On June 12, 2020, the official website of China Chamber of Commerce for Import & Export of Medicines & Health Products updated the list of medical materials manufacturers certified or registered based on foreign standards, and our novel coronavirus RNA 5-target detection reagents (ORF1ab, N, E, S, M) are in the list. So far, our novel coronavirus series reagents (RNA test, sample preservation solution, sample release agent, magnetic bead extraction reagent, column extraction reagent) have all obtained CE certification and export qualification, so we can provide overall solutions from preservation, extraction to amplification.
In particular, we have the largest number of genes (5 targets) for novel coronavirus RNA testing, which is different from the currently approved 2 or 3-target reagents, and are the most tolerant to the possible mutations caused by the spread of novel coronavirus.
Our novel coronavirus RNA 5-target detection reagents are divided into two amplification systems, and internal standard genes are introduced in each tube.
Why did we sacrifice throughput and adopt a two-tube amplification system? Considering the growing number of mutational variants of novel coronavirus, the primer and probe sites covered by the 5-target reagents are more tolerant to mutations. This is based mainly on the following analysis:
(1) As of yesterday's update of the GISAID database (Global Initiative on Sharing All Influenza Data, where the 2019-nCoV global sequences are gathered), from last December to this May, 2019-nCoV has now 2,664 pieces of genomic information and its phylogenetic tree is shown below. Note in particular that the different colored dots indicate different countries or regions, and we can see that there are already three major branches and that some mutational evolutionary differences have emerged among regions.
(2) The evolutionary origin and molecular characterization study of 2019-nCoV by Roujian Lu et al. (The Lancet, 2020) showed that 2019-nCoV is a Sarbecovirus subtype of Betacoronavirus, and the base sequence similarity between 2019-nCoV and the closely related Bat-SL-CoVZC45, Bat SL-CoVZXC21 and SARS-CoVGZ02 from high to low: E gene (98.7%, 98.7% and 93.5%), M gene (93.4%, 93.4% and 85.1%), N gene (91.1%, 91.2% and 88.1%), ORF1ab (88.9%, 88.7% and 79.5%), while the S gene had the lowest similarity (only 75.2%, 74.7% and 72.7%). It can be seen that the E region is highly homologous to these two bat species mentioned above, and primers and probes for the E region are difficult or impossible to be distinguished from these two species. The article cites Shuo Su's view that 2019-nCoV, a typical single-stranded RNA coronavirus, has an average evolutionary rate of about 10-4 nucleotide substitutions per site per year. The article also identified 10 SNP site mutations, five of which were located in the ORF1ab region.
(3) Ceraolo C et al. (Journal of Medical Virology, 2020.) compared 2019-nCoV with closely related virus species based on the whole genome sequences from the GISAID database, including SARS, BCoV, beta-CoV, and MERS.The collected 2019-nCoV genomes were detected to be very highly conserved among each other, with sequence identity higher than 99%. The sequence identity was 96.2% with the bat coronavirus genome (Gisaid EPI_ISL_402131) and 80.26% with the human SARS genome (NC_004718.3). The authors performed multiple genome alignments of approximately 30,000 nt on 54 complete 2019-nCoV genomes. Despite the low heterogeneity of the 2019-nCoV genomes, two hypervariable hotspot positions 8789 and 28151 were identified. 8789 confirms the presence of T(U) or C, belonging to the ORF1ab gene, which causes synonymous changes on the nucleotide triplet encoding serine 2839 (based on the amino acid coordinates of the reference genome NC_045512.2) and therefore is likely not to cause phenotypic differences between strains. 28151 is within ORF8, and is characterized by the presence of C or U, which would result in a serine/leucine (Ser/Leuchange) change in amino acid (aa)84. This could affect the conformation of the peptide. The genes encoding structural proteins are highly conserved in the genus β-coronavirus.
(4) On March 1, a study entitled First Report of COVID-19 in South America, published in the International Exchange Platform for Virology and co-authored by the National Institute of Adolfo Lutz, the National Reference Laboratory, the University of Sao Paulo, and the Institute of Tropical Medicine, University of Oxford, analyzed for the first time the gene sequence of the novel coronavirus in South America. The preliminary genetic analysis showed that the genome of this "Brazil/SPBR1/2020" strain differed from that of previously published "Hu-1 reference strain" in China in 3 places, indicating that the virus has started to mutate during transmission. Among these mutations, two are very close to the "Germany/BavPat1/2020 strain" extracted from the Munich cluster in Germany. This result indicates that the novel coronavirus transmitted in Europe is different from the original virus transmitted in China.
The paper On the Origin and Continuing Evolution of SARS-CoV-2 was published in the National Science Review sponsored by the Chinese Academy of Sciences on March 3, and the latest findings of the Chinese research team show that the novel coronavirus has recently mutated at 149 sites and evolved into two subtypes, subtype L and subtype S. The study found that these two subtypes showed great differences in geographical distribution and proportion in the population. Subtype S was a relatively older version, while subtype L was more aggressive and more contagious. A better understanding of different subtypes will help differentiated treatment and prevention and control of COVID-19. The paper says that the two subtypes differ at site 28144 of the viral RNA genome, T base for subtype L (corresponding to leucine, Leu) and C base for subtype S (corresponding to serine, Ser). By comparing with other coronaviruses, the authors found that the S-type novel coronavirus is closer to the bat-derived coronavirus in the evolutionary tree, leading to the conclusion that the S-type is relatively older. The authors of the paper suggest that there may be a significant difference in the transmission capacity and pathogenic severity between subtypes L and S based on the way the novel coronavirus evolved.
(5) From the above data we can see that the evolution of novel coronavirus is very rapid. The monitoring of sequence alignment revealed that the genes encoding structural proteins (e.g. S, E, M and N) are highly conserved in the genus β coronavirus, but the E gene is highly homologous to the bat species, and primers and probes for E are difficult or impossible to be distinguished from the two species. Mutations in the ORF1ab gene have been found at the relevant SNP sites, which need to be identified during RT-PCR assays. It is also foreseeable that the accumulation of novel coronavirus mutations to a certain extent will definitely affect the binding or amplification efficiency of primers or probes, which may lead to amplification failure and cause misjudgment.
Therefore, based on these considerations, we have sacrificed the advantage of high throughput of the current single-tube nucleic acid extraction corresponding to a single test, and adopted a two-tube amplification system for a single test. In addition, another advantage of two-tube amplification is that the results of two tubes can be mutually validated/verified to compensate for the abnormal curve caused by multiple factors.
A pseudo-viral quality control containing 5-target amplification fragments and the endogenous internal reference gene RNase P is introduced in our kit, which monitors and verifies the quality of RNA extraction. The amplification procedure of "10 (pre-amplification) + 35 (acquisition of fluorescence)" removes the background noise and allows for more comfortable adjustment of the Ct value baseline and more comfortable observation of weak positive amplification curves than the direct 40-cycle amplification condition.
