Research and production of rapid diagnostic kit for Corona Virus causing acute pneumonia (SARS-CoV-2) in Vietnamese by Realtime PCR technique
The COVID-19 respiratory disease caused by the SARS-CoV-2 virus (Severe acute respiratory syndrome corona virus 2), was first detected in December 2019 in Wuhan, China and is now a global pandemic with high levels of infection. SARS-CoV-2 is a virus of the order Nidovirales, family Coronaviridae, genus Sarbecovirus, an unfragmented positive-stranded RNA virus, ~30 kb in size, containing at least 6 open reading frames (ORFs) encoding for viral proteins, including RNA-dependent RNA polymerase, RdRP, spike protein S (spike), viral envelope protein E (envelope), membrane protein M (membrane), nuclear protein N (nucleocapsid) and other accessory proteins that the virus uses to infect cells (Figure 1).
Figure 1. Structural features of the SARS-CoV-2 virus genome (B)
sequenced 5′-leader-UTR- replicase-S (Spike) -E (Envelope) -M (Membrane) - N (Nucleocapsid) -3 UTR-poly(A) with complementary genes alternating in structural genes at the 3' end of the genome. (Source Chen and Zhong, 2020)
Faced with the complicated development of the COVID-19 epidemic, right after the outbreak began, the President of the Vietnam Academy of Science and Technology assigned the Institute of Biotechnology the project “Research on production of rapid diagnostic kits for Corona virus causing acute pneumonia (SARS-CoV-2) in Vietnamese patients by Realtime PCR technique”, code: CT0000.04/20-20. The project is chaired by Assoc.Prof.Dr. Dong Van Quyen. The goal of the project is to create a quick, specific diagnostic kit for the SARS-CoV-2 virus, contributing to the control of epidemic in Vietnam. The project was carried out for a period of 10 months, from February 2020 to December 2020.
At the time of the study, there was very little information about the SARS-CoV-2 genome in the world as well as in Vietnam. To develop the biological kit (Kit), the research team received genetic material, which is an RNA sample extracted from the SARS-CoV-2 virus that causes disease in Vietnamese patients, provided by the National Institute of Hygiene and Epidemiology; RNA samples of human respiratory viruses provided by the 108 Military Central Hospital and the Military Institute of Preventive Medicine to evaluate the sensitivity and specificity of the Kit.
The Kit is developed based on Realtime PCR technology. On the basis of primer pairs and probes, the research team designed and adjusted based on the sequence of important genes/genomic regions of the SARS-CoV-2 virus isolated in Vietnam that the research team cloned and sequenced (Figure 2), and hundreds of sequences of the SARS-CoV-2 virus have been published in international gene banks by countries. Around the world, at that time, some countries had developed SARS-CoV-2 test kits using real-time RT-PCR technology, as the kit recommended by WHO based on the detection of two target gene regions (Gen E and RdRp), the US CDC's Kit targets three target gene regions (N1, N2 and N3), the German Kit targets three target gene regions (N, E and RdRp). Through analyzing the genomic sequence data of the SARS-CoV-2 strain circulating in Vietnam and around the world, the research team has developed a Kit based on two target gene regions, the N and ORF1ab genes.
Regarding the results, the task sequenced and registered 5 gene sequences encoding the proteins S, M, E, N, RdRp and the ORF1ab region of SARS-CoV-2 on the international gene bank with codes MT127116, MT127115, MT127114, MT127113 and MT712208. Sequencing results have provided valuable information for diagnostic kit development. Simultaneously, these genes were cloned to test positive for the Kit.
Figure 2. Results of electrophoresis of PCR amplified gene fragments from SARS-CoV-2 isolated in Vietnam
Figure 3. Rapid diagnostic kit for Corona virus causing acute pneumonia (SARS-CoV-2) by real-time PCR technique
After successful development, the SARS-CoV-2 virus diagnostic kit has been evaluated and tested at the Institute of Biotechnology and externally evaluated by the Military Institute of Preventive Medicine, Ministry of Defense. The evaluation results show that the kit has the same sensitivity and specificity as the real-time RT-PCR Kit recommended by the World Health Organization (WHO), as well as the commercial kit used in the diagnosis of SARS-CoV -2 with technical parameters: clinical specificity is 100% (17/17), clinical sensitivity is 100% (10/10), detection threshold of 50 anticopies/reaction, stable after 6 months of storage at -20°C.
The results of the research to create the Kit were evaluated by the Scientific Council of VAST, experts from National Institute of Hygiene and Epidemiology and National Institute for Accreditation of Vaccines and Medical Biologicals - Ministry of Health.
Figure 4. Graph showing detection thresholds (A) for the N gene and (B) for the ORF1ab gene
Figure 5. Results of SARS-CoV-2 detection on clinical specimens performed on the Realtime PCR device DTprime (DNA Technology, Russia) at the Military Institute of Preventive Medicine
Besides, the mission also published 01 article in the Journal of Biology No. 43(2) DOI: 10.15625/2615-9023/15846; Developed a baseline standard for the Realtime RT-PCR kit to detect SARS-CoV-2, code TCCS 01:2020/VCNSH according to Decision 253/QD-CNSH signed on April 7, 2020 by the Institute of Biotechnology and trained 01 graduate student at the training institution of Hanoi University of Pharmacy. On April 5, 2021, the task was assessed and rated as Good by the Acceptance Council at the Vietnam Academy of Science and Technology.
The results of the project confirm that the Institute of Biotechnology, VAST has mastered modern technology and was completely proactive in creating a real-time RT-PCR Kit to detect SARS-CoV-2 virus in Vietnam.
Translated by Phuong Ha
Link to Vietnamese version
