Real-time PCR for food testing

Real-time PCR methods are ideal for fast investigations when urgent answers are needed (eg, contamination of a production plant). The recent E.coli O104:H4 German outbreak being a perfect example.

Since the discovery of the DNA structure by Watson and Crick in 1953, molecular biology has greatly evolved and played a major role in biology fundamental research. In 1983, K Mullis (Nobel Prize of Chemistry in1993) invented the polymerase chain reaction (PCR), which quickly became one of the most used molecular biology tools. Based on the physical properties of DNA and an enzymatic activity, this method amplifies specific DNA sequences in a very short time.

How does it work? The required components are: a DNA template (target), specific forward and reverse primers (specific short DNA sequences), a thermostable DNA polymerase (to copy DNA sequences), dNTPs (desoxyribonucleotides triphosphates), a buffer and magnesium chloride.

Typically, a PCR cycle is defined by three thermal steps: denaturation of DNA (95°C), annealing of the primers (55-60°C) and extension (72°C). In theory, each cycle allows duplication of the initial quantity of targeted DNA. Therefore, in optimised conditions, there’s an exponential amplification of the signal during the repetition of those cycles.

Figure 1: PCR schema and principle: denaturation - annealing - elongation.

Since the 1980s, technology has greatly evolved, at both chemistry, enzymatic and instrument levels. This evolution led to the development of real-time PCR (2nd-generation PCR) with new chemistries, based on fluorescence detection like SYBR Green (intercalating agent), TaqMan probes (lysis probes) or Double Strand probes (hybridisation probes) and newer real-time PCR detection systems.

 Figure 2: Principle of double strand probe. The fluorescence is emitted when it hybridises to the targeted amplicon, on both forward and reverse strand.

Multiplication of DNA is observed directly in each PCR well.

Figure 3: Example of real-time PCR curves. The target is declared positive when the fluorescence signal crosses the threshold.

The application scope of PCR and now real-time PCR is extremely wide from research, cloning, medical or genetic diagnostic to forensic investigations, as well as qualitative or quantitative applications. Food pathogen routine testing, as well as meat or fish identification, is one of those possible applications. The main advantages are:

• Specificity: At the genus, specie, serotype or at the strain level.
• Speed: Detection performed in less than two hours.
• Sensitivity: Only one DNA molecule can be detected in a PCR well.
• Simplicity: Ready-to-use reagents and automated analysis.
• Stability: PCR reagents can be shipped at room temperature.

Different ISO standards provide guidelines to handle PCR methods in routine labs. For emerging pathogens, the standard method can be based on real-time PCR, like for Shiga toxin-producing Escherichia coli (STEC) detection. It’s also a tool of choice for meat speciation testing. Considering foodborne pathogens detection, the analytical process usually includes the following steps in order to have equivalent results to the reference method (traditional culture method):

• Microbial enrichment, to reach the limit of detection of the molecular method (from 100 to 10⁴ cells/mL)
• DNA extraction
• PCR amplification and detection
• Data analysis
• Confirmation of presumptive positive results

For meat speciation, the analysis starts directly at the DNA extraction step. Overall, to reach good analytical performances, every step has to be optimal. As an example, the enrichment broth has to be efficient to properly recover stressed cells, or to apply enough selective pressure to avoid the overgrowth of interfering flora compared to the targeted organism.

These methods based on real-time PCR, called Alternative Methods, can be validated through independent and official organisations like NF Certification or the AOAC. Expert and/or independent laboratories will perform a complete evaluation of the performances of the alternative method compared to those of the reference method. For routine laboratories, the use of validated methods is required, especially when they perform official testing in an accredited environment.

Real-time PCR methods are ideal for fast investigations when urgent answers are needed (eg, contamination of a production plant). The recent E.coli O104:H4 German outbreak being a perfect example. The detection protocol was released after only a few days by the European Reference Laboratory for E. coli. (EURL VTEC - Department of Veterinary Public Health and Food Safety - Unit of Foodborne Zoonoses - Istituto Superiore di Sanità) and every analytical laboratory was to use it.

Those methods are also recognised for GMOs detection and quantification, and, more recently, they were highlighted in the horsemeat European scandal.

This technology is suitable for the detection and quantification of cumbersome cultivating bacteria like Legionella - in this case, results can be obtained in a few hours instead of the days required with cultural methods. This led to the recent standard: ISO/TS 12869:2012 Water quality. Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR).

The reverse transcriptase real-time PCR is also a suitable tool to detect Norovirus or Hepatitis A agents.

Nowadays, the 3rd-generation PCR known as Digital PCR is opening large perspectives in the diagnostic and research fields for quantitative applications or for single-cell or single-genome amplification.

Figure 4: Digital PCR represents a generation of PCR that enables absolute quantification of target sequences.