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Fluorescent detection‐polymerase chain reaction (FD‐PCR) assay on microwell plates as a screening test for salmonellas in foods
Author(s) -
Cano R.J.,
Rasmussen S.R.,
Fraga Gloria Sánchez,
Palomares J.C.
Publication year - 1993
Publication title -
journal of applied bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 0021-8847
DOI - 10.1111/j.1365-2672.1993.tb02773.x
Subject(s) - polymerase chain reaction , fluorescence , real time polymerase chain reaction , salmonella , microbiology and biotechnology , pcr test , chemistry , biology , chromatography , bacteria , genetics , gene , physics , quantum mechanics
This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100‐extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS 200 . The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55°C, the microwells were washed and an alkaline phosphatase‐labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 μl of 1 mmol 1 ‐1 AttoPhos TM (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1–10 cfu. A total of 172 food samples were tested, both by culture and FD‐PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD‐PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD‐PCR. assay described here can be useful to screen a large number of food samples for contamination by salmonellas.