A confirmation method for samples in the gray zone after nucleic acid amplification

A nucleic acid amplification reaction, confirmatory technology, applied in biomass post-processing, biochemical equipment and methods, biological material pretreatment, etc. Detection process, good specificity, and the effect of avoiding contamination

Active Publication Date: 2022-04-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in general, samples in the gray area cannot be directly judged as negative or positive, and often need to be re-tested by PCR amplification, which consumes a lot of manpower, material resources, and financial resources. The samples in the gray area will greatly shorten the time for judging samples and reduce the workload of experimenters

Method used

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  • A confirmation method for samples in the gray zone after nucleic acid amplification
  • A confirmation method for samples in the gray zone after nucleic acid amplification
  • A confirmation method for samples in the gray zone after nucleic acid amplification

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Taking the detection of a new coronavirus sample as an example to introduce a specific embodiment, the specific steps are as follows.

[0081] 1) According to the recommendation of China CDC, the gene loci for COVID-19 detection can be ORF or N gene fragments. Take the ORF gene PCR test recommended by CDC as an example.

[0082] If the Ct value of the sample is in the gray area after the PCR amplification, and it is difficult to determine whether it is a positive sample or a negative sample, the device is activated. First prepare the amplification product-specific detection reagent, its components include 0.2 micromolar per liter of specific nucleic acid probe corresponding to the PCR amplification product, 0.2 micromolar per liter of Cas protein, and 2.5 micromolar per liter of fluorescent Labeled single-stranded probes, in order to ensure better reaction, the system also includes 1×NEB buffer, 1U per microliter of RNA inhibitor. After preparation, take 25 microliter...

Embodiment 2

[0087] The specific embodiment is introduced by taking the detection of the transgene promoter CaMV335 gene in transgenic soybean as an example.

[0088] 1) Real-time quantitative PCR detection of the transgenic soybean promoter CaMV335.

[0089] If the Ct value of the sample is in the gray area after the PCR amplification, and it is difficult to determine whether it is a genetically modified soybean, the device is activated. Firstly, the amplification product-specific detection reagent is prepared. The use of Cas protein, fluorescently labeled single-stranded probe, NEB buffer, and RNA inhibitor is the same as in Example 1. The concentration of the specific nucleic acid probe corresponding to the PCR amplification product of the transgenic soybean is the same as that in Example 1.

[0090] 2) Put the PCR sample tube with the Ct value in the gray area after the PCR detection of the transgenic soybean into the PCR test tube nucleic acid amplification reaction tube rack, so th...

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Abstract

The invention discloses a confirmation method for a sample in a gray area after nucleic acid amplification. Add reagents into the detection reagent tube, put the nucleic acid amplification reaction tube into the nucleic acid amplification reaction tube rack, cover the sealing cap, the main body of the cartridge and the sealing cap form a closed space; then press down the sealing cap to form a pre-seal; continue Press the sealing cap downward, the blade contacts the nucleic acid amplification reaction tube, and cuts the bottom of the nucleic acid amplification reaction tube, the liquid in the nucleic acid amplification reaction tube flows out into the detection reagent tube and then reacts; the light source irradiation observation results confirm the detection; the device It includes a cartridge main body, a nucleic acid amplification reaction tube rack and a sealing cover, and the cartridge main body includes a shell, a knife holder and a blade. The present invention does not change any process of the existing PCR amplification or isothermal nucleic acid amplification operation, and can confirm and detect the detection samples in the gray area. The confirmation of the gray area signal has high sensitivity and good specificity, and the entire confirmation process is short in time and low in cost. Low and easy to operate.

Description

technical field [0001] The invention relates to a detection method for nucleic acid amplification, in particular to a method for confirmatory detection of samples in the gray area after nucleic acid amplification, which can avoid repeated detection of samples in the gray area and prevent aerosol pollution, and is suitable for the field of nucleic acid detection. It belongs to the fields of medical testing, analytical chemistry, food testing and so on. Background technique [0002] Nucleic acid is the carrier of genetic information, and the nucleic acid sequences of different organisms are specific, so nucleic acid detection can effectively identify and detect the substances to be tested. At present, nucleic acid detection technology has been widely used in food safety detection, clinical diagnosis, genotyping, molecular cloning and other aspects. Due to the low concentration of nucleic acid in the sample to be tested, it is often necessary to amplify the nucleic acid sample...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6848C12M1/34C12M1/00
CPCC12Q1/6848C12Q2521/327
Inventor 吴坚陈艳菊伍辉
Owner ZHEJIANG UNIV
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