A culture-independent and two culture-dependent real time PCR approaches were created to quantitatively identify L. monocytogenes in raw milk and soft cheeses.
The researchers investigated the suitability of a direct culture-independent rt-PCR approach versus two rt-PCR culture-dependent approaches, combining a liquid- or a solid-based enumeration with rt-PCR protocol.
Combination of culture-dependent methods with rt-PCR improved the limit of quantification and lowered the time of analysis compared to standard quali-quantitative protocols.
Researchers set up a TaqMan rt-PCR, targeting a 138 bp region of the hlyA gene, which encodes one of the most important and specific virulent factors for L. monocytogenes, the sulfhydryl-activated pore-forming cytolysin Listeriolisin O (LLO).
Culture independent and culture dependant
This was used directly (culture-independent approach) or after a liquid or solid-based cultivation step (culture-dependent approach) for the rapid identification and quantification of L. monocytogenes.
Applicability of their rt-PCR approaches was investigated by screening 13 real raw milk and 10 soft cheese samples for the presence of L. monocytogenes.
Rt-PCR was combined with a liquid- (MPN technique) or a solid- (ALOA and PALCAM) based enumeration.
The robustness and reliability of the rt-PCR assay is strengthened by the absence of cross reaction with the control strains tested, including L. innocua, the closest L. monocytogenes's relative, according to the study.
The MPN/rt-PCR approach was more sensitive than the solid-based culture-dependent rt-PCR assay.
MPN/rt-PCR best approach
MPN/rt-PCR was the best approach to enumerate low levels of L. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification was more effective than the PALCAM-based.
The rt-PCR culture-independent sensitivity in raw milk was higher than in soft cheeses.
Due to its limit of quantification, only up to 3.63 log CFU/g of raw milk could be quantitatively detected by the culture-independent rt-PCR approach.
The plate count-based rt-PCR approach allowed the detection of up to 1.61 and 2.61 CFU of L. monocytogenes per mL of raw milk, respectively, using PALCAM and ALOA media.
Advantages of this approach can be summarized as cell recovery on a liquid medium is usually better than that on a solid medium, easy lecture of results (in terms of growth/non growth) and speed.
The DNA-based confirmation of presumptive L. monocytogenes isolates, with an overall confirmation time of about six hours (including the DNA extraction) is less time- and labour-consuming than the reference method.
With biochemical identification and serological confirmation this takes five days for a negative result and up to 10 days to confirm a positive result.
“Our method demonstrated a high specificity and good diagnostic sensitivity even when complex matrices such as raw milk and soft cheeses, with a numerous background microflora, were used,” said the researchers.
“However, the combination of culture-dependent methods with rt-PCR allowed to improve the limit of quantification and to noticeably lower the time of analysis compared to standard quali-quantitative protocols for the detection and enumeration of L. monocytogenes in milk and milk-based products.”
Source: LWT - Food Science and Technology
Online ahead of print, DOI: 10.1016/j.lwt.2014.03.005
“Quantitative detection of Listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid- and solid-based culture-dependent real time PCR approaches “
Authors: Grazia Marina Quero, Elisa Santovito, Angelo Visconti, Vincenzina Fusco