Does PCR really meet the current needs of food quality control?
Does PCR deliver more reliable results than bacterial culture? Explore its benefits and limitations for more accurate pathogen detection in…
- November 15 2024
- Food safety
Does PCR really meet the current needs of food quality control?
In recent years, a number of pathogen detection methods have emerged in the food-beverage industry, including Polymerase Chain Reaction (PCR). This genomic amplification technique can be combined with other technologies to increase sensitivity, specificity and speed of detection1. However, to combine PCR with other technologies, it is essential to understand its application, its advantages and also its limitations.
The term PCR refers to the amplification of DNA, and is in fact a general term encompassing several technologies. The key methods used to detect pathogens will be presented to help you understand the principle. This article also covers the main issues faced by the sector: should PCR be used to increase test performance? Are the results more reliable than bacterial culture? What are its limitations?
Table of contents
1. What PCR methods are used to detect pathogens?
There are currently around twenty different PCR methods that can be combined. Some are adapted to meet technical constraints, such as Long Range PCR2 for long DNA fragments or GC-rich PCR3 for fragments rich in G-C nucleotides. Others are designed for higher test performance, including Touchdown PCR4 and Ligation-mediated PCR5 for bacterial typing.
Methods such as Nested PCR6 and Hot Start PCR7 aim to reduce the risk of aspecific amplification.
More recently, pathogen detection has been tested using Direct PCR8 (a method that performs PCR without DNA purification), the faster Fast PCR9, or Multiplex PCR (a method that simultaneously detects several genes in the same sample).
Given the technical and scientific characteristics outlined above, PCR offers a wide range of possible applications. For the detection of pathogenic bacteria in food matrices, PCR meets the challenges of speed, sensitivity and specificity10. As long as appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantifying genes and for gene expression11.
2. What are the advantages of using PCR to detect pathogenic agents?
In certain industries, such as dairy, studies show that real-time PCR (qPCR) is a fundamental tool for ‘food genomics ’12. qPCR incorporates continuous monitoring of the progress of the reaction, enabling quantification of the target DNA. This method is also used to quantify animal DNA and determine animal species. However, studies have shown that depending on the pathogen detected and the type of matrix (unpasteurised milk, pasteurised milk, yoghurt, cheese, infant milk powder, cream, etc.), the detection limit varies and can be doubled or even increased by a factor of ten13.
Detection methods present challenges and limitations, such as sample extraction and the design of specific primers, which have yet to be overcome. Advanced developments and improvements in molecular detection methods are essential to make the detection of food-borne pathogens more reliable, more sensitive and faster, to enable rapid data acquisition.
3. What makes food matrices complex and what impact do they have on PCR?
Because of their composition of various elements (carbohydrates, lipids, proteins, minerals, bacterial flora, additives, etc.), food matrices are undoubtedly the most complex to analyse. This can make some matrices incompatible with certain analytical methods14. In most cases, the consequence can be a false negative result.
Certain molecules are known to inhibit PCR. In particular, it has been shown that PCR is inhibited by certain quantities of sodium chloride, sucrose and lysine.
Milk is well known for its inhibitory effect on DNA extraction techniques and PCR. PCR inhibition depends mainly on calcium concentration, while fat content appears to have only a minor influence on amplification efficiency15. In addition, alkaline protease (also known as plasmin), which is naturally present in milk, degrades Taq polymerase and therefore acts as an inhibitor16.
Studies have shown that complex food matrices, such as ground beef17, chicken18, fruit, vegetables and seafood, can also inhibit nucleic acid amplification. Cationic polysaccharides present in vegetables, fruit and seafood can inhibit nucleic acid amplification by forming highly viscous DNA solutions. They can also interfere with the initiation of DNA amplification to inhibit DNA polymerase activity.
4. Are there any solutions to overcome the technical difficulties of matrices?
To overcome these technical constraints, certain pre-treatments such as homogenisation (fruit, vegetables and meat) can release enzymes or antimicrobial components that interfere with downstream analysis and detection. These elements will therefore not be found during the analysis and will be eliminated during the initial experimental stages.
