Evaluation of cost-effective total nucleic acids extraction protocols for cultured Mycobacterium tuberculosis; a comparison by PCR amplification of genes associated with drug resistance

Abstract

Background: The emergence of drug resistant strains of Mycobacterium tuberculosis complex has made the management of tuberculosis difficult. Also, Mycobacterium species has a peculiar cell wall, made of an impermeable complex structure rich in mycolate, making the lyses of its cell difficult. In order to apply a radio-labelled-probe based detection of mutations in selected genes leading to drug resistance, we concede that the evaluation and modifications of nucleic acid extraction protocols that are less sophisticated and less prone to contamination would be useful in the management of tuberculosis in a resource-constrained setting.

Findings: The average amount of nucleic acids was determined for different extraction treatments. High temperature treatment only, yielded the lowest amount of nucleic acids, i.e. 15.7 +/- 3.2 mug. The average amount of nucleic acids obtained with the addition of TE and triton-X100, was 133.7 +/- 8.9 mug, while that obtained with the addition of TE only, and TE and SDS were 68.4 +/- 22.7 mug and 70.4 +/- 20.3 mug respectively. Other treatments yielded 28.8 +/- 6.7 mug, 32.5 +/- 2.4 mug and 36.9 +/- 15.5 mug. The average amount of nucleic acids obtained with high temperature treatment in TE, and that obtained by freezing prior to high temperature treatment, successfully amplified for the genes of interest (rpoB, KatG, rrs).

Conclusion: We strongly recommend the use of 1x TE buffer, and freezing and heating for improved lysis of cultured M. tuberculosis, and therefore, as an effective method for the preparation of M. tuberculosis nucleic acid useful for PCR.

Figure 1

Amplification products for protocol 1, 2 and 5 respectively. a) rspL b) rrs c) KatG genes DNA extracted from five cell suspensions with each of the protocols 1, 2 and 5 were amplified by PCR for three genes. Amplification products of the rpoB, KatG and rrs genes obtained were each resolved at 70 Volts for 1 hour on a 2% agarose mini-gel containing 0.001 mg/mL ethidium bromide.

Figure 2

rpoB, KatG and rrs gene amplifications product of DNA obtained with protocol 4. DNA extracted from five cell suspensions with protocol 4 were amplified separately by PCR for three genes. Amplification products of the rpoB, KatG and rrs genes obtained separately by PCR were each resolved at 70 Volts for 1 hour on a 2% agarose mini-gel containing 0.001 mg/mL ethidium bromide.

Figure 3

rpoB, KatG and rrs gene amplifications product of DNA obtained with protocol 3. DNA extracted from five cell suspensions with protocol 3 were amplified by PCR for three genes. Amplification products of the rpoB, KatG and rrs genes obtained were each resolved at 70 Volts for 1 hour on a 2% agarose mini-gel containing 0.001 mg/mL ethidium bromide.