Both HCoV-229E and HCoV-OC43 were discovered in the 1960s, and have been recognized as the cause of the common cold for many years and cause frequent reinfections throughout their lives. In adults, these viruses account for 4% to 15% of acute respiratory diseases each year, and as high as 35% during peak periods. The annual incidence rate of children reaches 8%, and the highest incidence rate is as high as 20%. According to reports, the frequency of infection with the 229E and OC43 viruses in adults ranges from 15 to 25 per 100 people per year, and 80% of those infected are people who have previously had antibodies to the infection.

According to the introduction of CDC and WHO, nucleic acid diagnosis for HCoV-OC43 is generally for ORF1ab/RdRP (RNA-dependent RNA polymerase), Gene N and Gene E, and Gene S is more used in the development of corresponding drugs and vaccines. The NCBI included sequence is KX344031.1, with a total length of 30713bp, and the gene encodes 5 structural proteins: S, M, N, HE and E.

In the past, the performance evaluation of the detection limit of the kit generally used in vitro transcribed RNA or clinically positive virus as the standard product for performance evaluation. However, both have obvious certainty. First, the RNA transcribed in vitro is generally very pure and single, does not involve extraction, and does not meet the microenvironment of the clinical virus. Therefore, the detection sensitivity will inevitably be overestimated. This is also a high probability of false The main reason for the negative; clinically positive virus again, even if it is an inactivated virus, still has a great biological safety hazard. Therefore, the laboratory level is required to be P3-P4. Most of the units that develop diagnostic kits do not meet the requirements. The source of clinically positive viruses is also very limited and cannot be stably supplied, and is repeatedly used for performance evaluation of the kit.

It has the complete envelope structure of the virus and the nucleic acid sequence corresponding to HCoV-OC43, but the pseudovirus standard product with low biosafety risk perfectly overcomes the shortcomings of choosing in vitro transcribed RNA or clinically positive virus as the standard product. It is very suitable for the standard product of nucleic acid diagnostic performance evaluation.

Construction process: HCoV-OC43 gene synthesis → pseudovirus packaging → purification → QC detection (ddPCR)

ORF1ab/RdRP(2783bp)、Gene N(1347bp)、Gene E(255bp)

·According to the characteristics of the HCoV-OC43 coronavirus, a total of 3 sequences of ORF1ab/RdRP (RNA-dependent RNA polymerase), Gene E (envelope protein), and Gene N (nucleocapsid phosphoprotein) were selected for the packaging of pseudoviruses. The 3 segments well represent the characteristic sequence of HCoV-OC43;

·The primers and probe sequences of the ddPCR system are designed, and the QC copy number detection of pseudoviruses can be completed without constructing a standard curve, which can very accurately characterize the copy number of the standard product, and overcome the relative judgment of the CT value of Q-PCR analysis Standard inaccuracy;

·The pseudovirus standard product is non-pathogenic, renewable, reliable in quality control methods, stable between batches, and can be prepared and supplied stably for a long time. It also requires laboratory biosafety level P2 to meet the safety needs of many organizations.

ORF1ab/RdRP:

TCAAAAGATACTAATTTTTTAAACGGGTTCGGGGTACGAGTGTAGATGCCCGTCTCGTACCCTGCGCCAGTGGTTTATCTACTGATGTACAATTAAGGGCATTTGATATTTACAATGCTAGTGTTGCT

GGCATTGGTTTACATTTAAAAGTTAATTGTTGCCGTTTTCAGCGTGTTGATGAGAACGGTGATAAATTAGATCAGTTCTTTGTTGTTAAGAGGACAGATCTGACTATATATAATAGAGAGATGAAATG

CTATGAGCGTGTAAAAGATTGTAAGTTTGTGGCTGAACACGATTTCTTTACATTTGATGTAGAAGGTAGTCGTGTGCCACACATTGTACGCAAGGATTTAACAAAGTATACTATGTTGGATCTTTGCT

ATGCATTGCGACATTTTGATCGCAATGATTGCATGCTGCTTTGTGACATTCTCTCTATATATGCTGGTTGTGAACAATCCTACTTTACTAAGAAGGATTGGTATGATTTTGTTGAAAATCCTGATATT

