Development of tracking tool for p-nitrophenol monooxygenase genes from soil augmented with p-Nitrophenol degrading isolates: Bacillus, Pseudomonas and Arthrobacter

Abstract

The tracking of in situ-bioremediation of paranitrophenol through functional screening is time consuming. In this study we address a quick, reliable polymerase chain reaction-based method to detect the paranitrophenol monooxygenase gene from soil. All paranitrophenol degrading strains viz. Pseudomonas P19, Bacillus species C1, Bacillus species C2 and Arthrobacter species were isolated from contaminated agricultural soil. DNA was isolated from pure strains as well as soil inoculated with these strains. DNA extraction protocol was modified by including Cetyl Trimethyl Ammonium Bromide and sodium metabisulphite instead of polyvinylpolyprrolidone in polyethylene glycol method. Paranitrophenol monooxygenase gene was screened using degenerate primers designed to give 100 base pair amplification products. All strains used in study showed amplification product corresponding to target region using designed degenerate primers. Modified polyethylene glycol method leads to increase in DNA yield from soil. The assay technique is sensitive with broad specificity and has application in rapid identification of paranitrophenol monooxygenase gene. This method has advantage over DNA hybridization method as needs less DNA. The present study demonstrated the detection and monitoring of paranitrophenol monooxygenase gene from soil sample inoculated with either Gram-positive or Gram-negative bacteria using degenerate primers.