TaqMan, or both: highly sensitive, non-invasive detection of Cardicola blood fluke species in southern bluefin tuna ( Thunnus maccoyii )
2015
Three species of blood fluke from the genus Cardicola are known to parasitize and cause disease in
Bluefin Tunas – C. forsteri , C. orientalis , and C. opisthorchis. Although initially believed to be
separated by geography and host specificity, recent identification of at least two Cardicola spp.
concurrently present within all three Bluefin species has raised questions concerning pathogenicity,
relative abundance, and distribution of these parasites within Bluefin populations. Here, we present sensitive and differential real-time qPCR nucleic acid detection of these Cardicola
spp. by targeting the ITS2 region of the parasite rDNA for PCR amplification. A limit of sensitivity
was achieved to be between 1-5 genome copy equivalents for each of the three Cardicola species
tested without cross-species or host genomic amplification. Similar sensitivity was further achieved
in the presence of up to 20 ng/μL non-target host gDNA using SYBR Green chemistry alone, or in
the presence of up to 160 ng/μL host gDNA through the utilization of a TaqMan probe common-reporter
detection system. These methods were subsequently used to positively identify both C.
forsteri and C. orientalis DNA in preserved samples of serum, gill, and heart from ranched
Southern Bluefin Tuna Thunnus maccoyii . Both methods were more sensitive for positively and
differentially identifying the presence of Cardicola spp. than either histological or heart-flush
microscopy techniques previously employed, and also possess the ability to be applied in non-lethal
blood sampling of these highly-valued fish. This is the first report for rapid and differential molecular quantitative detection of Cardicola , and
opens the potential for effective monitoring of infection in cultured bluefin populations. Further, it
is anticipated that the use of SYBR Green for melt-curve analyses in conjunction with a common-reporter
TaqMan assay will present a flexible, accurate, and cost-effective approach for differential
detection of a variety of other pathogens in future.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
0
References
4
Citations
NaN
KQI