Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Abstract

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan ®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan ® MGB primer–probe pair using the Allele ID ® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer–probe pair’s sensitivity was 10 1 colony forming units (CFU) ml −1. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10 4 and 10 6 CFU ml −1. The designed TaqMan ® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.