**2. Advantages and disadvantages of real-time quantitative PCR for microbiological assessment of food quality**

For more than a century, the identification and isolation of pathogens in food and clinical samples were performed exclusively by microbiological culture techniques. The analyses use a wide variety of selective, nonselective, and differential media. The suspect microbial colonies in these media are selected and isolated and need to go through yet another confirmation step, the biochemical tests. If a pathogen is detected, serological typing and more detailed biochemical tests are performed, and the data from these tests facilitate epidemiological analyses. However, even though these conventional methods are valuable, there is a great need of time (around 1 week) and material, making this technique inadequate in the event of a food outbreak. One of the main criticisms of conventional methods is that the results are available relatively late in clinical disease, limiting the overall value of

**183**

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

the test. Treatment decisions are usually based on the clinical severity of the disease prior to receipt of confirmation of isolation of the microbial culture. This long period to diagnose foodborne diseases by traditional microbiological methods may have an impact on the clinical pathway for each patient. However, for isolation and identification of bacterial pathogens transmitted by food, these classical methods are still considered as the "gold standard," especially by regulatory agencies, since

In the last decades, several alternative methods have been developed with the purpose of producing fast microbiological results to ensure food safety and allowing manipulation of multiple samples in the same analysis [12]. These methods are based on chromogenic culture media, immunoassays for antigen detection, bacteriophage analysis, biosensors, or molecular methods that detect nucleic acids [11–15]. Among the molecular methods, polymerase chain reaction (PCR) is the

The real-time quantitative PCR technique (qPCR) is a variant of conventional PCR and offers the possibility of quantifying the pathogen DNA in a sample in real time, without the need of microbial growth steps. That is, the result can be expressed on the same day. In addition, it is possible to perform multiplex analyses, allowing simultaneous quantification of more than one pathogen in the same assay [16]. For absolute quantification of pathogens, it is necessary to design a standard curve through serial dilutions of a known amount of target DNA [17]. In this curve, the lowest DNA amount detectable by the technique may be included (< 10 copies of a target gene) to attest its sensitivity. The high sensitivity, specificity, and speed of results have allowed qPCR to be widely used for specific pathogen quantification

The use of qPCR, by reducing the time associated with generating quantitative data, offers the potential to increase the robustness of the quantitative microbial risk assessment, thus allowing a subsequent early intervention of control strategies. The quantification of a pathogen in a food product and the prevalence of contamination are important parts of the quantitative microbial risk assessment modeling process, because it needs to determine the probability of exposure as well as the

The quantification through qPCR is based on the exponential increase of the initial DNA amount during PCR amplification cycles. After amplifying a specific sequence, the amplification progress is monitored in real time using fluorescence technology. As soon as the fluorescent signal reaches a threshold level, correlation with the amount of original target sequence occurs, thus allowing DNA quantification in a sample. In addition, the final product may further be characterized by gradual raising temperature during a melt curve to determine the "melt temperature." This point is reached when half of the DNA strand is on single strand and the other half on double strands. It depends on the length and composition of nucleotide sequence of the target gene, which increases the specificity of technique [19]. Among fluorescent reagents, the DNA intercalating agents (such as SYBR Green) and hydrolysis probes (also known as TaqMan™ probes) are the most popularly used. SYBR Green dye is a nonspecific detection system that promotes intercalation, followed by surface binding to double strands of newly amplified DNA [20]. As the DNA is amplified, the fluorescent signal is emitted by the reagent and detected by the equipment. As any DNA amplification can be detected and quantified, to help ensure the reaction specificity, the melt curve of the amplified product can be analyzed to determine the melt temperature (Tm). If there are two

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

they are harmonized methods in worldwide [7, 11, 12].

most versatile and widely used amplification technique [12].

**2.1 Principle of real-time quantitative PCR techniques**

in which microbial amount is low.

amount of exposure to a pathogen [18].

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

the test. Treatment decisions are usually based on the clinical severity of the disease prior to receipt of confirmation of isolation of the microbial culture. This long period to diagnose foodborne diseases by traditional microbiological methods may have an impact on the clinical pathway for each patient. However, for isolation and identification of bacterial pathogens transmitted by food, these classical methods are still considered as the "gold standard," especially by regulatory agencies, since they are harmonized methods in worldwide [7, 11, 12].

