Sequencing of rpoB gene

Pallavi Sinha; G. N. Srivastava; Rajneesh Tripathi; Mukti Nath Mishra; Shampa Anupurba

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Sequencing of rpoB gene

PS Pallavi Sinha

GS G. N. Srivastava

RT Rajneesh Tripathi

MM Mukti Nath Mishra

SA Shampa Anupurba

This method is extracted from research article: BMC Microbiol, Sep 2020

Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens

DOI: 10.1186/s12866-020-01967-5

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DNA sequencing was performed on all clinical specimens found to be non-interpretable on LPA MTBDRplus assay by Sanger sequencing. The regions of rpoB associated with Rif^r were sequenced after amplification by PCR. The forward RSF (5′-GATGACCACCCAGGACGTGGAG-3′) and reverse RSR (5′-TCGATCGGCGAATTGGCCTGTG-3′) primers used for both PCR and sequencing to amplify a 438-bp fragment of the rpoB gene containing the 81-bp hypervariable region (Fig. (Fig.4).4). The 25 μl PCR reactions were performed with 5 μl 10X Q5 reaction buffer (New England BioLabs), 1 μl 200 μM of each dNTP, Q5 hot start Taq DNA polymerase (New England BioLabs), 5 μl Q5 GC enhancer (New England BioLabs), 10 μl of DNA from the specimen and 10 pmol of each primer (GeNei, Banglore, India) and added Milli Q to create a total volume of 25 μl.

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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