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Amplification primer composition and kit for detecting new coronavirus

A coronavirus and amplification primer technology, applied in the biological field, can solve the problems of high operation requirements of medical personnel, high operator requirements, and large investment in equipment costs, avoiding false positives, increasing annealing temperature, and high amplification efficiency. and specific effects

Active Publication Date: 2021-12-21
苏州蝌蚪生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the common methods for detecting 2019-nCoV are: enzyme-linked immunoassay, but this method has low sensitivity and high false positives, which is easy to cause false detection; fluorescent quantitative PCR method, this method has high sensitivity, but the cost of equipment is large, and it is difficult for operation. High personnel requirements; blood routine and CT detection method, this method is relatively intuitive, but also has high equipment cost, cumbersome operation, high requirements for medical personnel, and takes a long time, not convenient and fast enough

Method used

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  • Amplification primer composition and kit for detecting new coronavirus
  • Amplification primer composition and kit for detecting new coronavirus
  • Amplification primer composition and kit for detecting new coronavirus

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Effect test

Embodiment 1

[0088] 1. Primer design for PCR of 2019-nCoV

[0089] Referring to the 2019-nCoV genome sequence included in GenBank, select the conserved regions of ORF1ab gene and N gene of 2019-nCoV, and use ABI PrimerExpress 3.0 PCR primer design software to design and synthesize primers. The final sequences of each primer are as follows.

[0090]

[0091] 2. Locked Nucleic Acid Modified PCR Primers

[0092] The primer modification used in this embodiment is: each upstream primer and downstream primer has four bases modified by locked nucleic acid (+N bases are modified by locked nucleic acid).

[0093] 2.1 2019-nCoV-ORF1ab Locked Nucleic Acid Modified Primers

[0094] 2.1.1 2019-nCoV-ORF1ab four base-locked nucleic acid modified primers (see Table 2 above for details)

[0095] F: 5'-AATGT +T AATGC +A CTTTT +A TCTAC +T -3'

[0096] R: 5'-AACCG +T TCAA +T CATA +A GTGT +A -3'

[0097] 2.1.2 2019-nCoV-ORF1ab zero base-locked nucleic acid modified primers

[0098] F: 5'-AATGTTAA...

Embodiment 2201

[0181] Example 2 Detection Kit for 2019-nCoV PCR Amplification Combined with Nucleic Acid Capture Gold Standard Test Strip

[0182] Locked nucleic acid modified primers 2019-nCoV-ORF1ab-F (10 μM) 40 μL for PCR detection of 2019 novel coronavirus (2019-nCoV) nucleic acid, 2019-nCoV-ORF1ab-R (10 μM) 40 μL, 2019-nCoV- 40 μL of N-F (10 μM), 40 μL of 2019-nCoV-N-R (10 μM), 250 μL of master mix, 250 μL of 2mM deoxyribonucleotide, Mn 2+ 60 μL, 15 μL of amplification enzyme, 20 μL of 2019-nCoV plasmid DNA (30ng / μL), 1.5 mL of ultrapure water, 5 mL of loading sample diluent, and 48 finished test strips were packaged together to obtain the 2019 novel coronavirus (2019-nCoV ) PCR product nucleic acid capture gold standard test strip detection kit.

[0183] In summary, the kit and detection method provided by the present invention have the characteristics of high sensitivity, strong specificity, and good stability, and can be used for the rapid detection of 2019 novel coronavirus pneumo...

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Abstract

The invention discloses an amplification primer composition and a kit for detecting a new coronavirus. The amplification primer composition comprises a first primer group and a second primer group, the first primer group comprises a first forward primer and a first reverse primer, the second primer group comprises a second forward primer and a second reverse primer. In addition, .four basic groups in each sequence of the first forward primer, the first reverse primer, the second forward primer and the second reverse primer are modified by locked nucleic acid, one of the four basic groups is a 3'terminal basic group, and the distance between every two adjacent basic groups modified by the locked nucleic acid is 3-6 basic groups; the basic group modified by the locked nucleic acid is adenine and / or thymine. The invention also discloses the kit for detecting the new coronavirus. According to the amplification primer composition and the kit for detecting the new coronavirus, the kit has the characteristics of high sensitivity, strong specificity, good stability and the like, can be used for rapid detection of new coronavirus pneumonia, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to an amplification primer composition for detecting novel coronavirus, in particular to a novel coronavirus PCR amplification binding nucleic acid modified by locked nucleic acid and labeled with a chemical group. Specific primers for molecular capture, and corresponding kits. Background technique [0002] The 2019 novel coronavirus (2019-nCoV for short) belongs to the genus Coronavirus of the order Nidovirales, the family Coronaviridae, and the genus Coronavirus in terms of systematic classification. Viruses of the genus Coronavirus are RNA viruses with an envelope and a linear single-stranded positive strand genome. They are a large class of viruses that exist widely in nature. [0003] At present, the common methods for detecting 2019-nCoV are: enzyme-linked immunoassay, but this method has low sensitivity and high false positives, which is easy to cause false...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6853C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6853C12Q1/6848C12Q2531/113C12Q2527/107C12Q2527/101C12Q2563/131C12Q2565/625C12Q2525/121
Inventor 苏静沈强陆寿祥周莉质
Owner 苏州蝌蚪生物技术有限公司
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