Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.

What is PCR used for?

Once amplified, the DNA produced by PCR can be used in many different laboratory procedures. For example, most mapping techniques in the Human Genome Project (HGP) relied on PCR.

PCR is also valuable in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders. This technique has featured prominently in global testing for the novel Corona Virus (SARS-nCoV2).

How does PCR work?

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called “Taq polymerase” synthesizes – builds – two new strands of DNA, using the original strands as templates.

This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.

The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

PCR’s role in Covid19 testing

The info-graphic below will help you visualize the testing procedures for Covd19. This article will focus only on the PCR testing and not on the identification of antibodies with Serological Testing. PCR will assess if you are infected, whereas the antibodies test checks to see if you were infected.

The Diagnostic or PCR Test

This test uses a sample of mucus typically taken from a person’s nose or throat. The test may also work on saliva — that’s under investigation. It looks for the genetic material of the coronavirus. The test uses PCR (polymerase chain reaction), which greatly amplifies the viral genetic material if it is present. That material is detectable when a person is actively infected.

Generally speaking, in terms of producing a reliable result, these are the best tests. However, a few days may pass before the virus starts replicating in the throat and nose, so the test isn’t guaranteed to identify someone who has recently been infected. Swabs can also sometimes fail to pick up signs of active infection.

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Antigens are any substance that stimulates the immune system to produce antibodies. Antigens can be bacteria, viruses, or fungi that cause infection and disease. They can also be substances, called allergens, that bring on an allergic reaction. Common allergens include dust, pollen, animal dander, bee stings, or certain foods.

The Five Classes of Antibodies

There are five different kinds of antibodies, IgG, IgA, IgM, IgD, and IgE. Ig is the abbreviation for immunoglobulin, or antibody. Laboratories use Serological Testing to identify these antibodies in samples.

IgG antibodies are the most common and the most important. They circulate in the blood and other body fluids, defending against invading bacteria and viruses. The binding of IgG antibodies with bacterial or viral antigens activates other immune cells that engulf and destroy the antigens. The smallest of the antibodies, IgG moves easily across cell membranes. In humans, this mobility allows the IgG in a pregnant woman to pass through the placenta to her fetus, providing a temporary defense to her unborn child.

IgA antibodies are present in tears, saliva, and mucus, as well as in secretions of the respiratory, reproductive, digestive, and urinary tracts. IgA functions to neutralize bacteria and viruses and prevent them from entering the body or reaching the internal organs.

IgM is present in the blood and is the largest of the antibodies, combining five Y-shaped units. It functions similarly to IgG in defending against antigens but cannot cross membranes because of its size. IgM is the main antibody produced in an initial attack by a specific bacterial or viral antigen, while IgG is usually produced in later infections caused by the same agent.

IgD is present in small amounts in the blood. This class of antibodies is found mostly on the surface of B cells—cells that produce and release antibodies. IgD assists B cells in recognizing specific antigens.

IgE antibodies are present in tiny amounts in serum and are responsible for allergic reactions. IgE can bind to the surface of certain cells called mast cells, which contain strong chemicals, including histamine. When an allergen such as pollen binds with its specific IgE antibody, it stimulates the release of histamine from the mast cell. The irritating histamine causes the symptoms of an allergic reaction, such as runny nose, sneezing, and swollen tissues.

Can we produce antibodies in a laboratory?

Yes. Antibody production is carried out in the body by B cells. There are various methods for antibody production in the laboratory. Monoclonal antibodies can be produced through the use of hybridoma cells in vitro. Polyclonal antibodies are typically manufactured in rabbits.

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Several methods can be used to detect antibodies and antigens, including ELISA, agglutination, precipitation, complement-fixation, fluorescent antibodies and more recently chemiluminescence. Serological testing features prominently in Covid-19 testing.

It is used to test a patients sample for the presence of antibodies to the SARS nCoV2 virus that causes Covid-19. The presence of these antibodies tells us that the patient was infected with the virus. The test cannot be used to determine if you are currently or newly infected, that requires a NAAT based test that searches for actual viral material rather than antibodies and antigens

What do we use Serological Testing for?

Blood typing is typically performed using serologic methods. The antigens on a person’s red blood cells, which determine their blood type, are identified using reagents that contain antibodies, called antisera. When the antibodies bind to red blood cells that express the corresponding antigen, they cause red blood cells to clump together (agglutinate). These clumps can then be identified visually.

