On Monday, the Indian Council of Medical Research (ICMR) issued an advisory for using pooled samples for testing of COVID-19 in order to increase the number of tests conducted by laboratories across the country.Pool Testing can be explained as follows:
What is Pool Testing?
In a pooled testing algorithm, samples of multiple individuals are put together in a tube and screened through the PCR test. In case the pooled test turns out to be positive, individual samples are tested, which is referred to as pool de-convolution. If there’s no positive result, all individual samples in the pool are regarded as negative, resulting in substantial cost savingsIn other words, a pooled testing method involves putting multiple swab samples in a test tube and testing them using a single RT-PCR test.
If the test is positive, all persons are tested individually. This method can greatly enhance the capacity to test in a low-resources setting, as RT-PCR test is reliable but not scalable.
The number of samples that can be pooled(pool testing) will be determined by the infection rate. More samples can be pooled with less infection.ICMR has advised that while more than two samples can be pooled together, the number should not exceed five samples to avoid sample dilution, which can lead to false negatives.
This method can be used in areas where the prevalence of COVID-19 is low, which means a positivity rate of less than two percent. In areas with a positivity rate between two to five percent, sample pooling of PCR screening may be considered in a community survey of surveillance among asymptomatic individuals
Testing samples from multiple patients with a single PCR test, also known as pooled sampling, has been used previously in the early stages of the HIV epidemic when PCR costs were high.
Advantages of Pool Testing:
1.Cost Friendly-Using this method, substantial costs and testing kits are saved. For instance, if a pooled sample consists of the samples of five individuals and it tests negative, the cost of four testing kits is saved and more number of people are covered with fewer resources
2.Mass Screening:In particular, the “door-to-door” pooled-sample approach can facilitate mass screening in early stages of COVID-19 outbreaks, especially in low- and middle-income settings, and in containing foreseeable second wave outbreaks worldwide
3..Pooled screening can also help in tracing asymptomatic cases of the disease, thereby tracking community transmission.
4.Saves Time:This makes it possible for the implementation of expanded testing in larger population groups
What is PCR?
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient in that untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:
Two “primers”, short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
A pile of DNA building blocks that the polymerase needs to make that copy.
How is PCR (polymerase chain reaction) done?
As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step — denatauration (alteration of structure), annealing (joining), and extension — takes place at a different temperature:
- Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
- Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded “template” (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
- Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.
With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.
What is RT PCR?
RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA).
Reverse transcription polymerase chain reaction (RT-PCR) is a variation of standard PCR that involves the amplification of specific mRNA obtained from small samples.
RT-PCR eliminates the need for the tedious mRNA purification process required for conventional cloning techniques. In RT-PCR, reverse transcriptase and an RNA sample are used in addition to the standard PCR reagents. The reaction mixture is heated to 37 ˚C, which enables the production of cDNA from the RNA sample by reverse transcription
The technique consists of two parts:
The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and
The amplification of a specific cDNA by the polymerase chain reaction (PCR).
RT-PCR has been used to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.