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Novel coronavirus detection kit as well as application and use method thereof

A detection kit and coronavirus technology, applied in the field of new coronavirus detection kits, can solve the problems of infection of healthy people, death of patients, affecting the sensitivity of detection and analysis, and reproducibility of specific detection, so as to increase the sensitivity , avoid false negatives, good specificity

Active Publication Date: 2021-08-20
ZYBIO INC
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AI Technical Summary

Problems solved by technology

[0003] The sequence of the 2019 novel coronavirus has been announced successively by the Chinese Center for Disease Control and Prevention and the World Health Organization WTO. Like other coronaviruses, the novel coronavirus is an ssRNA virus. The sequencing results show that its full length is about 30Kb. The novel coronavirus belongs to For RNA virus, the research on this virus has just started. At present, most manufacturers use two regions for detection. However, in actual detection, there is a certain proportion of positive results for a single target region, which shows that the reagent is sensitive to different regions. There are differences in the sensitivity, which may also be caused by the competition between the targets. In addition, other components of the kit, the reaction system and the amount of sample added will affect the sensitivity, specificity and reproducibility of the detection analysis.
[0004] At present, most test kits have internal quality control substances in them. These quality control substances are directly added to the samples after sampling. The internal quality control substances can only test whether the entire extraction and detection process is operated correctly, but they cannot guarantee sampling. Whether the process is successful, and whether the sample collection is successful is related to the test results of the patient, and the amount of sampling is closely related to the successful detection of the sample
The use of external sources and internal standards for detection will lead to a large number of potential coronaviruses being missed due to sampling failures, patients will not be isolated and treated in time, and will eventually lead to the death of patients and the infection of healthy people

Method used

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  • Novel coronavirus detection kit as well as application and use method thereof
  • Novel coronavirus detection kit as well as application and use method thereof
  • Novel coronavirus detection kit as well as application and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 A kind of novel coronavirus detection kit

[0037] 1. PCR reaction solution: Tris-HCl pH=8.5 1mM, KCl 1mM, MgCl 2 6mM, dATP 0.5mM, dCTP 0.5mM, dGTP 0.5mM, dUTP 2mM, DTT 1mM.

[0038] 2. PCR enzyme solution: Hot Start Taq 10%, UNG 1%, HiScript Reverse Transcriptase 1%, TaqSSB 10%, Anti-Taq 1%, glycerol 10%.

[0039] 3. Gene primer pairs and probe sequences are as follows:

[0040] The sequence of the forward primer of the Orf1ab gene is shown in SEQ ID NO: 1; the sequence of the reverse primer of the Orf1ab gene is shown in SEQ ID NO: 2;

[0041] The sequence of the forward primer of the N gene is shown in SEQ ID NO: 3; the sequence of the reverse primer of the N gene is shown in SEQ ID NO: 4;

[0042] The sequence of the forward primer of the S gene is shown in SEQ ID NO: 5; the sequence of the reverse primer of the S gene is shown in SEQ ID NO: 6;

[0043] The sequence of the probe of the Orf1ab gene is shown in SEQ ID NO: 7;

[0044] The sequence of t...

Embodiment 2

[0052] Embodiment 2 A kind of novel coronavirus detection kit

[0053] 1. PCR reaction solution: Tris-HCl pH=8.5 10mM, KCl 5mM, MgCl 2 12mM, dATP 2mM, dCTP 2mM, dGTP 2mM, dUTP 4mM, DTT 5mM.

[0054] 2. PCR enzyme solution: Hot Start Taq 50%, UNG 20%, HiScript Reverse Transcriptase 10%, TaqSSB 50%, glycerol 50%, Anti-Taq 50%.

[0055] 3. Gene primer pair and probe sequence (same as in Example 1)

[0056] The fluorescent groups corresponding to the Orf1ab gene, N gene and S gene are ROX, TET and CY5 respectively, and the corresponding quenching groups are all BHQ3.

[0057] 4. The primer and probe sequence (same as in Example 1) of internal quality control product

[0058] The fluorophore corresponding to the internal quality control is VIC.

[0059] The PCR reaction system is as follows: 50% PCR reaction solution, 20% PCR enzyme solution, 4 μM forward and reverse primers for single gene and internal quality control, 0.8 μM for both probes, 30% for nucleic acid extraction p...

Embodiment 3

[0060] Embodiment 3 A kind of novel coronavirus detection kit

[0061] 1. PCR reaction solution: Tris-HCl pH=8.5 5mM, KCl 4mM, MgCl 2 8mM, dATP 0.8mM, dCTP 0.8mM, dGTP 0.8mM, dUTP 2mM, DTT 2mM.

[0062] 2. PCR enzyme solution: Hot Start Taq 15%, UNG 7%, HiScript Reverse Transcriptase 8%, TaqSSB 15%, Anti-Taq 15%, glycerol 40%.

[0063] 3. Gene primer pair and probe sequence (same as in Example 1)

[0064] The fluorescent groups corresponding to Orf1ab gene, N gene, and S gene are ROX, FAM, and CY5, respectively, and the corresponding quenching groups are all BHQ1.

[0065] 4. The primer and probe sequence (same as in Example 1) of internal quality control product

[0066] The fluorophore is VIC.

[0067] The PCR reaction system is as follows: 25% of PCR reaction solution, 10% of PCR enzyme solution, 0.8 μM of forward primer and reverse primer of single gene and internal quality control, 0.5 μM of probe, 20% of nucleic acid extraction product .

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Abstract

The invention provides a novel coronavirus detection kit as well as an application and use method thereof. The kit mainly comprises a primer probe mixture, wherein the primer probe mixture comprises Orf1ab gene, N gene and S gene specific forward and reverse primers and specific probes. The kit provided by the invention has the advantages of rapidness, high sensitivity, good specificity and good reproducibility. An amplified fragment in a primer sequence of an internal quality control product is gene in a human genome, so that the sensitivity of judging whether sampling is successful or not can be improved, a false negative detection result can be avoided, and potential patients can be isolated and treated in time. Research and development personnel accidentally discover that the S gene in the novel coronavirus has good conservative property in the research and development process, and has good specificity for detection of the novel coronavirus.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a kit for detecting novel coronavirus by using multiple real-time fluorescent RT-PCR technology, its application and the usage method of the kit. Background technique [0002] Winter and early spring are influenza outbreak seasons, and the common cold in adults at this time of year is mainly caused by coronaviruses. Coronavirus is a large family of viruses found in animals and humans. It was first isolated from chicken embryos. So far, about 15 different coronaviruses have been discovered. Among them, there are six kinds of coronaviruses that can infect humans, HCoV-229E, HCoV-0C43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Among the 6 viruses, the first four can only cause common colds, and common viral colds will have corresponding symptoms within 2-5 days, and can recover after 1-2 weeks of treatment. The latter two are Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/101C12Q2563/107
Inventor 董璐陈威汪瑶
Owner ZYBIO INC
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