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Evaluation of yeast and mould contamination of industrial pastries by qPCR

Evaluation of yeast and mould contamination of industrial pastries by qPCR

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.

Does your company want to carry out a study? Do you want to set up a new sanitary control protocol? Please fill in the following form or contact our laboratory directly on +33(0)4 78 56 79 36

qPCR versus petri dish

qPCR versus petri dish

PCR versus petri dishReal-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,...

The qPCR to fight against the pathological risks of our bees

Until the end of the fifties, beekeeping was essentially a family beekeeping organized in small non-transhumant apiaries. The “flight hole” observation was the absolute reference to evaluate the health of a colony. The bees of autochthonous races in balance with their environment, which was then rich in biodiversity, were carriers of only a limited number of viruses (1 to 2), the main pathogens being rather bacterial (foulbrood) and fungal (nosemosis). Since the fifties, beekeeping practices have evolved and the mobility of the flocks has become the rule both in transhumance to ensure the harvest of honey and in the worldwide exchange of genetics.

Viral carriage has increased with no less than thirty-two viruses identified to date, with the same bee being able to harbor 7 to 8 different viruses. The dominant presence of Nosema apis, which is not very pathogenic, to the benefit of Nosema ceranae, which is much more deleterious, constitutes a new serious threat to the health of colonies.

What research has already shown:

The scientific literature on the multiple roles of pathogens is particularly abundant and we will limit ourselves to a few of the most decisive for beekeeping practices.

Nosema ceranae

Its presence in the gut can at any time cause the collapse of the colony:

– either by the development of the pathogenic fungus in case of dearth, especially of protein dearth or in case of indigestibility of the sugars offered to the bees

– or by collapse of the immune system in synergy with pesticides

– or by stimulation of the viral populations.

About viruses

Many facts must be kept in mind: the transmission of viruses can be vertical:

-all queens carry one or more viruses that they transmit to their offspring, the same observation is made about spermatozoa,

-the nurse bees excrete a lot of virus through their hypopharyngeal glands and thus contaminate the larvae,

– Varroa destructor disseminates a large number of viruses, either by passive carriage or as a multiplication host,

– Many viruses are not expressed directly but induce the development of other viruses present. Thus the virus of deformed wings may not produce alterations in the bees but may cause symptoms of sacciform brood because it has stimulated the multiplication of S.B.V. viruses.

– severe symptoms of contamination appear with a thousand times less virus particles if the penetration is done by the hemolymph route rather than by the oral route.

Efficiency of analytical means

The great revolution of the last few years has been the development of a new analytical method, qPCR (Polymerase Chain Reaction), which makes it possible to identify a pathogen by its DNA or RNA (RT-PCR).

The improvement of the different steps of the analysis and the multiplication of the primers allow to identify any living being in only a few hours.

Solu’Nature has partnered with the DNAucleis Laboratory to offer a PathoBEE 1 analytical pack that can routinely identify the presence of 10 pathogens.

Fungi Nosema apis and Nosema ceranae
Bacteria American foulbrood Paenibacillus larvae
APV (=ABPV) Acute paralysis virus
SBV Sacciform brood virus
CBPV Chronic paralysis virus = black disease
DWV Deformed wing virus phenotype A
VDV Varroa virus = DWV phenotype B
KBV Kashmir virus
SBPV Slow paralysis virus
BQCV Black royal cell virus

The analysis is carried out on 30 to 50 bees in a mixture because individual analyses do not always show the systematic carrying of pathogens per bee and the result is not representative in this case of the state of infection of their colony.

Thus, we can better understand the collapse of the densest colonies, those for which the observation “at the flight hole” is the most reassuring. Indeed, in case of overpopulation, the rubbing of bees against each other provokes micro lesions of the cuticle (in the same way as organic acids) which prove to be the most efficient entry door for the dissemination of viruses.

Be careful, the risk of collapse is important when going from a normal population to a colony with a large population. Thus, the removal of the supers may cause a sudden increase in the population density and initiate the development of various pathogens.

Dr Gilles Grosmond, veterinary expert, solu’nature

qPCR versus petri dish

qPCR versus petri dish

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.