Construction of internal control for quantitation of Vibrio parahaemolyticus DNA with the PCR

Lee, C.Y.; Chen, C.N.; Sheu, D.S.

Journal of the Chinese Agricultural Chemical Society 33(4): 504-513


ISSN/ISBN: 0578-1736
Accession: 008379640

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An internal control DNA was constructed for quantitation of V. parahaemolyticus DNA with PCR. The internal control DNA was generated by amplification of a 400 bp of tdh gene fragment using two composite primers. Each composite primer had a target gene primer sequence attached to the tdh gene primer sequence. Serial dilutions of the known amount of an internal control DNA were coamplified with a constant amount of target DNA. Following PCR, the amount of products amplified by the internal control DNA and target DNA were compared. Logarithmic values of the molecules of internal control before amplification and their corresponding ratio of target DNA and internal control DNA products were plotted to generate a standard curve. A ratio of the PCR products as 1 could then be used to determine the initial quantity of target DNA. This constructed internal control could overcome the difficulty of possibly present inhibitors on the PCR. Furthermore, the internal control could be linked to a previously reported PCR-ELOSA system to accurately quantify the amount of V. parahaemolyticus DNA in the various samples.