O-26: Rapid Detection of Mycobacterium Tuberculosis Complex by Loop Mediated Isothermal Amplification Technique
Objective: Despite continuous effort in preventing and treatment of tuberculosis (TB), the disease remains the second leading cause of death due to a single infectious agent. Rapid and efficient diagnosis is one of the crucial issues in the control of this disease. Loop mediated isothermal amplification (LAMP) is a simple and rapid technique for nucleic acid amplification. In this study, we evaluate a LAMP assay targeting IS6110 gene for diagnosis of M. tuberculosis (MTB), comparing it with PCR in clinical samples. Materials and Methods: 155 clinical sputum specimens were collected. A set of six specific primers were designed for LAMP assay. LAMP reaction was performed under optimal condition. At the end of reaction, LAMP products were identified by adding 0.1% SYBR Green. Sensitivity and specificity of LAMP were determined. Results: A successful LAMP reaction with newly designed primers was carried at 66ºC for 60 minutes. The detection limit of LAMP was one copy of the MTB DNA per tube. This detection sensitivity was greater than that for PCR, which can detect 20 copies of DNA. The sensitivity and specificity of this assay in clinical sputum samples were 100% and 95.9% (70/73, 95% confidence interval 91.3-98.7%) respectively. Conclusion: Tuberculosis remains an important global public health problem. One of the important reasons for failure to control TB is the lack of affordable simple diagnostic methods that have better sensitivity and specificity than conventional mthods commonly used in clinical mycobacteriology laboratories. Among the Nucleic acid amplification tests, the PCR has been most widely used for the detection of MTB in clinical specimens. LAMP has several advantages in comparison with PCR. LAMP amplify DNA under isothermal conditions, requiring only a regular water bath or heating block for maintaining the temperature at 66ºC, and make it more economical and practical than PCR. In addition, LAMP is more effective and rapid than conventional PCR. Our results showed that LAMP reaction was able to detect 5fg µl-1of DNA (one copy of MTB DNA). This detection sensitivity was greater than that for PCR, which can detect 100 fg µl-1 DNA (20 copies of MTB DNA). So, our study develops the LAMP assay with newly designed primer that showed high specificity and sensitivity in comparison with LAMP methods used in previous studies.