hCov-HKU1 was discovered in Hong Kong in 2005 and is a common cause of self-limiting infections of the upper and lower respiratory tracts, most commonly in young children. There is no laboratory diagnosis, usually through reverse transcription polymerase chain reaction (RT-PCR) of respiratory samples, HCoV-HKU1 respiratory tract infection cannot be distinguished from infections caused by other respiratory viruses. Treatment should be symptomatic and consistent with general guidelines that support viral respiratory infections.

According to the introduction of CDC and WHO, nucleic acid diagnosis for hCov-HKU1 is generally for RdRP (RNA-dependent RNA polymerase), Gene N and Gene E, and Gene S is more used for the development of corresponding drugs and vaccines. The NCBI included sequence is KF686346, with a full length of 29982, 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-HKU1, 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-HKU1 gene synthesis→ pseudovirus packaging→ purification→ QC detection (ddPCR)

ORF1ab/RdRP(2783bp)、Gene N(1326bp)

·According to the characteristics of the HCoV-HKU1 coronavirus, a total of 2 segments of ORF1ab/RdRP (RNA-dependent RNA polymerase) and Gene N (nucleocapsid phosphoprotein) were selected for the packaging of pseudoviruses. The 2 segments are good Characterizes the characteristic sequence of HCoV-HKU1;

·Coby Bio has designed the primers and probe sequences of the ddPCR system, which can complete the QC copy number detection of pseudovirus without constructing a standard curve, which can very accurately characterize the copy number of the standard product, and overcome the Q-PCR analysis of CT Inaccuracy of the relative judgment standard of the value;

·Pseudovirus standard products, non-pathogenic, renewable, reliable quality control methods, stable between batches, can be prepared and supplied stably for a long time, and require laboratory biosafety level P2 to meet the safety needs of many organizations.

ORF1ab/RdRP:

TCTAAAGATTTAAATTTTTTAAACGGGTTCGGGGTACTAGTGTGAATGCCCGGCTAGTACCCTGTGCTAGTGGTTTATCTACTGATGTTCAATTAAGGGCATTTGATATTTGTAATACCAATAGAGCT

GGTATAGGTTTATATTATAAAGTGAATTGTTGCCGTTTTCAGCGTATAGATGACGACGGTAATAAATTGGATAAGTTCTTTGTTGTCAAAAGAACTAATTTAGAAGTTTATAATAAAGAGAAAACTTA

TTATGAGTTGACTAAAAGTTGTGGTGTTGTGGCTGAACATGATTTCTTTACATTTGATATTGATGGTAGTCGCGTGCCACATATAGTTCGTAGGAATCTTTCAAAGTATACTATGTTAGATCTTTGCT

ATGCATTGCGTCATTTTGATCGTAATGATTGTTCAATATTGTGTGAAATTCTTTGTGAGTATGCTGATTGTAAAGAATCCTACTTTTCTAAGAAAGATTGGTATGATTTTGTTGAAAATCCTGATATT

ATTAATATATATAAAAAATTAGGCCCTATTTTTAATAGAGCTTTACTTAATACTGTCATTTTTGCAGACACCTTAGTTGAAGTAGGTTTAGTTGGTGTTTTAACTTTAGATAACCAAGATTTGTATGG

TCAATGGTATGATTTTGGTGATTTTATACAAACAGCCCCAGGATTTGGTGTGGCAGTCGCAGATTCTTACTATTCTTATATGATGCCTATGTTGACTATGTGTCATGTATTAGATTGTGAATTATTTG

TTAATGATAGTTATAGACAATTCGATCTTGTGCAGTATGATTTTACTGATTACAAGTTAGAGTTGTTTAATAAGTATTTTAAGTATTGGGGTATGAAGTATCATCCTAATACTGTGGATTGTGATAAT

GATAGGTGTATTATTCATTGTGCTAATTTTAATATACTATTTAGTATGGTTTTACCTAATACTTGTTTTGGTCCCCTTGTTAGACAAATTTTTGTAGATGGTGTACCGTTTGTTGTTTCTATTGGTTA

CCATTACAAAGAGTTAGGTGTAGTTATGAACTTAGATGTTGACACACACCGTTATCGTTTGTCTCTTAAAGATTTACTTCTTTATGCAGCAGATCCTGCTATGCATGTTGCATCTGCTAGTGCTCTGC

TTGATTTACGAACTTGTTGTTTTAGTGTAGCTGCCATTACAAGTGGTATAAAATTTCAAACTGTAAAACCAGGTAACTTTAACCAAGACTTTTACGAGTTTGTTAAAAGTAAAGGCTTGTTTAAAGAG

GGTAGTACAGTTGATTTGAAACATTTTTTCTTTACTCAAGATGGTAATGCTGCAATTACTGATTATAATTATTATAAGTATAATTTACCTACTATGGTTGATATTAAGCAGTTATTGTTTGTATTAGA

AGTTGTTTATAAATATTTTGAAATTTATGATGGTGGTTGTATACCAGCATCACAAGTTATTGTTAATAATTATGATAAAAGTGCTGGTTATCCATTTAATAAATTTGGTAAAGCCAGACTTTATTATG

AGGCATTATCATTTGAAGAACAGAATGAAATTTATGCATATACTAAACGTAATGTTCTGCCCACCTTAACTCAAATGAATTTAAAATATGCTATCAGTGCTAAGAATAGAGCTCGCACTGTAGCAGGT

