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Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method

A detection kit and detection method technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial determination/inspection, etc., can solve the problem that the work of identifying sea cucumbers is limited to, etc., to improve detection efficiency and sensitivity, overcome the The effect of limitation and simple operation

Inactive Publication Date: 2013-10-02
曹际娟 +3
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Problems solved by technology

Therefore, the application of traditional methods to identify sea cucumbers is limited to professionals with rich identification experience

Method used

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  • Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method
  • Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method
  • Apostichopus japonicus detection primer, kit and real-time fluorescent PCR (polymerase chain reaction) detection method

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Embodiment 1

[0047] 1. Primer and probe design

[0048] In the present invention, DNAMAN V6 is used for sequence alignment, and Primer Express3.0 is used for primer and probe design. System 1 and System 2 are designed according to the sequence of the A. japonicus COX1 gene (Table 3), and system 3 is designed according to the sequence of the NAD4 gene of A. japonicus. (Table 3), system 4 and system 5 were designed according to the 16S rRNA genes of the above-mentioned 12 kinds of sea cucumbers (Table 3). Among them, system 1, system 2, and system 3 (Table 3) are used to specifically identify sea cucumbers, and system 4 and system 5 ((Table 3) are used to monitor the nucleic acid extraction of 12 species of sea cucumber. All detection systems passed Confirm by BLAST, in order to avoid the possibility of non-specific amplification.In addition, the present invention compares system 4, system 5 with other sea cucumber sequences, and finds that the two are identical with the corresponding site s...

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Abstract

The invention discloses an apostichopus japonicus detection primer, a kit and a real-time fluorescent PCR (polymerase chain reaction) detection method. On the basis of design systems 1 to 3 of apostichopus japonicus parameter mitochondria COX1 gene and NAD4 gene, a probe method in the fluorescent PCR technology is used for carrying out the specificity identification experiment of the apostichopus japonicus. On the basis of design systems of 12 sea cucumber mitochondria 16S rRNA genes, a green I embedded fluorescent method in the fluorescent PCR technology is used for monitoring the sea cucumber nucleic acid extraction situation. The invention particularly designs a primer for detection, namely SEQ ID NO. 1 to No. 13, and a method for quickly detecting the apostichopus japonicus is established by utilizing the real-time fluorescent PCR technology. The manual operation time is less than 60 minutes in the entire detection process. The method has a wide application prospect in the authenticity identification of the apostichopus japonicus.

Description

technical field [0001] The invention belongs to the technical field of sea cucumber gene detection, and in particular relates to a sea cucumber-imitation detection primer, a kit and a real-time fluorescence PCR (Real time fluorescence PCR) detection method. Background technique [0002] Sea cucumbers belong to the class Echinodermata and are a general term for a class of invertebrates in the ocean. According to the classification principle of the Pawson et Fell classification system, the whole sea cucumber class includes 3 subclasses, 6 orders, and 24 families. So far, more than 900 species of sea cucumbers have been discovered all over the world, of which more than 40 species have long-term consumption records in human society, so they can be used as edible sea cucumbers. [1][2][3] . Apostichopus japonicus (Apostichopus japonicus), commonly known as Liaoshen, belongs to the order Apostichopus, Apostichopaceae, and Apostichopus genus. It is the species of sea cucumber with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 曹际娟徐君怡徐扬陈颖
Owner 曹际娟
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