In the last decades, several alternative methods have been developed with the purpose of producing fast microbiological results to ensure food safety and allowing manipulation of multiple samples in the same analysis [12]. These methods are based on chromogenic culture media, immunoassays for antigen detection, bacteriophage analysis, biosensors, or molecular methods that detect nucleic acids [11–15]. Among the molecular methods, polymerase chain reaction (PCR) is the most versatile and widely used amplification technique [12].

### **2.1 Principle of real-time quantitative PCR techniques**

The real-time quantitative PCR technique (qPCR) is a variant of conventional PCR and offers the possibility of quantifying the pathogen DNA in a sample in real time, without the need of microbial growth steps. That is, the result can be expressed on the same day. In addition, it is possible to perform multiplex analyses, allowing simultaneous quantification of more than one pathogen in the same assay [16]. For absolute quantification of pathogens, it is necessary to design a standard curve through serial dilutions of a known amount of target DNA [17]. In this curve, the lowest DNA amount detectable by the technique may be included (< 10 copies of a target gene) to attest its sensitivity. The high sensitivity, specificity, and speed of results have allowed qPCR to be widely used for specific pathogen quantification in which microbial amount is low.

The use of qPCR, by reducing the time associated with generating quantitative data, offers the potential to increase the robustness of the quantitative microbial risk assessment, thus allowing a subsequent early intervention of control strategies. The quantification of a pathogen in a food product and the prevalence of contamination are important parts of the quantitative microbial risk assessment modeling process, because it needs to determine the probability of exposure as well as the amount of exposure to a pathogen [18].

The quantification through qPCR is based on the exponential increase of the initial DNA amount during PCR amplification cycles. After amplifying a specific sequence, the amplification progress is monitored in real time using fluorescence technology. As soon as the fluorescent signal reaches a threshold level, correlation with the amount of original target sequence occurs, thus allowing DNA quantification in a sample. In addition, the final product may further be characterized by gradual raising temperature during a melt curve to determine the "melt temperature." This point is reached when half of the DNA strand is on single strand and the other half on double strands. It depends on the length and composition of nucleotide sequence of the target gene, which increases the specificity of technique [19].

Among fluorescent reagents, the DNA intercalating agents (such as SYBR Green) and hydrolysis probes (also known as TaqMan™ probes) are the most popularly used. SYBR Green dye is a nonspecific detection system that promotes intercalation, followed by surface binding to double strands of newly amplified DNA [20]. As the DNA is amplified, the fluorescent signal is emitted by the reagent and detected by the equipment. As any DNA amplification can be detected and quantified, to help ensure the reaction specificity, the melt curve of the amplified product can be analyzed to determine the melt temperature (Tm). If there are two

*Synthetic Biology - New Interdisciplinary Science*

total quality of food can be reached [3–5].

pathogenic microorganisms [8].

reverse transcription-qPCR (RT-qPCR).

**microbiological assessment of food quality**

Food safety practices aim to ensure the appropriate physical, chemical, and microbiological conditions for product quality. For food industries, the safety aspect is always a determining factor about quality, since any problem can compromise the consumer health, culminating in serious financial losses and diminishing the reliability of their products. Thus, the industry advocates the application of food safety management system in the entire food production chain, as a preventative approach toward identifying, preventing, and reducing foodborne hazards, to ensure the food is safe for consumption and with nutritional value. Only then, the

One of the main parameters that determine the food quality is its microbiological characteristic, since microbial contamination is responsible for most foodborne disease (FBD) outbreaks in worldwide, affecting individuals of all ages, particularly children under 5 years of age and persons living in low-income regions of the world [6]. The microbiological evaluation is performed with the objective of establishing the absence of pathogens or their toxins and to enumerate total or indicator microorganisms that provide information about the conditions of processing, storage, distribution, shelf life, and the health risk of the population [7]. As such examples, we can cite the increased numbers of *Staphylococcus aureus* when detected in a food processing step might imply in contamination by handling, the increased numbers of *Escherichia coli* might suggest fecal contamination, and the detection of *Salmonella* spp. might indicate that the processing has not been able to eliminate

