Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

Abstract

Background:Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive.Materials and Methods:The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples.Results:The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions.Conclusions:According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

Tuberculosis Outbreak in an Educational Institution in Henan Province, China

“…WGS of the 9 isolates was performed at Institute of Microbiology, Chinese Academy of Science. Briefly, DNA from the strains was extracted using the CTAB method (20,21). DNA sequencing libraries were prepared as previously described and sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA), with all reads performed at 2,150 bp at a median depth of 352 (range, 52-706).…”

A PCR-Free Genome Detection of Mycobacterium Tuberculosis Complex in Clinical Samples using MWCNT/PPy/KHApNps Modified Electrochemical Nano-Biosensor

“…71 The boiling method was used for the extraction of DNA sputum. 72 The total genome of M. tb H37Rv was extracted by the Van-Soolingen method. 73 The PCR amplification was done on the IS 6110 fragment gene as a target.…”

Testing of Biological Products: A Review of Alternative Non-microbiological Techniques for Mycobacterial Testing

“…Different methods can be combined to increase the efficiency of cell lysis. Mechanical lysis is the simplest and one of the most efficient techniques to break down the mycobacterial cell walls [17,38]. The most commonly used mechanical lysis methods include bead beating [30,39], boiling [40,41], or sonication [39].…”

“…A chemical lysis of mycobacterial cell wall can be performed with cetyltrimethylammonium bromide, which induces cell lysis by precipitation of cell wall components such as free lipoglycans and polysaccharides. However, the technique needs to be coupled with organic solvent extraction (phenol-chloroform), and thus needs specialized equipment and skilled personnel [28,38]. Detergents such as sodium dodecyl sulphate and Triton X-100 can be used for dissolving the bacterial cell wall [29].…”