Nucleic acid testing test kit, method and application thereof

A detection method and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as safety hazards

Pending Publication Date: 2020-11-24
SHANGHAI EPIPROBE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, genetically modified technology also brings some potential safety hazards. Illegal cultivatio...

Method used

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  • Nucleic acid testing test kit, method and application thereof
  • Nucleic acid testing test kit, method and application thereof
  • Nucleic acid testing test kit, method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Nucleic acid detection of Salmonella

[0081] The nucleic acid detection method for Salmonella mainly includes the following steps:

[0082] 1.1. Preparation of test samples: use a bacterial nucleic acid extraction kit to extract the nucleic acid of Salmonella, and then prepare the following three samples, positive sample 1 (sample 1) containing a sample of Salmonella nucleic acid fragment, and positive sample 2 (sample) containing a sample of Salmonella nucleic acid fragment. 2) Negative samples (sample 3) that do not contain samples of Salmonella nucleic acid fragments.

[0083] 1.2. Design primers and probes: For the extracted genome sequence of Salmonella, design isothermal amplification primers and detection probes. The amplification forward primer sequence is SEQ ID NO.01, the amplification reverse primer sequence is SEQ ID NO.02, and the detection probe sequence is SEQ ID NO.03.

[0084] 1.3. Constant temperature amplification: The RNA constant temper...

Embodiment 2

[0093] Example 2 Detection of African swine fever virus

[0094] The nucleic acid detection method of African swine fever virus mainly includes the following steps:

[0095] 2.1. Preparation of test samples: use a viral nucleic acid extraction kit to extract the nucleic acid of African swine fever virus, and then prepare the following three samples, positive samples containing African swine fever virus nucleic acid fragments 1. Positive samples containing African swine fever virus nucleic acid fragments 2. Negative samples that do not contain African swine fever virus nucleic acid fragments 3.

[0096] 2.2. Design primers and probes: For the extracted genome sequence of African swine fever virus, design isothermal amplification primers and detection probes. The amplification forward primer sequence is SEQ ID NO.04, the amplification reverse primer sequence is SEQ ID NO.05, and the detection probe sequence is SEQ ID NO.06.

[0097] 2.3. Constant temperature amplification: Use...

Embodiment 3

[0105] Example 3 Detection of transgenic nucleic acid

[0106] The detection of transgenic nucleic acid takes the detection of NOS gene as an example. The detection method of NOS gene mainly includes the following steps:

[0107] 3.1. Preparation of test samples: prepare the following three kinds of samples, positive sample 1 (sample 1) containing nucleic acid fragments of NOS gene, positive sample 2 (sample 2) containing nucleic acid fragments of NOS gene, and nucleic acid fragments not containing NOS gene of negative sample 3 (sample 3).

[0108] 3.2. Design primers and probes: Design isothermal amplification primers and detection probes for the extracted nucleic acid sequence of NOS gene. The amplification forward primer sequence is SEQ ID NO.07, the amplification reverse primer sequence is SEQ ID NO.08, and the detection probe sequence is SEQ ID NO.09.

[0109] 3.3. Constant temperature amplification: Use the RNA constant temperature rapid amplification kit (Item No.: WL...

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Abstract

The invention provides a nucleic acid testing test kit, method and application thereof. The method comprises the following steps: S1, isothermal amplification: carrying out isothermal amplification onnucleic acid to be detected; S2, secondary amplification: carrying out secondary amplification on a product obtained after isothermal amplification, the product obtained after secondary amplificationbeing a nucleic acid single strand, or generating a single strand in other modes; S3, probe hybridization: hybridizing a product obtained after secondary amplification with a probe; S4, probe degradation: adding nuclease into the hybridized product to degrade redundant single-stranded nucleic acid; and S5, detecting a product, namely detecting the product obtained in the step S4. The test kit comprises an isothermal amplification primer, a secondary amplification enzyme, a probe, nuclease and a test strip; the application comprises various kinds of nucleic acid testing such as pathogenic microorganism nucleic acid testing, virus nucleic acid testing, transgenic nucleic acid testing, genetic disease nucleic acid testing, tumor nucleic acid testing, DNA methylation nucleic acid testing andthe like. The invention provides a rapid, specific and sensitive nucleic acid testing test kit without complex instruments and equipment, a method and application thereof.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid detection kit, method and application thereof. Background technique [0002] Nucleic acid detection is a method of detecting whether there are target nucleic acid substances in samples such as viruses, bacteria, and cells. In terms of nucleic acid detection in the prior art, there are mainly three types: (1) Conventional real-time quantitative fluorescent PCR detection, which is the most common method at present. One or more pairs of primers are designed for the sequence of the new coronavirus, and the nucleic acid is extracted. Reverse transcription and qPCR, the test result is the number of cycles; (2) On-site detection methods that do not rely on large instruments, such as the CRISPR / Cas12-based method DETECTER developed by American researchers, the samples used in this method are nasal swabs or pharyngeal swabs The nucleic acid extracted from the swab cannot meet ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6816
CPCC12Q1/6844C12Q1/6816C12Q1/689Y02A50/30C12Q1/701C12Q2521/307C12Q2521/319C12Q2521/507C12Q2527/101C12Q2531/113C12Q2531/119C12Q2531/125C12Q2545/114C12Q2563/107C12Q2563/131
Inventor 李振艳董世华徐鹏
Owner SHANGHAI EPIPROBE BIOTECH CO LTD
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