title: Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.
abstract: A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.
authors.affiliation: Biological Sciences Division, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI 49931, USA. rsaha@mtu.edu
authors.forename: Ratul
authors.initials: R
authors.lastname: Saha
authors.forename: Robert S
authors.initials: RS
authors.lastname: Donofrio
authors.forename: Susan T
authors.initials: ST
authors.lastname: Bagley
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D007220:Industrial Waste; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D007220:Industrial Waste; D015345:Oligonucleotide Probes; D027081:DNA Gyrase
references.citation: J Microbiol Methods. 2008 Jun;73(3):266-8
references.pmid: 18423662
references.citation: J Clin Microbiol. 2003 Sep;41(9):4438-41
references.pmid: 12958287
references.citation: J Clin Microbiol. 2004 Aug;42(8):3827-30
references.pmid: 15297540
references.citation: AIHAJ. 2000 Mar-Apr;61(2):205-13
references.pmid: 10782192
references.citation: J Bacteriol. 1941 Mar;41(3):373-86
references.pmid: 16560407
references.citation: Appl Environ Microbiol. 2002 Nov;68(11):5580-4
references.pmid: 12406752
references.citation: J Microbiol Methods. 2004 May;57(2):259-68
references.pmid: 15063066
references.citation: Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
references.pmid: 9396791
references.citation: Nucleic Acids Res. 2007;35(19):e127
references.pmid: 17897966
references.citation: AIHA J (Fairfax, Va). 2002 Mar-Apr;63(2):178-83
references.pmid: 11975654
references.citation: Appl Environ Microbiol. 2005 Apr;71(4):2190-4
references.pmid: 15812058
references.citation: Microbiology (Reading). 2002 Jan;148(Pt 1):257-266
references.pmid: 11782518
references.citation: Curr Microbiol. 2010 Apr;60(4):294-300
references.pmid: 19936829
references.citation: Microb Ecol. 1987 Mar;13(2):103-14
references.pmid: 24213209
references.citation: Mol Cell Probes. 2004 Feb;18(1):67-73
references.pmid: 15036372
references.citation: Microbiology (Reading). 2000 Oct;146 ( Pt 10):2385-2394
references.pmid: 11021915
references.citation: AIHAJ. 2000 Nov-Dec;61(6):788-97
references.pmid: 11192210
references.citation: Environ Microbiol. 2003 Jun;5(6):453-61
references.pmid: 12755712