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Primer-probe, kit and rapid detection method for detecting novel coronavirus

A coronavirus, primer probe technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of nucleic acid instability, no primer-probe screening rules, and mismatching detection systems of extraction reagents, etc.

Pending Publication Date: 2021-09-10
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem of existing extraction-free nucleic acid release agents is that cell lysis is not complete, resulting in incomplete release of nucleic acid, resulting in instability of nucleic acid, especially RNA, and is easily degraded by temperature, enzymes and other factors; in addition, the protein content in the cell is relatively high , and contains a large number of substances that inhibit nucleic acid detection, resulting in a mismatch between the extraction reagent and the detection system, resulting in inaccurate detection results
[0003] RT-RPA, recombinase polymerase amplification, is the most similar detection technology to fluorescent quantitative PCR. This method has high sensitivity and specificity, but it does not have a mature primer and probe screening rule. Screen a large number of primers and probes to form detection reagents with high sensitivity and specificity
There are many reports on detection methods combining trace nucleic acid release agents and PCR, but due to the difficulty of designing primers and screening, there are currently no literature reports on the combination of trace nucleic acid release agents and RT-RPA methods for the detection of new coronaviruses

Method used

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  • Primer-probe, kit and rapid detection method for detecting novel coronavirus
  • Primer-probe, kit and rapid detection method for detecting novel coronavirus
  • Primer-probe, kit and rapid detection method for detecting novel coronavirus

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The present invention provides a primer and a probe for detecting the S gene of the novel coronavirus based on recombinase polymerase amplification technology, and after a large number of experimental verifications, it is finally determined that the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are selected The upstream primer and downstream primer, and the probe containing the nucleotide sequence shown in SEQ ID NO.3. Among them, in the nucleotide sequence shown in SEQ ID NO: 3, the 32nd base from the 5' end is labeled with a FAM luminescent group, the 33rd base is connected with an abasic site, and the 35th base is Mark the quenching group BHQ1, the sequence is shown in Table 1. Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to synthesize the primer probes as shown in Table 1.

[0035] Table 1 Summary of primers and probes for detection of novel coronavirus E gene RPA

[0036]

[0037] (2) Specificity verification experiment

[0038] Select...

Embodiment 2

[0049] Establish the RT-RPA detection method that is used for novel coronavirus, it comprises the steps:

[0050] The sample to be tested can be serum, whole blood, plasma, throat swab, feces as a 10% liquid solution, and a small amount of nucleic acid release agent is mixed with the sample to be tested at 1:1 to extract the nucleic acid in the sample to be tested.

[0051] The RPA primer probe concentration and reaction system are as follows: Among them, the RT-RPA kit used is a fluorescent RNA constant temperature rapid amplification kit.

[0052] 1) Add 29 μL A buffer to each dry powder tube by the pipetting mechanism;

[0053] 2) Add 5 μL of primer-probe mixture (2 μL of the upstream primer shown in Example 1, 2 μL of the downstream primer shown in Example 1, and 1 μL of the probes at a concentration of 10 μM).

[0054] 3) Add 13.5 μL of the micro-nucleic acid releasing agent and the sample into the reaction tube by the pipetting mechanism.

[0055] 4) Add 2.5 μL of B buf...

Embodiment 3

[0060] Embodiment 3 repeatability verification experiment

[0061] According to the method described in Example 2, RT-RPA amplification was performed on the new coronavirus indoor quality control products S2 (median value) and S1 (low value) (diluted with PBS), and each sample was repeated three times for verification. The repeatability of this method.

[0062] Result table 2, the amplification figure is shown in figure 2 , the results show that the new coronavirus indoor quality control products S2 (median value) and S1 (low value) have good repeatability in three experiments.

[0063] Table 2 Repeatability verification experiment of RPA detection of novel coronavirus E gene

[0064]

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Abstract

The invention relates to a primer-probe, kit and rapid detection method for detecting novel coronavirus. The primer-probe comprises an upstream primer and a downstream primer with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and a probe containing a nucleotide sequence as shown in SEQ ID NO.3, wherein in the nucleotide sequence as shown in SEQ ID NO.3, the 32nd base from the 5'-terminal is marked with a FAM luminous group, an abasic site is connected behind the 33rd base, and the 35th base is marked with a quenching group BHQ1. According to the rapid detection method for the novel coronavirus, provided by the invention, the novel coronavirus is detected by combining a trace amount of nucleic acid releasing agent and RT-RPA, and a whole process of nucleic acid extraction and detection can be completed within 30 minutes, so that the detection time is greatly shortened, the detection efficiency is improved, and the labor cost and the time cost are effectively reduced.

Description

technical field [0001] The invention relates to a primer probe, a kit and a rapid detection method for detecting a novel coronavirus, belonging to the technical field of biological detection. Background technique [0002] At present, nucleic acid extraction is an important part of nucleic acid detection. Traditional nucleic acid extraction methods include physical methods, including boiling, magnetic beads, ultrasonic methods, etc.; chemical methods include surfactants, alkali lysis, etc.; biological methods mainly include enzymes. law etc. The main problem of existing extraction-free nucleic acid release agents is that cell lysis is not complete, resulting in incomplete release of nucleic acid, resulting in instability of nucleic acid, especially RNA, and is easily degraded by temperature, enzymes and other factors; in addition, the protein content in the cell is relatively high , and contains a large number of substances that inhibit nucleic acid detection, resulting in a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6806C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q1/6806C12Q2521/507C12Q2563/107C12Q2527/125
Inventor 常宇桐胡孔新刘丽娟张丽萍杨爽
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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