Development of a rapid procedure of real-time PCR to detect Listeria monocytogenes in cheese

From January 2015 to March 2016, an outbreak of 23 human cases of listeriosis in the Marche region and one human case in the Umbria region of Italy was caused by Listeria monocytogenes strains showing a new pulsotype never described before in Italy. A total of 37 clinical strains isolated from patients exhibiting listeriosis symptoms and 1374 strains correlated to the outbreak were received by the Italian National Reference Laboratory for L. monocytogenes (It NRL Lm) of Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) for outbreak investigation. A real-time PCR assay was purposely designed for a rapid screening of the strains related to the outbreak. PCR-positive strains were successively typed through molecular serogrouping, pulsed field gel electrophoresis (PFGE), and Next Generation Sequencing (NGS). Applying the described strategy, based on real-time PCR screening, we were able to considerably reduce time and costs during the outbreak investigation activities.

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Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probe-based real-time PCRs

Journal of Veterinary Research ◽

10.1515/jvetres-2017-0069 ◽

2017 ◽

Vol 61 (4) ◽

pp. 427-432 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Marek Krupa ◽

Anna Szczawińska ◽

Monika Reksa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Biological Samples ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye

AbstractIntroduction:The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene ofListeriaspp. and thehlyA gene ofListeria monocytogenesin biological samples of the liver, brain, and blood.Material and Methods:Five strains ofL. monocytogenesand single strains of each speciesL. ivanovii,L. innocua,L. grayi,L. welshimeri,andL. seeligeriwere used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification thehlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging toListeriaspp. andL. monocytogeneswere conducted.Results:The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA andhlyA genes which confirm their belonging toListeriaspp. andL. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.Conclusion:Both real-time PCR methods for the detection ofListeriaspp. andL. monocytogenesin biological samples demonstrated a significant sensitivity and high specificity.

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