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Duplex real-time PCR method with internal amplification control for quantification of verrucosidin producing molds in dry-ripened foods.

Abstract：

:Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain.

journal_name

Int J Food Microbiol

journal_title

International journal of food microbiology

authors

Rodríguez A,Córdoba JJ,Werning ML,Andrade MJ,Rodríguez M

doi

10.1016/j.ijfoodmicro.2011.10.020

subject

Has Abstract

pub_date

2012-02-01 00:00:00

pages

85-91

issue

1-2

eissn

0168-1605

issn

1879-3460

pii

S0168-1605(11)00630-1

journal_volume

153

pub_type

杂志文章

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Duplex real-time PCR method with internal amplification control for quantification of verrucosidin producing molds in dry-ripened foods.