**3. Potential of qPCR for the monitoring microbiological quality of foods: the challenge of differentiating viable cells from nonviable cells**

The changes in consumption, diversity, and food mobility, due to globalization, world population growth, and increasing purchasing power, have increased the need of analyzing food qualitatively and quantitatively, especially from the perspective of standardization, authentication, and certification. In this sense, real-time PCR is undergoing continuous improvement and becoming a method present in food analysis both to detect and quantify pathogens, allergens, and plant species or animals that are present in food, with high sensitivity and specificity. Many fluorescent probes are available, and nowadays, nanoparticles are opening up new diagnostic opportunities using this methodology due to it high sensitivity and providing results in a short time [31].

As already mentioned, the inability of qPCR to differentiate viable cells from nonviable (dead) cells is one of its main limitations in microbiological food analysis [30]. As DNA persists in samples even after the cell have lost its viability, the DNA-based detection methods cannot differentiate whether positive signals originate from living or dead bacterial targets. Thus, in order to detect only viable microorganisms in foods, DNA intercalating dyes, such as propidium monoazide (PMA) or ethidium monoazide (EMA), have been used in a step prior to PCR methods (**Table 2**). These agents selectively penetrate in damaged cell membranes and cross-link to DNA, thereby reducing the amplification capacity of the DNA template [32]. Both EMA and PMA are being used for detection of viable cells from different human pathogens, including those that assume the physiological status of "viable but non-culturable" (VBNC), such as *Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium*, *Shigella dysenteriae*, and *Vibrio cholerae*, which may be viable, but cannot grow outside their natural habitat [33].

PMA has been reported to be more effective than EMA in eliminating qPCR signals from dead cells [32]. Studies comparing EMA and PMA have shown that EMA can also penetrate in living cells of some bacterial species, such as *Anoxybacillus flavithermus* [34], *Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Mycobacterium avium, Streptococcus sobrinus*, and *Escherichia coli* O157: H7 [32],

**187**

*Real-Time Quantitative PCR as a Tool for Monitoring Microbiological Quality of Food*

**Food matrix Microorganisms Cell viability** 

Poultry *Campylobacter jejuni*; *Campylobacter coli*

Broiler carcass rinses *Campylobacter jejuni; Campylobacter coli*

Milk and milk products *Cronobacter sakazakii; Bacillus cereus*; *Salmonella* spp*.*

Raw shrimp *Vibrio parahaemolyticus; Listeria* 

*monocytogenes*

Chicken breasts and legs *Campylobacter jejuni* EMA-qPCR [37]

Chicken carcasses *Campylobacter* spp*.* PMA-qPCR [40] Ground beef *E. coli* O157:H7 EMA-qPCR [41]

Meat products *Staphylococcus aureus* PMA-qPCR [45] Meat exudates *Listeria monocytogenes* PMA-qPCR [46]

Ground beef meatballs *E. coli* O157:H7\* PMA-qPCR [48]

Gouda cheese *Listeria monocytogenes* EMA-qPCR [49] Infant formula *Cronobacter sakazakii* EMA-qPCR [50] Pasteurized milk Coliform bacteria; Enterobacteriaceae EMA/PMA-qPCR [51]

Milk powder *Staphylococcus aureus* PMA-qPCR [45] Ice cream *Salmonella typhimurium* PMA-qPCR [54]

Probiotic yogurt *Bifidobacterium* EMA-qPCR [57]

Fish fillets 16S rDNA EMA-qPCR [59, 60]

Smoked salmon juice *Listeria monocytogenes* PMA-qPCR [46]

UHT milk *Bacillus sporothermodurans* PMA-semi-nested

Milk *E. coli* O157: H7; *Salmonella* spp*.* PMA-multiplex

*Salmonella* spp. EMA-qPCR [38]

*Salmonella* spp*.* PMA-qPCR [43]

*Campylobacter* spp*.* PMA-qPCR [47]

*Bacillus cereus* group PMA-qPCR [53]

*Lactobacillus paracasei* PMA-qPCR [58]

*Vibrio parahaemolyticus* PMA-qPCR [62]

*Vibrio parahaemolyticus*\* PMA-qPCR [64]

PCR

qPCR

PMA-multiplex qPCR

PMA-qPCR [61]

PMA-multiplex qPCR

**dye-PCR method**

EMA/PMA-qPCR [39]

PMA-qPCR [42]

PMA-qPCR [44]

[52]

[55]

[56]

[63]

**References**

*DOI: http://dx.doi.org/10.5772/intechopen.84532*

**Meat**

broth

Chicken rinses and egg

Frozen and chilled broiler

carcasses

**Seafood**

crab)

Raw seafood (oyster, scallop, shrimp, and

Shrimp, pomfret fish,

and scallop

**Dairy products**


*Real-Time Quantitative PCR as a Tool for Monitoring Microbiological Quality of Food DOI: http://dx.doi.org/10.5772/intechopen.84532*

*Synthetic Biology - New Interdisciplinary Science*

industries, leading to faster product release for sale [8].

culture methods for microbiological analysis of food.

providing results in a short time [31].

