PCR-mediated detection of acidophilic, bioleaching-associated bacteria

Abstract

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.

Effect of simulated rhizodeposition on the relative abundance of polynuclear aromatic hydrocarbon catabolic genes in a contaminated soil

“…Nested PCR [19] reactions were performed on a Biometra thermocycler (Model T-gradient; Goettingen, Germany) with the following temperature conditions: 50ЊC for 2 min, followed by 95ЊC for 10 min and then 30 cycles at 95ЊC for 15 s and 55ЊC for 1 min, with a final extension at 70ЊC for 10 min. The PCR mixtures contained 1ϫ Taqman buffer, 500 M of each universal bacterial primer BACT1369F and PROK1492R, 2 l of the sample DNA, and sterile DNase-free water to make up a final volume of 25 l. The amplified PCR product (2 l) was then mixed with 1ϫ Taqman buffer, 500 M of the forward and reverse primers coding for oxygenase genes (Table 2), and sterile DNAase-free water to make up a final volume of 25 l. Temperature cycle was set as described above.…”

“…Neither naphthalene diox‐ygenase ( nahAc ) nor toluene monooxygenase ( bmoA ) were detected by RTQ‐PCR in our samples (detection limit, ∼10 2 copies/g soil). The detection of these genes using nested PCR methods as described elsewhere [19], however, suggests that these genes were present at very low copy numbers.…”

“…Nested PCR [19] reactions were performed on a Biometra thermocycler (Model T‐gradient; Goettingen, Germany) with the following temperature conditions: 50°C for 2 min, followed by 95°C for 10 min and then 30 cycles at 95°C for 15 s and 55°C for 1 min, with a final extension at 70°C for 10 min. The PCR mixtures contained 1 × Taqman buffer, 500 ηM of each universal bacterial primer BACT1369F and PROK1492R, 2 μl of the sample DNA, and sterile DNase‐free water to make up a final volume of 25 μl.…”

Sulfur Oxygenase Reductase (Sor) in the Moderately Thermoacidophilic Leaching Bacteria: Studies in Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus

“…Purity tests of At. caldus S1 and S2 strains were done by a two-stage nested polymerase chain reaction (PCR)-mediated detection method [ 45 ]. Additionally, At.…”

Insights into the characterization of ars genes in the biomining bacterium Acidithiobacillus caldus SM-1

“…Conventional plate count methods and biochemical identification methods described previously could not circumvent the problems linked to the long wait for the colony to develop and/or the inability of some bacteria to grow on solid media (Johnson, 1991;Ahmad, 1993). In recent years, various nucleic acid-based molecular methods, such as Polymerase chain reaction (PCR) method (Feng et al, 2012;Escobar et al, 2008;Kamimura et al, 2001;DeWulfDurand et al, 1997) and fluorescent in situ hybridization (FISH) (Mahmoud et al, 2005), have been developed for the rapid detection and identification of Acidithiobacillus strains because of simplicity in operation, stable detection results, and savings in time. A high level of marker specificity is crucial for various nucleic acid-based molecular methods.…”