ATTAATGTGTATAAAAAGCTAGGACCTATTTTTAATAGAGCCCTAGTTAGCGCTACTGAGTTTGCGGACAAATTGGTGGAGGTAGGCTTAGTAGGCGTTTTAACACTTGATAATCAAGATTTAAATGG

TAAATGGTATGATTTTGGTGACTATGTTATTGCAGCCCCAGGATGTGGTGTTGCTATAGCAGATTCTTATTATTCTTATATCATGCCTATGCTGACCATGTGTCATGCATTGGATTGCGAATTGTATG

TGAATAATGCTTATAGACTATTTGATCTTGTACAGTATGATTTTACTGATTACAAGCTTGAATTGTTTAATAAGTATTTTAAGCACTGGAGTATGCCATATCATCCTAACACTGTTGATTGTCAGGAT

GATCGGTGTATTATACATTGTGCTAATTTTAACATACTTTTTAGTATGGTTTTACCTAATACATGTTTTGGGCCTCTTGTTAGGCAAATTTTTGTGGATGGTGTGCCTTTTGTTGTTTCAATTGGCTA

CCATTATAAAGAACTTGGTATTGTGATGAATATGGATGTGGATACACATCGTTATCGCTTGTCTTTAAAAGACTTGCTTTTATATGCTGCTGATCCAGCTTTGCATGTAGCTTCTGCTAGTGCATTGT

ATGATTTACGCACTTGCTGTTTTAGTGTTGCCGCTATAACAAGCGGTGTAAAATTTCAAACAGTTAAACCTGGTAATTTTAATCAGGATTTTTATGATTTTGTTTTAAGTAAAGGCCTGCTTAAAGAG

GGTAGCTCAGTTGATCTGAAGCACTTTTTCTTTACACAGGATGGTAATGCTGCTATTACTGATTATAATTATTATAAGTATAATTTGCCCACCATGGTGGACATTAAGCAGTTGTTGTTTGTTTTGGA

AGTTGTTTATAAGTATTTTGAGATTTATGATGGTGGGTGTATACCGGCATCACAAGTCATTGTTAATAATTATGATAAGAGTGCTGGCTATCCATTTAACAAATTTGGAAAAGCCAGGCTCTATTATG

AAGCATTATCATTTGAGGAACAGGATGAAATTTACGCTTATACTAAGCGTAATGTCCTGCCAACACTTACTCAAATGAATTTGAAATATGCTATTAGTGCTAAGAATAGAGCCCGCACTGTTGCTGGT

GTTTCCATACTTAGTACTATGACTGGCAGAATGTTTCATCAAAAATGTTTGAAAAGTATAGCAGCTACACGTGGTGTTCCTGTAGTTATAGGCACCACTAAATTTTATGGTGGCTGGGATGATATGTT

ACGCCGCCTTATTAAAGATGTTGACAATCCTGTACTTATGGGTTGGGATTATCCTAAGTGTGATCGTGCTATGCCAAACCTACTACGTATTGTTAGTAGTTTGGTATTAGCCCGAAAACATGAGACAT

GTTGTTCGCAAAGCGATAGGTTTTATCGACTTGCGAATGAATGCGCACAAGTTTTGAGTGAAATTGTTATGTGTGGTGGCTGTTATTATGTTAAGCCTGGTGGCACTAGTAGTGGTGATGCAACTACT

GCTTTTGCTAATTCAGTCTTTAACATATGTCAAGCTGTTTCAGCCAATGTATGTGCCTTAATGTCATGCAATGGCAATAAGATTGAAGATCTTAGTATACGTGCTCTTCAGAAGCGCTTATACTCACA

TGTGTATAGAAGTGATAAGGTTGATTCAACCTTTGTCACAGAATATTATGAATTTTTAAATAAGCATTTTAGTATGATGATTTTGAGTGATGATGGGGTTGTGTGTTATAATTCTGATTATGCGTCCA

AAGGGTATATTGCTAATATAAGTGCCTTTCAACAGGTATTATATTATCAAAATAACGTTTTTATGTCAGAATCCAAATGTTGGGTTGAACATGACATAAATAATGGACCTCATGAATTCTGTTCACAA

CACACAATGCTTGTAAAGATGGATGGTGACGATGTCTACCTTCCATATCCTAATCCTAGTCGTATATTAGGAGCTGGATGTTTTGTAGATGATTTGTTAAAGACTGATAGTGTTCTTTTAATAGAACG

