Effective qPCR methodology to quantify the expression of virulence genes in Aeromonas salmonicida subsp salmonicida

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Rivera, L. (author)

Effective qPCR methodology to quantify the expression of virulence genes in Aeromonas salmonicida subsp salmonicida

Article/chapterEnglish2015

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2015-01-26
Oxford University Press (OUP),2015
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LIBRIS-ID:oai:DiVA.org:umu-102103
https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-102103URI
https://doi.org/10.1111/jam.12740DOI

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Language:English
Summary in:English

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Aims This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida). Methods and Results Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns. Conclusions The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency. Significance and Impact of the Study The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.

Subject headings and genre

MEDICIN OCH HÄLSOVETENSKAP Medicinska och farmaceutiska grundvetenskaper Mikrobiologi inom det medicinska området hsv//swe
MEDICAL AND HEALTH SCIENCES Basic Medicine Microbiology in the medical area hsv//eng
MEDICIN OCH HÄLSOVETENSKAP Medicinsk bioteknologi Medicinsk bioteknologi hsv//swe
MEDICAL AND HEALTH SCIENCES Medical Biotechnology Medical Biotechnology hsv//eng
NATURVETENSKAP Biologi Mikrobiologi hsv//swe
NATURAL SCIENCES Biological Sciences Microbiology hsv//eng
Aeromonas salmonicida subsp
salmonicida
gene expression
normalization
qPCR
reference genes

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Lopez-Patino, M. A. (author)
Milton, Debra L.Umeå universitet,Institutionen för molekylärbiologi (Medicinska fakulteten)(Swepub:umu)demi0001 (author)
Nieto, T. P. (author)
Farto, R. (author)
Umeå universitetInstitutionen för molekylärbiologi (Medicinska fakulteten) (creator_code:org_t)

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In:Journal of Applied Microbiology: Oxford University Press (OUP)118:4, s. 792-8021364-50721365-2672

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