|Year : 2013 | Volume
| Issue : 4 | Page : 366-369
Diagnostic appraisal of simultaneous application of two nested PCRs targeting MPB64 gene and IS6110 region for rapid detection of M. tuberculosis genome in culture proven clinical specimens
KL Therese1, R Gayathri1, L Dhanurekha1, R Sridhar2, N Meenakshi3, HN Madhavan1
1 Larsen & Toubro Microbiology Research Centre, Vision Research Foundation, Chennai, Tamil Nadu, India
2 Department of Thoracic Medicine, Stanley Medical College, Chennai, Tamil Nadu, India
3 Institute of Thoracic Medicine, Chetpet, Chennai, Tamil Nadu, India
|Date of Submission||22-Mar-2013|
|Date of Acceptance||09-Aug-2013|
|Date of Web Publication||25-Sep-2013|
K L Therese
Larsen & Toubro Microbiology Research Centre, Vision Research Foundation, Chennai, Tamil Nadu
Source of Support: Chennai Willingdon Corporate Foundation,
Chennai, Conflict of Interest: None
Background: Early diagnosis of tuberculosis is critical for its effective management and prevention. Several gene amplification methods are used in the detection of tubercle bacilli from clinical specimens. MPB64 gene and IS6110 region have been identified as potential gene targets for the specific detection of Mycobacterium tuberculosis from direct clinical specimens. Objective: The present study was conducted to evaluate the diagnostic utility of simultaneous application of two nested polymerase chain reaction (nPCRs) targeting MPB64 and IS6110 region for the detection of M. tuberculosis genome. Materials and Methods: A total of 100 and 354 clinical specimens from the control group and clinically suspected tuberculosis patients, respectively, were included in the study. nPCRs targeting MPB64 and IS6110 region were performed. Results and Conclusion: All of the 100 clinical specimens from the control group were negative for both nPCRs. Out of the 354 clinical specimens, 339 were positive for both culture and nPCRs, 10 and 5 were positive for culture, and nPCR targeting IS6110 and MPB64 regions, respectively. To conclude, nPCRs targeting MPB64 and IS6110 region are reliable and specific targets when applied simultaneously on clinical specimens to attain 100% sensitivity for the detection of M. tuberculosis genome.
Keywords: BACTEC, culture, IS6110, M. tuberculosis, MPB64, MicroMGIT, Nested PCR
|How to cite this article:|
Therese K L, Gayathri R, Dhanurekha L, Sridhar R, Meenakshi N, Madhavan H N. Diagnostic appraisal of simultaneous application of two nested PCRs targeting MPB64 gene and IS6110 region for rapid detection of M. tuberculosis genome in culture proven clinical specimens. Indian J Med Microbiol 2013;31:366-9
|How to cite this URL:|
Therese K L, Gayathri R, Dhanurekha L, Sridhar R, Meenakshi N, Madhavan H N. Diagnostic appraisal of simultaneous application of two nested PCRs targeting MPB64 gene and IS6110 region for rapid detection of M. tuberculosis genome in culture proven clinical specimens. Indian J Med Microbiol [serial online] 2013 [cited 2019 Jun 19];31:366-9. Available from: http://www.ijmm.org/text.asp?2013/31/4/366/118887
| ~ Introduction|| |
Accurate and early diagnosis of tuberculosis is important for its effective management. Various gene amplification based methods have been applied for the rapid detection and identification of mycobacterial species. Gene amplification and restriction assays targeting several genes have been developed, and the most important targets for the detection of Mycobacterium tuberculosis (M. tuberculosis) genome are MPB64 , and IS6110.  The conventional diagnostic tests that include tuberculin test, radiological examination, and other imaging methods, sputum smear and characteristic histopathology are important approaches, but there could be problems with the non-specific features of these tests.
