Skip to content

Research at St Andrews

Evaluation of the efficacy of two methods for direct extraction of DNA from Mycobacterium tuberculosis sputum

Research output: Contribution to journalArticle

DOI

Open Access permissions

Open

Author(s)

Victor Ndhlovu, Wilson Mandala, Derek Sloan, Mercy Kamdolozi, Maxine Caws, Gerry Davies

School/Research organisations

Abstract

Introduction: Whole genome sequencing (WGS) has shown superiority over other bacterial typing methods and can be used to monitor disease transmission. The long culture period hinders use of WGS as a diagnostic tool for TB. The ideal situation would be to efficiently sequence directly from clinical specimens such as sputum. Attempts to sequence directly from Mtb clinical samples have achieved very low coverage (less than 0.7X). We compared DNA extraction methods for direct extraction from Mycobacterium tuberculosis positive sputum and assessed their suitability for Single Molecule Real Time sequencing.

Methodology: We evaluated the extraction efficiency of the PrimeXtract kit and an in-house CTAB method by extracting DNA from Mtb sputum. We evaluated the methods on these parameters: ease of use, efficiency (quantity and purity) and the cost per extraction.

Results: The PrimeXtract kit was able to isolate 5.93 mu g/mL ± 0.94, (Mean ± SEM) concentration of DNA and a yield of 0.2975 mu g ± 0.04723, (Mean ± SEM). Comparatively, the CTAB method isolated 1.88 mu g/mL ± 0.38 DNA and a yield of 0.09 mu g ± 0.02. Both concentration and yield from the kit were significantly (p = 0.0002) higher than those from CTAB. The PrimeXtract kit had a DNA purity ratio of 1.69 ± 0.09 compared to the CTAB's 1.73 ± 0.14 and this difference was not statistically different.

Conclusion: PrimeXtract kit has a superior extraction efficiency than the CTAB method on Mtb sputum in terms of DNA yield although no significant difference by DNA purity was seen.

Close

Details

Original languageEnglish
Pages (from-to)1067-1072
Number of pages6
JournalJournal of Infection in Developing Countries
Volume12
Issue number12
DOIs
Publication statusPublished - 31 Dec 2018

    Research areas

  • Mycobacterium tuberculosis, SMRT sequencing, PrimeXtract, CTAB

Discover related content
Find related publications, people, projects and more using interactive charts.

View graph of relations

Related by author

  1. PrimeStore MTM and OMNIgene sputum for the preservation of sputum for Xpert MTB/RIF testing in Nigeria

    Bimba, J. S., Lawson, L., Kontogianni, K., Edwards, T., Ekpenyong, B. E., Dodd, J., Adams, E. R., Sloan, D. J., Cresswell, J., Dominguez, J. & Cuevas, L. E., 4 Dec 2019, In : Journal of Clinical Medicine. 8, 12, 9 p., 2146.

    Research output: Contribution to journalArticle

  2. Defining the roles of Data Manager and Epidemiologist in emergency medical teams

    Bartolucci, A., Jafar, A. JN., Sloan, D. J. & Whitworth, J., 16 Oct 2019, In : Prehospital and Disaster Medicine. First View, 7 p.

    Research output: Contribution to journalArticle

  3. Genetic diversity of Mycobacterium tuberculosis clinical isolates in Blantyre, Malawi

    Ndhlovu, V., Kiran, A., Sloan, D., Mandala, W., Kontogianni, K., Kamdolozi, M., Caws, M. & Davies, G., Oct 2019, In : Heliyon. 5, 10, 5 p., e02638.

    Research output: Contribution to journalArticle

  4. A tuberculosis molecular bacterial load assay (TB-MBLA)

    Sabiiti, W., Mtafya, B. A., Alferes De Lima, D., Dombay, E., Baron, V. O., Azam, K., Orascova, K., Sloan, D. J. & Gillespie, S. H., 6 Sep 2019, (Accepted/In press) In : Journal of Visualized Experiments.

    Research output: Contribution to journalArticle

  5. Protocol for the development of the STrengthening the Reporting of Pharmacogenetic Studies (STROPS) guideline: checklist of items for reporting pharmacogenetic studies

    Richardson, M., Kirkham, J. J., Dwan, K. M., Sloan, D. J., Davies, G. & Jorgensen, A., 11 Jul 2019, In : BMJ Open. 9, 7, 5 p., e030212.

    Research output: Contribution to journalArticle

Related by journal

  1. Journal of Infection in Developing Countries (Journal)

    Muge Cevik (Member of editorial board)
    2019 → …

    Activity: Publication peer-review and editorial work typesEditor of research journal

ID: 257477852

Top