Evaluation of yeast and mould contamination of industrial pastries by qPCR

The objective of the study conducted by ADNucleis at the request of Groupe O* is to evaluate an automated detection method (including DNA extraction-quantification and quantitative PCR) of microorganisms (yeasts and moulds) that can contaminate industrial pastries or alter their organoleptic qualities.

To date, the industrial reference method used for the detection of fungal contamination is based on culture on Petri dishes containing Sabouraud agar with chloramphenicol added. It requires an incubation period of 3 to 7 days at 28°C in order to identify the presence or absence of colonies.

The automated method of the Séquence PRO robot makes it possible to detect these contaminants even in very small quantities (less than 10 copies) by detecting their DNA using quantitative PCR or qPCR (Quantitative Polymerase Chain Reaction).

The use of this machine in the food industry for the identification of contaminating microorganisms is in full development thanks to its major advantage: its speed and simplicity. Indeed, the results of the amplification of the target DNA sought are obtained in less than four hours and allow a significant saving of time for a release of the finished products; possibly in the event of nonconformity, it authorises a fast implementation of the necessary corrective measures.

To meet the study requested by the O Group, several extraction and amplification kits were tested and developed.

In addition, a correlation study between the “Ct” (thermal cycle) values obtained in qPCR and the number of CFU (Colony Forming Units) obtained on Petri dishes was carried out. This comparative study makes it possible to determine precisely at what “Ct” level the finished products can be released or returned, within a short period of time of a few hours.

Several types of kits were selected and developed for this study: 2 broad spectrum kits and 2 specific kits.

The first step in this study was to specifically identify the microorganisms that could be isolated on the production lines. This step allowed us to develop a relevant study model in relation to the problems actually encountered in an industrial environment and to select the most effective detection kit.

These results showed the importance of using broad-spectrum kits given the variability of the contaminants.

For this study, Adnucleis developed different sampling and pre-treatment protocols for the samples in order to achieve a homogeneous extraction of nucleic acids. Our results show that qPCR is able to detect viable contaminating cells that traditional Petri methods do not detect.

The second part of our work consisted in establishing a correlation between the results obtained via the traditional Petri dish method and the results provided by the SEQUENCE PRO in qPCR.

The results of the study show that qPCR is more sensitive than the traditional Petri dish methods, probably due to its wider spectrum of detection of the desired targets.

The search for very low contamination, 1 to 2 colonies per g of sample, can lead to the use of an enrichment step of a few hours: this method allows to obtain more discriminating results for the detection of fungal contaminants.

In conclusion, the use of the qPCR method, possibly coupled with enrichment, makes it possible to obtain a high level of sensitivity to guarantee the quality of the pastry while improving its shelf life. The sensitivity of the method makes it possible to better define or even increase the shelf life while guiding manufacturers towards better production practices.

Yannick Lequette, molecular biology expert at Adnucleis

* For confidentiality reasons, we will not mention the name of the O group.

PCR versus petri dish

Real-time PCR is known to allow the isolation, identification and quantification of DNA from bacteria, viruses, parasites, allergens, GMOs in a very short time of about 1 to 3 hours in all fields: food hygiene, animal health and/or human health, cosmetic industries…

For a long time restricted for price reasons to the detection of a few pathogens in food hygiene or to short-lived products, the objective of Professor Michel Franck, CEO of ADNucleis, is to broaden the indications of this PCR technique to the identification and quantification of traditional hygiene indicators (Total Flora, E.coli, coliforms, Pseudomonas spp., Listeria spp., molds, yeasts…) and/or pathogens (Salmonella, Listeria monocytogenes, Cronobacter sakazakii, STEC such as E.coli O157H7, viruses, parasites…). This objective is now achieved thanks to a new “breakthrough” method at a price equivalent to the price of cultural methods.

Associated with the SEQUENCE PRO PCR robot – much less expensive than traditional pipetting machines – a compact robot (65cm x 65cm) that can be directly positioned on production lines, real-time PCR analysis can be performed by personnel not qualified in molecular biology, in situ and in real-time.

Thanks to its industrial partners such as Pasquier, ADNucleis continues to make real-time PCR an industrial standard, notably by simplifying and robotizing the entire analytical process.

Adnucleis