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Real-time fluorescent PCR (polymerase chain reaction) specific detection system of gadus morhua and application

A real-time fluorescent and specific technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. High sensitivity and good specificity

Inactive Publication Date: 2016-07-06
蒋丹 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Judging by morphological characteristics is the traditional method of identifying Atlantic cod, 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 of 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 fluorescent PCR (polymerase chain reaction) specific detection system of gadus morhua and application
  • Real-time fluorescent PCR (polymerase chain reaction) specific detection system of gadus morhua and application
  • Real-time fluorescent PCR (polymerase chain reaction) specific detection system of gadus morhua and application

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Embodiment 1

[0026] The design of embodiment 1 fluorescent PCR specific detection primer sequence

[0027] 1. Analysis of the CoⅠ gene of the relative species of Atlantic cod

[0028] Log in to NCBI (http: / / www.ncbi.nlm.nih.gN program to search for similar sequences in the nr database, and the species distribution of the returned similar sequences is as follows figure 1 As shown, all the sequences were searched in the nr database using Gadusmorhua and CoI as keywords, and after the sequences of the search results were downloaded and saved, the Blast sequence was downloaded and saved. figure 1 is the species distribution of similar sequences, where the species in the box is the location of Atlantic cod.

[0029] 2. Design of Atlantic cod species-specific detection primers

[0030] The downloaded sequence is subjected to sequence similarity analysis and sequence analysis, and specific primers are designed for sequence differences.

[0031] 3. Specificity analysis of Atlantic cod species-s...

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 Atlantic cod species, that is, to use the real-time fluorescent PCR specific detection system for Atlantic cod species to amplify the CoI gene of Atlantic cod species, and to perform real-time fluorescent PCR specific detection for Atlantic cod species. The specific steps are as follows :

[0046] Table 3 Reaction system of real-time fluorescent PCR specific detection system for Atlantic cod species

[0047]

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

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

[0050]

[0051] When the Atlantic cod species real-time fluorescent PCR specific detection composition is used for real-time fluoresc...

Embodiment 3

[0052] Embodiment 3 specific detection

[0053] Utilize the Atlantic cod species real-time fluorescent PCR specific detection system to carry out Atlantic cod species component real-time fluorescent PCR specific detection Atlantic cod and other cod samples, use 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 the Atlantic cod , other samples showed amplification curves or no amplification curves after 30 cycles. This method can accurately identify the components of Atlantic cod and has good specificity, such as 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 gadus morhua 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 gadus morhua by adopting gadus morhua specific detection primers, thus specifically identifying the components of gadus morhua. The specific primers are reasonable in design, are used for detecting the gadus morhua species, have good specificity and high detection sensitivity, can be used for accurately identifying the components of gadus morhua 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 Atlantic cod species and its application. Background technique [0002] Atlantic cod (scientific name: Gadusmorhua) is a species of cod in the cod family. It is native to the cold waters of the North Atlantic from northern Europe to Canada and the eastern United States, and has a wide range of habitats, from the coast to the continental shelf, as well as many open seas. Atlantic cod is one of the fishes with the largest annual catch in the world. It is a food fish with high economic value. As a commercial fish species, its main export countries are Canada, Iceland, Norway and Russia. The main production area in Japan is Hokkaido. [0003] Atlantic cod has no dorsal fin spines, 40-55 dorsal soft rays; 33-45 anal soft rays; 51-55 vertebrae. Maxillary prominence, lower jaw with prominent barbels. The lateral line ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 蒋丹尹祺李莉于珂
Owner 蒋丹
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