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

BackgroundThe 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.FindingsThe 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 μg. The average amount of nucleic acids obtained with the addition of TE and triton-X100, was 133.7 ± 8.9 μg, while that obtained with the addition of TE only, and TE and SDS were 68.4 ± 22.7 μg and 70.4 ± 20.3 μg respectively. Other treatments yielded 28.8 ± 6.7 μg, 32.5 ± 2.4 μg and 36.9 ± 15.5 μg. 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).ConclusionWe strongly recommend the use of 1× 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.

Mycobacterial Phenolic Glycolipids Selectively Disable TRIF-Dependent TLR4 Signaling in Macrophages

“…TLR4-driven inside–out activation of CR3 was previously shown to dampen TLR4 signaling in BMDMs through a negative feedback mechanism involving Src-mediated degradation of MyD88 and TRIF by the ubiquitin:proteasome system ( 31 ). In parallel, it was reported that Leukadherin (LA)-1, a CR3 agonist, downregulates MyD88 protein levels in TLR7/8-stimulated THP-1 macrophages ( 32 ).…”

Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

“…Procedures commonly used for the isolation of nucleic acids do not obtain good results for M. leprae DNA extraction [68,73,74] and pretreatment steps with: freezing/thawing [75], bead beater with pearls [27,35] proteinase K [8,30,66,76] minimum 16 hours incubation [33], tris-HCL or tris-EDTA [44], detergents such as triton X-100 [22,77], and SDS [8,66,[76][77][78], alone or in combination should be included.…”

Draft Genome Sequence of Multidrug-Resistant Mycobacterium tuberculosis Strain CWCFVRF MDRTB 670, Isolated from the Sputum of a Patient from Chennai, India, with Clinically Suspected Tuberculosis

“…This isolate was resistant to all the first-line anti-tuberculous drugs, streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide, which was confirmed through phenotypic drug susceptibility testing using a micro BACTEC MGIT culture system. DNA was extracted from the isolate using the sodium dodecyl sulfate extraction method ( 4 ) and was further purified using a DNeasy miniprep kit (Qiagen, Hilden, Germany). Whole-genome sequencing was performed using an Ion Torrent (PGM) sequencer.…”