Multiple nucleic acid detection method, combination and kit

A multiple nucleic acid and signal detection technology, applied in the field of molecular biology, can solve the problems of increased detection cost and unfavorable clinical promotion and use

Pending Publication Date: 2020-12-22
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in addition to using PCR amplification detection equipment, this method also needs to use capillary electrophoresis equipment for product analysis, which greatly increases the detection cost and is not conducive to clinical promotion and use.

Method used

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  • Multiple nucleic acid detection method, combination and kit
  • Multiple nucleic acid detection method, combination and kit
  • Multiple nucleic acid detection method, combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Example 1 is designed to detect primer sets and flexible probes for respiratory-associated pathogens

[0178] In order to verify the combination of the present invention in the detection of influenza A virus (Influenza A virus, IAV), influenza B virus (Influenza B virus, IBV), respiratory syncytial virus and (Respiratory Syncytial Virus, RSV), adenovirus (Adenovirus, ADV), Mycoplasma pneumoniae (Mycoplasma pneumonia, MP) and novel coronavirus (SARS-CoV-2), designed the primers and probes in Table 1 below.

[0179] Table 1

[0180]

[0181] Wherein, upstream primer 1 and downstream primer 1 are specific primers designed for the target sequence of adenovirus; upstream primer 2 and downstream primer 2 are specific primers designed for the target sequence of influenza B virus; upstream primer 3 and downstream primer 3 are Specific primers designed for the target sequence of Mycoplasma pneumoniae; upstream primer 4 and downstream primer 4 are specific primers designed fo...

Embodiment 2

[0185] Example 2 Single detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, Mycoplasma pneumoniae and novel coronavirus

[0186] Sample preparation: Use the in vitro transcribed RNA of each target sequence as a positive sample, and test each target sequence separately to verify the detection ability of a single virus infection. Pure water was used as a no-template control (NTC).

[0187] Reaction preparation:

[0188] After the sample preparation is completed, configure the reaction system according to the proportions described in Table 3 below.

[0189] table 3

[0190]

[0191] PCR amplification and melting curve analysis:

[0192] After sealing the PCR tube cap, mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The PCR tube was placed in the palm centrifuge again, and after a short centrifugation, it was transferred to the tray of a fluorescent quantitative PCR instrument (Suzhou...

Embodiment 3

[0197] Example 3 Multiple detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, Mycoplasma pneumoniae and novel coronavirus

[0198] Sample preparation: Use the in vitro transcribed RNA of each target sequence as a positive sample, mix the two target sequence templates of the same fluorescent channel in Table 2, and then perform detection to verify the detection ability of virus mixed infection. Ultrapure water was used as a no-template control (NTC).

[0199] Reaction preparation:

[0200] After the sample preparation is completed, configure the reaction system according to the proportions described in Table 3.

[0201] PCR amplification and melting curve analysis:

[0202] After sealing the PCR tube cap, mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The PCR tube was placed in the palm centrifuge again, and after a short centrifugation, it was transferred to the tray of a fluorescent...

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Abstract

The invention relates to a multiple nucleic acid detection method, combination and kit. The method comprises the following steps of mixing a combination containing a first primer group and a first probe with a sample from a subject; amplifying a target sequence possibly existing in the sample; carrying out melting curve analysis on an amplified product in a corresponding detection channel; and judging whether one or more targets exist or not according to a result of the melting curve analysis.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a solution for detecting melting curves of multiple targets. Background technique [0002] The polymerase chain reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without the use of living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as diagnosis of infectious diseases, gene cloning, phenotyping of experimental animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, paternity testing, etc. . Due to its unparalleled ability to replicate and be precise, PCR is considered by molecular biologists to be the method of choice for nucleic acid detection. In the late 1990s, the real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) technology and related products launched by the American ABI company developed PCR in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143C12Q2527/107C12Q1/6876C12Q2600/16C12Q2531/107
Inventor 赵雨航詹浩淼黄梅
Owner SICHUAN MACCURA BIOTECH CO LTD
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