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Nucleic Acid Resistance Lab Report

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AMPLIFICATION OF rpoB, kat G & mab A (fab G1)- inh A PROMOTOR DNA SEQUENCES BY PCR IN MULTIPLE DRUG RESISTANCE TUBERCULOSIS
Bhanu Mehta, Afreen Siddiquie, Rakhi Kaushik, Rahul Bisht, Narotam Sharma1*,
1Central Molecular Research Laboratory, Department of Biochemistry, SGRR Institute of Medical & Health Sciences, Dehradun, India.

*Address for Correspondence:
Dr. Narotam Sharma, Scientist,
Central Molecular Research Laboratory,
Biochemistry Department
Shri Guru Ram Rai Institute of Medical and Health Sciences, Patel Nagar
Dehradun-248001(Uttarakhand), India.
Email: sharmanarotam5@gmail.com

ABSTRACT
Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose …show more content…

Nested PCR for the detection of Mycobacterium tuberculosis complex targeting IS6110 gene. Further Nested PCR was performed on the DNA template isolated from various specimens. This test is based on the principles of single-tube nested PCR method, which is a powerful and sensitive diagnostic tool for the identification of Mycobacterium Tuberculosis complex. This assay is a two-step sequential assay. In the first step, the Insertion sequence region of Mycobacterium tuberculosis complex DNA sequence, a 220 bp is amplified by specific external primers. In the second step, the nested primers are added to further amplify a 123 bp amplification product. In this assay, false positive reactions that may be caused by previous amplicon contamination are prevented by the use of uracil DNA glycosylase (UDG) and dUTP instead of dTTP added in the premix [11, 12]. Nested PCR. An amplimer of size 123 bp is indicative of infection with Mycobacterium tuberculosis complex. The amplification product of internal control DNA is 340 bp which is used for the validation of the results (as depicted in …show more content…

Nautiyal, Rakhi Kaushik, Amanpreet Singh, Akhil Pratap Singh, Jivesh K. Shaw, Shilpi Dixit, and R. K. Singh, 2013. Nested-PCR and Uracil-N-Glycosylase-significant approach to prevent amplicon contamination in tuberculosis PCR performing laboratories." Archives of Applied Science Research 5(2): 177-179.
12) Narotam Sharma, Mukesh Kumar, Natasha K Ahmed, Shivani Sailwal, Deepti Singh, Subhash Sharma, Danish Kalim, Application of Nested Polymerase Chain Reaction using IS6110 Molecular Target to Reveal Mycobacterium Tuberculosis DNA from Extra Pulmonary Tuberculosis Patient, International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013, 43-49.
13) Sekiguchi, J. I., Miyoshi-Akiyama, T., Augustynowicz-Kopeć, E., Zwolska, Z., Kirikae, F., Toyota, E., ... & Kirikae, T. (2007). Detection of multidrug resistance in Mycobacterium tuberculosis. Journal of clinical microbiology, 45(1), 179-192.
14) Siddiqi, N., M. Shamim, S. Hussain, R. K. Choudhary, N. Ahmed, Prachee, S. Banerjee, G. R. Savithri, M. Alam, N. Pathak, A. Amin, M. Hanief, V. M. Katoch, S. K. Sharma, and S. E. Hasnain. 2002. Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India. Antimicrob. Agents Chemother.

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