I talked to a microbiologist (https://twitter.com/drsaranshmittal) working at a Tertiary Government Hospital in Jodhpur to try and understand RT-PCR and covid kits better. Before we understand his experiences, we need to understand how RT-PCR works, here is a summary:
The first thing we need to understand about RT-PCR reaction or in general most of molecular diagnostics is that it works by identifying genes present in the sample we have. DNA is just a double helix physical manifestation of a complementary strands of sequences of genes. The (DNA) genes themselves are a sequence of base-pairs (A-T and G-C are base pairs). The way RT-PCR is a generic genetic material detection platform. First the designer of the test decides which genees to identify. This is the first value added/R&D heavy step. One can choose to identify many genes which are specific to the virus. Each would provide differing levels and types of difficulty in its identification (the process we will discuss in subsequent part of the post). I did cover this part briefly in previous posts. One can identify Covid-19 virus with 3 types of genes: RDRP, N-gene, E-gene. But these are only the most popular ones. A kit designer can choose to do R&D and identity other genes which might provide higher specificity or ease of detection. Once a gene for identification is chosen, scientist needs to decide which specific sequence of base-pairs in that gene will be identified using their test. As an example, the N-gene in SARS-COV-2 is of length 419. Scientists need to figure out which specific subsequence of this 419-length sequence will their test detect. As a toy-example, suppose the single helix of the RNA is:
Gene: A-T-G-G-G-C-A-A-C-G-T-T-A-A-G-A-T-T-C-G-G-A-T-T-A-C-G
And the target sequence is:
Gene: A-T-G-G-G-C-A-A-C-G-T-T-A-A-G-A-T-T-C-G-G-A-T-T-A-C-G
Then, the scientist needs to design what are called probes which attach to the left and right side of the target and help “identify the target”.
Example Probes (in italics):
Gene: A-T-G-G-G- C-A-A-C-G -T-T-A-A-G-A-T-T- C-G-G-A-T- T-A-C-G
This design of target sequence in the gene and the subsequence probes constitutes the second open ended R&D problem which the scientists must solve. The probes cannot be too long, otherwise, they bind with the target RNA helix even if there are mismatches or errors. The probes cannot be too short otherwise they are not specific enough.
Another critical part of PCR reactions is is target concept of cycles. In each cycle, we double the concentration of the target RNA gene by initiating a replication process. The primer molecule is used to detect the beginning of the gene and used to initiate the replication process. This is the third critical part of the design process. The design of the primer molecule.
Here is a blow-by-blow account of how RT-PCR happens:
- We work to extract the viral RNA from the sample.
- Then we convert it to DNA using a reverse transcriptase reaction. (RT Part)
- (PCR part begins): DNA is again broken down into the individual strands.
- Probe molecules attach to the left and right of the target sequence in the gene.
- Fluorescence contained modified A/T/G/C molecules attach to the target sequence bases.
- Fluorescence molecules are released.
- PCR machine checks if fluorescence is above threshold
- If not, cycle is repeated after initiating doubling of the RNA concentration initiating a replication with the primer molecule.
- Number of cycles after which we get fluorescence of enough intensity that PCR machine detects it is known as CT value.
Here is a summary of the knowledge i gained from saransh about his experiences:
- The key contribution of the kit maker, the actual R&D part is the target gene, target sequence, Primer & probe design. These are proprietary.
- Thermofisher and Applied Biosciences are the gold standard for kit design and manufacture. Thermofisher aquired quigen : netherlands company acquired in 2019 mid. Worked out well for them.
- We were already using TruPCR kits in our hospital/college even before Covid. It was being used for Japanese encephalitis and so we knew that they would have good quality.
- Before covid, primary use-case for Rt-PCR was for research. One needs very good reliability and accuracy of tests to ensure paper gets accepted at top tier conferences.
- Thermofisher was a default choice for diagnostic kits due to their brand value. Everybody trusts Thermofisher with closed eyes.
- In Covid RNA extraction kit quigen (thermofisher) is superior/best quality.
- Dr rachna tripathi CNBC interview is worth watching to understand this space.
- Rajasthan government has created covid testing facility in each district. Swine flu has not gone away. We see regular outbreaks once in 2-3 years due to Genetic shift, & genetic drift in swine flu virus. Expect similar pattern in Covid-19 too. So testing infra cannot go away entirely. Besides, government has already bought PCR machines worth millions of rupees. WHat will they do with it? PCR machines can be a good growth trigger for molecular diagnostics if it can be priced at a level where government colleges/labs can afford it.
- Fluorescent molecule quality is also important for overall specificity/sensitivity.
- Many low quality covid kits just import everything from China se, package it and sell it. We even found many kits where the stuff inside was the same meaning that the 2 kit sellers had same chinese supplier.
- RT-PCR is a platform/technique and can be used to detect any viral or microbial disease. Already in use for HIV, hepatitis etc.
- Over time antigen or card tests will replace RT-PCR tests because they are cheaper, but it takes more time to become as precise as RT-PCR because they measure antibodies which are incredibly difficult to isolate and measure. We have many types of antibodies for each microbe and it is difficult to create a good quality anti-body test but this eventually happens for each disease. For example for dengue as well we first had RT-PCRs then got replaced by antigen tests.
- 3BBB makes antigen tests as well. TruRapid got rejected recently but initially even Covid TruPCR was rejected. Company can iterate on it to create successful product. It takes time.
- Reason why only some regions have viral endemics (eg: swine flu) is due to virus residing in Animal reservoirs, seasonal outbreaks, immunity patterns of population.
- Kit can be mistaken, so we repeat the test. Repeatability is good for TruPcr. It is a good quality kit. Also able to produce it cheaply and supply is cheaply so that they are able to meet government tenders.
- Their college lab has processed 7 lakh samples of covid-19 in 1.5 years of covid. Supplying the kit Pan-india is difficult due to Cold Chain logistics being involved in supplying the kit.
- Huwel lifesciences also made good kits. Drastic improvement recently
- Thermofisher & Applied Biosciences kits are high quality but are imported. Difficult for them to meet government tenders.
- CoviSure Rt-PCR is bad quality kit.
- Government tender is also a large customer. One cannot ignore it. At the end of the day, the decision on the tender is by professors and heads and department. If relationship is established, then it can be mined in the future. Building brand is important. If people trust TruPCR then would be more likely to approve it in future.
- 3BBB has many good non covid kits too that we have used. Hepatitis kit is good. Japanese encephalitis.
- Most important ingredient for success in this business is R&D, need to employ and retain good scientists.
Disc: Invested and biased