ABSTRACT

DETERMINATION OF PRESENCE/ABSENCE OF LISTERIA MONOCYTOGENES

BY REAL-TIME PCR IN FROZEN FISH DESTINED FOR EXPORTATION AT A
FISH PROCESSING PLANT, 8TH REGION - CHILE.

GARCES-AVILES, R1‡; ESTEBAN, D.1 and FRIAS, J.2

1.
Veterinary Faculty. Univ. San Sebastian. Gral Cruz Str. Nº 1577. Campus Concepción. Chile
2.
School of Biotechnology. Faculty of Engineering and Technology. Univ. San Sebastian.
Gral Cruz Str. Nº 1577. Campus Concepción. Chile
‡
Corresponding author rgarces@uss.cl rgarces@mail.com

From different areas of Jack Mackerel (Trachurus declivis) five samples of approximately 5 g
were taken. A total of 25 g sample was obtained. Each sample was placed in a 250 mL sterile
bottle contained 225 mL of Half Frasier Broth. Bottle flask was sealed and incubated at 37 °C/
24 h. Post-incubation the samples were manually shaken to achieve a uniform mixture. Two
samples of 1 mL each were taken. Each sample was placed in 1 mL Eppendorf tube. One of
the tubes was properly identified as backup template sample and stored at –20 °C. The other
tube was processed for DNA extraction using the DNeasy Plant Mini Method.
A pure culture sample of L. monocytogenes (ATCC 7644) as positive control was used.
Positive control DNA extraction was performed with the same methodology as the fish
samples. The Loop was cultivated in Half Frasier Broth at 37 °C/ 24 h. Negative controls
were: a) PCR water-DEPC; b) PCR water-DEPC + primers and c) Brilliant SYBR Green
QPCR Master Mix.
Target gene was listeriolysinO (hlyA). The primers were designed and acquired at Tib
Molbiol. Primers concentrations were 200, 100 and 50 nM. Each reaction mixture (25 µL)
contained 12.5 µL of Master Mix, 1.25 µL upstream primer, 1.25 µL downstream primer and
1 µL DNA template (sample) or purified DNA (positive controls). Nuclease free water for
PCR - DEPC was added to complete 25 µL.
PCR reaction was performed on an Applied Biosystems 7300 Real-Time PCR System.
Temperature profile used was: Hold cycle, 95 °C /10 min. followed by 40 cycles. Each cycle
included: 1) denaturation: 30seg/95 °C; 2) annealing: 1 min/55-60 °C and 3) extension: 1 min/
71 °C.
Listeria monocytogenes was absenting in 25g of frozen fish in all sampled lots.
According to the hygienic quality of frozen fish samples is possible to suggest them as food
safety products. Study’s hypothesis was therefore accepted.

Keywords: Listeriosis, DNA, Polymerase, Mackerel.