1st International Conference on Emerging Trends in Scientific Research 15-16 March, 2014 Pearl International Hotel Kuala Lumpur, Malaysia

1. Abstract of Emerging Trends in Scientific Research, 2014, Vol.1
DOI: 10.18488/journal.1002/2014.1/1002.1
1st
International Conference on Emerging Trends in
Scientific Research
15-16 March, 2014
Pearl International Hotel Kuala Lumpur, Malaysia
Conference Website: www.pakrdw.com
2
Paper ID: 11/14/1
st
ICETSR
Loop Mediated Isothermal Amplification (LAMP)- Emerging
New Technology for Diagnosis of Pulmonary Tuberculosis
Sunil Sethi1
--- Rakesh Yadev2
--- Sunil Dhatwalia3
--- Dheeraj Gupta4
Abstract
Design: Loop-mediated isothermal amplification (LAMP) is a recently developed
molecular method that has been successfully implemented in the detection of
Mycobacterium tuberculosis in clinical specimens. LAMP has several advantages, such
as rapidity, high sensitivity, ease of application and cost-effectiveness. In this study we
developed LAMP and compared with IS6110 PCR and conventional techniques in TB
endemic country Methods ; LAMP assay in the present study targets 16s rRNA gene of
M. tuberculosis complex, using one set of each- Inner primer (FIP & BIP), Outer primer
(F3 &B3) and Loop primer (FLP & BLP). LAMP was carried out in a total volume of 25 μl,
containing 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 0.8 μM each of
FLP and BLP, 20 mM Tris-HCl (pH: 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 9 mM MgSO4,
1.4 mM dNTP, 0.8 M Betaine (Sigma- Aldrich), 8 U Bst DNA polymerase (New England
Biolabs, USA) and 5 μl DNA sample. To find the optimum time and temperature for
LAMP assay, the reactions were carried out at 60 to 68°C for 35 to 90 min. A positive
and negative control was included in each run. LAMP testing was blinded as compared to
smear microscopy, culture and PCR. LAMP amplicons were directly detected with the
naked eye by adding 0.1% SYBR Green I (Invitrogen lot: 49743A, USA) to the tube and
observing the color of the solution under UV light. The solution turned green in the
presence of a LAMP amplicon, while it remained orange with no amplification. To confirm
the structure of the LAMP products, the amplicons were analyzed by gel electrophoresis
in 2% agarose gel. Sputum samples were collected from 100 patients. The samples were
subjected to microscopy, culture, IS 6110 and LAMP assay which used set of six specific
primers targeting 16s rRNA gene of M.tuberculosis. Results: Overall, LAMP positivity was
observed in 82% (82/103) and IS6110 could detect TB in 71 (71) patients. The positive
and negative predictive values of LAMP was 100 and 65.2% respectively.Both IS6110
and LAMP were negative in all the 30 Non TB patients giving the specificity of 100%. The
proportion of agreement among IS6110 and LAMP by using kappa was approximately
significant i.e 0.6 Conclusions:. The study showed that the LAMP assay is emerging new
technology which is cost effective sensitive and specific specially for resource poor
settings for diagnosis of tuberculosis