Development of a quantitative real-time PCR for the detection of Tenacibaculum maritimum and its application to field samples.
The development and the application of a quantitative real-time PCR for the detection of Tenacibaculum maritimum are described. A set of primers and probe was designed to amplify a 155-bp fragment specific to the T. maritimum 16S rRNA gene. The test was shown to be very sensitive, able to detect as little as 4.8 DNA copies number μL(-1) . In addition, the assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R(2)  = 0.999) extending over 6 log(10)  dilutions and a high efficiency (100%). The assay was applied to DNA samples extracted from 48 formalin-fixed paraffin-embedded (FFPE) Atlantic salmon, Salmo salar, gill tissues showing varying degrees of gill pathology (scored 0-3) and from 26 jellyfish samples belonging to the species Phialella quadrata and Muggiaea atlantica. For each sample, the bacterial load was normalised against the level of the salmonid elongation factor alpha 1 (ELF) detected by a second real-time PCR using previously published primers and probe. Tenacibaculum maritimum DNA was detected in 89% of the blocks with no signs of gill disease as well as in 95% of the blocks with mild-to-severe gill pathology. Association between bacterial load and gill pathology severity was investigated. T. maritimum DNA was detected at low level in four of the 26 jellyfish tested.
Veterinary Sciences Division, Agri-food and Biosciences Institute of Northern Ireland, Stormont, Belfast, UK. elena.fringuelli@afbini.gov.uk
E
E
Fringuelli
P D
PD
Savage
A
A
Gordon
E J
EJ
Baxter
H D
HD
Rodger
D A
DA
Graham
D000818:Animals; D058491:Bacterial Load; D004784:Environmental Monitoring; D005393:Fish Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology; D045826:Flavobacteriaceae Infections / Q000175:diagnosis / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D005880:Gills / Q000382:microbiology / Q000473:pathology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020317:Salmo salar; D037401:Scyphozoa / Q000382:microbiology*; D060493:Tenacibaculum / Q000235:genetics*
D012336:RNA, Ribosomal, 16S