If I’ve timed this correctly, tonight’s show is dropping on Monday 27 February 2017.
So, what is PCR? PCR stands for polymerase chain reaction. It’s now a technique that has been used in research and diagnostics for more than two decades. I remember doing my fifth-year medical school elective at the Sir Albert Sakzewski Virus Research Laboratory at the Royal Children’s Hospital and seeing PCR in action using buckets of hot and cold water. This was before the availability of taq polymerase. I’ll explain that soon.
PCR is a technique we use to amplify short segments of DNA in clinical specimens or from cultured material to develop millions of copies of these short DNA segments to make it easier to identify microorganisms including bacteria, viruses and parasites.
Apart from clinical microbiology, the technique is used to investigate genetic diseases, forensic science and DNA research.
The repeated heating and cooling, known as thermal cycling, allows DNA to melt and enzymes to work to replicate more DNA. As more DNA is produced, the thermal cycling occurs and more DNA melts and so on, so after a dozen or more cycles you end up with very large quantities of the identical DNA segments.
As the name suggests the thermal cycling is a chain reaction with an exponential multiplication of the DNA segments.
The enzymatic replication requires a DNA polymerase. Most enzymes are heat labile, so in the early days, buckets of hot and cold water were required and you had to manually move the reaction vessel between the buckets. This changed when the heat-stable DNA polymerase known as taq polymerase was discovered from a bacterium found in the deep ocean near active undersea volcanos. This meant the thermal cycling could occur in a single reaction chamber, which could be a test tube which is mounted into a metal block which can have its temperature changed rapidly electronically. So instead of big buckets of water, PCR can be done in a benchtop instrument and some PCR is now done in test systems the size of a fist or smaller.
In clinical microbiology, we regularly use PCR to diagnose infections from normally sterile sites like cerebrospinal fluid. If a patient presents to the emergency department with sudden onset of a high fever, headache, neck rigidity, sore eyes when the lights are on and a skin rash, most emergency physicians and their trainees will immediately think of meningococcal meningitis. Antibiotics will often be started as blood is drawn for culture, and after a CT scan of the head to rule out a space-occupying lesion, a lumbar puncture may be performed so we can get some CSF for microscopy, culture, glucose, protein and PCR for commonly found causes of acute bacterial meningitis.
We can also use PCR now to quickly determine if a golden staph is a MRSA or if an Enterococcus is vancomycin resistant.
PCR is also used in faeces microbiology in what is known as multiplex PCR so that multiple DNA targets are sought and can be identified. That makes diagnosis faster but sometimes at the expense of other valuable information if culture is not performed. There is currently a debate in public health circles about so-called culture-independent diagnostic testing and the ramifications for public health.
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