# An Overview of the Public Health Challenges in Diagnosing and Controlling Human Foodborne Pathogens
## 4 Diagnostic Approaches of Foodborne Pathogens
### Methods Based on Nucleic Acids
#### 4.2.1 Polymerase Chain Reaction (PCR)  
A number of benefits are associated with multiplex PCR, including its ability to rapidly identify multiple species of harmful microorganisms, the high level of sensitivity and high specificity for the target organism [124; 125; 126]. By using this method, it is possible to quickly detect and magnify a wide range of bacteria in a single response, and multiple bacteria can be viewed at the same time. Using this approach, it would appear that the basic concept behind it is that a mixture of numerous primer pairs with varying target gene segments amplify the target gene simultaneously. There are several main applications of multiplex PCR, such as gene knockouts, mutation analyses and RNA detection. In doing so, it becomes possible to ensure that food is of high quality and safe to consume. In an example of multiplex PCR, [PERSON] et al. [127] used multiplex PCR technique in order to identify _Escherichia coli_ in food samples by detecting _lacZ_ and _yaiO_ genes. The multiplex PCR method was also successfully applied to identify _Listeria monocytogenes_ in vegetables [128]. Using a multiplex PCR technique coupled with a membrane chip, [PERSON] et al. [124] developed a method that simultaneously detected nine types of foodborne pathogens. A number of other methods have also been created, such as the FilmArrayTM Gastrointestinal Panel (BioFire Diagnostics(r), Salt Lake City, UT, USA) and the Gastrointestinal Pathogen Panel (GPP) created by Applied BioCode(r) (Santa Fe Springs, CA, USA) to detect the 22 and 17 most commonly identified foodborne pathogens. With the aid of multiplex PCR, [PERSON] et al. [129] revealed the presence of three genes in _Vibrio parahaemolyticus_, which are named _tll_, _tdl_ and _trl_, respectively. There was a 19% prevalence of _Vibrio parahaemolyticus_ in this investigation, indicating that this bacterium has the potential to cause serious health problems, as it grew and multiplied rapidly. Consequently, multiplex PCR has proved to be an effective tool for the detection of a wide range of foodborne pathogens. However, to improve the efficiency of multiplex PCR analysis, it is important to determine if there is a correlation between the primers used in the different PCRs that might lead to poor amplification rates as the design of the primers plays a crucial role. A proper primer sequence should be constructed with a single annealing temperature in order to differentiate amplicons after a thermal cycle [130]. In addition, the inability to differentiate between alive and dead bacteria makes it possible to result in a false-positive result [131]. Therefore, multiplex PCR is frequently reported to produce subpar results because of its complexity. An integral part of the quantitative PCR reaction mechanism is the use of fluorescent reporter signals as a means of detecting PCR products. As a result of the presence of DNA that carries pathogenic properties, we are able to regularly determine whether the mixture contains pathogens by checking its fluorescence. Using a standard curve for calibration, it is possible to determine the quantity of pathogenic organisms present in a sample by measuring the degree of fluorescence within the sample [132]. Quantitative PCR is one of the most precise and selective methods of genetic testing. There is no need for post-PCR DNA analysis since real-time detection of PCR products eliminates that requirement. It can be argued that due to these factors, the quantitative PCR technique has become one of the most commonly used methods for identifying and classifying foodborne pathogens for its accuracy and effectiveness. As a result of the rapid development of TaqManTM and LightCyleTM probes, quantitative PCR procedures are becoming more frequent [124]. It has previously been demonstrated that quantitative PCR has been used to identify three subtypes of the _stx1_ and seven subtypes of the _stx2_ associated with _Shigella_ toxins [133]. Similarly, [PERSON] et al.