ATTTGTAAGTCTTGCAATAGATGCTTATCCACTTGTGTATCATGAAAATGAAGAATACCAAAAGGTTTTTCGTGTTTATTTGGCGTATATAAAGAAGTTGTACAATGACCTGGGTAATCAGATCTTGG

ATAGCTACAGTGTTATTTTAAGTACTTGTGATGGACAAAAGTTCACTGATGAGTCCTTTTACAAGAACATGTATTTAAGAAGTGCAGTTATGCAG

Gene N:

ATGTCTTTTACTCCTGGTAAGCAATCCAGTAGTAGAGCGTCCTCTGGAAATCGTTCTGGTAATGGCATCCTCAAGTGGGCCGATCAGTCCGACCAGTTTAGAAATGTTCAAACCAGGGGTAGAAGAG

CTCAACCCAAGCAAACTGCTACCTCTCAGCAACCATCAGGAGGGAATGTTGTACCCTACTATTCTTGGTTCTCTGGAATTACTCAGTTTCAAAAGGGAAAGGAGTTTGAGTTTGTAGAAGGACAAGG

TGTGCCTATTGCACCAGGAGTCCCAGCTACTGAAGCTAAGGGGTACTGGTACAGACACAACAGACGTTCTTTTAAAACAGCCGATGGCAACCAGCGTCAACTGCTGCCACGATGGTATTTTTACTAT

CTGGGAACAGGACCGCATGCTAAAGACCAGTACGGCACCGATATTGACGGAGTCTACTGGGTCGCTAGCAACCAGGCTGATGTCAATACCCCGGCTGACATTGTCGATCGGGACCCAAGTAGCGATG

AGGCTATTCCGACTAGGTTTCCGCCTGGCACGGTACTCCCTCAGGGTTACTATATTGAAGGCTCAGGAAGGTCTGCTCCTAATTCCAGATCTACTTCGCGCACATCCAGCAGAGCCTCTAGTGCAGG

ATCGCGTAGTAGAGCCAATTCTGGCAATAGAACCCCTACCTCTGGTGTAACACCTGACATGGCTGATCAAATTGCTAGTCTTGTTCTGGCAAAACTTGGCAAGGATGCCACTAAACCTCAGCAAGTA

ACTAAGCATACTGCCAAAGAAGTCAGACAGAAAATTTTGAATAAGCCCCGCCAGAAGAGGAGCCCCAATAAACAATGCACTGTTCAGCAGTGTTTTGGTAAGAGAGGCCCTAATCAGAATTTTGGTG

GTGGAGAAATGTTAAAACTTGGAACTAGTGACCCACAGTTCCCCATTCTTGCAGAACTCGCACCCACAGCTGGTGCGTTTTTCTTTGGATCAAGATTAGAGTTGGCCAAAGTGCAGAATTTATCTGG

GAATCCTGACGAGCCCCAGAAGGATGTTTATGAATTGCGCTATAACGGCGCAATTAGGTTTGACAGTACACTTTCAGGTTTTGAGACCATAATGAAGGTGCTGAATGAGAATTTGAATGCCTATCAA

CAACAAGATGGTATGATGAATATGAGTCCAAAACCACAGCGTCAGCGTGGTCATAAGAATGGACAAGGAGAAAATGATAATATAAGTGTTGCAGTGCCCAAAAGCCGCGTGCAGCAAAATAAGAGTA

GAGAGTTGACTGCAGAGGACATCAGCCTTCTTAAGAAGATGGATGAGCCCTATACTGAAGACACCTCAGAAATATAA

Gene E:

ATGTTTATGGCTGATGCTTATCTTGCAGACACTGTGTGGTATGTGGGGCAAATAATTTTTATAGTTGCCATTTGTTTATTGGTTACAATAGTTGTAGTGGCATTTTTGGCAACTTTTAAATTGTGTA

TTCAACTTTGCGGTATGTGTAATACCTTAGTACTGTCCCCTTCTATTTATGTGTTTAATAGAGGTAGGCAGTTTTATGAGTTTTACAATGATGTAAAACCACCAGTCCTTGATGTGGATGACGTTTAG