Microbiology Testing - Real-time PCR
PCR is a method for synthesizing multiple copies of a specific piece of DNA. The first stage in the PCR process is to raise the temperature to 95oC. This causes the double stranded DNA to denature into single strands. The temperature is then reduced to about 60°C to allow the two target specific primers to bind, at specific points on the single-stranded DNA of the target sequence. Finally, the temperature is raised to 72°C and Taq polymerase catalyzes the duplication of the target sequence. This results in two double-stranded DNA fragments that are identical copies of the original target sequence. The temperature cycling process is then repeated several times, typically 40 times, creating a doubling of the number of copies of the target sequence at each cycle. This gives an exponential increase in target DNA concentration and produces sufficient DNA for reliable detection from a single target sequence in less than 1h30.
Real-time PCR detection methods use (beside the two specific primers) a fluorescent reporter probe, which also binds specifically to the target DNA sequence during annealing. Probes have a fluorescent reporter dye at one end and a quencher dye, which inhibits fluorescence, at the other. During the extension stage the probe is broken apart by the DNA-polymerase and begins to fluoresce more strongly. The fluorescence emitted can be measured at each cycle and increases in proportion to the number of target sequence copies produced. To quantify the assay, the cycle at which the fluorescence intensity rises above the background level is recorded for each test sample and for a set of standards run at the same time. A standard curve can then be constructed. The amount of target DNA present in the sample can be calculated from the standard curve.
Typical Work Flow
I - Sample enrichment
- 25g of sample added to 225ml of enrichment broth and incubated overnight. Enrichment times can be as little as 8 hours and up to 24 hours according to the target micro-organism. An average of 16 hours enrichment time is normal.
II - Sample preparation
- Collect enriched samples and lyse cells to extract target DNA.
- Add extracted DNA to PCR tubes/wells containing PCR assay reagents. Time ≈0h30
III - DNA amplification and analysis
- Place the samples in the real-time PCR thermocycler and start PCR reaction.
- Dedicated software manages cycling and detection and calculates results. Time ≈1h30.
Advantages of Realtime PCR
- The main advantage for PCR systems over other methods is in time saving: total time from sampling to result is significantly reduced. Total time for assay can be as little as 10 hours, but commonly between 20-30 hours (including the pre-enrichment period!).
- PCR systems are flexible, allowing for several pathogens to be assayed in a single run.
- Real-time PCR assays can also be made quantitative without adding additional stages or operations to the assay.
- PCR has very high specificity and generally requires fewer repeat tests than most other rapid methods, leading to further time saving.
- There is a clear cost benefit in rapid test results allowing faster HACCP verification and positive release of finished food products.
Implementation of alternative methods
- CellMadeTM Laboratories selects alternative methods from trusted suppliers and only implements alternative methods that are certified according to internationally recognized certification bodies such as AFNOR or AOAC.
- The implementation process of a novel method within our laboratory is performed according to ISO 16140-3. The method verification report(s) and data are available under the Resources Centre of this website.
- CellMadeTM Laboratories regularly participates in international proficiency tests organized by recognized companies and institutions.
- The results of the proficiency test(s) are available under the Resources Centre of this website.
Below table summarizes the available testing services using real-time PCR.
|MBPCR0001||Campylobacter spp. (AOAC 031209)||
Food pathogen. Specific detection of Campylobacter spp. by real-time PCR after 24h-30h pre-enrichment.
|MBPCR0002||Cronobacter spp. (BRD 07/23-01/13)||
Food pathogen. Specific detection of Cronobacter spp. by real-time PCR after 18h-24h pre-enrichment.
|MBPCR0003||Enterobacteriaceae (AOAC 082003)||
Quality indicator. Specific detection of Enterobacteriaceae. by real-time PCR after 18h pre-enrichment.
|MBPCR0004||Escherichia coli 0157:H7 (BRD 07/15-06/08)||
Food pathogen. Specific detection of Escherichia coli O157:H7 by real-time PCR after 8h-24h pre-enrichment.
|MBPCR0005||STEC (VirX et SerO) (AOAC 121203)||
Food pathogen. Specific detection of stx/eae virulence genes and identification of Shiga-toxin producing E.coli (STEC) major O-groups.
|MBPCR0006||Listeria monocytogenes (BRD 07/10-04/05)||
Food pathogen. Specific detection of Listeria monocytogenes by real-time PCR after 18h-24h pre-enrichment.
|MBPCR0007||Listeria spp. (BRD 07/13-05/07)||
Food pathogen. Specific detection of Listeria spp. by real-time PCR after 18h-24h pre-enrichment.
|MBPCR0007||Salmonella spp. (BRD 07/06-07/04)||
Food pathogen. Specific detection of Salmonella spp. by real-time PCR after 8h-24h pre-enrichment.
|MBPCR0008||Salmonella enteritidis (AOAC 081903)||
Food pathogen. Specific detection of Salmonella enteritidis by real-time PCR after 18h-24h pre-enrichment.
|MBPCR0009||Salmonella typhimurium (AOAC 081904)||
Food pathogen. Specific detection of Salmonella typhimurium by real-time PCR after 8h-24h pre-enrichment.
|MBPCR0010||Vibrio spp. (AOAC 032002)||
Food pathogen. Specific detection of Vibrio spp (V. cholerae, V. parahaemolyticus, V. vulnificus) by real-time PCR after 7h pre-enrichment.
Cell culture contamination. Detection of >130 mollicute species by amplifcation of 16S rDNA.