Wednesday, March 25, 2020

How SARS-CoV-2 (aka Corona Virus, aka COVID-19) Test Kit work

SARS-CoV-2 stands for Severe Acute Respiratory Syndrome-Coronavirus-2

How Testing is Done?

I. Collecting the sample
Healthcare worker collects material from a patient's nose or back of the throat. This sample is later shipped in a cold container to the testing lab.

II. Separate RNA from everything else in the sample (RNA Extraction)

This can be done either manually or automatically.

a. Manually: Add chemicals and centrifuge the sample. During this process, RNA will go to a different layer.

b. Automatic:
Here instead of mixing the chemicals manually, samples are put into a diagnostic machine. The robot arm in the machine will do the pipetting process and mixes the liquids on several tests at once. According to Seegene (a South Korean company which is a lead producer of these kits), "this method takes only four hours to test samples from 94 patients - four times faster than the manual method. It also reduces the risk of human error or contamination."

III. Convert to DNA

RNA, we got from above step will be mixed with reverse transcriptase enzyme. This enzyme converts it to DNA (changing it from one strand RNA to two stand DNA). Later this DNA will be mixed with nucleotides and short synthesized DNA fragments called primers in a test tube.[1]

What primers will do?

Their job is to find specific segments of the viral genome and bind to it. What these specific segments are? These are the genetic materials from the virus. These primers should not mix with anything other than virus's materials. In this way selecting a primer is very important for these tests to work fine. 

IV. Polymerase Chain Reaction (PCR)

Well, these are not done by humans. Generally, these are done by PCR machines. This is a lab equipment which is used to amplify segments of DNA via polymerase chain reaction (aka PCR). "device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps" [4]

When equipment is heated, DNA's double helix will separate into two strands. Now these strands are exposed. Now the temperature will be lowered, this time our primers will start doing their job. Primers will be locked into their targeted segments of the exposed DNA. How to identify these target segments? We will discuss it later. 

Now our enzyme uses these primers and starts building a complementary stance of DNA according to the exposed sequence. After 30-50 cycles a single DNA will be multiplied to hundreds of crores. This way we have enough DNA to detect something. [1]

V. Fluorescent dye

Now the question is how to detect viruses from this mix. This is not something we can identify using a naked eye or microscope. Here we use the concept of dye and tracing. Apply a fluorescent dye to the solution which produce fluorescence in the presence of certain agents; in this case, affected DNA. Higher the count of DNAs higher will be fluorescence. PCR machine contains an instrument that measures these light patterns which in turn tell us whether the sample is infected or not. [1]


As a matter of fact, this procedure this very generic. RT-PCR is used to identify the specific genetic segment. What changes here is the components we need to add to detect each virus,
 - RNA extraction kit
 - Which PCR machine
 - Which primers to use

This combination is known as protocols. 

Well, that means someone must standardize and publish the protocols for these tests to work. For formulating the protocols, we need genetic sequences of virus. 


The world rushed to sequence SARS-COV-2 virus and did it successfully. Chinese sequenced and published the first full sequence of SARS-COV-2 genome in January. Other major labs across the world also did sequencing. Once the sequence is available to major labs across the world began designing, testing and publishing the protocols for detecting virus via RT-PCR. 

An extract from one of the papers,

"Sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends... 

The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%)

2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting beta corona virus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans." [6]

Protocol By CDC-US

Their protocol has four sets of primers.

N1, N2: Target unique regions of SARS-CoV-2 genome (a protein) that encapsulates and protects the genetic material of the virus.
Third Primer: Targets common gene for whole family of SARS-like virus. Remember nCov-19 (SARS-COV-2) falls in to SATS group.
Fourth: Targets the human genome. 

CRISPER (Clustered Regularly Interspaced Short Palindromic Repeats)

Companies like Sherlock Biosciences and Mammoth Biosciences are also trying to use CRISPR based technique to identify the virus. If this works then testing will be as simple as a pregnancy test using strips. [1]

Serological Test

This test looks for antibodies produced by humans against the virus. This will also tell us if a person is infected and then got cured. Even though this is less accurate than molecular tests, SARS-COV-2 also show up in this test.

Please note that the above article draws heavily from below mentioned reference links, especially Wired. In case you have more doubts and want to know more about this procedure please go through reference links given below.


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