GTTTCTATTCTTAGTACTATGACAGGCCGAATGTTCCATCAAAAATGTTTGAAGAGTATAGCAGCTACCCGAGGTGTTCCTGTTGTTATAGGAACCACTAAATTTTATGGTGGTTGGGACGATATGTT

ACGTCATCTTATAAAGGATGTTGACAACCCTGTTCTTATGGGTTGGGATTATCCTAAATGTGATCGTGCTATGCCAAATATTTTGCGTATTGTTAGTAGTTTAGTTTTGGCCCGCAAACATGAATTTT

GTTGTTCACATGGTGATAGATTTTATCGCCTTGCGAATGAATGTGCTCAAGTTTTGAGTGAAATAGTTATGTGTGGCGGTTGCTATTATGTTAAGCCTGGTGGTACTAGCAGTGGTGATGCAACTACT

GCTTTTGCTAATTCTGTTTTTAATATATGTCAGGCTGTTACTGCTAACGTTTGTTCTCTTATGGCCTGTAATGGCCATAAGATTGAAGATTTAAGTATACGCAATTTACAAAAACGCTTATACTCTAA

TGTTTATCGTACAGATTATGTTGATTATACATTTGTTAATGAGTATTATGAATTTTTATGTAAGCATTTTAGTATGATGATTTTGAGTGATGATGGTGTTGTCTGTTATAACTCTGATTATGCTAGTA

AGGGTTATATAGCTAATATAAGTGTTTTTCAACAAGTTTTGTACTATCAGAATAATGTTTTTATGTCTGAATCTAAATGTTGGGTTGAAAATGATATTACTAATGGTCCTCATGAATTTTGTTCCCAA

CATACTATGTTGGTTAAGATAGATGGTGATTATGTTTATTTACCATATCCAGATCCTTCTAGAATTCTAGGAGCTGGTTGTTTTGTTGATGATTTATTGAAGACTGACAGTGTTCTTTTGATAGAGCG

CTTTGTAAGTCTAGCTATAGATGCTTACCCTTTAGTATATCATGAAAATGAAGAATACCAAAAAGTCTTTCGTGTATATTTAGAATATATAAAAAAACTGTATAATGATCTTGGTACTCAGATCTTAG

ATAGTTATAGTGTTATTTTAAGTACTTGTGATGGTTTAAAGTTTACTGAAGAATCATTTTACAAGAATATGTATTTAAAAAGTGCCGTGATGCAG

Gene N:

ATGTCTTATACTCCCGGTCATTATGCTGGAAGTAGAAGCTCCTCTGGAAATCGTTCAGGAATCCTCAAGAAAACTTCTTGGGCTGACCAATCTGAGCGAAATTACCAAACCTTTAATAGAGGCAGA

AAAACCCAACCTAAATTCACTGTGTCTACTCAACCACAAGGAAATACTATCCCACATTATTCCTGGTTCTCCGGGATCACTCAATTTCAAAAAGGTAGAGACTTTAAATTTTCAGATGGTCAAGGA

GTTCCCATTGCTTTCGGAGTCCCCCCTTCTGAAGCAAAAGGATATTGGTATAGACACAGCCGGCGTTCTTTTAAAACAGCTGATGGTCAACAAAAGCAGTTGTTACCGAGATGGTATTTCTACTAT

CTCGGTACCGGCCCATATGCCAATGCATCCTATGGTGAATCCCTCGAAGGGGTCTTCTGGGTTGCTAATCACCAAGCTGACACTTCTACTCCCTCCGATGTTTCGTCAAGGGATCCTACTACTCAA

GAAGCTATCCCTACTAGGTTTCCGCCTGGTACGATTTTGCCTCAAGGCTATTATGTTGAAGGCTCAGGAAGGTCTGCTTCTAATAGTCGACCAGGTTCACGTTCTCAATCACGTGGACCCAATACT

CGTTCATTAAGTAGAAGTAATTCTAATTTTAGACATTCAGATTCTATAGTAAAACCTGATATGGCTGATGAGATCGCTAATCTTGTTTTAGCCAAGCTTGGTAAAGATTCTAAACCTCAGCAAGTC

ACTAAGCAAAATGCCAAGGAAATCAGGCATAAAATTTTAACTAAACCTCGCCAAAAGCGAACTCCTAATAAACATTGTAATGTTCAACAGTGTTTTGGTAAAAGAGGACCTTCTCAAAACTTTGGT

AATGCTGAAATGTTAAAGCTTGGTACTAATGATCCTCAGTTTCCTATTCTTGCAGAATTAGCTCCTACACCAGGTGCTTTTTTCTTTGGTTCTAAATTAGAGTTGGTTAAAAGAGAGTCCGAGGCT

GACTCACCTGTTAAAGATGTTTTTGAACTTCGTTATTCTGGTTCTATTAGGTTTGATAGTACTTTACCTGGTTTTGAGACAATTATGAAAGTTCTTAAAGAGAATTTAGATGCTTATGTTAATTCT

AATCAGAACACTGTTTCTGGTTCGCTGAGTCCTAAACCTCAGCGTAAAAGAGGTGTTAAACAATCACCTGAATTGTTTGACTCTCTTAATTTAAGTGCTGATACTCAGCACATTTCAAATGATTTT

ACTCCTGAGGATCATAGTTTACTTGCTACTCTTGATGATCCTTATGTAGAAGACTCTGTTGCTTAA