Journal of Microbial & Biochemical Technology

Rapid detection of M. tuberculosis complex with LAMP PCR method combined with lateral flow dipstick (LFD)

12^th World Congress on Biotechnology and Microbiology

June 28-29, 2018 | Amsterdam, Netherlands

H Esra Agel

TUBITAK Marmara Research Center, Turkey

Posters & Accepted Abstracts: J Microb Biochem Technol

Abstract:

Loop mediated isothermal amplification (LAMP) method is preferred for the development of diagnosis kit for the rapid and sensitive diagnosis of tuberculosis infection. In this study, we were developed a detection system for M. tuberculosis complex with LAMP method combined with Lateral Flow Dipstick (LFD). DNA isolations were performed with Qiagen Qiamp DNA minikit. LAMP reaction was performed in 25 μl, containing 0.2 μM each outer primers (F3:5â??-CGAATTGCGAAGGGCGA-3â?? and B3:5â??-GTAGGTCGATGGGGCGA-3â??), 0.8 μM each inner primers (FIP:5â??-Biotin-ACCGTTAATTAGCGTGCTGGCCATTTTAAAGACCGCGTCGGC- 3â?? and BIP: GATCATCAGGGCCACCGCGAG-CGGTCAGCTGTGTGCAGAT), 1X NEB buffer, 5 mM MgSO4, 0.4 mM dNTP, 2 U Bst polymerase and 5 μl DNA sample. Samples were incubated at 71oC for 60 minutes and 80oC for five minutes. LAMP amplified products were then analyzed using 2% agarose gel electrophoresis. The LAMP primer set and probe (5â??-FAM-ATGGCCGTACAGGTT-3â??), which recognize six distinct regions IS6110 gene, was design by Primer Explorer V5 software (Eiken Genome; https://primerexplorer.jp/e/). LAMP products were analyzed using LFD (Milenia Biotec Germany), according to the manufacturerâ??s instructions. Biotin labelled FIP and FAM labelled probe were used for LFD. The detection time which including DNA isolation, is only 90 minutes. LAMP method which combined with LFD was faster than conventional PCR that consumes nearly three hours. The LOD was detected as 102 cfu/mL. Gel electrophoresis was not required in this method. Also like other methods, there were no needs for sophisticated laboratory equipment and qualified personnel. High sensitivity and specificity, relatively short analysis time, and relatively inexpensive equipment use were significant advantages of the LAMP-LFD assay.