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 obsta...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.展开更多
文摘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.
