: A Low Cost 30-Minute Diagnostic Test for SARS-CoV-2 (Covid-19) Using Colorimetric Isothermal Amplification and Open Source Technologies
: For Bangladesh to get back to work and gradually end the lock-down, a rapid diagnostic test for Covid-19 infection is extremely important. This is of big significance at airports for testing of incoming passengers. This is how Covid-19 originally spread in Bangladesh and may spread in a 2nd wave if the ‘detect, test and isolate’ principle is not in place. In the absence of specific drugs or suitable vaccines, simple, rapid and reliable detection of infection is crucial for prevention and control, optimal treatment, isolation, rapid contact tracking and investigation of outbreaks as well. So far total 46,485 tests have been done by the RT-PCR technique (from 4 March 2020 to 26 April, 2020) or roughly 3,473 per day. If we are to contain this virus, we need to do at least 20,000 in a day and maybe even 50,000 or more in a day for highly populated country like us. The RT-PCR technique requires 3-4 hours for the actual assay and then another hour or so to analyze the results. It needs an expensive machine costing >30 lakhs taka and trained personnel along with standard set-up to conduct the test.
A simple test has been reported by scientists in Wuhan in conjunction with a commercial Molecular biology company called New England Biolabs based in USA. The test is Loop-mediated isothermal amplification assay (LAMP test). They have already published the paper for general use for anyone in the world (Zhang et al. 2020, preprint in MedRxiv). Another open source website https://foundry.bio/coronavirus-covid-19/ has also provided details for what to do and ideas about how to make the test cheap so that it can be used by millions.
Loop-mediated isothermal amplification assay (LAMP test):
This SARS-CoV-2 RNA detection test however has not been done in Bangladesh. So our idea is to first test the feasibility of the simple test using a commercial kit and then scale it up using home grown enzymes and bulked reagents so that it can be produced cheaply and for many samples at a time and at the point of care. The original sample from patients would consist both of swabs and/or saliva. We would want to test and establish saliva as the source for sample, which can be collected more readily. CDC has recently approved of the use of saliva for sampling.
The steps in the establishment:
1. Establish the isothermal test, which is called LAMP (Loop-mediated isothermal amplification) using control RNA synthesized from a clone. Negative controls will also be used. Time frame 1 month if commercial kit is at hand. If commercial kit needs to be imported, then 2 months. The tests are likely to need only 30 minutes to perform according to Zhang et al. 2020.
• This will involve synthesizing gene blocks corresponding to ORF1a-A, GeneN-A (Integrated DNA Technologies) and adding T7 polymerase promoters by PCR (NEB). Positive control RNA would be produced by in vitro transcription. Alternately positive RNA will be used from Covid-19 RT-PCR kits already available in Bangladesh.
• The LAMP kit from NEB will be used to establish the test for the positive RNA. The kit contains WarmStart LAMP 2X Master Mix, which contains a blend of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase in an optimized LAMP buffer solution. There is a fluorescent dye in the mixture as well. Amplification will result in a visible detection of the fluorescent color, if RNA is present and no color will be visible in the negative control.
2. Once the test is established with positive control RNA, the commercial kit will be used to test samples from patients. Comparative test would be done at the same time using RT-PCR kits. Both saliva and swabs will be tested. The samples can now be collected by medical professionals under appropriate Biosafety conditions.
3. Swabs/saliva will be collected from patients immediately placed into sterile tubes containing 3ml of viral transport media (VTM). The tubes will be sealed for transportation to our laboratory. The samples will be deactivated in Biosafety Level 2 clean benches available in our laboratory by heating at 56°C for 30 min and cooled before sampling for the LAMP test. The tests will be established with crude lysate without isolating the RNA as demonstrated in the paper by Zhang et al. 2020
4. The tests will be adapted for 96 well plate tubes so that at least 90 or 45 in duplication. Samples with positive and negative controls can be runs at once. It is envisaged that sample processing and testing will not take more than 2 hours. So the reactions will be timed for repeated conduction throughout the day. In an 8-hour day, the reaction for 90 samples can be done by one person 4 times or 360 tests. With 3 technicians a single institute can perform approximately 1000 tests. Since the test just need one incubator set at 65°C, the test can be easily performed by all diagnostic centers, university laboratories and medical colleges or even at the sample collection point like airport if necessary. So if 50 institutes are used all over Bangladesh, 50,000 tests per day can be targeted.
5. Once the tests are established, the component enzymes can be produced in-house by negotiating with NEB for the clones which can be used to express and purify the necessary enzymes: Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx Reverse Transcriptase.
• The components of the buffer and viral transport media will be purchased in bulk and aliquoted in vials for use in the different laboratories.
• Once established the process can be transferred to a suitable Pharmaceutical company for production (cost reduction).