Department of Veterinary Public Health & Pharmacology

Development of the diagnostic assay platform (in a form of test kit) which detects SARS-COV-2 viral nucleic acids in a shorter time (30 min, excluding time taken for RNA extraction) with a high specificity (>99%) and sensitivity (>95%) compared to real time RT-PCR (Reverse Transcription-Polymerase Chain Reaction).

The Covi-2 Loop Test Kit COVID-19 is an extremely contagious and deadly viral disease caused by SARS-CoV-2. Diagnosis of COVID-19 through clinical signs alone is ineffective due to the extreme variability in time taken for manifestation of clinical signs and the non-specificity of clinical signs that are mostly shared with that of a common flu. Therefore, confirmatory identification of a suspected individual even before the manifestation of clinical signs (based on contact tracing) is essential as it provides the opportunity to isolate the infected patients and control the spread of the virus.
The current standard for confirmatory diagnosis is real time RT-PCR; which requires a real time PCR machine and many other proprietary laboratory consumables. Further, the availability of a limited number of real time PCR machines is a hindrance for establishing regional/field level testing facilities in many developing nations. Even though Rapid Antigen Tests (RAT) are more convenient, cost effective and field deployable, their sensitivity can be as low as12-71% compared to PCR assay which will leave an alarming number of high false negatives thronging the streets freely and spreading the virus.
The diagnostic assay platform developed (in a form of test kit) by the University of Peradeniya, detects SARS-COV-2 viral nucleic acids in a shorter time (30 min, excluding time taken for RNA extraction) with a high specificity (>99%) and sensitivity (>95%) compared to real time RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in identifying infected individuals without the need for expensive equipment such as real time PCR machines. This has been achieved by specific designing of LAMP primers targeting SARS-CoV-2 RNA. Further, this target region of the virus has not mutated in any of the variants described to date. This colorimetric RT-LAMP kit comes with a separate internal control assay targeting human beta actin mRNA so that each sample is subjected two parallel assays in two different tubes: first assay in the tube with the red lid amplifies SARS-CoV-2 specific RNA and second assay in the tube with the green lid amplifies human beta actin m-RNA. The latter assay is important to ensure the integrity of the sample and quality of the RNA extraction process. The internal control assay primer design only allows the amplification of the cDNA coming from mRNA, but not the beta actin gene. As a result, this assay can be used to confirm the presence of beta actin m-RNA in the sample, and successfully convert mRNA into cDNA. Hence this assay indirectly provides evidence on the integrity of the RNA in the sample and the ability for the reaction to successfully convert RNA to cDNA and amplify cDNA. The cost of each test is about LKR 1500 (USD 6). The simplicity of the Test, complemented by the significantly short time and cost it takes would enable health officials to screen a much larger number of suspected individuals in a shorter time period even at the level of field stations and/ or peripheral hospitals.
Currently, the COVI-2 Loop Test Kit has obtained technical clearance from the committee appointed by the National Medicines Regulatory Authority (NMRA) of Sri Lanka. This technical clearance was granted after testing the performance of the test kit at two independent clinical facilities in Sri Lanka. After receiving the NMRA technical clearance, Medivesta Remedies Pvt Ltd. undertook the task of constructing a facility to manufacture the test kit at the Homagama Industrial Zone. This facility will be operating under the GMP certification provided by the National Medicines Regulatory Authority of Sri Lanka. The final product is expected to reach the local market within the next few weeks. At the same time, Medivesta Remedies Pvt Ltd. with the University of Peradeniya (which has the patent rights) will be seeking FDA approval in the near future. This product will make quick and reliable COVID-19 testing available at a reasonable cost, which is a top priority need in the world. Cost effective, reliable and faster testing methods which can be performed in the field stations is essential to operate factories at their full capacity, reopen schools in many places, allow people to travel more freely without a long waiting time to get screening results, and to keep our global fight against the SARS-CoV-2 virus effective until an effective cure is invented.

Further developments expected in the near future:

Research is undertaken to validate the method to be used with a simplified RNA extraction, as RT-LAMP methods are relatively insensitive to enzyme inhibitors present in the sample matrix. Further, this kit is shown to be compatible with saliva samples (in addition to any sample taken for PCR) avoiding the necessity of taking painful nasal and pharyngeal swabs while providing an opportunity for self-sampling. With the simplified RNA extraction, this kit can provide a diagnostic accuracy of almost similar to RT-PCR while reducing the testing time to only 30 min. At present, the COVI-2 Loop test kit developers are working on developing a new version of the test kit which runs both the internal control assay in the same tube as the SARS-CoV-2 assay. Once these developments are incorporated into the test kit the cost of a test will come down to nearly LKR 750 (USD 3). Further, the research team is currently working on developing similar diagnostic assays for other human and animal diseases.

Research Team

1. Dr. Ruchika Fernando / Senior Lecturer /Department of Veterinary Public Health and Pharmacology, Faculty of Veterinary Medicine and Animal Science
2. Dr. Saranga Sumathipala / Consultant Medical Virologist / Teaching Hospital Anuradhapura
3. Dr. Rohitha Muthugala / Consultant Medical Virologist / National Hospital Kandy

Acknowledgements

1. Prof. M.D. Lamawansa / Vice Chancellor - University of Peradeniya
2. Prof. Upul B. Dissanayake / Former Vice Chancellor - University of Peradeniya
3. Prof. Parakrama Karunaratne / Former Deputy Vice Chancellor - University of Peradeniya
4. Prof. Roshan Dharshana Yapa / Director, Business Linkage, Innovation, Incubation and Technology Transfer Office (BLII-TTO), University of Peradeniya.

Financial Support
University Grants Commission and the AHEAD Project of the Ministry of Higher Education