Development and evaluation of a quantitative real-time PCR assay for the detection of saltwater Bacteriovorax.
Bdellovibrio-and-like-organisms (BALOs) are small, Gram-negative predatory bacteria with the ability to prey on a wide variety of Gram-negative bacteria, and which may have a significant ecological role. Detection and quantification of BALOs by culture-dependent methods are complicated, as their reproduction is dependent upon the use of appropriate prey. For this reason, a sensitive and specific molecular detection method was developed. This paper describes a SYBR Green-based real-time PCR (quantitative PCR) assay that combines the use of a specific 16S rDNA primer with a universal primer for quantitative detection of halophilic Bacteriovorax. 16S rDNA sequences from 174 BALO strains, including both halophilic and freshwater, were aligned and a consensus region was identified that is unique to the halophilic Bacteriovorax strains. A specific primer was designed and analysed for specificity. The PCR conditions were optimized to obtain high specificity and sensitivity. The specificity was evaluated by testing a series of halophilic Bacteriovorax samples and prey specimens, including both pure cultures and environmental saltwater samples. A linear and reproducible standard curve was obtained over a range of 10(1)-10(6) gene copies and the detection limit was determined to be 10 copies of 16S rRNA gene per reaction. The results presented in this study validate the procedure as a rapid, sensitive and accurate method for the detection and quantification of halophilic Bacteriovorax in environmental saltwater samples. It is anticipated that this culture-independent method will facilitate future investigations of the distribution and population dynamics of these interesting predatory bacteria, leading to a better understanding of their ecological role.
Zheng
Guili
G
School of Medicine, University of Maryland Baltimore, Baltimore, MD 21201, USA.
Wang
Cynthia
C
Williams
Henry N
HN
Pineiro
Silvia A
SA
D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D020564:Deltaproteobacteria / Q000302:isolation & purification*; D003959:Diamines; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D052160:Benzothiazoles; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I