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A kind of real-time fluorescent PCR specific detection system and its application

A real-time fluorescent and specific technology, which is applied in the measurement/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of large environmental impact, identification errors, identification difficulties, etc., to achieve easy operation, good The effect of high specificity and detection sensitivity

Inactive Publication Date: 2019-07-23
刘淑艳 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Judging by morphological characteristics is the traditional method of identifying A. hulus, but these morphological characteristics are highly plastic, are greatly affected by the environment, and have artificial subjective tendencies. The morphological differences are subtle, so the traditional morphological feature recognition methods have the problems of recognition difficulties and recognition errors

Method used

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  • A kind of real-time fluorescent PCR specific detection system and its application
  • A kind of real-time fluorescent PCR specific detection system and its application
  • A kind of real-time fluorescent PCR specific detection system and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The design of embodiment 1 fluorescent PCR specific detection primer sequence

[0028] 1. Analysis of the CoⅠ gene of the similar species of A. hulus

[0029] Log in to NCBI (http: / / www.ncbi.nlm.nih.gov), use Huso huso and CoI as keywords to search in the nr database, and then run the Blast N program in the nr database to search for similar sequences, and the returned The distribution of species with similar sequences is as follows figure 1 As shown in , the species in the box are the locations of A. hulus, and all similar sequences are downloaded and saved.

[0030] 2. Design of species-specific detection primers for A. hulus

[0031] The downloaded sequence is subjected to sequence similarity analysis and sequence analysis, and specific primers are designed for sequence differences.

[0032] 3. Specificity analysis of species-specific detection primers for A. hulus

[0033] Use the primer blast program to select the nr database of the designed primers in NCBI, and...

Embodiment 2

[0045] Embodiment 2 Application of real-time fluorescent PCR specific detection system

[0046] The application of the real-time fluorescent PCR-specific detection system for the species of A. hulus is to use the real-time fluorescent PCR-specific detection system for the species of hulus to amplify the CoI gene of the species of hulus, and to perform the specific detection of the species of huso by real-time fluorescent PCR. The specific steps are as follows :

[0047] Table 3 The reaction system of the real-time fluorescent PCR specific detection system for Hulus hulus species

[0048]

[0049] The reaction parameters of the real-time fluorescent PCR specific detection system for the species of Hulus are as follows in Table 4:

[0050] Table 4 Reaction parameters of real-time fluorescent PCR-specific detection system for Hulus hulus species

[0051]

[0052] When using the real-time fluorescent PCR specific detection composition of A. Premix Ex Taq DNA polymerase f...

Embodiment 3

[0053] Embodiment 3 specific detection

[0054] Utilize the real-time fluorescent PCR specific detection system for species of A. hulus to detect species components of A. hulus in real-time fluorescent PCR to specifically detect samples of A. hulus and other sturgeons. DNA extraction kits are used to extract the template DNA of each sample to be tested. The concentration of the extracted template DNA was detected by a photometer to be 50 ng / μL, and PCR amplification was carried out according to the reaction system and reaction parameters described in the above step 4, and the specific amplification curve generated below 30 cycles was the A. , other samples showed amplification curves or no amplification curves after 30 cycles, this method can accurately identify the components of A. Figure 5 shown.

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Abstract

The invention belongs to the technical field of biology and in particular relates to a real-time fluorescent PCR (polymerase chain reaction) specific detection system of huso huso species and an application. Primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. A specific amplification curve can be generated after carrying out real-time fluorescent PCR amplification on the Co I gene of huso huso by adopting huso huso specific detection primers, thus specifically identifying the components of huso huso. The specific primers are reasonable in design, are used for detecting the huso huso species, have good specificity and high detection sensitivity, can be used for accurately identifying the components of huso huso through fluorescent PCR analysis and have good specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a real-time fluorescence PCR specific detection system for beluga and its application. Background technique [0002] Huso huso (scientific name: Huso huso) is the largest freshwater fish in the world, with a body length of 7.2 meters and a weight of more than 1,000 kilograms. It is mainly distributed in the Pacific, Atlantic and Arctic waters of the northern hemisphere. system, Mississippi River, Black Sea, Caspian Sea, Aral Sea region. Ob River to Kolyma River and other arctic water systems; in my country, it is mainly distributed in the Bohai Sea, Yellow Sea, East China Sea, Heilongjiang water system, Yellow River, Yangtze River, Qiantang River, Minjiang River to Pearl River and Xinjiang Irtysh River Basin. [0003] The beluga is huge, with a spindle-shaped body, tapering toward the tail; the tail is crooked, the upper lobe is long, and the lower lobe is short; the mou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2561/113C12Q2563/107
Inventor 刘淑艳丁健蒋丹肖珊珊
Owner 刘淑艳