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A novel coronavirus detection kit, application and method of use 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: 2022-03-25
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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  • A novel coronavirus detection kit, application and method of use thereof
  • A novel coronavirus detection kit, application and method of use thereof
  • A novel coronavirus detection kit, application and method of use 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 corresponding fluorescent groups of Orf1ab gene, N gene and S gene are ROX, TET, CY5, and the corresponding quenching group is BHQ3.

[0057] 4. Primer and probe sequences of the internal quality control substance (same as in Example 1)

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

[0059] The PCR reaction system is as follows: PCR reaction solution 50%, PCR enzyme solution 20%, forward primer and reverse primer of single gene and internal quality control product are 4μM respectively, probe is 0.8μM, nucleic acid extraction product 30%...

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 corresponding fluorescent groups of Orf1ab gene, N gene and S gene are ROX, FAM, CY5, and the corresponding quenching group is BHQ1.

[0065] 4. Primer and probe sequences of the internal quality control substance (same as in Example 1)

[0066] The fluorophore is VIC.

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

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Abstract

The present invention provides a novel coronavirus detection kit, application and method of use thereof. The kit mainly includes a primer probe mixture, and the primer probe mixture consists of Orf1ab gene, N gene and S gene-specific forward and reverse primers. and specific probe composition, the kit of the present invention has the advantages of rapidity, high sensitivity, good specificity and good reproducibility; the amplified fragment in the primer sequence of the internal quality control product in the present invention is human genome In the gene, it can increase the sensitivity of judging whether the sampling is successful, can avoid false negative test results, and allow potential patients to be isolated and treated in time; during the research and development process, researchers accidentally discovered that the S gene in the new coronavirus has a very good It is conservative and has good specificity for the detection of new coronaviruses.

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 a method for using the kit. Background technique [0002] Winter and early spring are the seasons for influenza outbreaks, and the common cold among adults at this time of year is primarily caused by coronaviruses. Coronaviruses are a large family of viruses found in animals and humans. They were first isolated from chicken embryos. So far, about 15 different coronaviruses have been discovered. Among them, there are six coronaviruses that can infect humans, HCoV-229E, HCoV-0C43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Among these 6 viruses, the first four can only cause common colds. Common viral colds will have corresponding symptoms within 2-5 days, and can be recovered after 1-2 weeks of treatment. The latter two are the globally feared severe acute respirator...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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