PMID- 20495940
OWN - NLM
STAT- MEDLINE
DCOM- 20110302
LR  - 20211020
IS  - 1476-5535 (Electronic)
IS  - 1367-5435 (Linking)
VI  - 37
IP  - 9
DP  - 2010 Sep
TI  - Quantitative real-time PCR and fluorescence in situ hybridization approaches for 
      enumerating Brevundimonas diminuta in drinking water.
PG  - 909-18
LID - 10.1007/s10295-010-0738-1 [doi]
AB  - Brevundimonas diminuta is a small Gram-negative bacterium used for validation of 
      membranes and filters used in the pharmaceutical and drinking water treatment 
      industries. Current assays are time consuming, nonselective, and may be subject 
      to interference by competing indigenous microorganisms. The focus of this study 
      is to develop rapid and specific enumeration methodologies for B. diminuta. 
      Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ 
      hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD 
      (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and 
      probes were designed, and a 100-200 bp segment of each gene was targeted in the 
      qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was 
      selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole 
      Quencher). Probe specificity studies, conducted against Gram-negative and 
      Gram-positive reference strains as well as environmental strains, revealed high 
      specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR 
      reactions using purified genomic DNA from B. diminuta were determined to be 0.89 
      pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta 
      suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest 
      sensitivity observed for B. diminuta was 1 x 10(3) colony forming units (CFU) per 
      mL when tryptic soy broth was used as the growth medium. When compared with 
      direct microscopic enumeration using a 5' 6-FAM FISH probe, traditional plating 
      methods showed significant underestimation of B. diminuta concentration (P = 
      0.01) when this organism was cultivated in saline lactose broth. The results of 
      this investigation demonstrate that qPCR and FISH are effective methods for rapid 
      (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when 
      validating drinking water filtration systems.
FAU - Donofrio, Robert S
AU  - Donofrio RS
AD  - NSF International, 789 Dixboro Road, Ann Arbor, MI 48105, USA. Donofrio@nsf.org
FAU - Bestervelt, Lorelle L
AU  - Bestervelt LL
FAU - Saha, Ratul
AU  - Saha R
FAU - Bagley, Susan T
AU  - Bagley ST
LA  - eng
PT  - Journal Article
DEP - 20100522
PL  - Germany
TA  - J Ind Microbiol Biotechnol
JT  - Journal of industrial microbiology & biotechnology
JID - 9705544
RN  - 0 (Bacterial Proteins)
RN  - 0 (Sigma Factor)
RN  - EC 5.99.1.3 (DNA Gyrase)
SB  - IM
MH  - Bacterial Load/*methods
MH  - Bacterial Proteins/genetics
MH  - Caulobacteraceae/*isolation & purification
MH  - DNA Gyrase/genetics
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Polymerase Chain Reaction/*methods
MH  - Sensitivity and Specificity
MH  - Sigma Factor/genetics
MH  - *Water Microbiology
MH  - *Water Supply
EDAT- 2010/05/25 06:00
MHDA- 2011/03/03 06:00
CRDT- 2010/05/25 06:00
PHST- 2010/03/05 00:00 [received]
PHST- 2010/04/28 00:00 [accepted]
PHST- 2010/05/25 06:00 [entrez]
PHST- 2010/05/25 06:00 [pubmed]
PHST- 2011/03/03 06:00 [medline]
AID - 10.1007/s10295-010-0738-1 [doi]
PST - ppublish
SO  - J Ind Microbiol Biotechnol. 2010 Sep;37(9):909-18. doi: 
      10.1007/s10295-010-0738-1. Epub 2010 May 22.