Reagent and kit for quickly detecting microbial pollution of saliva DNA, and application thereof

A technology of microbial contamination and kit, applied in the field of molecular biology, to achieve the effect of convenient operation, low difficulty and guaranteed reliability

Inactive Publication Date: 2017-07-04
SUZHOU ZHONGXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The main purpose of the present invention is exactly for above present situation, provides a kind of reagent, test kit and application thereof for rapid detection of microbial contamination in saliva DNA, to overcome the deficiencies in the prior art

Method used

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  • Reagent and kit for quickly detecting microbial pollution of saliva DNA, and application thereof
  • Reagent and kit for quickly detecting microbial pollution of saliva DNA, and application thereof
  • Reagent and kit for quickly detecting microbial pollution of saliva DNA, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] 1. Reagent preparation:

[0071] 6×Loading Dye (Thermo), 100bp gene ruler (Thermo), Escherichia coli DNA (Promega), human genomic DNA (Agilent);

[0072] Reagent I: 2x KAPA HiFi HotStart ReadyMix 12.5uL, Oligo 1 (1uM) 5uL, Oligo 2 (1uM) 5uL.

[0073] 2. Instruments for testing:

[0074] PCR instrument (LongGene), Liuyi electrophoresis instrument, gel imager (Furi).

[0075] 3. Experimental operation process:

[0076] (1) Take 5 ng of 3 samples to be tested, positive control Escherichia coli DNA, and negative control human genomic DNA;

[0077] (2) Take a 1.5ml EP tube, and mark the reagent I mixture on the EP tube;

[0078] (3) Reagent I mixed solution preparation (N=5 in this experiment):

[0079]

[0080] (4) Reaction system:

[0081]

[0082] (5) Add 25 μl of PCR reaction solution to the 0.2mL PCR tube according to the samples;

[0083] (6) Put the sample into a microcentrifuge, set at 1000×g, and centrifuge for 30s;

[0084] (7) After the centrifugatio...

Embodiment 2

[0094] 1. Reagent preparation:

[0095] 6×Loading Dye (Thermo), 100bp gene ruler (Thermo), Escherichia coli DNA (Promega), human genomic DNA (Agilent);

[0096] Reagent I: 2x KAPA HiFi HotStart ReadyMix 12.5uL, Oligo 1 (1uM) 5uL, Oligo 2 (1uM) 5uL.

[0097] 2. Instruments for testing:

[0098] PCR instrument (Mycycler, Biorad), Liuyi electrophoresis instrument, gel imager (Ecosai).

[0099] 3. Experimental operation process:

[0100] (1) Take 5 ng of 3 samples to be tested, positive control Escherichia coli DNA, and negative control human genomic DNA;

[0101] (2) Take a 1.5ml EP tube, and mark the reagent I mixture on the EP tube;

[0102] (3) Reagent I mixed solution preparation (N=5 in this experiment):

[0103]

[0104] (4) Reaction system:

[0105]

[0106] (5) Add 25 μl of PCR reaction solution to the 0.2mL PCR tube according to the samples;

[0107] (6) Put the sample into a microcentrifuge, set at 1000×g, and centrifuge for 30s;

[0108] (7) After the cen...

Embodiment 3

[0118] 1. Reagent preparation:

[0119] 6×Loading Dye (Thermo), 100bp gene ruler (Thermo), Escherichia coli DNA (Promega), human genomic DNA (Agilent);

[0120]Reagent I: 2x KAPA HiFi HotStart ReadyMix 12.5uL, Oligo 1 (1uM) 5uL, Oligo 2 (1uM) 5uL.

[0121] 2. Instruments for testing:

[0122] PCR instrument (S1000, BioRad), BioRad electrophoresis instrument, and gel imager (Ecosa).

[0123] 3. Experimental operation process:

[0124] (1) Take 5 ng of 3 samples to be tested, positive control Escherichia coli DNA, and negative control human genomic DNA;

[0125] (2) Take a 1.5ml EP tube, and mark the reagent I mixture on the EP tube;

[0126] (3) Reagent I mixed solution preparation (N=5 in this experiment):

[0127]

[0128]

[0129] (4) Reaction system:

[0130]

[0131] (5) Add 25 μl of PCR reaction solution to the 0.2mL PCR tube according to the samples;

[0132] (6) Put the sample into a microcentrifuge, set at 1000×g, and centrifuge for 30s;

[0133] (7) A...

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Abstract

The invention discloses a reagent and a kit for quickly detecting microbial pollution of saliva DNA, and applications thereof. The reagent includes a first primer and a second primer. A detection method includes the steps of: 1) providing the reagent or the kit for quickly detecting microbial pollution of saliva DNA; 2) uniformly mixing a to-be-detected saliva DNA sample, the reagent for quickly detecting the microbial pollution of saliva DNA and a regular component for PCR amplification detection, and performing PCR amplification. The reagent has simple configuration, is simple in operation and short in time consumption, is low in cost, is free of special technical training and special instruments and devices, can effectively detect the degree of the microbial pollution in saliva DNA, effectively guarantees reliability of application of a next-generation sequencing targeted capturing technology in medical examination, and greatly reduces experiment and cost risks due to failure of subsequent experiment, which is caused by the microbial pollution of a saliva DNA sample, thereby ensuring that the saliva DNA is used for downstream molecular biological detection.

Description

technical field [0001] The invention relates to the technical field of molecular biology, more specifically, to the technical field of DNA, in particular to a reagent, kit and application thereof for rapidly detecting microbial contamination in saliva DNA. Background technique [0002] In May 2013, Hollywood actress and director Angelina Jolie, who has 6 children, confirmed that she had inherited the breast cancer susceptibility gene BRCA1 through genetic testing, making her breast cancer risk as high as 87%. The probability of suffering from ovarian cancer is 50%. At the same time, her mother and aunt died of breast cancer. Therefore, in April 2014 and March 2015, Julie voluntarily underwent double breast and ovary resection operations to reduce the risk of cancer as much as possible. [0003] With the development of molecular genetics, according to the research of Nobel Prize winners McHale and Harold, it was found that the fundamental mechanism of all tumors is the damag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/04
CPCC12Q1/689C12Q1/686C12Q2545/113C12Q2565/125
Inventor 魏冬凯丁国徽刘佳
Owner SUZHOU ZHONGXIN BIOTECH
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