Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.
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.
Saha
Ratul
R
Biological Sciences Division, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI 49931, USA. rsaha@mtu.edu
Donofrio
Robert S
RS
Bagley
Susan T
ST
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
D001426:Bacterial Proteins; D017931:DNA Primers; D007220:Industrial Waste; D015345:Oligonucleotide Probes; D027081:DNA Gyrase
J Microbiol Methods. 2008 Jun;73(3):266-8
18423662
J Clin Microbiol. 2003 Sep;41(9):4438-41
12958287
J Clin Microbiol. 2004 Aug;42(8):3827-30
15297540
AIHAJ. 2000 Mar-Apr;61(2):205-13
10782192
J Bacteriol. 1941 Mar;41(3):373-86
16560407
Appl Environ Microbiol. 2002 Nov;68(11):5580-4
12406752
J Microbiol Methods. 2004 May;57(2):259-68
15063066
Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
9396791
Nucleic Acids Res. 2007;35(19):e127
17897966
AIHA J (Fairfax, Va). 2002 Mar-Apr;63(2):178-83
11975654
Appl Environ Microbiol. 2005 Apr;71(4):2190-4
15812058
Microbiology (Reading). 2002 Jan;148(Pt 1):257-266
11782518
Curr Microbiol. 2010 Apr;60(4):294-300
19936829
Microb Ecol. 1987 Mar;13(2):103-14
24213209
Mol Cell Probes. 2004 Feb;18(1):67-73
15036372
Microbiology (Reading). 2000 Oct;146 ( Pt 10):2385-2394
11021915
AIHAJ. 2000 Nov-Dec;61(6):788-97
11192210
Environ Microbiol. 2003 Jun;5(6):453-61
12755712