A method previously developed for direct (non-enrichment) detection of Escherichia coli O157:H7 was adapted for Listeria monocytogenes. The sample treatment protocol was successful in concentrating bacteria from 10 mL raw milk samples and reducing PCR inhibition, but qPCR detection sensitivity and reproducibility was poor. Two DNA extraction reagents and multiple extraction conditions were tested to identify an efficient and reproducible DNA extraction method. Two primer/probe sets were evaluated at two concentrations and three annealing temperatures to minimize false-positive results and optimize sensitivity and reproducibility of qPCR detection. Under the selected conditions, DNA was extracted efficiently from the entire milk sample in a volume of 10 mL, and subsequently quantitated by a 50 nuclease qPCR assay lasting 50 min. The method provided detection of 1 cfu mL1 L. monocytogenes in 10 mL raw milk and quantitation from 10 to 1000 cfu mL1 with a total time to result of <3 h.
Moushumi Paul, Gian Marco Baranzoni, Sabrina Albonetti, Jeffrey D. Brewster (2015). Direct, quantitative detection of Listeria monocytogenes in fresh raw whole milk by qPCR. INTERNATIONAL DAIRY JOURNAL, 41, 46-49 [10.1016/j.idairyj.2014.09.008].
Direct, quantitative detection of Listeria monocytogenes in fresh raw whole milk by qPCR
ALBONETTI, SABRINA;
2015
Abstract
A method previously developed for direct (non-enrichment) detection of Escherichia coli O157:H7 was adapted for Listeria monocytogenes. The sample treatment protocol was successful in concentrating bacteria from 10 mL raw milk samples and reducing PCR inhibition, but qPCR detection sensitivity and reproducibility was poor. Two DNA extraction reagents and multiple extraction conditions were tested to identify an efficient and reproducible DNA extraction method. Two primer/probe sets were evaluated at two concentrations and three annealing temperatures to minimize false-positive results and optimize sensitivity and reproducibility of qPCR detection. Under the selected conditions, DNA was extracted efficiently from the entire milk sample in a volume of 10 mL, and subsequently quantitated by a 50 nuclease qPCR assay lasting 50 min. The method provided detection of 1 cfu mL1 L. monocytogenes in 10 mL raw milk and quantitation from 10 to 1000 cfu mL1 with a total time to result of <3 h.File | Dimensione | Formato | |
---|---|---|---|
Albonetti.pdf
accesso aperto
Tipo:
Versione (PDF) editoriale
Licenza:
Licenza per Accesso Aperto. Creative Commons Attribuzione - Non commerciale - Non opere derivate (CCBYNCND)
Dimensione
986.35 kB
Formato
Adobe PDF
|
986.35 kB | Adobe PDF | Visualizza/Apri |
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.