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Primer pair for identifying plectropomus leopardus, probe, kit and detection method

A technology of leopard gills and detection methods, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effects of high sensitivity, rapid sensitivity and high sensitivity

Inactive Publication Date: 2014-02-19
陈双雅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, domestic and foreign scholars have done very little research on the species DNA identification of Leopard gillfish, and there is no report at home and abroad on a method that can quickly identify Leopard gillfish

Method used

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  • Primer pair for identifying plectropomus leopardus, probe, kit and detection method
  • Primer pair for identifying plectropomus leopardus, probe, kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: the preparation of primer, probe and kit

[0029] 1. Design of primer pairs and probes: designed and synthesized based on the COI gene sequence of leopard gillfish (Genbank accession number JF750763) and other grouper COI sequences. The specific primer pair sequences are as follows:

[0030] SEQ ID No: 1:5'-GCTTCTACCTCTCTTCTTTCCTC-3'

[0031] SEQ ID No: 2: 5'-CTGCTAGAGGAGGGTAAACTG-3'

[0032] The probe sequences are as follows:

[0033] SEQ ID No: 3: 5'-TCCTCCTTTCTAGCCTCATCAGGCGTT-3';

[0034] A fluorescent reporter group FAM is connected to the 5' end of the probe, and a fluorescent quencher group TAMRA is connected to the 3' end;

[0035] 2. Synthesis of primer pairs and probes and preparation of kits:

[0036] Primer pairs and probes were synthesized by TAKARA Company based on the above nucleotide sequence information.

[0037] The above-mentioned synthesized primer pairs and probes were respectively prepared into a solution with a concentration...

Embodiment 2

[0039] Embodiment 2: real-time fluorescent PCR amplification detection

[0040] 1. Extraction of genomic DNA from the sample (preparing the genomic DNA solution of the sample according to the QIAGEN DNeasy Blood and Tissue DNA Extraction Kit):

[0041] 1) After the muscle tissue of the fish is cut into pieces, it is fully ground and mixed with liquid nitrogen. Take about 25 mg from it and place it in a sterilized 1.5 mL centrifuge tube;

[0042] 2) Add 180 μL of ATL buffer and 20 μL of proteinase K, vortex to mix, and place in a water bath at 56 °C for about 1 hour, and shake continuously until the tissue is completely digested;

[0043] 3) Add 200 μL AL buffer and vortex to mix. Then add 200 μL absolute ethanol and mix again;

[0044]4) Transfer the solution in the centrifuge tube to a DNeasy Mini spin column in a 2 mL collection tube, centrifuge at 12 000 rpm for 1 min, discard the collection tube and the centrifuged liquid in the tube;

[0045] 5) Put the spin column ...

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Abstract

The invention discloses a primer pair for identifying plectropomus leopardus, a probe, a kit and a detection method. The primer pair comprises primers F289 and R380 with sequences as shown in SEQ ID No:1 and SEQ ID No:2 respectively; the probe P309 has a sequence as show in SEQ ID No:3, a fluorescent reporter group FAM is connected with the 5' end of the probe sequence, and a fluorescent quenching group TAMRA is connected with the 3' end of the probe sequence. The invention further discloses the kit comprising the primer pair and the probe as well as the detection method for identifying the plectropomus leopardus by the primer pair, the probe or the kit. The method has the advantages of quickness, accuracy and sensitiveness.

Description

technical field [0001] The invention relates to the technical field of marine biology, in particular to a primer pair, a probe, a kit and a real-time fluorescent PCR detection method for identifying leopard gillfish. Background technique [0002] Species identification is an important issue related to many aspects of food safety, quality, quality and nutrition. Foods of unknown species origin may pose huge food safety risks to consumers due to the presence of allergens and toxic ingredients. In addition, due to the difference in value of different fish species, the phenomenon of illegal substitution of economically important fish species with cheap species is becoming more and more serious. Some unscrupulous traders frequently make huge profits through wrong labeling, shoddy and false labeling, which damage the interests and health of consumers, making the quality and safety of fish food gradually prominent. Regulation 104 / 2000 of the European Commission and my country's "...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 陈双雅张永祥李宏王嘉鹤陈伟玲周昱
Owner 陈双雅
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