The microbiological quality should be effectively focused on traceability, with emphasis on the "farm-to-fork" approach, reaching the entire food chain. For this, rapid diagnostic methods are highly recommended so that early interventions of control strategies can be applied, ensuring the consumer's health and reducing the financial losses of the industry, as well as the costs with public health in cases of FBD outbreaks. In addition, these methods are essential for assessment of food safety objectives (maximum levels of hazards at the point of consumption) in food safety management, which require results in a shorter time than those obtained by culture cultivation [9]. Among rapid methods, real-time quantitative polymerase chain reaction (qPCR) has been shown to be a good tool for monitoring microbiological quality of food, since this technique is evolving to improve the sensitivity and specificity in detection and quantification of pathogens. According to "MIQE guidelines" [10], we chose to use the abbreviation qPCR for real-time quantitative PCR in entire chapter, avoiding confusion with other abbreviations that designate

**2. Advantages and disadvantages of real-time quantitative PCR for** 

For more than a century, the identification and isolation of pathogens in food and clinical samples were performed exclusively by microbiological culture techniques. The analyses use a wide variety of selective, nonselective, and differential media. The suspect microbial colonies in these media are selected and isolated and need to go through yet another confirmation step, the biochemical tests. If a pathogen is detected, serological typing and more detailed biochemical tests are performed, and the data from these tests facilitate epidemiological analyses. However, even though these conventional methods are valuable, there is a great need of time (around 1 week) and material, making this technique inadequate in the event of a food outbreak. One of the main criticisms of conventional methods is that the results are available relatively late in clinical disease, limiting the overall value of

**182**

or Tm peaks, it is suggested that more than one sequence was amplified, and one may not be the specific DNA target [19] or even primer dimer.

The principle of detection system using the hydrolysis probes is based on Förster resonance energy transfer, when a non-radiative energy is transferred from a fluorescent donor (the fluorophore) to a lower energy acceptor (the quencher) via longrange dipole-dipole interactions [21, 22]. It occurs because the hydrolysis probes are small dual-labeled oligonucleotide sequences: in one side, it is labeled by a specific fluorophore, and the other side by the quencher. As the fluorophore and quencher are in close proximity, the quencher adsorbs the reporter fluorophore signal. When the DNA amplification occurs during qPCR reaction, the probe is hydrolyzed by the Taq DNA polymerase, due to its 5′-nuclease activity, and the fluorophore and quencher are separated, emitting fluorescence that corresponds to specific amplification of the target DNA [19]. The great advantage of qPCR using hydrolysis probes is that when the probes are labeled with fluorophores that emit fluorescence at different wavelengths, there is the possibility of performing a multiplex qPCR reaction in which more than two targets are detected and quantified simultaneously in a specific way [23]; therefore, it is a good alternative for use as a rapid test in large number of samples, providing real-time results, and to diminish the cost of analyses.

#### **2.2 qPCR** *versus* **traditional culture method in food microbiology**

Some studies comparing qPCR and microbial culture observed that qPCR for the detection of a single pathogen (singleplex assay) demonstrated to be statistically more sensitive than the conventional technique. Real-time PCR assay specific for detection of *Salmonella enterica* serotype enteritidis analyzed 422 naturally contaminated environmental samples from integrated poultry houses, being the same samples also evaluated by traditional microbiology. The diagnostic sensitivity of the qPCR assay for these samples was significantly higher than those using the culture method. In addition, the result of real-time PCR was obtained in 2 days, while the traditional method took 4–8 days [24]. Another study comparing standard culture methods, conventional PCR, and real-time PCR for the detection of *Listeria monocytogenes* in milk, cheese, fresh vegetables, and raw meat showed that the real-time PCR assay was statistically more sensitive, reducing the time of analysis and laborious work [25]. The targeted gene coding for a protein of the ribosome large subunit was used in qPCR for quantifying Enterobacteriaceae in 51 food products naturally contaminated. The results showed high specificity to differentiate Enterobacteriaceae of non-Enterobacteriaceae based on the cycle threshold (Ct) values; by comparing qPCR and culture methods, only a < 1log difference between methods was obtained in 81.8% of these samples [26]. In seafood products and sediments, conventional PCR, real-time PCR, and culture methods were used to detect pathogenic *Vibrio* spp. (*V. parahaemolyticus*, *V. cholerae*, and *V. alginolyticus*) in 113 fish, 83 clams, 30 seawater samples, and 21 sediment samples. Of the 247 samples analyzed, 41.3% were positive for traditional microbiological method, while 51% were positive for the molecular methods, without prior isolation of pathogens [27].

