it is not uncommon to see news about the low accuracy of the PCR test kit. Why WHO insisted that the standard method of testing is real-time reverse transcription polymerase chain reaction (rRT-PCR). This article will talk about what the test kit is, how it works and compare with other detection methods. I believe you will have your own judgment on the practicality of the detection reagent after reading.
Table of Contents
Novel Coronavirus(2019-nCoV) RT-PCR Detection Kit
What’s in the kit
Novel Coronavirus(2019-nCoV) RT-PCR Detection Kit has five main components (take the kit sold by Heal Force as an example):
1. CoVID-19 Reaction: d NTPs, MgCL2, Primers( ORF1ab gene, E gene and N gene of 2019-nCoV) and Probes
2. RT-PCR Enzyme: Taq DNA polymerase, Reverse Transcriptase, UNG enzyme
3. Positive Control of CoVID: RNA template
4. Negative Control: NaCL
5. Internal Reference A : RNA template
What’ the use of these components
I believe followers who have read the previous article about PCR already understand the reaction process of RT-PCR
RT-PCR Enzyme: Reverse Transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template
RT-PCR Enzyme: Taq DNA polymerase
In Denaturation, after each round of replication the mixture needs to be heated above 90 °C to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. Taq polymerase is well-suited for this application because it is able to withstand the temperature of 95°C which is required for DNA strand separation without denaturing or losing function.
CoVID-19 Reaction: Primers
A primer is a short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis.
CoVID-19 Reaction: ORF1ab gene, E gene and N gene of 2019-nCoV
We all know that In biology, a gene is a sequence of nucleotides in DNA or RNA that encodes the synthesis of a gene product, either RNA or protein. To generate complementary DNA (cDNA) from an RNA template, we need conserved sequences from COVID-19. They are ORF1ab gene, E gene and N gene.
CoVID-19 Reaction: Probes
Use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR.
Positive Control and Negative Control
In an enzyme assay to measure the amount of an enzyme in a set of extracts, a positive control would be an assay containing a known quantity of the purified enzyme (while a negative control would contain no enzyme). The positive control should give a large amount of enzyme activity, while the negative control should give very low to no activity.
Internal Reference
The most important issue in this analysis is the variation in genetic material between samples caused by changes in mRNA isolation and reverse transcription efficiency. Therefore, internal reference genes are needed to standardize sample variation.
Collectively，Novel Coronavirus(2019-nCoV) RT-PCR Detection Kit is a fluorescent probe-based Taqman RT-PCR assay system. It is a test method that detect the presence of virus itself. With internal reference and three conserved sequences, there are almost no false positives.
Why the news reports say the accuracy is low?
There are four reasons for the low accuracy
Unqualified test kits: Since testing kits are in short supply, it is inevitable that some manufacturers produce unqualified products. The test kit is the first line of defense to control the epidemic. Buyers need to select manufacturers carefully.
Immature test kits: The kits were launched urgently at the beginning of the epidemic, so the early kits are Immature, and some products only use two conserved sequences even one conserved sequence, which is less accuracy compared to three conserved sequences.
Irregular sampling:Common errors include incorrect collection location and insufficient sample collection. what’s more, at different stages of infection, the collection location will also change. For example, if virus infects the trachea, it is difficult to collect the virus in the throat.
Irregular operation methods: Large-scale screening leads to the need for operators to test a large number of samples every day. Inadvertent operation between samples is prone to sample contamination, resulting in inaccurate data (but the latest batch of kits equip UNG enzyme and dUTP can effectively reduce the contamination between samples)
Rapid diagnosis only need ten minutes？
I believe you have heard of a test strip. It is said that this test strip can quickly diagnose within ten minutes. Actually, It is serology tests. Its essence is the Colloidal gold method and most commonly used by ordinary people on pregnancy test sticks.
Part of the immune response to infection is the production of antibodies including IgM and IgG. But according to the FDA, IgM antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although levels over the course of infection are not well characterized. IgG antibodies to SARS-CoV-2 become detectable later and normally peak around 28 days after the onset of infection.
So it is no difficult to find that this test method has lower sensitivity than rRT-PCR method. Although it has lower sensitivity, it can test fast which means it can be used in preliminary screening.
Medical imaging
In addition to the first two detection methods, We can also use CT to visually see whether the lungs are infected. Typical features on CT initially include bilateral multilobar ground-glass opacities with a peripheral, asymmetric and posterior distribution. Subpleural dominance, crazy paving, and consolidation may develop as the disease evolves.
However, only observing image is easy to coincide with the feathers of other diseases, resulting in misjudgment.
So Chest CT scans may be helpful to diagnose COVID-19 in individuals with a high clinical suspicion of infection based on risk factors and symptoms but is not recommended for routine screening.
Conclusion
RT-PCR Detection Kit：Relatively fast and high accuracy, but requires high operation
serology tests：Fast, simple operation,low sensitivity
CT: Be helpful to diagnose COVID-19 in individuals with a high clinical suspicion of infection but not recommended for routine screening.
So the best way is use serology tests to do Large-scale preliminary screening, then use RT-PCR Detection Kit to further screening and make a definite diagnosis, finally use CT image diagnose individuals with a high clinical suspicion.
