TITLE:
Non Specific Amplification with the LAMP Technique in the Diagnosis of Tuberculosis in Sri Lankan Settings
AUTHORS:
K. D. Senarath, R. B. Usgodaarachchi, V. Navaratne, A. Nagahawatte, C. D. Wijayarathna, J. Alvitigala, C. L. Goonasekara
KEYWORDS:
Tuberculosis, LAMP Assay, Nonspecific Amplification
JOURNAL NAME:
Journal of Tuberculosis Research,
Vol.2 No.4,
November
19,
2014
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.