Some commercial DNA extraction kits have increased extraction yields and modified their reagent composition to adapt to inhibition mechanisms19.
These studies are still insufficient and have not been clearly established. The elimination strategies mentioned in the studies to inhibit nucleic acid amplification are rather limited as they only target specific inhibitors and have not been proven to be applicable to various inhibitors. Therefore, further studies on nucleic acid amplification inhibitors and inhibitor removal strategies are needed to detect pathogens.
5. Which bacterial physiological states should be detected for quality control?
Before examining the methods available for detecting food-borne pathogens, it is important to remember the meaning of the word ‘viable’ and the metabolic states to which it refers. In practice, viability is defined as the ability of bacterial cells to replicate in liquid or solid culture media20.
Microorganisms present in foods exposed to different stresses or environmental conditions may exist in various metabolic states or growth phases. Some of these states may be transient and lose the ability to grow on or in laboratory culture media. In addition to the fully competent and viable state, the scientific literature suggests that micro-organisms present in foods, particularly bacteria, can exist in three other metabolic or physiological states:
Sublethal lesions
Prolonged exposure to chemical or physical treatments20. Essential cellular functions are then partially destroyed20.
Viable but non-cultivable (VBNC) or ‘’persistent‘’
Low metabolic activity, which may be due to osmotic stress, pH changes, exposure to low temperatures, pasteurisation of milk, addition of food preservatives or exposure to disinfectants21, 23, 24. They are detectable on non-selective culture media (genes are expressed and proteins are produced)22. All these phenomena are found during the industrial transformation process. This is why the majority of pathogenic bacteria are present in this form.
Dormancy
Have a rather unusual metabolic form due to their metabolic arrest, which cannot be detected by viability tests25. These strains therefore have the ability to ‘fly under the radar’, making them all the more dangerous. This state can be caused by severe osmotic stress or a lack of nutrients.
6. How can Viable Cultivable Bacteria (VCB) be distinguished from Viable Non-Cultivable Bacteria (VBNC)?
These three states can lead to a viable physiological state that can be cultivated under optimum temperature and growth conditions. Detecting them in food is therefore crucial to ensure consumer health.
Several methods associated with PCR for detecting viable pathogens have been reported in recent years, including the use of phage cell viability stains (plate assay or phage amplification and lysis plus PCR/qPCR), immunocaptures or enzymatic assays26. However, these studies are few in number and need to be developed further.
It is for this reason that most manufacturers continue to go through a pre-enrichment stage to ensure that only growing bacteria are detected. However, this process is time-consuming and tedious because of the repetitive movements involved and the risk of musculoskeletal disorders (MSD).
What are the current PCR challenges for the food industry?
The detection of pathogens from a complex food matrix is a crucial technical challenge for analytical technologies based on nucleic acid amplification. It is therefore necessary to evaluate and develop pre-treatment techniques associated with pathogen detection technologies so that the level of sensitivity and specificity is not affected by the complexity of food matrices. Comparing the performance of different PCR methods remains a challenge due to intra-sample variability and the pathogen detected. What's more, the conditions tested in the literature vary, making harmonisation difficult.
Conclusion : Is it simple for a quality control laboratory to switch from bacterial culture to PCR?
Setting up a PCR method in a quality control laboratory requires upstream compatibility and matrix/bacteria/reagent interaction tests. Sample preparation remains essential for high-quality PCR.
At the moment, completely eliminating microbiology, particularly the pre-enrichment stage, is still a risk when it comes to discriminating between viable and non-viable bacteria. In fact, using it in synergy with alternative molecular biology methods remains the most appropriate strategy until there is sufficient research and studies to apply it on an industrial scale in a simple, rapid, intuitive and reliable way. Commercial methods are also attempting to improve the user experience, making it easier to manage change after more than 50 years of bacterial culture.
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