The present study was undertaken with the aim to apply the nested PCRs (nPCRs) targeting MPB64 gene and IS6110 region simultaneously on DNA extracted directly from clinical specimens positive for M. tuberculosis by using the micro Mycobacteria Growth Indicator Tube (MGIT) system. The main purpose of this study was to evaluate the diagnostic value of combining the two nPCRs as it is well-known that some strains lack the IS6110 insertion sequences in M. tuberulosis strains circulating in Indian and Asian population.  Sputum smear microscopy has both the problems of sensitivity and specificity. Differentiation of Mycobacterium species has traditionally relied upon biochemical test profiles of pure cultures; these methodologies are cumbersome and require 3-6 weeks for even skilled microbiology technicians to report results. Standard biochemical tests may yield ambiguous and misleading results, as the tests used may not be highly reproducible and the phenotype of a species is not an absolute property and may exhibit quite remarkable variability. Thus, the present study focused on the detection of M. tuberculosis genome by nPCR targeting MPB64 gene and IS6110 region in culture-positive clinical specimens.
| ~ Materials and Methods|| |
The study was conducted with approval from the institutional ethics committee. An informed consent was obtained from the patients after explaining the study purpose. A total of 354 clinical specimens collected from (November 2009 to August 2011) clinically suspected tuberculosis patients who were BACTEC culture positive (MicroMGIT culture system) for the isolation of M. tuberculosis were subjected to nPCRs for the detection of M. tuberculosis genome targeting MPB64  gene and IS6110  region. A portion of the clinical specimens were preserved in −80°C freezer for molecular biological study. A majority of the clinical specimen were from the respiratory system (283-87.9%), followed by fine needle aspiration biopsy (FNAB) (36-10.1%) and miscellaneous (3 from pus from cervical lymph node, 1 each from pus from cold abscess-sternal region, pus from the right axillary lymph node, pus from the right wrist joint and sinus tract) [Table 1]. Also, 100 sputum specimen from the control group (radiologically negative and acid fast bacteria [AFB] smear negative) were included in the study.
|Table 1: Distribution of 354 M. tuberculosis culture‑positive clinical specimens|
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DNA extraction and nPCR targeting MPB64 gene and IS6110 region
DNA extraction was performed by using Qiagen DNA extraction mini kit (Germany) according to the manufacturer's instructions. A 50 μl reaction was set with 200 μM of each dNTPs (dATP, dTTP, dGTP, dCTP), 1 μM of each primer, 1X buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl 2 ), 1 unit of Taq DNA polymerase, and 10 μl of DNA. nPCR was performed in the thermal cycler (Eppendorf Master cycler), with the thermal profile given in [Table 2]. In each of the PCR amplification experiment, M. tuberculosis H37Rv ATCC DNA was used as a positive control.
|Table 2: Thermal profile for nPCR targeting MPB64 gene and IS6110 region|
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Detection of amplified products
After the nPCRs were completed, 10 μl of the amplified product was subjected to electrophoresis on 2% agarose gel incorporated with 0.5 μg/ml ethidium bromide for visualization by using UV transilluminator.
| ~ Results|| |
Out of the 354 clinical specimens, 339 (95.8%) were culture and nPCRs positive both, 10 each were positive for culture and nPCR targeting IS6110 region alone, and 5 were positive for culture and nPCR targeting MPB64 gene alone [Table 3].
|Table 3: Results of nPCR targeting MPB64 gene and IS6110 region and mycobacterial culture by BACTEC MicroMGIT system|
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Among the 339 culture and nPCRs positive specimens, 273 were from sputum, 33 from FNAB, 21 from bronchial wash, and 6 each from pus aspirates and pleural fluid. The remaining distribution of the clinical specimens among the other categories of nPCRs and culture positive are mentioned in [Table 4]. All of the 100 controls were negative for BACTEC culture and both nPCRs.
It is interesting to note that all the 354 clinical specimens positive for the isolation of M. tuberculosis by BACTEC culture was positive for the nPCRs. However, in 15 clinical specimens, either MPB64 or IS6110 alone was positive. The average turnaround time for the isolation of M. tuberculosis isolates by BACTEC MicroMGIT culture system was 22 days, whereas for nPCRs targeting MPB64 and IS6110 region, only 8 hrs is required to get the report.
| ~ Discussion|| |
The identification of the mycobacteria responsible for disease has important ramifications for infection control and the selection of antimicrobial therapy. Identification is hampered by the slow growth of most mycobacteria, which may take as long as 2 months, even if the organism is present in pure culture.  Several rapid diagnostic modalities for the identification of mycobacteria have been developed. The first major advance in the rapid identification of mycobacteria was the commercial availability of genetic probes. Although these assays significantly diminish the time to identification, the recommended use of this product requires the growth of an organism in culture, and it is limited to only a few species of Mycobacteria.
