hCov-NL63 was first isolated from an aspirate from a 7-month-old baby in the Netherlands in early 2004. Since then, HCoV-NL63 infection has been proven to be a universal disease worldwide and is related to many clinical symptoms. Diagnosis includes severe lower respiratory tract infection, buttitis and bronchiolitis. HCoV-NL63 causes childhood diseases. The elderly and immunocompromised children account for 1.0-9.3% of childhood respiratory infections.

According to the introduction of CDC and WHO, nucleic acid diagnosis for HCoV-NL63 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 JX504050.1, with a total length of 27553bp, and the gene encodes 4 structural proteins: S, M, N 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-NL63, 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-NL63 gene synthesis → pseudovirus packaging → purification → QC detection (ddPCR)

ORF1ab/RdRP（2780bp）、Gene N（1134bp）、Gene E（234bp）

·The pseudovirus standard of HCoV-NL63 is different from RNA synthesized in vitro and clinically positive live virus. It perfectly overcomes the shortcomings of both, and is really suitable for performance evaluation of the kit, including detection limit, specificity, repeatability, etc. ；

·According to the characteristics of the HCoV-NL63 coronavirus, a total of 3 segments of ORF1ab/RdRP (RNA-dependent RNA polymerase), Gene E (envelope protein), and Gene N (nucleocapsid phosphoprotein) were selected for the packaging of pseudovirus , 3 segments well represent the characteristic sequence of HCoV-NL63;

·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: