Detection of quantitative and qualitative for p.aeruginosa in contaminated and non-contaminated soil with oil residues targeting gyrb gene by QPCR

Abstract

Comparison of P. aeruginosa counts is found in contaminated and non- contaminated soil with oil residue using a real-time quantitative PCR by Direct DNA extraction from soil samples as well as indirect DNA extraction from bacterial isolates. Sampling were collected randomly from ten different regions of the Ministry of Science &Technology during the period from October to December 2019. Twenty soil samples were investigated for hydrocarbon tolerance in selective broth containing hydrocarbon source, and then isolates diagnosed based on phenotype, microscopic, and biochemical tests, were used. Direct DNA extraction from soil samples using DNeasy Power Soil Pro Kit for the rapid detection of P. aeruginosa from soil, as well as wizard DNA extraction method from bacterial isolation. Quantitative and Qualitative PCR was performed with the Magnetic Induction Cycler (Mic), DNA extraction from soil and bacterial isolates amplified and detected using fluorescent reporter dye probes specific for Pseudomonas aeruginosa DNA and Internal Control IC. A total of twenty bacterial isolates were obtained from the different soil samples according to phenotyping tests, and also, we obtain 20 DNA samples from direct DNA extraction from soil, the results of purity were (1.70- 1.95) for wizard method and (1.46- 2.35) for direct extraction from soil. Pseudomonas aeruginosa counts in contaminated soil samples (8 x 107 Copies/μL) showed higher compared with the non- contaminated soil samples (2 x 102 Copies/μL) at maximum use of qPCR and soil DNA. Rapid and specific test (less than 4 hours) using Rt-PCR by targeting gyrB gene for Pseudomonas aeruginosa detection in contaminated and non-contaminated soil samples, combined with correctly adjusted samples processing methods, as well as soil DNA extraction were made which were applied within the sensitivity required for methods.