Several factors play important roles in standardizing a sensitive and specific nPCR for the detection of M. tuberculosis genome. The primers used in the amplification of target gene play an important role in optimizing a sensitive PCR. In the present study, nPCR targeting MPB64 gene and IS6110 region was performed in both pulmonary and extra pulmonary specimens. nPCR targeting both the gene targets was positive in 95.8% of the culture-positive clinical specimens. However, the present study emphasizes on the need for including two targets in simultaneously attaining 100% sensitivity for the detection of M. tuberculosis genome directly from clinical specimens and avoiding the false negativity of 2.8% (10) targeting IS6110 and 1.4% (5) with MPB64 gene, as encountered in this study. All the culture positive clinical specimens were positive for either both the nPCRs or any one of the gene targets. Moreover, 4.2% (n = 15) of the specimens may have been missed if both PCRs were not applied simultaneously on the clinical specimens. In a study by Singh et al.,  nPCR targeting MPB64 and IS6110 regions were performed in lymphadenitis patients and they showed that 77.8% of specimens by either one or both the target gene and 7 clinical specimens were positive by MPB64 alone. In a study by Drews et al.,  the Seeplex™ TB detection assay that detects M. tuberculosis from clinical specimens targeting MPB64 and IS6110 region have shown a sensitivity and specificity of 95% and 100%, respectively, from paraffin-fixed human biopsy specimens.
As reported in our earlier study,  nPCR has a significant advantage over conventional methods for the detection of M. tuberculosis from both pulmonary and extra pulmonary clinical specimens. The novelty in this study is that both nPCRs were applied simultaneously to prove 100% sensitivity in a large number of clinical specimens, including pulmonary and extra pulmonary specimens, which were culture-positive for M. tuberculosis and reappraising the utility of these two target genes to get 100% sensitivity and specificity. The important contribution of the study is that more than one target gene should be included for PCR for the detection of M. tuberculosis genome to rule out false negative results.
The future scope of the study would be converting the two nPCRs targeting MPB64 and IS6110 region into a duplex nPCR by designing newer sets of primers for gene targets that yields amplified products of different sizes. To conclude, application of nPCR targeting MPB64 and IS6110 region simultaneously are reliable and specific target genes for the detection of M. tuberculosis genome from clinical specimens.
| ~ Acknowledgment|| |
The authors gratefully acknowledge Chennai Willingdon Corporate Foundation, Chennai for funding the research project.
| ~ References|| |
|1.||Shankar P, Manjunath N, Mohan KK, Prasad K, Behari M, Shriniwas, et al. Rapid diagnosis of tuberculous meningitis by polymerase chain reaction. Lancet 1991;337:5-7. |
|2.||Therese KL, Jayanthi U, Madhavan HN. Application of nested polymerase chain reaction (nPCR) using MPB64 gene primers to detect Mycobacterium tuberculosis DNA in clinical specimens from extrapulmonary tuberculosis patients. Indian J Med Res 2005;122:165-70. |
|3.||Gunisha P, Madhavan HN, Jayanthi U, Therese KL. Polymerase chain reaction using IS6110 primer to detect M. tuberculosis in clinical samples. Indian J Pathol Microbiol 2001;44:97-102. |
|4.||Narayanan S. Molecular epidemiology of tuberculosis. Indian J Med Res 2004;120:233-47. |
|5.||Wang JY, Lee LN, Chou CS, Huang CY, Wang SK, Lai HC, et al. Performance assessment of a nested-PCR assay (the RAPID BAP-MTB) and the BD ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical specimens. J Clin Microbiol 2004;42:4599-603. |
|6.||D'Amato RF, Wallman AA, Hochstein LH, Colaninno PM, Scardamaglia M, Ardila E, et al. Rapid diagnosis of pulmonary tuberculosis by using Roche AMPLICOR Mycobacterium tuberculosis PCR test. J Clin Microbiol 1995;33:1832-4. |
|7.||Singh HB, Singh P, Jadaun GP, Srivastava K, Sharma VD, Chauhan DS, et al. Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis. J Commun Dis 2006;38:274-9. |
|8.||Drews SJ, Eshaghi AR, Pyskir D, Chedore P, Lombos E, Broukhanski G, et al. The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens. Diagn Pathol 2008;3:37. |
[Table 1], [Table 2], [Table 3], [Table 4]