Painting a pathogenic picture
17 Sep 2013 by Evoluted New Media
This year’s Lab Innovations show will include a special Campden BRI theatre featuring talks on the latest tests and technologies available to food and drink manufacturers. By way of a taster, here we learn of the challenge of PCR pathogen testing
Since the discovery of the DNA structure by Watson and Crick in 1953, molecular biology has greatly evolved and played a major role in fundamental biology research.
In 1983, K. Mullis (Nobel Prize of Chemistry in 1993) invented the Polymerase Chain Reaction (PCR) and demonstrated its feasibility – it quickly became one of the most used molecular biology tools in many research labs around the world. PCR, based on the physical properties of DNA and an enzymatic activity, is a method that amplifies specific DNA sequences in a very short time.
How does it work? As a general description, the required components are: A DNA template (target), specific forward and reverse primers (single strand oligonucleotides with given sequences), a thermostable DNA polymerase (Ex. from Thermophilus aquaticus), dNTPs (desoxyribonucleotides triphosphates), a buffer and magnesium chloride.
Typically, a PCR cycle is defined by 3 steps: Denaturation of DNA at 95°C, Annealing of the primers at 55°C – 60°C and Extension at 72°C. In theory, each cycle allows to duplicate the initial quantity of targeted DNA. Therefore, in optimised conditions, there’s an exponential amplification of the signal during the repetition of those cycles.
Since the invention of this method, technology has greatly evolved, at both chemistry, enzymatic and instruments levels. DNA polymerases have been improved to be more stable, accurate, rapid, depending of specified application. A large variety of enzymes and chemistries are available to meet all kinds of requirements.
This evolution led to the development of real-time PCR (2nd generation PCR) with new chemistries, based on fluorescence detection like i.e. SYBR Green (intercaling agent), TaqMan probes (lysis probe), Molecular Beacons or Double Strand probes. This evolution has also been possible with the development of real-time PCR thermal cyclers. The main advantages of real-time PCR are:
- Specificity: by selecting the genetic target and optimising the reaction conditions, the specificity can be at the genus, species, serotype, or at the strain level.
- Speed: while at least a day of culture is needed to get one colony on an agar plate (10^8 to 10^9 cells), this level of amplification can be reached easily in less than two hours with PCR.
- Sensitivity: only one DNA molecule can be amplified and detected in a PCR well.
- Simplicity: more and more assays and methods are ready to use and the results are analysed automatically.
- Stability: PCR reagents are now extremely stable and can be shipped at room temperature or kept at 2-8°C.
For emerging pathogens, the standard method can be based on real-time PCR. For example, Shiga toxin-producing Escherichia coli (STEC) detection – ISO13136: Microbiology of food and animal feed – Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens – Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups is relevant.
Considering foodborne pathogen detection, the analytical process usually includes the following steps in order to have equivalent results to the reference method (traditional culture method):
Microbial enrichment This initial microbiological step is still needed in order to reach the limit of detection of the molecular method. Usually, from 100 to 104 cells/mL are needed at the end of the enrichment to be properly detected, depending of the downstream protocols.
DNA extraction This important step for the PCR analysis has to be efficient to reach the proper sensitivity and to avoid PCR inhibition. From the simple lysis of cells to the DNA purification, different technical solutions are available and fit most of the laboratory needs.
PCR amplification and detection The optimisation of amplification and detection are critical to reach the expected performances. Moreover, the integration of an internal positive control is mandatory to properly monitor PCR inhibitions and to validate negative results with confidence.
Analysis Analysis criteria must be clearly defined for a correct automated or manual result interpretation.
Confirmation of presumptive positive results The presumptive positive results could be confirmed by a second method based on a different principle. This confirmation can be easily performed by using the corresponding chromogenic media.
Overall, to reach good analytical performances, every step has to be optimal, the sensitivity of PCR is not sufficient. 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 compare to the targeted organism.
These methods based on real-time PCR, called Alternative Methods, can be validated through independent and official organisations, for example NF Certification, NordVal, AOAC-RI or Health Canada. 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.
Finally, real-time PCR methods are also ideal for fast investigations when urgent answers are needed (i.e. contamination of a production plant), the recent E.coli O104:H4 German outbreak is a perfect example. The detection protocol for this 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). As such every analytical laboratory now has the chance to use it. Real-time PCR methods are also recognised for GMOs detection and quantification, as well as for meat speciation with, for example, the recent horse meat European scandal.
This technology is ideal for the detection and quantification of cumbersome cultivating bacteria like Legionella. In this case, results can be obtained in few hours instead of days 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. Today, the 3rd generation PCR known as Digital PCR opens large perspectives in the diagnostic and research fields for quantitative applications or, for single cell or single genome amplification.
