Evaluation of DNA preparation methods combined with different PCR-based assays for Coxiella burnetii detection in milk
2016
Capuano, F. | Proroga, Y. T. R. | Mancusi, A. | Perugini, A. G. | Berri, Mustapha
Introduction - Coxiella burnetii (Cb) is the causative agent of Q fever, a zoonosis that occurs worldwide. Due to health concerns, unpasteurized cow's milk and a number of dairy products produced by unpasteurized milk may contain virulent Cb. PCR method is commonly employed for sensitive, specific and rapid test for Cb detection in biological samples including milk. Aim - In this study, six DNA purification methods for recovering Cb DNA from experimentally contaminated cow's milk were evaluated, together with three PCR-based assays targeting the IS 1111 Cb-repeated element. Materials and methods - For DNA extraction, the cetyltrimethylammonium bromide method was implemented and the following commercial kits were used: QIAamp DNA Mini kit; DNeasy Mericon Food kit; NucliSENS miniMAG; NucleoSpin Food; Wizard Genomic DNA Purification Kit. The three assays considered were standard PCR, TaqMan real-time PCR and SYBR Green combined with the evaluation of the melting temperature of the amplicon. Results and discussion - The best extraction methods, QIAamp DNA Mini kit, DNeasy Mericon Food kit and NucleoSpin Food, combined with the TaqMan real-time PCR assay, allowed us to detect the presence of 5 Cb cells per mu L of milk. Conclusion - The analysis of bulk milk seems to be a suitable means of monitoring the Q fever health condition in cows' herds, as long as efficient extraction methods and sensitive amplification assays are used.
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