Year: 2022 | Month: October | Volume 12 | Issue 5

Evaluation of Various Methods for Genomic DNA Extraction from Pure Cultures of Lysis Resistant Campylobacters Isolated from Wild Animals

Nawal Kishor Singh Ajay Kumar Upadhyay Aman Kamboj Maansi Shukla Tanuj Kumar Ambwani Rajesh Kumar Himani Sharma Namita Shukla
DOI:10.30954/2277-940X.05.2022.22

Abstract:

Campylobacter is one of the important foodborne zoonotic bacterial pathogen that causes enteric disorders in animals, birds as well as in humans. The organism is fastidious in nature, requires microaerophilic environment for its growth and survival. Morphologically, it is gram- negative rods with spiral and gull wing appearance. Polymerase chain reaction (PCR) is gold standard method for the detection of Campylobacter from clinical and food samples. For performing PCR, extraction of DNA to be used as template is a challenging task due to lysis resistant nature of bacteria. The genomic DNA isolation was attempted from pure cultures of Campylobacter by three methods viz. Snap-chill, Salt-Tris EDTA (STE) and Columns based commercial kit method. The average concentration of extracted DNA was highest in STE method (03 -3500 ng/µl) followed by Kit method (03 -2000 ng/µl) and Snap-chill method (00-20 ng/µl). The absorbance ratio at 260 nm and 280 nm (A260/A280) was high up to 1.90 in STE method followed by up to 1.80 with column-based kit and 1.5 with snap-chill method which reflects the high purity of isolated DNA by STE and kit-based protocol. As per the results STE method and Kit method was comparable to each other but due to high cost of commercial kit, STE method is proposed to be desirable and may be used routinely for extraction of DNA of lysis resistant bacteria. The PCR results also advocate the preference of STE method over kit method while Snap-chill method was not found effective for lysis resistant Campylobacter isolates.

Highlights

  • STE method yielded comparatively better results than other two methods for DNA extraction of lysis resistant thermophilic Campylobacters.
  • Higher DNA yield gives better results in multiplex PCR for Campylobacter species identification.


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