Primer set, probe set and kit for detecting COVID-19 virus, and application of kit

A COVID-19 and kit technology, applied in the field of microbial detection, can solve the problems of high operator skill requirements, large sequencing data volume, high sample requirements, etc., achieve large market prospects, wide application scenarios, and improve sensitivity.

Pending Publication Date: 2020-09-11
广州中科抗体生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used methods for detecting the new coronavirus COVID-19 are real-time fluorescent RT-PCR method and sequencing method. Negative cases also require a professional PCR laboratory to carry out, which requires high skills for operators, which is not conducive to large-scale promotion
However, the method of gene sequencing requires special sequencing instruments, the time period for sequencing is long, and the amount of sequencing data is large. It also requires professional bioinformatics analysts to analyze the sequence information. The cost is high and it is not conducive to clinical application.

Method used

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  • Primer set, probe set and kit for detecting COVID-19 virus, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] (1) Preparation of luminescent label-No. 1 probe complex:

[0070] 1) Select acridinium ester as a luminescent marker, and the acridinium ester solution is prepared by the following method:

[0071] Dissolve 5 mg of acridinium ester with 1.58 mL of DMSO to obtain a mother solution of acridinium ester with a concentration of 3.164 mg / mL, which is set aside. The acridinium ester mother liquor was diluted 10 times with DMSO to obtain an acridinium ester solution with a concentration of 0.3164 mg / mL.

[0072] 2) Preparation of No.1 probe solution:

[0073] Take probe No. 1 bound with biotin and dissolve in 0.1M NaHCO 3 , and the volume was adjusted to 90 μl to obtain the No. 1 probe solution.

[0074] 3) Mix the acridinium ester solution in step 1) with the No. 1 probe solution in step 2), control the mass ratio of the acridinium ester to the No. 1 probe to be (4-16): 1, and then place the mixed solution at room temperature The reaction was carried out for 5 hours, and ...

Embodiment 2

[0079] A kit, comprising: PCR amplification reaction system, No. 1 probe, No. 2 probe, acridinium ester solution, magnetic beads, cleaning solution, and excitation solution.

[0080] The No.1 probe is: 5’-ctcgttgact ttcaggttac tatagcagag at-Biotin-3’

[0081] The second probe is: 5'-NH 2 -ctaactgaga ataaatattc tcaattagat ga-3';

[0082] Among them, the No. 1 probe and the acridinium ester solution can be prepared into a luminescent marker-No. 1 probe complex by the method described in Example 1; the No. 2 probe and the magnetic beads can be formed by the method described in Example 1. Magnetic Beads - No. 2 Probe Complex.

[0083] The PCR amplification reaction system includes: 1.0 μL 10×Buffer, 0.2 μL dNTPs, 1.0 μL 2U / μL Taq enzyme, 0.1 μL 1.25 μmol / L upstream primer, 1.0 μL 1.25 μmol / L downstream primer, template, etc., ddH 2 O to make up to 10 μL;

[0084] The upstream primer is: 5'-at ctcgttgact ttcaggttac-3'

[0085] The downstream primer is: Phos phosphate group-5'-...

Embodiment 3

[0088] Adopt the kit detection COVID-19 virus of embodiment 2, comprise the following steps:

[0089] (1) Asymmetric PCR amplification

[0090] Using the serum of a patient with COVID-19 virus infection confirmed by sequencing as a sample, RNA was extracted, and asymmetric PCR amplification was performed after reverse transcription to obtain the target single-stranded DNA.

[0091] Among them, the PCR amplification program is: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 15s, 55°C annealing for 30s, 72°C extension for 60s, 40 cycles, and purification to obtain PCR amplification products.

[0092] Adopt 15% agarose electrophoresis to detect PCR product, the result is as follows figure 1 As shown, an obvious band appeared at about 150bp.

[0093] (2) One hybridization reaction

[0094] According to the method described in Example 1, the luminescent marker-No. 1 probe complex was prepared, wherein the mass ratio of the luminescent marker acridinium ester to the N...

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Abstract

The invention discloses a primer set for detecting the COVID-19 virus. The primer set comprises an upstream primer and a downstream primer, and the primer set has relatively low sample requirements and strong amplification specificity, and greatly improves the sensitivity of clinical detection results. The invention also discloses a probe set for detecting the COVID-19 virus. The probe set comprises a first probe and a second probe, and the probes can bind to target single-stranded DNA by base complementary pairing respectively, and are captured by magnetic beads, so that the purpose of amplifying optical signals and performing quantitative detection is realized. The invention also discloses a kit for detecting the COVID-19 virus. The kit uses the primer set and the probe set, combines with the magnetic bead capture and optical signal amplification technology, has the characteristics of high sensitivity and high specificity, has simple operation, has low cost, and can be widely used inprimary medical institutions. The kit can be used to detect the COVID-19 virus, has stable and reliable detection results, has wide applicable scenarios, and has great market prospects.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a primer set, a probe set and a kit for detecting COVID-19 virus and applications thereof. Background technique [0002] The novel coronavirus was named 2019-nCoV by the World Health Organization on January 12, 2020, and officially named COVID-19 by the World Health Organization on February 11, 2020. Professor Zhang Yongzhen from Fudan University led a research team to decipher its gene sequence and publish it on the website. Shi Zhengli's team found that the whole genome of 2019-nCoV has a sequence homology of 76% with SARS-CoV. By comparing its seven conserved non-structural proteins, it was confirmed that 2019-nCoV and SARS-CoV belong to the genus of coronavirus beta. The spike glycoprotein (S) invades the body by binding to the host cell receptor angiotensin-converting enzyme II (ACE-2). As a large virus family, coronaviruses are single-stranded positive-sense R...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682C12N15/11
CPCC12Q1/701C12Q1/682C12Q2531/107C12Q2563/107C12Q2563/143C12Q2563/149Y02A50/30
Inventor 周宁
Owner 广州中科抗体生物技术有限公司
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