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Real-time fluorescence PCR specific detection system and applications of Merluccius Merluccius

A real-time fluorescent and specific technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. The effect of high detection sensitivity

Inactive Publication Date: 2016-08-10
刘淑艳 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Judging by morphological characteristics is the traditional method to identify hake, but these morphological characteristics are highly plastic, are greatly affected by the environment, have artificial subjective tendencies, and there are abundant related species, and the morphology between related species The difference is subtle, so the traditional morphological feature recognition method has the problems of recognition difficulty and recognition error

Method used

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  • Real-time fluorescence PCR specific detection system and applications of Merluccius Merluccius
  • Real-time fluorescence PCR specific detection system and applications of Merluccius Merluccius
  • Real-time fluorescence PCR specific detection system and applications of Merluccius Merluccius

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Effect test

Embodiment 1

[0025] The design of embodiment 1 fluorescent PCR specific detection primer sequence

[0026] 1. Analysis of the CoⅠ gene of the relative species of hake

[0027] Log in to NCBI (http: / / www.ncbi.nlm.nih.g) The N program searches for similar sequences in the nr database, and the species distribution of the returned similar sequences is as follows figure 1 shown, and searched all the sequences in the nr database using Merluccius Merluccius and CoI as keywords, downloaded and saved the sequences of the search results, and then downloaded and saved the Blast sequence. figure 1 is the species distribution of similar sequences, where the species in the box is the location of hake.

[0028] 2. Design of Hake Species-Specific Detection Primers

[0029] The downloaded sequence is subjected to sequence similarity analysis and sequence analysis, and specific primers are designed for sequence differences.

[0030] 3. Specificity analysis of hake species-specific detection primers

[0...

Embodiment 2

[0044] Embodiment 2 Application of real-time fluorescent PCR specific detection system

[0045]The application of the real-time fluorescent PCR specific detection system for hake species is to use the real-time fluorescent PCR specific detection system for hake species to amplify the CoI gene of hake species, and perform real-time fluorescent PCR specific detection for hake species. The specific steps are as follows :

[0046] Table 3 The reaction system of the real-time fluorescent PCR specific detection system for hake species

[0047]

[0048] The reaction parameters of the real-time fluorescent PCR specific detection system for hake species are as follows in Table 4:

[0049] Table 4 Reaction parameters of the real-time fluorescent PCR specific detection system for hake species

[0050]

[0051] When the CoI gene of hake is amplified by real-time fluorescent PCR using the real-time fluorescent PCR specific detection composition of hake species, the Premix Ex Taq ...

Embodiment 3

[0052] Embodiment 3 specific detection

[0053] Utilize the real-time fluorescent PCR specific detection system for hake species to perform real-time fluorescent PCR specific detection of hake species components of hake and other cod samples, use a DNA extraction kit to extract the template DNA of each sample to be tested, and use microspectrometry 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 hake , other samples showed amplification curves or no amplification curves after 30 cycles. This method can accurately identify the components of hake and has good specificity, such as Figure 5 shown.

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Abstract

The invention belongs to the field of biotechnologies, and in particular relates to a real-time fluorescence PCR specific detection system and applications of Merluccius Merluccius. The primer sequences are shown as SEQ ID NO.1 and SEQ ID NO.2. A specific detection primer of Merluccius Merluccius is adopted for carrying out real-time fluorescence PCR amplification on the Co I genes of Merluccius Merluccius, and after the real-time fluorescence PCR amplification, a specific amplification curve is generated, so that the ingredients of Merluccius Merluccius are subjected to specificity identification. The specific primer is reasonable in design, and is good in specificity and high in detection sensitivity when used for species detection of Merluccius Merluccius; through fluorescence PCR analysis, the ingredients of Merluccius Merluccius can be accurately identified, and the specific primer has good specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a real-time fluorescent PCR specific detection system for hake species and its application. Background technique [0002] Hake, scientific name Merluccius Merluccius, belongs to Codaceae, Hake genus, long body, big head, big and pointed teeth. There are two dorsal fins, the second dorsal fin is long, and there is a shallow concave near the middle. The anal fin is also long and concave. Pelvic fins anterior, before pectoral fins. Fast-swimming carnivorous fish. [0003] Judging by morphological characteristics is the traditional method to identify hake, but these morphological characteristics are highly plastic, are greatly affected by the environment, have artificial subjective tendencies, and there are abundant related species, and the morphology between related species The difference is subtle, so the traditional morphological feature recognition method has the problems of reco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124
Inventor 刘淑艳孙晓飞宋大贺万超
Owner 刘淑艳
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