However, by using multiplex qPCR assay for detection/quantification of more than one pathogen, the sensitivity of the technique may decrease compared to the traditional culture technique (or even compared with singleplex assay), probably due to the competitive nature of the process [8]. In our lab, we compared multiplex qPCR assay for quantification of *Escherichia coli, Staphylococcus aureus,* and *Salmonella* spp. with singleplex assays (by hydrolysis probes and by SYBR Green) in 28 naturally contaminated oyster samples containing pools of 40 oysters collected from natural estuarine environment (1120 in total). The multiplex assay presented lower sensitivity and higher specificity than both singleplex assays

**185**

**Table 1.**

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

(data not published). This can be caused by the competition of the primers by the reagents available in the reaction mix or by the non-varying concentrations of the reaction components (which are used in the same way in the singleplex and multiplex reactions). In addition, the amplification of one target DNA present in the reaction can be overcome by more efficient amplification of other targets (including nonspecific products), thereby reducing the efficiency of the multiplex reaction and consequently decreasing its sensitivity [28]. The same methodology was applied in different food matrices (ground beef, milk, and oyster samples) artificially contaminated by *E coli, S. aureus*, and *Salmonella enteritidis*. Differences <1log in *E. coli* and *S. aureus* quantities were observed comparing multiplex qPCR and traditional culture method in milk and ground beef, with no statistic difference. However, in oyster samples, the multiplex qPCR demonstrated to be more sensitive than culture methods for *E. coli* quantification [8]. Thus, we can affirm that the food matrix can interfere in the sensitivity of the results due to the intrinsic

Shorter analysis time ++ +++ 0 Specificity +++ ++ + Sensitivity +++ + ++ Reproducibility +++ +++ ++ Monitoring the results in real time +++ +++ 0 Simultaneous quantification of different pathogens 0 +++ 0 Distinguishing of living cells from dead cells 0 0 +++

Potential of automation +++ +++ 0 Standardized method in worldwide + 0 +++ "Gold standard" for regulatory agencies 0 0 +++ Fast screening of large number of samples +++ ++ 0 Useful for microbiological quality control +++ +++ + Useful for the quantitative microbial risk assessment +++ ++ +

Cost of material, equipment, and infrastructure −−− −−− −−− Competitive amplification (decrease of the efficiency) 0 −−− 0 Interference of food sample −−− −−− 0 Labor-intensive analysis −−− −−− −−− Need for qualified personnel −−− −−− −−−

*Advantage and disadvantage scores of real-time quantitative PCR (singleplex/multiplex qPCR) and* 

**Singleplex qPCR**

**Multiplex qPCR**

+++ ++ 0

0 0 +++

**Traditional culture**

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

nature of PCR inhibitors present in such food.

Detection of "viable but non-culturable" (VBNC)

Colony isolation for further genotyping/phenotyping

*(+) advantage score; (−) disadvantage score; (0) no score for such characteristic.*

*traditional culture methods for microbiological analysis of food\*.*

**Advantages**

microorganisms

**Disadvantages**

*\*Based on Refs. [8, 12].*

analysis

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

(data not published). This can be caused by the competition of the primers by the reagents available in the reaction mix or by the non-varying concentrations of the reaction components (which are used in the same way in the singleplex and multiplex reactions). In addition, the amplification of one target DNA present in the reaction can be overcome by more efficient amplification of other targets (including nonspecific products), thereby reducing the efficiency of the multiplex reaction and consequently decreasing its sensitivity [28]. The same methodology was applied in different food matrices (ground beef, milk, and oyster samples) artificially contaminated by *E coli, S. aureus*, and *Salmonella enteritidis*. Differences <1log in *E. coli* and *S. aureus* quantities were observed comparing multiplex qPCR and traditional culture method in milk and ground beef, with no statistic difference. However, in oyster samples, the multiplex qPCR demonstrated to be more sensitive than culture methods for *E. coli* quantification [8]. Thus, we can affirm that the food matrix can interfere in the sensitivity of the results due to the intrinsic nature of PCR inhibitors present in such food.


