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A method for the detection and identification of wrinkled plate abalone by fluorescent quantitative PCR

A wrinkled abalone, fluorescence quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. , The result is accurate and convenient to determine the effect

Active Publication Date: 2022-03-22
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no fluorescent quantitative PCR technology for the detection and identification of wrinkled disc abalone

Method used

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  • A method for the detection and identification of wrinkled plate abalone by fluorescent quantitative PCR
  • A method for the detection and identification of wrinkled plate abalone by fluorescent quantitative PCR
  • A method for the detection and identification of wrinkled plate abalone by fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: wrinkled plate abalone fluorescent quantitative PCR primer specificity test

[0022] 1. Preparation of materials

[0023] The abalones for testing are as follows: wrinkled pan abalone, Japanese pan abalone; black lip abalone; green lip abalone; variegated abalone; red abalone; big abalone; stone sulfur (earth abalone).

[0024] The above-mentioned abalones were obtained through domestic investigation and collection, and after being confirmed by the National Mollusc Quarantine and Identification Key Laboratory of the former General Administration of Quality Supervision, Inspection and Quarantine, they were stored at -20°C for later use.

[0025] 2. Establishment of fluorescent quantitative PCR method

[0026] 2.1. Design and synthesis of primers: Based on the sequence of the Abalone rugosa COI gene, the primer design software PrimerExpress3 was used to assist in the design and analysis of primers. After the specificity was tested by NCBI Blast, the primers...

Embodiment 2

[0034] Example 2: Sensitivity test of fluorescent quantitative PCR detection of wrinkled disc abalone

[0035] The DNA stock solution (100 ng / μL) extracted in Example 1 was diluted into 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 Different concentration gradients of fg / μL, 10 fg / μL and 1 fg / μL.

[0036] Fluorescent quantitative PCR amplification reaction system: the total volume is 25 μL, including 12.5 μL of 2×Premix Ex TaqTM PCR (ProbeqPCR), 1 μL of upstream and downstream primers and probes, 2 μL of 100 ng / μL DNA template, 0.5 μL of ROXII, and the rest with inert ddH2 O make up. After mixing, put it into a fluorescent quantitative PCR amplification instrument for amplification.

[0037] The two-step amplification reaction program of fluorescent quantitative PCR is: 95°C pre-denaturation for 5 minutes; 95°C for 10s, 60°C for 34s, 40 cycles; end the reaction.

[0038] Sensitivity test results: when the DNA concentration is 10 pg / μL, the amplification curve still a...

Embodiment 3

[0039] Example 3: Reality identification test of abalone and its products by fluorescence quantitative PCR

[0040] 1. Preparation of materials

[0041] The tested abalone and its products are as follows: wrinkled plate abalone (fresh abalone; dried abalone; steamed abalone; canned abalone); black lip abalone (steamed); ddH2O was used as a blank control.

[0042] The above-mentioned abalones and their products were purchased from the market, and after being confirmed by the National Mollusc Quarantine and Identification Key Laboratory of the former General Administration of Quality Supervision, Inspection and Quarantine, they were stored at -20°C for later use.

[0043] 2. Identification of authenticity by fluorescent quantitative PCR

[0044] According to the method 2.2 in Example 1, the whole genome DNA of abalone and its products was extracted, and the DNA was diluted to 100 ng / μL with TE solution, and stored at -20°C for later use (the DNA concentration of canned abalone ...

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Abstract

The invention provides a method for detecting and identifying wrinkled abalone by fluorescent quantitative PCR, and belongs to the technical field of species resource identification. The method of the invention comprises using specific primers and probes to identify the molecular features of the abalone wrinkled. The method of the invention can quickly and accurately detect and identify wrinkled pan abalone, not only has the advantages of strong specificity, high sensitivity, short time consumption, accurate and convenient result determination, but also does not need PCR post-processing, and can effectively avoid false positives and cross-contamination. The invention provides a new technical means for rapid and accurate identification of wrinkled plate abalone in the inspection of inbound and outbound species resources, fishery production, consumption and trade.

Description

technical field [0001] The invention relates to a method for detection and identification of wrinkled abalone by fluorescent quantitative PCR, which belongs to the technical field of species resource identification and is suitable for rapid and accurate identification and authenticity identification of wrinkled abalone in the inspection of entry and exit species resources, fishery production, consumption and trade. Background technique [0002] wrinkled plate abalone Haliotis discuss hannai Ino belongs to Mollusca Mollusca, Gastropoda Gastropoda, Prosobranchia Prosobranchia, Archaeogastropoda Primitive Gastropoda, Haliotidae Baalaceae, Bao Genus Haliotis , is a rare aquatic product with high economic value. Abalone shell is a traditional Chinese medicinal material that was used very early in the history of Chinese medicine. , insomnia and other diseases, it can also treat ulcers and golden sores externally. Abalone meat is warm in nature and salty in taste. It has the fu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2563/107C12Q2545/114
Inventor 王沛胡美玲周卫川
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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