PCR-based procedures in detection and DNA-fingerprinting of Salmonella from samples of animal origin |
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Authors: | A del Cerro S M Soto E Landeras M A Gonzlez-Hevia J A Guijarro M C Mendoza |
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Affiliation: | a Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, c/Julián Clavería 6, 33006, Oviedo, Spain;b Laboratorio de Salud Pública, Consejería de Sanidad del Principado de Asturias, Carretera del Rubín s/n, 33001, Oviedo, Spain |
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Abstract: | The usefulness of selected PCR-protocols for the detection of Salmonella in 117 samples of animal origin (17 raw minced meat, 27 raw chicken meat, 8 raw sausages, and 25 egg samples, as well as 18 poultry faecal, and caecal swabs samples) and DNA-fingerprinting typing is shown. To establish an accurate PCR-procedure for Salmonella detection the following parameters were evaluated: two pre-PCR concentration procedures, centrifugation and immunomagnetic separation (IMS) using Dynabeads anti- Salmonella; the specificity and sensitivity of 10 sets of primers; and different conditions of the amplification reaction. In light of the results obtained from the use of PCR-based procedures alone or in combination with conventional methods, the following findings can be underlined: First, IMS is more efficient than centrifugation in the recovery of Salmonella. Second, the selected IMS/PCR-detection protocol is less time-consuming (45 h) than the IMS/culture procedure (90 h), and a good concordance between them was found when the Kappa coefficient was calculated (0·87). Third, PCR-ribotyping technique showed a very low discrimination power, being able to differentiate only three profiles. Fourth, RAPD technique using specific primers supports previous works in which it was proposed as a simple and useful tool for discriminating isolates between and within serotypes. Fifth, The efficiency, rapidity, and flexibility of the PCR-protocols applied were high, and they can be performed using two PCR-programs and the same basic equipment. |
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