*(+) advantage score; (−) disadvantage score; (0) no score for such characteristic.*

#### **Table 1.**

*Synthetic Biology - New Interdisciplinary Science*

or Tm peaks, it is suggested that more than one sequence was amplified, and one

The principle of detection system using the hydrolysis probes is based on Förster resonance energy transfer, when a non-radiative energy is transferred from a fluorescent donor (the fluorophore) to a lower energy acceptor (the quencher) via longrange dipole-dipole interactions [21, 22]. It occurs because the hydrolysis probes are small dual-labeled oligonucleotide sequences: in one side, it is labeled by a specific fluorophore, and the other side by the quencher. As the fluorophore and quencher are in close proximity, the quencher adsorbs the reporter fluorophore signal. When the DNA amplification occurs during qPCR reaction, the probe is hydrolyzed by the Taq DNA polymerase, due to its 5′-nuclease activity, and the fluorophore and quencher are separated, emitting fluorescence that corresponds to specific amplification of the target DNA [19]. The great advantage of qPCR using hydrolysis probes is that when the probes are labeled with fluorophores that emit fluorescence at different wavelengths, there is the possibility of performing a multiplex qPCR reaction in which more than two targets are detected and quantified simultaneously in a specific way [23]; therefore, it is a good alternative for use as a rapid test in large number of samples, providing real-time results, and to diminish the cost of analyses.

may not be the specific DNA target [19] or even primer dimer.

**2.2 qPCR** *versus* **traditional culture method in food microbiology**

Some studies comparing qPCR and microbial culture observed that qPCR for the detection of a single pathogen (singleplex assay) demonstrated to be statistically more sensitive than the conventional technique. Real-time PCR assay specific for detection of *Salmonella enterica* serotype enteritidis analyzed 422 naturally contaminated environmental samples from integrated poultry houses, being the same samples also evaluated by traditional microbiology. The diagnostic sensitivity of the qPCR assay for these samples was significantly higher than those using the culture method. In addition, the result of real-time PCR was obtained in 2 days, while the traditional method took 4–8 days [24]. Another study comparing standard culture methods, conventional PCR, and real-time PCR for the detection of *Listeria monocytogenes* in milk, cheese, fresh vegetables, and raw meat showed that the real-time PCR assay was statistically more sensitive, reducing the time of analysis and laborious work [25]. The targeted gene coding for a protein of the ribosome large subunit was used in qPCR for quantifying Enterobacteriaceae in 51 food products naturally contaminated. The results showed high specificity to differentiate Enterobacteriaceae of non-Enterobacteriaceae based on the cycle threshold (Ct) values; by comparing qPCR and culture methods, only a < 1log difference between methods was obtained in 81.8% of these samples [26]. In seafood products and sediments, conventional PCR, real-time PCR, and culture methods were used to detect pathogenic *Vibrio* spp. (*V. parahaemolyticus*, *V. cholerae*, and *V. alginolyticus*) in 113 fish, 83 clams, 30 seawater samples, and 21 sediment samples. Of the 247 samples analyzed, 41.3% were positive for traditional microbiological method, while 51% were positive for the molecular methods, without prior isolation of pathogens [27]. However, by using multiplex qPCR assay for detection/quantification of more than one pathogen, the sensitivity of the technique may decrease compared to the traditional culture technique (or even compared with singleplex assay), probably due to the competitive nature of the process [8]. In our lab, we compared multiplex qPCR assay for quantification of *Escherichia coli, Staphylococcus aureus,* and *Salmonella* spp. with singleplex assays (by hydrolysis probes and by SYBR Green) in 28 naturally contaminated oyster samples containing pools of 40 oysters collected from natural estuarine environment (1120 in total). The multiplex assay presented lower sensitivity and higher specificity than both singleplex assays

**184**

*Advantage and disadvantage scores of real-time quantitative PCR (singleplex/multiplex qPCR) and traditional culture methods for microbiological analysis of food\*.*

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

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 culture methods for microbiological analysis of food.
