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Triple real-time fluorescent RT-PCR primer, probe and detection method for detecting novel coronavirus

A coronavirus and detection method technology, applied in the field of triple fluorescent RT-PCR primers, can solve the problems of low positive separation rate, long detection cycle, delay of disease course and control of epidemic situation, so as to reduce case fatality rate, high sensitivity and control epidemic situation The effect of gaining time

Active Publication Date: 2021-07-23
SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional detection method has a long detection cycle and a low positive rate of isolation, which often delays the course of the disease and the control of the epidemic

Method used

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  • Triple real-time fluorescent RT-PCR primer, probe and detection method for detecting novel coronavirus
  • Triple real-time fluorescent RT-PCR primer, probe and detection method for detecting novel coronavirus
  • Triple real-time fluorescent RT-PCR primer, probe and detection method for detecting novel coronavirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, the design of triple real-time fluorescent RT-PCR primers and probes for detecting novel coronavirus (2019-nCoV)

[0017] For the Orf1ab gene, N gene, and human ribozyme P gene specific conserved region of the new coronavirus (2019-nCoV) as the target region, specific primers and fluorescent PCR probes were designed. The sequences are:

[0018] SEQ ID No.: 1

[0019] 5′-TGGAAAGGTTATGGCTGTAGTTGTG-3′

[0020] SEQ ID No.: 2

[0021] 5′-CACTTACACCGCAAACCCGTT-3′

[0022] SEQ ID No.: 3

[0023] 5′FAM-CAACTCCGCGAACCCATGCTTCAGTC-BHQ1-3′

[0024] SEQ ID No.: 4

[0025] 5′-ATTGGCCGCAAATTGCACA-3′

[0026] SEQ ID No.: 5

[0027] 5′-TCTTCTGTCTCTGCGGTAAGGC-3′

[0028] SEQ ID No.: 6

[0029] 5'HEX-CCCCCAGCGCTTCAGCGTTCTTC-BHQ1-3'

[0030] SEQ ID No.: 7

[0031] 5′-CTGTTTGGGCTCTCTGAAAGTG-3′

[0032] SEQ ID No.: 8

[0033] 5′-AGAAGCGCTGCTCGGCA-3′

[0034] SEQ ID No.: 9

[0035] 5′CY5-CGCCAAGGCTGCGGTGTCCACC-BHQ1-3′

Embodiment 2

[0036] Embodiment 2, the establishment of a kind of triple fluorescent RT-PCR method for detecting novel coronavirus (2019-nCoV)

[0037] (1) Triple fluorescent RT-PCR reaction

[0038] 1) Extraction of sample RNA: The sample RNA can be extracted by using the viral RNA nucleic acid extraction kit from Kaiser Bioengineering Co., Ltd., Germany, according to the kit instructions.

[0039] 2) Using the sample RNA prepared in step 1) as a template, add the RT-qPCR Buffer reaction solution and enzyme mixture in the Quanti Probe One-Step RT-qPCR kit of Shenzhen United Medical Technology Co., Ltd. to make a triple RT -PCR reaction system for amplification reaction.

[0040] The reaction system is 25 μl, as follows:

[0041]

[0042]

[0043] (2) Fluorescence detection

[0044] Place the above-mentioned reaction tube in a fluorescent PCR detector with three fluorescent channels for fluorescence detection. The specific reaction procedure is as follows:

[0045]

[0046] Neg...

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Abstract

The invention provides a fluorescent RT-PCR primer and a fluorescent probe for detecting a novel coronavirus (2019-nCoV) by using a triple real-time fluorescent RT-PCR nucleic acid amplification technology and application of the fluorescent RT-PCR primer and the fluorescent probe in detecting the novel coronavirus (2019-nCoV). The fluorescent RT-PCR primer and the fluorescent probe can be used for rapidly and qualitatively detecting the novel coronavirus (2019-nCoV) in a throat swab sample, two target genes of the novel coronavirus (2019-nCoV) can be detected simultaneously and the sampling effectiveness can be effectively monitored. The fluorescent RT-PCR primer and the fluorescent probe has stronger specificity and higher sensitivity, which can completely meet rapid diagnosis and whole-process monitoring of epidemic situations, and can buy time for early diagnosis and early treatment of the epidemic situations, reduction of case fatality rate and control of the epidemic situations.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a triple fluorescent RT-PCR primer, probe and detection method for detecting novel coronavirus (2019 novel Coronavirus, 2019-nCoV) in throat swab samples. Background technique [0002] 2019-nCoV acute respiratory disease (2019-nCoV acute respiratory disease) is a serious respiratory disease caused by 2019 novel coronavirus (2019-nCoV), and it is a disease after SARS-CoV and MERS-CoV. A newly discovered coronavirus that is highly transmissible and can infect humans. The main symptoms of the novel coronavirus (2019-nCoV) are fever, fatigue, and dry cough. A few are accompanied by symptoms such as nasal congestion, runny nose, and diarrhea, which are similar to other respiratory virus infection symptoms and difficult to distinguish. Moreover, some patients only showed low-grade fever, mild fatigue, etc., without pneumonia, clinically asymptomatic, but con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107Y02A50/30
Inventor 周冬根罗洁俞雪钧
Owner SHANDONG BOSIYUAN BIOLOGICAL TECH CO LTD
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