Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Apr;53(4):1137-43.
doi: 10.1128/JCM.03073-14. Epub 2015 Jan 28.

Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures

Antonina A Votintseva  1 , Louise J Pankhurst  2 , Luke W Anson  2 , Marcus R Morgan  3 , Deborah Gascoyne-Binzi  4 , Timothy M Walker  2 , T Phuong Quan  2 , David H Wyllie  5 , Carlos Del Ojo Elias  2 , Mark Wilcox  4 , A Sarah Walker  6 , Tim E A Peto  6 , Derrick W Crook  6
Affiliations

Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures

Antonina A Votintseva et al. J Clin Microbiol. 2015 Apr.

Abstract

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Protocol for mycobacterial DNA extraction from the MGIT cultures by ethanol precipitation with the MolYsis Basic5 kit (version A) or saline wash (version B) pretreatment steps for removal of human DNA.
FIG 2
FIG 2
DNA extraction from early positive MGIT cultures with QIAmp minikit with MolYsis (QIAmp-MolY) and QIAmp minikit (QIAmp), QuickGene with MolYsis (QG-MolY) and QuickGene (QG), ethanol precipitation protocol with MolYsis (EtOH-MolY), and ethanol precipitation protocol with saline wash (EtOH-SW). Each gray dot represents a single extraction; crossed horizontal lines represent median DNA concentration for each extraction method; solid black line represents the threshold amount of DNA of 0.2 ng/μl required for the MiSeq library preparation.
See this image and copyright information in PMC

References

    1. Balasingham SV, Davidsen T, Szpinda I, Frye SA, Tonjum T. 2009. Molecular diagnostics in tuberculosis: basis and implications for therapy. Mol Diagn Ther 13:137–151. doi:10.1007/BF03256322. - DOI - PubMed
    1. Sharma N, Sharma V, Singh PR, Jawed B, Babu V, Kandpal J, Nautiyal SC, Singh RK. 2013. Tuberculosis and molecular diagnosis. WebmedCentral Biotechnology 4:WMC003992. doi:10.9754/journal.wmc.2013.003992. - DOI
    1. Luetkemeyer AF, Kendall MA, Wu X, Lourenco MC, Jentsch U, Swindells S, Qasba SS, Sanchez J, Havlir DV, Grinsztejn B, Sanne IM, Firnhaber C. 2014. Evaluation of two line probe assays for rapid detection of Mycobacterium tuberculosis, tuberculosis (TB) drug resistance, and non-TB Mycobacteria in HIV-infected individuals with suspected TB. J Clin Microbiol 52:1052–1059. doi:10.1128/JCM.02639-13. - DOI - PMC - PubMed
    1. Morgan M, Kalantri S, Flores L, Pai M. 2005. A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. BMC Infect Dis 5:62. doi:10.1186/1471-2334-5-62. - DOI - PMC - PubMed
    1. Raizada N, Sachdeva KS, Chauhan DS, Malhotra B, Reddy K, Dave PV, Mundade Y, Patel P, Ramachandran R, Das R, Solanki R, Wares DF, Sahu S, O'Brien R, Paramasivan CN, Dewan PK. 2014. A multi-site validation in India of the line probe assay for the rapid diagnosis of multi-drug resistant tuberculosis directly from sputum specimens. PLoS One 9:e88626. doi:10.1371/journal.pone.0088626. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources