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.
Pseudomonas aeruginosa was isolated from fifty soil samples were collected from different sites of contaminated soil with oil residues in Ministry of science& technology. Morphological, biochemical tests and 16S rRNA genes sequencing was performed for bacterial isolates identification. Thirty (30) isolates of P. aeruginosa bacteria were confirmed according to morphological, biochemical tests. For molecular analysis, all isolates that tested positive for P. aeruginosa underwent amplification of the 16S rRNA gene using previously described primers that amplified a particular DNA fragment of 956 bp. PCR product was delivered to Macrogen Corporation - Korea for Sanger sequencing utilizing an automated DNA sequencer called the ABI3730XL. The results were emailed to us, and we used geneious software to analyze them. The investigation of phylogenetic relationships between different strains of Pseudomonas aeruginosa has frequently been conducted on the sequencing of the 16S rRNA gene region, which is a viable technique for species identification. To determine the degree of genetic similarity between the organisms, a distance tree was built. Consequently, gene sequencing of the 16S rRNA region was an appropriate method for isolating isolates at the molecular level. The a novel local strain of Ps. aeruginosa which isolated from contaminated soil with oil residues samples (MSZ. IRQ20) and we will registration of isolate in GenBank under accession number of MT832126.1. Distance Tree Using Blast Tool in the Geneious software.
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