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Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal

A technology for amplifying signals and detection methods, applied in the fields of molecular biology and nucleic acid detection, can solve the problems of restricting the application of ECL technology, less sequence disclosure, and restricting the combination of technologies, etc., and achieves the effect of convenient automatic detection, simple equipment, and convenient operation.

Inactive Publication Date: 2017-05-24
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the principle of bDNA technology has been proven to a certain extent, it has not been widely promoted in actual use. The reason is that there are many technical difficulties from theory to actual use, such as buffering in actual use. The construction of the liquid system and the design of the amplified probe set will directly affect the effect of use. In reality, although there are some related products, they are limited to only a few manufacturers. The composition of the buffer solution is not specified, and the design method and specific sequence of the amplified probe set involved are not disclosed, but a technical secret protection method is adopted. In addition, the specific experiment of the current electrochemiluminescence (ECL) technology The practice is not open, and the specific synthesis method of the luminescent labeling probe involved in it is rarely disclosed, resulting in many difficulties in the actual research and use of electrochemiluminescence that need to be faced and solved. At the same time, electrochemiluminescence also requires the use of special electrochemiluminescence. Chemiluminescence instrument, and its development requires knowledge of physics, chemistry, biology and electronics, which also greatly limits the application of ECL technology
Furthermore, the combination of nucleic acid detection, bDNA technology and ECL technology requires that the required probe sequence be very clear to avoid problems such as non-specific hybridization and identification. For now, the sequences involved in these two technologies are rarely disclosed. , which limits the combination of these two technologies, especially in the field of nucleic acid detection

Method used

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  • Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal
  • Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal
  • Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal

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Effect test

Embodiment 1

[0067] Foodborne Staphylococcus aureus detection

[0068] At present, harmful pathogenic microorganisms not only affect people's health and endanger people's lives, but also cause panic in the whole society and affect normal social operation. The traditional identification of pathogenic microorganisms mainly relies on methods such as bacterial culture, serology, biochemistry, and colony morphology for classification and identification. While reliable, these methods often take days or even weeks to obtain results; and colony morphology can also be affected by the environment. Nucleic acid detection of microorganisms can quickly and accurately identify pathogenic microorganisms, thereby minimizing the damage of pathogens to the human body.

[0069] 1) Strain culture

[0070] Staphylococcus aureus is cultured in 7.5% NaCl broth, Vibrio cholerae and Vibrio parahaemolyticus are cultured in alkaline peptone water, Streptococcus pyogenes is cultured in glucose meat infusion broth, ...

Embodiment 2

[0083] CaMV35S Promoter Screening Kit

[0084] The safety of genetically modified products is controversial. Most transgenic organisms contain CaMV35S promoter and NOS terminator, and these two genes are commonly used regulatory elements for exogenous gene expression in plants, which can be used as markers of transgenic organisms. In this example, the CaMV35S promoter was selected as the target gene to be tested for the transgenic product.

[0085] The kit includes the following components: Taq DNA polymerase, MgSO 4 , dNTP, 10×PCR Buffer, Marker, positive control and negative control, the positive control is a recombinant plasmid containing the CaMV35S promoter gene sequence, and the negative control is ddH 2 O.

[0086] Use of the kit: the kit selects the detection object of the CaMV35S promoter, and combines the detection method of branched DNA electrochemiluminescence for the detection of transgenic products.

[0087] Preservation of the kit: the contents in the kit sh...

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Abstract

The invention discloses an electrochemical luminescence nucleic acid detection method based on branched a DNA (Deoxyribonucleic Acid) amplification signal. The electrochemical luminescence nucleic acid detection method comprises the following steps: acquiring single-chain target DNA by using asymmetric PCR (Polymerase Chain Reaction) amplification target genes, designing an electrochemical luminescence nucleic acid detection primer group for target DNA specificity sequences according to branched DNA amplification signal principles, performing hybridization on the single-chain target DNA and the electrochemical luminescence nucleic acid detection primer group, performing electrochemical luminescence nucleic acid detection on a hybridization product, and performing qualitative and quantitative analysis on target DNA according to existence and intensity of an electrochemical luminescence signal, thereby finally achieving detection on a target gene. The invention further provides a kit for nucleic acid detection by using the method. The electrochemical luminescence nucleic acid detection method has the advantages of being sensitive in reaction, high in detection speed and accurate in result.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and nucleic acid detection, in particular to an electrochemiluminescence nucleic acid detection method based on branched DNA amplification signals, and also relates to an electrochemiluminescence nucleic acid detection kit based on branched DNA amplification signals. Background technique [0002] In recent years, nucleic acid testing (NAT) has been used in the prevention and control of infectious diseases, early diagnosis and precise treatment of malignant tumors, early screening of genetic diseases, prevention and control of animal and plant viruses, and detection of genetically modified genes. play an important role. [0003] At present, the main methods for nucleic acid detection include gene chips, in situ hybridization, and PCR. Gene biosensors based on nucleic acid hybridization are one of the most rapidly developed nucleic acid detection methods in recent years. Among them, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6851C12Q2531/107C12Q2563/143C12Q2563/149
Inventor 朱德斌刘岩曹玉娟
Owner SOUTH CHINA NORMAL UNIVERSITY
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