RT-PCR in Food Testing Explained: A Simple Guide for Food Industry Professionals

PCR (Polymerase Chain Reaction) is the technique that was awarded a Nobel Prize in Chemistry in 1993 for solving major testing bottlenecks in the medical and forensic sciences. The same technology that has tracked COVID variants in past years is now used in food testing to analyse and detect food adulteration. Contamination from pathogens, toxic chemicals, and malicious food mislabelling that allow harmful substances to reach consumers are the primary drivers of the majority of the foodborne illnesses and fatalities worldwide. According to the WHO (World Health Organization), approximately 600 million individuals (~1 in 10) worldwide become ill from unsafe food each year; consequently, 420,000 people die, with 30% of those fatalities occurring in children under the age of five. The only way to prevent such mishaps is to rigorously identify food contamination in the food industry before it reaches the consumer. Implementation of molecular food testing techniques, such as RT PCR (Real-time Polymerase Chain Reaction), which targets the DNA in the food sample, acts as an early warning system that prohibits unsafe products from going out of the plant. 

The journey of PCR in food analysis

Decades ago, PCR was just an academic tool that was complex and required technical expertise to run and analyse food samples.The food industries mostly used to rely on traditional biochemical or plating techniques for food analysis, which usually take 5-7 days to depict the results. Then came the endpoint PCR, which was quite unsuccessful in the food industry due to its cost, time, and complex methodology. The technical experts had to manually cast agarose gels, inject toxic dyes like ethidium bromide, and look for bands under UV light. The emergence of RT-PCR caused a real turning point in food analysis. Experts used fluorescent dyes that illuminated while the DNA was copied, thereby saving them from waiting hours to run a gel. After this, the testing window has significantly dropped from 7 days to under 24 hours. Subsequently, PCR emerged as the ultimate tool for transparency during the Horsemeat Scandal (2013), when inexpensive horsemeat was substituted for beef across Europe. Eventually, in food industries, PCR was deployed to detect allergens such as peanuts, gluten, soy, etc., to avoid recalls. Today, Seqlo simplifies molecular food testing methods with modern RT PCR setups that use a complete package of reagents altogether and automated software. A quality expert now doesn't require a technically dense, sophisticated approach to obtain a clear result for detecting pathogens, exposing species adulteration, identifying GMOs, and pinpointing allergens in food. 

What is RT-PCR, and why does it matter for food? 

In modern food testing, time and precision are the two most important factors, because these are closely related to shelf life and financial aspects such as expensive product recalls. RT-PCR is a molecular food testing method that uses DNA as an extremely stable, unique molecular template with high precision standards and produces results in a few hours. 

Imagine finding sugar crystals in a tub of salt. This is the way it feels to detect a trace of an allergen, like a peanut or a dangerous pathogen, such as Salmonella, in a pool of processed food products. 

In food testing, a very small part of the massive production is taken as a sample. The problem with the traditional or swab-based method is that the evidence of the target is usually very small. This can be compared to collecting rainwater with teacups. For example, our target is to find Salmonella in a bulk tanker of milk. As a sample, we take a few drops from the tanker and try to grow bacteria on a Petri dish, but the Salmonella in these few drops of milk takes 3-5 days to grow and multiply. There is a chance that in these few drops of milk taken as a sample, Salmonella is not able to grow even on the fifth day because we have only limited Salmonella present in the sample as compared to the tanker.  

On the other hand, the RT-PCR acts as a DNA photocopier. When the DNA is extracted from these few drops of the milk sample, even if a single strand of Salmonella DNA is present, RT-PCR makes multiple copies of this DNA, which makes detection simpler. 

Step-by-step procedure of how RT-PCR food testing works

Step 1: Sample preparation

The process begins with collecting a sample of the food product from the production line. The food sample is usually very complex, with fats, sugars, proteins, and other substances trapping the targets (pathogen, allergen, etc.) inside very tightly, making them completely invisible at this stage. The sample is homogenised, i.e., ground if it is solid; for fluid-like samples, it is mixed thoroughly. 

Step 2: Pre-Enrichment

This step is crucial when the food sample is analysed, especially for pathogen detection. This involves mixing the homogenised sample with a nutrient-rich liquid broth (generally, buffered peptone water) and incubating the mixture at a warm temperature. This encourages the minute number of pathogens present in the sample to multiply rapidly. Assume this is giving an injured and stressed pathogen a therapy so that it recovers and wakes up fresh to increase its productivity. 

Step 3: DNA Extraction

This step is basically extracting or isolating the genomic DNA from the food sample. Imagine this as cracking the eggshell to use the egg whites and yolk inside for cooking. 

DNA extraction involves multiple processes, starting with the lysis of cells that contain the DNA. A special chemical solution containing salts, detergents, and some denaturing agents is added to the homogenised sample. This breaks and opens the cell walls of the target (e.g., E. coli or gluten allergen) present in the sample. This follows a crucial enzymatic step called "protein digestion" using a specialised enzyme called proteinase K. This step helps to digest the cellular or food proteins or other tissues that are tightly bound to DNA and also protects the target DNA from damage by rapidly degrading endogenous enzymes (DNases). This follows an incubation period in a hot water bath or a heating block, which facilitates activation of the enzyme. After this, centrifugation is done to separate the target DNA from all the unwanted matter, like debris, fats, denatured proteins, and other food particulates. Using some binding buffers (salts) and silica-based columns, which act as a solid-phase magnet for DNA, DNA gets isolated. Once the DNA binds with the columns, rigorous washing is done to purify and discard the unnecessary elements while keeping the target DNA bound to the silica membrane. Lastly, the DNA is eluted with a low salt buffer, producing a crystal-clear liquid, which is “pure target DNA".

