nucleic acid target. PCR-based methods have the potential to be faster, more accurate, and the most efficient means of detecting M bovis. The detection of M bovis by PCR will contribute to the more efficient detection and control of tuberculosis.

• Tuberculosis and Respiratory Diseases
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• v.39 no.6
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• pp.517-525
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• 1992

Background: Since its development by Saiki et al, polymerase chain reaction (PCR) has been very useful in various fields of molecular biology. PCR can be used for the detection of a very small amount of microbial agent, and is especially useful in those patients who are difficult to diagnose microbiologically or serologically. Mycobacterium tuberculosis is a very slowly growing organism and AFB staining frequently shows false negative results, and therefore PCR would be a very rapid, easy, and sensitive diagnostic method for the diagnosis of Mycobacterium tuberculosis. Method: To compare PCR with conventional methods in diagnosing Mycobacterium tuberculosis in sputum, we used sputa of patients who visited or were admitted to Seoul National University Hospital. The amplification targets were 383 base pair DNA, a part of 2520 base pair DNA encoding 65 kD Mycobacterium tuberculosis specific protein (the primers are TB-1, -2), and 123 base pair DNA, a part of IS6110 fragment, which multiple copies are known to exsist PCR one genome (the primers are Sal I-1, -2). We also requested AFB staing and culture to the lab of Seoul National University Hospital with the same sample and compared the results. Results: 1) Using TB-1, -2 primers, PCR was positive in 73.1% (19/26) of culture positive sputa, in 12.5% (1/8) of culture negative. but clinically diagnosed tuberculous sputa, and was negative in all sputa of patients who were clinically diagnosed as non-tuberculous etiology. 2) Using Sal I-I, -2 primers, PCR was positive in 94.1% (32/34) of culture positive sputa, in 23.1% (6/26) of culture negative, but clinically diagnosed tuberculous sputa, and was negative in 87.5% (14/16) of sputa from patients who were clinically diagnosed as non-tuberculous etiology. Conclusion: PCR could be a very rapid, sensitive and specific method for the diagnosis of Mycobacterium tuberculosis in sputa, and further studies should be followed for the development of easier method.

• Tuberculosis and Respiratory Diseases
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• v.40 no.5
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• pp.509-518
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• 1993

Background: By amplifying small amount of DNA, polymerase chain reaction (PCR) can be used for the detection of very small amount of microbial agent, and may be especially useful in certain cases which are difficult to be diagnosed microbiologically or serologically. Tuberculous pleurisy is a disease that can be diagnosed in only 70% of cases by conventional diagnostic tools, and PCR would be a very rapid, easy, and sensitive diagnostic method. Method: The specificity and sensitivity of PCR to detect Mycobacterium tuberculosis DNA were evaluated using various strains of Mycobacteria. To evaluate the diagnostic usefulness of PCR in tuberculous pleurisy, we used PCR to detect Mycobacterium tuberculosis DNA in pleural fluid. The amplification target was 123 base pair DNA, a part of IS6110 fragment, 10~16 copies of which are known to exist per genome. The diagnostic yield of PCR was compared with conventional methods, including pleural fluid adenosine deaminase (ADA) activity. Also, the significance of PCR in undiagnosed pleural effusion was evaluated prospectively with antituberculosis treatment. Results: 1) Using cultured Mycobacterium tuberculosis and other strains, PCR could detect upto 1 fg DNA and specific for only Mycobacterium tuberculosis and Mycobacterium bovis. 2) Using pleural effusions of proven tuberculosis cases, the sensitivity of PCR was 80.0% (16/20), and the specificity 95.0% (19/20). 3) Among 13 undiagnosed, but suspected tuberculous effusion, the positive rate was 60% in 10 improved cases after antituberculosis medications, and 0% in 3 cases of proven malignancy later. 4) Adenosine deaminase level of proven and clinically diagnosed tuberculous pleurisy patients was significantly higher than that of excluded patients, and correlated well with PCR results. Conclusion: We can conclude that PCR detection of Mycobacterium tuberculosis in pleural effusion has acceptable sensitivity and specificity, and could be an additional diagnostic tool for the diagnosis of tuberculous pleurisy.