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Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method

A constant temperature gene amplification and real-time detection technology, applied in the biological field, can solve the problems of inability to quantify, easy to pollute, and high detection cost

Inactive Publication Date: 2016-07-27
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Purpose of the invention: In view of the problems of high detection cost, easy pollution, strong subjectivity and inability to quantify in the prior art, the purpose of the invention is to provide a new detection method to solve the above problems

Method used

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  • Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method
  • Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method
  • Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1 A kind of specificity result of nucleic acid (DNA / RNA) real-time constant temperature gene amplification detection

[0058] LAMP amplification was performed on the negative controls of other microorganisms that had homology to the 9 HBV nucleic acid sequences, were likely to cause the same or similar clinical symptoms, and were normally parasitized at the sampling site or were likely to be concurrent. The results were as follows: figure 1 As shown, the 5 HBV sample reaction tubes showed an upward trend in the amplification curve in about 30 minutes, which was a positive result, and the amplification curves of the 9 negative control sample reaction tubes had no upward trend, which was a negative result.

Embodiment 2

[0059] Example 2 Sensitivity results of a nucleic acid (DNA / RNA) real-time constant temperature gene amplification detection method

[0060] The concentration of the initial HBV sample for fluorescence quantification was 8.1x10 7 copies / μl. Take the 10-fold diluted sample as the template for detection, and the results are as follows: figure 2 As shown, the detection limit of this method is 8.1copies / system, and the detection limit of PCR method is generally 10 2 copies / system.

Embodiment 3

[0061] Example 3 Visible light visual detection results of a nucleic acid (DNA / RNA) real-time constant temperature gene amplification detection method

[0062] According to the optimized conditions monitored by the microplate reader, add a color indicator, react at 63°C for 30 minutes, and observe with the naked eye. image 3 In order to observe the results, the left tube is the negative control, and the right tube is the reaction using the DNA of the HBV sample as the template. The left tube is pale yellow after the reaction, and the right tube is brownish red after the reaction. Matching kits and related Lamp primers are designed. After adding the samples to be tested, use an inexpensive water bath to react at 63°C for 30 minutes, and the results can be quickly observed without opening the cover, avoiding pollution.

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Abstract

The invention discloses a nucleic acid (DNA / RNA) real-time constant-temperature gene amplification detection method. The method is a rapid and efficient novel isothermal amplification detection method taking microplate reader detection as a core. The method particularly comprises the following steps that a to-be-detected sample DNA / RNR template is added into a 15 microliters-25 microliters of LAMP amplification system with certain neutral red concentration and then put into a microplate reader with the temperature ranging from 60 DEG C to 65 DEG C to detect change of absorbance of a reaction system in real time, and therefore the reaction is indirectly monitored. A reaction signal is expressed in the form of a curve, the curve enters a logarithmic phase within 30 min-60 min to express that a detected sample is positive, and the curve extends all the time but not enters the logarithmic phase to express that the detected sample is negative. The nucleic acid (DNA / RNA) real-time constant-temperature gene amplification detection method is simple, rapid and convenient, a great push function is achieved for subsequent basic-level popularization, compared with a traditional PCR detection method, rapid and safe detection of a gene is achieved, and an important significance in the aspects of blocking transmission routes, effectively controlling and tracking infection sources and effectively controlling outbreak and prevalence of superbacteria is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid (DNA / RNA) real-time constant temperature gene amplification detection method. Background technique [0002] At present, the detection methods for pathogenic bacteria mainly include: pathogen isolation method, immunological method, and PCR detection method based on nucleic acid level. Pathogen isolation method is the gold standard for clinical diagnosis, but it has the disadvantages of long time-consuming and cumbersome operation; immunological method is not widely used because of its non-specific amplification and low sensitivity; PCR detection method can be used from the nucleic acid level Direct detection has high specificity and sensitivity, but it has disadvantages such as requiring expensive experimental instruments and a stable experimental environment, high cost of consumables, and limited operating skills of testing personnel, so it needs to be furt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2561/113
Inventor 王升启王娈娈刘琪琦陈苏红肖瑞王琼
Owner ACADEMY OF MILITARY MEDICAL SCI
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