Step 4: Preparation of Mastermix

Once the DNA is extracted, it is mixed with specialised chemicals such as primers and dyes to proceed with making millions of copies of DNA and amplification. Let's understand the process behind this. So the master mix contains an enzyme called "Taq polymerase", which acts as a photocopier that makes the actual copies of the target DNA. Then, there are dNTPs, which are molecular building blocks or loose genetic materials (A, T, C, and G) responsible for assembling new DNA strands. Primers: These are small pieces of engineered target DNA (e.g., E. coli or gluten allergen) that search and attach only to the target DNA. The dye (SYBR Green) refers to an object that floats around invisibly until it finds double-stranded DNA. When it locks onto the DNA, it lights up. 

Step 5: Real-time PCR and amplification

Once the master mix is prepared, it is then immediately set up in the RT-PCR machine. The process, like temperature changes, at this point is fully automated. In this, there are three major steps: denaturation, annealing, and extension, which run in a cycle up to 40 times. 

In denaturation, high-temperature incubation is used to “melt” double-stranded DNA into single strands and loosen secondary structure in single-stranded DNA. This is basically an unzipping process of the double-stranded DNA (zipper) that takes place in the machine to convert it into single strands. 

During annealing, the temperatures are cooled so that the primers have an opportunity to bind or anneal to their specific, complementary sequences on the single-stranded DNA templates. This can be assumed to be a lock-on process, where the temperatures dropped to allow primers to lock onto the single-stranded DNA if the target is present in the sample. 

In the extension process, the temperatures rise to 70-72°C so that the enzyme Taq DNA polymerase binds to the primers and moves along the single DNA strand, adding free-floating bases (A, T, C, G) to build a brand-new double-stranded DNA molecule. Imagine this as adding new zipper teeth one by one; it runs down the line until it finishes building a brand-new second half for both open zippers.

Step 6: Results

Inside the RT-PCR machine, there is an optical sensor, which monitors the above process for each cycle and plots data into a visual graph on the screen. 

Negative Result: The graph shows a straight, flat, horizontal line without any peaks. This represents that the primers have not found anything that they can lock onto. When there is nothing to lock on to and no copies are made. This means there was no target DNA (e.g., E. coli or gluten allergen) spotted. 

Positive Result: The graph ascends and forms a peak, i.e., amplification. This represents that the primers found the target DNA, and the gene was duplicated to several billion copies. This means that the target DNA (e.g., E. coli or gluten allergen) was spotted. 

What can RT-PCR detect in food?

RT-PCR can detect pathogens, GMOs, allergens, and species in food in a few hours. As this method involves scrutinising the DNA—the ultimate barcode of the samples it eliminates the risk of false results and larger waiting times. This, in turn, helps to prevent threats of product recalls or food contamination, contributing to a safer supply chain and distribution.

1. Pathogens: Almost 20-30% of food product recalls worldwide are due to microbial contamination in the food, impacting public health and causing outbreaks. The major culprits of the situation are Salmonella, E. coli, norovirus, Campylobacter, Listeria monocytogenes, and many more. 

2. Allergens: The highest overall food recalls are due to undeclared food allergens, which contribute to almost 45-60% of product recalls worldwide. The big 9 allergens, such as milk, eggs, gluten, peanuts, soy, seafood, mustard, sesame, and celery, account for serious health issues worldwide. 

3. GMOs: The detection of GMOs is necessary to comply with labelling laws in many countries to ensure transparency. Mostly GMOs are detected in soybeans, cotton, corn, canola, and a few other crops like papaya and beets. During the GMO screening, elements like 35S Promoter, NOS Terminator, EPSPS, PAT, etc. are detected to get the status. 

4. Species: One of the major concerns related to food fraud is species adulteration, substituting cheaper animal sources with expensive ones. This happens mostly with milk, meat, seafood, and sometimes herbal products for a potential financial gain. This not only violates dietary and religious beliefs but also can lead to adverse health effects. 

Is in-house RT-PCR right for your facility? 

An in-house testing facility is indispensable for manufacturers that produce perishable food products such as dairy, raw meats, RTEs (ready-to-eat), or freshly baked goods. Because the product holding in such cases affects the shelf life of the product. Similarly, for the manufacturers with high testing volumes, 4-5+ batches per week, outsourcing the tests drastically increases the spend, and waiting for the clearance holds the product in inventory, which again increases the costs. This is where the food manufacturers need rapid and accurate in-house testing, which can give results within hours, eliminating the waiting times and holding costs. Seqlo’s RT-PCR delivers a definitive answer to this. With an affordable and robust system that can be used from the farm to lab scale, Seqlo's RT-PCR delivers results in a matter of hours without requiring a technical expert to run it. With easier data interpretation, food manufacturers can access food testing with molecular precision. 

Ready to bring RT-PCR into your lab? Start with a free sample kit. 

FAQs:

Q: What food testing can be done using RT-PCR?

A: RT-PCR can detect pathogens, GMOs, allergens, and species in food in a few hours.

Q: How is RT-PCR different from traditional or swab-based tests?

A: RT-PCR utilises DNA-based food testing to detect targets such as pathogens, allergens, GMOs, etc., thus guaranteeing extreme sensitivity in a few hours. Whereas swab tests run on samples from the surface.

Q: What does a negative or positive result in Seqlo’s RT-PCR look like?

A: A straight, horizontal line in the amplification curve represents negative, whereas a peak represents positive.

Q: Who are the right ones to take up in-house Seqlo's RT-PCR testing?

A: The Seqlo RT-PCR is a valuable system for any food manufacturer operating from a farm to a lab. 

Q: Can I detect allergens, pathogens, GMOs, and species adulteration using the same system?

A: Yes, allergens, pathogens, GMOs, and species can be detected using the same Seqlo RTPCR system.