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Detection of Koi herpesvirus: impact of extraction method, primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations
Author(s) -
Meyer Kirsten,
Bergmann Sven Michael,
van der Marel Marian,
Steinhagen Dieter
Publication year - 2012
Publication title -
aquaculture research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 89
eISSN - 1365-2109
pISSN - 1355-557X
DOI - 10.1111/j.1365-2109.2011.02897.x
Subject(s) - biology , primer (cosmetics) , polymerase chain reaction , primer dimer , taq polymerase , hot start pcr , microbiology and biotechnology , dna polymerase , dna extraction , dna , polymerase chain reaction optimization , virology , genomic dna , polymerase , multiplex polymerase chain reaction , genetics , gene , chemistry , thermus aquaticus , organic chemistry
Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV ‐infected koi, one KHV ‐infected common carp, one KHV ‐infected goldfish and one non‐infected common carp. DNA was extracted with DNA zol Reagent, High Pure PCR Template DNA Preparation Kit and QIA amp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets – KHV ‐F and ‐R, KHV ‐Gray‐2F and ‐2R and KHV ‐ TK f and ‐ TK r – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.