Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods.
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 IS200. 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 degrees 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 microliters of 1 mmol 1(-1) AttoPhos (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.
Cano
R J
RJ
Biological Sciences Department, California Polytechnic State University, San Luis Obispo 93407.
Rasmussen
S R
SR
Sánchez Fraga
G
G
Palomares
J C
JC
D000469:Alkaline Phosphatase; D000818:Animals; D002417:Cattle / Q000382:microbiology; D002614:Chelating Agents; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D005470:Fluorometry; D005504:Food Analysis / Q000295:instrumentation / Q000379:methods*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D008460:Meat / Q000382:microbiology; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011200:Poultry / Q000382:microbiology; D012117:Resins, Synthetic; D012475:Salmonella / Q000302:isolation & purification*; D012478:Salmonella Food Poisoning / Q000517:prevention & control; D012680:Sensitivity and Specificity; D013552:Swine / Q000382:microbiology
D002614:Chelating Agents; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012117:Resins, Synthetic; C024997:Chelex 100; D000469:Alkaline Phosphatase