To increase sensitivity, a pre-enrichment step may be applied prior to qPCR reaction. However, this stage favors microbial growth making it impossible to quantify the pathogens in the original sample; only their detection is possible [29]. Therefore, for simultaneous quantification of pathogens in food, multiplex qPCR can be a potential tool for rapid screening of large number of samples in food

The high cost of equipment investment and its maintenance can be an obstacle to qPCR implementation in routine food analysis laboratories. We must not forget the training of skilled labor. This is because, despite the potential of automation of the technique, the interpretation of the results must be done in a thorough way, so that the "noises" produced by the technique are not interpreted as real signals. However, what really limits the use of this technique in microbiological analysis of foods is the impossibility of distinguishing living cells from dead cells [30]. That is, this technique is able of amplifying any target DNA present in the sample, even being from nonviable cells, which can generate false-positive results by overestimating the number of pathogens present in the food. The **Table 1** summarizes some advantages and disadvantages of qPCR (singleplex and multiplex) and traditional

**3. Potential of qPCR for the monitoring microbiological quality of foods: the challenge of differentiating viable cells from nonviable cells**

The changes in consumption, diversity, and food mobility, due to globalization, world population growth, and increasing purchasing power, have increased the need of analyzing food qualitatively and quantitatively, especially from the perspective of standardization, authentication, and certification. In this sense, real-time PCR is undergoing continuous improvement and becoming a method present in food analysis both to detect and quantify pathogens, allergens, and plant species or animals that are present in food, with high sensitivity and specificity. Many fluorescent probes are available, and nowadays, nanoparticles are opening up new diagnostic opportunities using this methodology due to it high sensitivity and

As already mentioned, the inability of qPCR to differentiate viable cells from

PMA has been reported to be more effective than EMA in eliminating qPCR signals from dead cells [32]. Studies comparing EMA and PMA have shown that EMA can also penetrate in living cells of some bacterial species, such as *Anoxybacillus flavithermus* [34], *Staphylococcus aureus, Listeria monocytogenes, Micrococcus luteus, Mycobacterium avium, Streptococcus sobrinus*, and *Escherichia coli* O157: H7 [32],

nonviable (dead) cells is one of its main limitations in microbiological food analysis [30]. As DNA persists in samples even after the cell have lost its viability, the DNA-based detection methods cannot differentiate whether positive signals originate from living or dead bacterial targets. Thus, in order to detect only viable microorganisms in foods, DNA intercalating dyes, such as propidium monoazide (PMA) or ethidium monoazide (EMA), have been used in a step prior to PCR methods (**Table 2**). These agents selectively penetrate in damaged cell membranes and cross-link to DNA, thereby reducing the amplification capacity of the DNA template [32]. Both EMA and PMA are being used for detection of viable cells from different human pathogens, including those that assume the physiological status of "viable but non-culturable" (VBNC), such as *Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium*, *Shigella dysenteriae*, and *Vibrio cholerae*, which

may be viable, but cannot grow outside their natural habitat [33].

**186**


#### **Table 2.**

*Summary of the studies using PMA or EMA prior to PCR methods for microbiological analysis applied in different food matrices.*

causing loss of genomic DNA during extraction [35] and reducing the efficiency of PCR. However, PMA has been shown to be highly selective in penetrating only bacterial cells with compromised membrane integrity, but not in cells with intact cell membranes. After the DNA intercalation of nonviable cells, the azide group, present in the dye molecule, forms a covalent grid and when exposed to halogen light makes the DNA insoluble, which results in its loss during the extraction process of the genomic DNA. Thus, exposing a bacterial population composed of living and dead cells to PMA treatment results in the selective removal of DNA from dead cells [32]. Nevertheless, the dose of PMA must be carefully adjusted because this reagent becomes increasingly toxic to cells at higher concentrations. It is important to note that the cost of method may become prohibitive in the case of increasing concentration of PMA for its use in different food matrix, or its use in large scale [36].
