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Polymerase chain reaction, or PCR, is kind of like a copy machine. In the most general sense, a PCR reaction makes copies and enlarges the amount of a virus or any microbe so it can be easily detected. The value of this relatively new testing process cannot be underestimated. It is versatile, sensitive and broadly applicable. It improves patient care by giving physicians the answers they need to prescribe the best treatment regimen.
If you aren’t doing PCR yet, you will be soon.
What is PCR?
All living things have RNA and DNA. A virus has either RNA or DNA as its genetic code. PCR can take the smallest piece of DNA or RNA and amplify it a billion times or more. Once that happens, you can detect that DNA or RNA and see it very easily. This makes it possible to determine if a certain virus or other microbe is present in a clinical sample, such as blood.
PCR was discovered in the mid-1980s, it was available in a few clinical labs in the early '90s, but it has become increasingly commercialized and thus more widely available to laboratories in the last few years.
According to Gregory A. Storch, MD, Director of the Virology Laboratory at St. Louis Children’s Hospital, the technology is developing very rapidly right now and commercial players are creating different PCR platforms that are simple to perform. There are even multiplex PCR assays that detect multiple microbes at the same time. With more and more commercial applications available, more labs are getting into PCR testing.
“But you do need some specific molecular training and expertise for most PCR testing,” said Dr. Storch. “Anyone coming out of college with a biology or medical technician degree would learn something about molecular biology, but it is also important to get some practical hands-on experience. This could be in a research lab or a clinical lab.”
In most cases, a doctoral-level lab director is involved in setting up and overseeing PCR testing, either an MD or Ph.D. clinical pathologist. A medical technologist actually runs the tests.
“In the future, a large portion of infectious disease testing and microbiology testing will be based on PCR. Basically all labs will have to get involved,” said Dr. Storch.
Qualitative vs. Quantitative
With qualitative testing, the answer is yes or no. Either the virus is there or it’s not. With quantitative testing, you are looking to see how much of a microbe is there. This is crucial information to have in cases such as CMV, HIV or hepatitis C.
With quantitative testing, there are more stringent quality controls. A lab has to monitor every assay to make sure the performance is uniform.
“There can be drift over time,” said Dr. Storch. “If you analyzed the same sample every day over two weeks, the value could go down if there is drift in the system. You should have measures in place to recognize whether there is drift in a quantitative assay. This kind of monitoring is new to microbiology labs, but is well known in other areas of the clinical laboratory, especially chemistry.”
The Patient Benefit
The benefit of PCR for patients is often very great. Compared to conventional methods, PCR can offer definitive answers that guide the best treatment for illness and disease.
Herpes Simplex is a virus that causes cold sores, but can also cause deadly encephalitis (the irritation and inflammation of the brain). In the past, the only way to confirm the diagnosis was to do a biopsy of the brain. Now you can do a spinal tap, perform a PCR reaction on the spinal fluid, and make a diagnosis. With conventional methods, the level of the virus that is present is just too low to detect.
“PCR testing allows medications to be targeted for patients who really need it. Before PCR, patients who may or may not have had herpes encephalitis were treated with acyclovir. Often they would receive two to three weeks of treatment. Now we can be more confident in the diagnosis, which allows us to prescribe the right treatment,” said Dr. Storch.
Another area where PCR has changed the course of medicine is stem cell and solid-organ transplant patients. According to Dr. Storch, CMV was a very serious, sometimes fatal illness in these patient populations. Now, with PCR and effective medication to treat CMV, it is under much better control than in the past.
Whooping cough, also known as pertussis, was a difficult diagnosis to make pre-PCR. PCR has greatly improved the diagnosis of pertussis. This benefits patients of any age.
Designing a Lab for PCR
If you are in the process of designing a new molecular lab, you will want to create a successful workflow for PCR. In the early years, contamination of the PCR reaction with the amplification product from a previously performed PCR reaction was a potential problem. Even the tiniest amount of DNA can cause contamination and inaccurate results.
In order to combat contamination it was originally recommended that PCR labs have three separate rooms: one for preparing the reaction, one for amplification, and one for analysis for amplified products. “These days, many PCR platforms are closed platforms in which the reaction is never opened,” said Dr. Storch. “The potential for contamination is much less. Still, it requires good facilities that are well designed to minimize risk of contamination.”
To detect contamination, the lab must run controls with PCR reactions. Dr. Storch recommends running a known positive (positive control) to show the PCR reaction worked. The lab should also include mock samples that are expected to be negative (negative controls).
“If you run a patient sample side by side with a negative control in which water is substituted for the patient sample, and the reaction containing water yields a positive result, then you know there is a problem. It is very important to detect contamination and make sure the reaction is working properly,” he said.
PCR testing will continue to grow as the demand increases. Quicker turn around times; faster diagnosis and treatment; and better patient outcomes will continue to be factors that drive commercialization and innovation in this market. With PCR offerings, your lab can keep up the pace.