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A probe primer set, kit and detection method for rapid differential detection of mink coronavirus and novel coronavirus

A coronavirus and kit technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of unreported identification and detection methods, and achieve good specificity

Active Publication Date: 2022-06-17
山东省动物疫病预防与控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the human new coronavirus detection method has been applied on a large scale, the differential detection method for simultaneous detection of new coronavirus and mink coronavirus has not been reported

Method used

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  • A probe primer set, kit and detection method for rapid differential detection of mink coronavirus and novel coronavirus
  • A probe primer set, kit and detection method for rapid differential detection of mink coronavirus and novel coronavirus
  • A probe primer set, kit and detection method for rapid differential detection of mink coronavirus and novel coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Design of primers and probes

Embodiment 1

[0046] 1. Primer and probe design

[0047] 1.1 Principles of primer and probe design

[0048] In order to ensure the conservation and specificity of primers and probes, the specific sequence of the N gene coding region of mink coronavirus and new coronavirus was selected as the target region, and the primer length was about 20 bases and the GC content was 50%-60%. , no secondary structure and repeatability within primers, no complementary sequences between primers and within primers, and the difference in melting temperature (Tm value) between primers is less than 2°C. The length of the probe is about 25 bases, and the Tm value is about 5°C higher than the Tm value of the primer to design specific primers and probes.

[0049] Most primer designs in this field are directly synthesized by software, and the optimal primer sequences are selected. The primer sequences involved in the examples of this application are the results of artificial design on the basis of software design;...

Embodiment 2

[0067] Example 2 The establishment of the fluorescent RT-PCR reaction system of the rapid identification and detection kit of mink coronavirus and novel coronavirus

[0068] On the basis of multiple sequence alignment, a probe primer set that can simultaneously and rapidly identify and detect mink coronavirus and 2019-nCoV was designed, a fluorescent RT-PCR detection method was initially established, and the Taguchi method was used for optimization to find out primers and probes. , Mg 2+ The optimal range of concentration, adjust accordingly according to the optimal range, and finally obtain primers, probes, Mg 2+ To determine the optimal conditions of the detection method: the reaction system is 25 μL in total, including fluorescent RT-PCR reaction solution, enzyme mixture, and template. Among them, a detection reaction solution consists of 2 μL of dNTP Mixture (adenine, guanine, thymine, cytosine deoxynucleotide mixture) (2.5 mmol / L), 1 μL of primer MF (10 μmol / L), 1 μL of...

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Abstract

The invention specifically relates to a probe primer set, a kit and a detection method for rapid identification and detection of mink coronavirus and novel coronavirus, and belongs to the technical field of animal quarantine. The present invention selects the specific sequence of the N gene coding region of mink coronavirus as the target region, and designs mink coronavirus primers and probes on the basis of multiple sequence comparisons; selects the specific sequence of the N gene coding region of the new coronavirus as the target region, On the basis of sequence comparison, the design and selection of new coronavirus primers and probes are carried out. The probe primer designed and synthesized by the present invention has the advantages of rapidity, sensitivity, strong specificity, good stability, and is not easy to be polluted.

Description

technical field [0001] The invention relates to a probe primer set, a kit and a detection method for rapid identification and detection of mink coronavirus and novel coronavirus, belonging to the technical field of animal quarantine. Background technique [0002] The description of the background technology in the present invention belongs to the related art related to the present invention, and is only used to illustrate and facilitate the understanding of the invention content of the present invention, and should not be construed as the applicant's explicit belief or presumption that the applicant believes that the present invention is the first application of the present invention. the prior art of the filing date. [0003] Mink coronavirus disease, also known as mink coronavirus enteritis, is a viral infectious disease caused by mink coronavirus that endangers mink aquaculture. The main symptom is diarrhea. The disease is another important viral infectious disease that ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/166C12Q2521/107C12Q2545/113C12Q2563/107C12Q2527/127C12Q1/6883
Inventor 王贵升张鹤晓苏莹兰邹然李玉杰马慧玲杨鹏
Owner 山东省动物疫病预防与控制中心
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