JTR  Vol.2 No.4 , December 2014
Non Specific Amplification with the LAMP Technique in the Diagnosis of Tuberculosis in Sri Lankan Settings
Abstract: Background: Tuberculosis (TB) remains a burden to Sri Lanka, where the incidence of the disease has been increasing over the past decade. The lack of early and accurate detection of the disease has been the main obstacle to its control. Microscopy or the culturing of mycobacteria from clinical samples is the most commonly used TB diagnostic tools in Sri Lanka. All these methods have their own limitations. Alternative diagnostic methods are therefore of high importance. Objectives: In this study, an attempt was made to validate loop mediated isothermal amplification (LAMP), which specifically amplifies a DNA sequence very rapidly at a low cost with limited resources. Methods: Crude DNA extractions of fifty culture isolates prepared from sputum samples, which were collected from patients with suspected TB extracts, were subjected to three separate LAMP assays. One assay was specific for 16S ribosomal RNA (16S rRNA) gene in genus Mycobacterium, and could detect the bacteria up to the genus level. The other two contained MTB specific primers targeting rimM or gyrB gene sequences in Mycobacterium tuberculosis (MTB), which enabled detection up to the species level. The sensitivity and specificity of the LAMP assays in the identification of mycobacteria or MTB were compared to microscopy and culture techniques. Results: Forty three out of the 47 Mycobacterium cultures were Mycobacterium-positive for LAMP assays with universal primers indicating a sensitivity of 92% in identifying Mycobacterium genus. However, thirteen out of 14 culture negatives were also positive with LAMP assays, which showed a specificity of only 7% in identifying MTB. The results suggested a high percentage of false positives by LAMP assays as compared to culture. Based on the colour changing of ZYBR Green dye and gel electrophoresis of the LAMP-amplified product, the detection of a non-specific amplification, even in the absence of target DNA, was recurrently observed. The result was the same even after following strict safety operations and laboratory practices to avoid the possibility of a cross-over contamination of MTB. Interestingly, this nonspecific DNA amplicon did not respond to digestion with BsaI restriction enzyme, suggesting that the false positives are not due to the presence of MTB. Conclusion: Under the tested conditions, the specificity of the LAMP method to identify MTB is low as compared to culture technique. Further investigations into optimizing the LAMP assay technique are required before it can be used, in its simple form, to diagnose TB in local clinical settings.
Cite this paper: Senarath, K. , Usgodaarachchi, R. , Navaratne, V. , Nagahawatte, A. , Wijayarathna, C. , Alvitigala, J. and Goonasekara, C. (2014) Non Specific Amplification with the LAMP Technique in the Diagnosis of Tuberculosis in Sri Lankan Settings. Journal of Tuberculosis Research, 2, 168-172. doi: 10.4236/jtr.2014.24021.

[1]   World Health Organization (2014) Communicable Diseases, TB in South East Asia.

[2]   Dunlap, N.E., Bass, J., Fujiwara, P., Hopewell, P., Horseburgh, P.R., Salfinger, M. and Simone, P.M. (2000) Diagnostic Standards and Classification of Tuberculosis in Adults and Children. American Journal of Respiratory and Critical Care Medicine, 161, 1376-1395.

[3]   Ling, D.I., Flores, L.L., Riley, L.W. and Pai, M. (2008) Commercial Nucleic-Acid Amplification Tests for Diagnosis of Pulmonary Tuberculosis in Respiratory Specimens: Meta-Analysis and Meta-Regression. PLoS One, 3, e1536.

[4]   Boehme, C.C., Nabeta, P., Henostroza, G., Raqib, R., Rahim, Z., Gerhardt, M., Sanga, E., Hoelscher, M., Notomi, T., Hase, T. and Perkins, M.D. (2007) Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries. Journal of Clinical Microbiology, 45, 1936-1940.

[5]   Geojith, G., Dhanasekaran, S., Chandran, S.P. and Kenneth, J. (2010) Efficacy of Loop Mediated Isothermal Amplification (LAMP) Assay for the Laboratory Identification of Mycobacterium Tuberculosis Isolates in a Resource Limited Setting. Journal of Microbiological Methods, 165, 211-220.

[6]   Tomita, N., Mori, Y., Kanda, H. and Notomi, T. (2008) Loop-Mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nature Protocols, 3, 877-882.

[7]   Poon, L.L., Wong, B.W., Ma, E.H., Chan, K.H., Chow, L.M., Abeyewickreme, W., Tangpukdee, N., Yuen, K.Y., Guan, Y., Looareesuwan, S. and Peiris, J.S. (2006) Sensitive and Inexpensive Molecular Test for Falciparum Malaria: Detecting Plasmodium Falciparum DNA Directly from Heat-Treated Blood by Loop-Mediated Isothermal Amplification. Clinical Chemistry, 52, 303-306.

[8]   Iwamoto, T., Sonobe, T. and Hayashi, K. (2003) Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium Tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples. Journal of Clinical Microbiology, 41, 2616-2622.

[9]   Zhu, R.Y., Zhang, K.X., Zhao, M.Q., Liu, Y.H., Xu, Y.Y., Ju, C.M., Li, B. and Chen, J.D. (2009) Use of Visual Loop-Mediated Isotheral Amplification of rimM Sequence for Rapid Detection of Mycobacterium tuberculosis and Mycobacterium bovis. Journal of Microbiological Methods, 78, 339-343.