跳至主要内容

Non Specific Amplification with the LAMP Technique in the Diagnosis of Tuberculosis in Sri Lankan Settings

Read full paper at:
http://www.scirp.org/journal/PaperInformation.aspx?PaperID=51522#.VG2xZWfHRK0

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.
http://www.who.int/gho/tb/en/
[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.
http://dx.doi.org/10.1164/ajrccm.161.4.16141
[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. http://dx.doi.org/10.1128/JCM.02352-06
[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.
http://dx.doi.org/10.1038/nprot.2008.57
[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.
http://dx.doi.org/10.1373/clinchem.2005.057901
[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.
http://dx.doi.org/10.1128/JCM.41.6.2616-2622.2003
[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.
http://dx.doi.org/10.1016/j.mimet.2009.07.006           eww141120lx
标签：

评论

发表评论

此博客中的热门博文

Does Immigration Promote the Investment of the Monopolistic Firm?

In the present paper, we examine the effect of increasing uncertainty of immigrants’ growth on the optimal timing of investment of a firm that has a monopolistic power over the labor market. It is revealed that when the uncertainty of immigrants’ growth is more than a threshold level, increasing uncertainty of immigrants’ growth accelerates the optimal timing of firms’ investment and enhances the economic growth, even if the uncertainty of immigrants’ growth is formulated by the geometric Brownian motion, which is in sharp contrast to the standard result that an increase in the uncertainty postpones the optimal timing. With an increase in the immigrants over the past ten years, workforces in the host countries have been growing significantly to the extent that the immigrants represent 70% of the increase in the workforce in Europe, and 47% in the United States as OECD indicates. In the present paper, we attempted to investigate the effect of increased uncertainty caused by the growi...
发表评论
阅读全文

Education Policy Implementation: A Mechanism for Enhancing Primary Education Development in Zanzibar

Education is one of the fundamental rights of individuals; therefore, the government of a country needs to develop and strengthen educational policy and quality as well as to ensure that everyone has equal access to basic education. The improvement of access and quality of education in the world is becoming as an essential factor in development, whereas the basic education (primary school), is acknowledged as a foundation of the higher educational development for every country. To fulfill this goal, governments introduce several policies and procedures; however, it requires some reforms and participation from the politician, policymakers, and other stakeholders to re-examine educational policy so that it can lead to multiplication and betterment of the reforms. Educational reforms actually focus on accountability. A positive educational development and reform is very challenging and needs more effort and strategy on how to use and utilize the resources effectively as such it can achie...
发表评论
阅读全文