Serologic tests also play a key role in transfusion medicine. Prior to a blood transfusion,the test is used for crossmatching, a process that ensures a donor and recipient are compatible. The direct antiglobulin test is performed to detect if antibodies are bound to red blood cells inside the person’s body, which is abnormal and can occur in conditions like autoimmune hemolytic anemia, hemolytic disease of the newborn and transfusion reactions.

In Immunology, Serologic tests can help to diagnose autoimmune disorders by identifying abnormal antibodies directed against a person’s own tissues.

The importance of Serology

Serological surveys are often used by epidemiologists to determine the prevalence of a disease in a population. Such surveys are sometimes performed by random, anonymous sampling from samples taken for other medical tests or to assess the prevalence of antibodies of a specific organism or protective titre of antibodies in a population.

These surveys are potentially the most direct and informative technique available to infer the dynamics of a population’s susceptibility and level of immunity. The emergence of the tests usefulness in tracking viral outbreaks and measuring prevalence in communities across the globe has been made evident by the Coronavirus Pandemic of 2019/20

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A nucleic acid test (NAT) is a technique used to detect genetic material (DNA or RNA) in samples you provide for testing. It differs from conventional tests that focus on detecting antibodies (Serological Test) or antigens for their results. NAT’s detect a particular nucleic acid sequence and thereby identify a particular species or subspecies of organism, often a virus or bacteria that acts as a pathogen in blood, tissue, urine, etc.

How Does NAAT Work?

Many NATs include a step that amplifies the genetic material—that is, makes many copies of it. This is a crucial step as genetic materials occur in very small amounts making identifying them difficult. Such NATs are called nucleic acid amplification tests (NAATs).

There are several ways of amplification or copying, including polymerase chain reaction (PCR), strand displacement assay (SDA), or transcription mediated assay (TMA).

One of the most common uses of a NAAT test is to establish the presence of Chlamydia in a patient. NAAT is used extensively for detecting STD’s. Because of the amplification process, doctors can now detect STD’s in urine samples that were previously only detectable by swab.

As NAAT’s are incredibly sensitive to even minute quantities of viral DNA, previously undetectable traces of HIV and other blood borne pathogens in blood samples can also be detected, enabling early treatment.

The simplicity of testing urine rather than relying on invasive genital or other swabs, has allowed for wide scale screening across the US to determine the prevalence of diseases such as asymptomatic STD’s in the general population. NAAT has been extensively used in the Covid-19 outbreaks to ascertain if the tested patient is infected with the SARS-nCoV2 virus.

How Reliable is NAAT?

Nucleic acid amplification tests are the most sensitive tests for the screening and diagnosis of genital chlamydial infections. NAATs are more sensitive than previously available diagnostic tests (culture, antigen detection, or nucleic acid hybridization) by at least 20 to 30%. That’s a significant difference, but according to an NIH report, not all NAAT’s are created equally and depending on the assays used, varying specificity occurs.

in other words, the different testing methods employed by the NAAT determine the outcome, but the NAAT still produces far better results than any other test we currently have access to. Confirming its results in large test groups becomes difficult when you have no other measure by which to compare the results, other testing measures being far less reliable.

If you are diagnosed as positive via a NAAT, then you’ve a very slim chance the results are not correct (a false positive) and depending on the severity of the diagnosis you can always request the test be run again using a different assay. According to the NIH report, the most reliable assay proven by their trial was the APTIMA COMBO 2 assay followed by the APTIMA CT assay. Both are manufactured by Gen-Probe, Inc.

Why NAAT is so Important

To understand this more clearly, lets use the example of a person who has contacted covid-19. Lets assume they’ve just been infected and that they are asymptomatic (not showing any symptoms). A swab is taken and sent off for testing.

A conventional Serological Test could be run on the swab, but ti will come back as negative. The reason is simple. A Serological test works by identifying antibodies in the sample. Your body produces these in reaction to the virus, but this takes time. If you’ve just been infected, your body isn’t going to be producing the antibodies yet.

The NAAT will enable technicians to find and correctly identify the Covid-19 virus itself, rather than the antibodies which you aren’t producing yet, and correctly diagnosing you as being infected. This means you can be isolated, even if you aren’t displaying any symptoms as you’re still infectious to others.

The value to both doctors and patients of being able to detect and treat diseases in their early stages cannot be over-stressed.

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