Real Time rtPCR Test to Detect Covid 19 Disease

Covid 19 disease is caused by a virus named SARS-COV-2. The test to detect it is called Real Time Reverse Transcriptase Polymerase Chain Reaction(Real time rtPCR}. PCR was concieved by a California surfer named Kary Mullis and he was awarded the nobel prize for that. PCR is a way of making copies of DNA. If a double standed helix of DNA is heated it will separate into two single strands. After cooling, and if there are complementary bases in solution, a enzyme called  DNA polymerase rebuilds the strands into the double strands. So,each cycle in PCR doubles the amount of DNA. 10 cycles will create about 1000 copies and 20 cycles will make over 1,000,000 copies. Heating the copies destroys the original polymerase so each cooling cycle had to have fresh enzyme added. Then, it was discovered that the polymerase in a hot spring bacteria called Thermophilus aquatica did not denature. Using that meant the test could be done faster because it did not have to be paused to add the polymerase enzyme in each cycle.
The virus that causes covid 19 is not DNA based.  Rather,  it is single stranded RNA. To run it on a PCR machine it must be converted into DNA. So another enzyme called reverse transcriptase must be used used to covert it into DNA.
PCR only copies DNA. The test was called rtPCR.  To ID the DNA, another test must be done and that test was called gel electrophoresis.   In gel electrophoresis the amplified  DNA is placed in a gel cell and an electric current is applied.  This causes sections of the DNA to migrate.  Then, it can be compared to a known DNA.  It took much longer to do these tests and had more errors.
A new technique was developed that was faster and more accurate, It could also not only ID the virus early on, it could quantify it. This test is called Real Time rtPCR, It is a sophisticated test using probes and fluorescent markers to look for sections on the RNA strand that are unique to the virus.  Early in the test,  the virus can be identified and even quantified.  It can tell how many copies of RNA were in the sample taken from the patient.  It can not only ID the virus, it can give an idea of the load of virus the patient has and how much the patient is putting into the environment.  Real time means that one can look at the ongoing test in real time rather than waiting for the PCR and electrophoresis to be completed.

On December 31st, the Chinese government reported to the World Health Organization that there was a pneumonia in patients in Wuhan China of unknown origin.  A few days later it had been sequenced and identified as a novel(new) corona virus.  The virus and it’s disease quickly spread in  China.   A Real Time rtPCR test was developed to ID the virus.

In the US,  the CDC developed it’s own test for the virus but used different sections on the viral strand to ID the virus.  The test kits the CDC sent out for running the tests were faulty and that was a problem that had to be corrected.  Also,  there were few labs approved that could do the tests.  Another problem was  that  the CDC requirements for who could be tested was too narrow.  The patient had to show symptoms and a history of contact  to someone from China.

Since then,  more labs can do the test and there are fewer restrictions on who gets tested.  A company called Roche has a machine and test procedure that is very fast and can run many  tests and that has been approved.  Because the US is behind on testing there could be many  more cases out there and  the virus could be more widespread.  Catching up on testing is imperative.  It needs to be done along with all the other things being done in a situation that is getting worse here and much of the rest of the world.

by Paul Snoey

 

About Paul Snoey

I have a degree in Biology and Environmental Science from WSU Vancouver
I am very fond of Gee Creek and Allen Canyon Creek and do a lot of volunteer work to restore these creeks.

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