Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis

Esteban Soto Martinez, Kimberly Bowles, Denise Fernandez, John P. Hawke

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Members of the genus Francisella are small Gram-negative facultative intracellular bacteria that cause francisellosis in a wide variety of fish species worldwide. F. noatunensis subsp. orientalis has been recently described as a warm-water pathogen of tilapia Oreochromis spp. In this study, a quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay was developed to rapidly and accurately detect and quantify F. noatunensis subsp. orientalis from fish tissue. The target region of the assay was the F. tularensis iglC gene homologue previously found in F. noatunensis subsp. orientalis. Probe specificity was confirmed by the lack of signal and cross-reactivity with 12 common fish pathogens, 2 subspecies of F. tularensis, F. noatunensis subsp. noatunensis, and tilapia tissue. The range of linearity was determined to be 50 fg to 1.4 mg, and the lower limit of detection was 50 fg of DNA (equivalent to ∼25 genome equivalents) per reaction. A similar sensitivity was observed with DNA extracted from a mixture of F. noatunensis subsp. orientalis and fish tissue. The assay was also able to detect and quantify F. noatunensis subsp. orientalis from the spleens of experimentally infected tilapia. No signal was observed in the control groups. In conclusion, we have developed a highly sensitive and specific assay that can be used for the specific identification and quantification of F. noatunensis subsp. orientalis.

Original languageEnglish (US)
Pages (from-to)199-207
Number of pages9
JournalDiseases of Aquatic Organisms
Volume89
Issue number3
DOIs
StatePublished - Apr 9 2010
Externally publishedYes

Fingerprint

Francisella
quantitative polymerase chain reaction
pathogen
assay
pathogens
assays
tilapia (common name)
fish
probe
Tilapia (Cichlidae)
Oreochromis
DNA
linearity
warm water
cross reaction
polymerase chain reaction
subspecies
spleen
detection limit
genome

Keywords

  • Francisella
  • QPCR
  • Tilapia

ASJC Scopus subject areas

  • Aquatic Science
  • Ecology, Evolution, Behavior and Systematics

Cite this

Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis. / Soto Martinez, Esteban; Bowles, Kimberly; Fernandez, Denise; Hawke, John P.

In: Diseases of Aquatic Organisms, Vol. 89, No. 3, 09.04.2010, p. 199-207.

Research output: Contribution to journalArticle

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