Not All PCR Methods are Created Equal
PCR is a technique used to make copies of DNA outside of a cell. Exponential multiplication occurs, with millions of copies created from one DNA strand. The process consists of cycles of heating and cooling. The first heating cycle is where denaturation or DNA melting occurs. Denaturation unwinds the two complementary strands of the DNA double helix and separates it into single strands through the breaking of hydrogen bonds between the bases. During the subsequent cooling phase the primers (previously synthesized short pieces of single-stranded DNA that are complementary to the target DNA) find and associate with the target DNA strand (i.e. Salmonella, Listeria, E.coli).
Next, DNA polymerase reads the template strand one piece at a time and adds dNTPs (basic building blocks of DNA) to the end of the primers making it double stranded again. The same thing happens to the other original strand creating two double-stranded pieces of DNA, which are identical copies to the original double-stranded target DNA. With well-optimized PCR there is very close to a doubling of the specific target DNA each cycle, creating millions of copies after completing 30-45 cycles. In the process of real-time PCR fluorescent molecules emit specific colors of light in response to the amplification. This addition to the basic process of PCR really enables the detection to be done rapidly, routinely, and at lower cost.
Most PCR systems use standard block thermocycling for the various temperature cycles. This method uses a metal block (often heated by a Peltier device) for heat transfer to the samples. This method requires longer cycle times because of the time necessary to heat and cool the block and transfer the temperature to the samples. There can also be a temperature variance of about 2-3oC with this method. Air thermocycling is a much faster and accurate method for conducting the temperature changes. This method uses heating coils (think of a blow dryer) to heat and cool the air surrounding the samples. It allows for faster cycle times and provides more accurate temperature control, within 1oC of the reported temperature.
So why is cycle time and temperature control important? When it comes to rapid pathogen detection, the importance is in the name 'rapid'. Faster cycling means true, real-time analysis of samples; the faster the time to results, the better. But there's more to love about faster cycle times than just fast. Lingering at the temperature stage where primers anneal to the target DNA could affect the specificity of the primers and many other DNA strands could be amplified along with the target DNA.
Faster temperature cycling enables more specific reactions and takes some of the strain off extremely good primer design for specific results. For similar reasons, accurate temperature control and temperature uniformity both within a sample and between samples are important. For the reaction to work optimally, all the samples need to be at the reported temperature. If one sample is off by a couple of degrees, one test could fail and another work in the same carousel. This is especially important when control samples are in the carousel and expected to produce a known result. If the actual temperature is a few degrees lower than the reported temperature, priming to entirely wrong parts of DNA could occur. There are millions of base pairs in bacterial DNA, if temperature control is inaccurate, primers can start attaching incorrectly and amplify non-targeted DNA.
Dr. Roy P. Radcliff is Director and Chief Scientific Officer of Marshfield Food Safety, which has been using PCR technology developed by Idaho Technology since 1999. The true real-time analysis is what sets their air thermocycling PCR system apart from the standard block method. 'I've had experience with 'real-time' systems that were very slow because they used block thermocycling instead of air thermocycling. Newer systems have fast cycling speeds and offer faster process to results for the client,' says Dr. Radcliff. This technology also provides a quantitative number to a positive result quickly; adding tremendous value to the information. In addition to this, the speed of Idaho Technology's PCR system provides cost savings for Marshfield Food Safety allowing them to get through all samples in a nine hour workday without extra staff or machines.
Marshfield Food Safety and Idaho Technology worked together to develop and validate a pooling technique for Idaho Technology's R.A.P.I.D. ® LT PCR instrument. This allows food producers to test more samples at once, enabling faster and better decisions at a lower cost. Pooling is unique in that it relies on post-enrichment pooling as opposed to conventional pre-enrichment sample pooling. Post-enrichment pooling maintains 100% sample integrity and traceability. Up to five samples can be pooled and is most effective when predominately negative results are expected. Pooling samples presents a substantial cost reduction of over 60%. This pooling technique combined with the rapid air-thermocycling method employed by the R.A.P.I.D. LT, makes this system one of the most cost and time efficient PCR systems available.
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Source : Microbiology International View Company Information
Posted on May 12, 2009