Method for detecting and identifying haliotis discus hannai ino through fluorogenic quantitative PCR

A kind of wrinkled abalone, fluorescence quantitative technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Strong and sensitive effect

Active Publication Date: 2019-04-26
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

Method used

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  • Method for detecting and identifying haliotis discus hannai ino through fluorogenic quantitative PCR
  • Method for detecting and identifying haliotis discus hannai ino through fluorogenic quantitative PCR
  • Method for detecting and identifying haliotis discus hannai ino through fluorogenic 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 COI gene sequence of Abalone rugosa, the primer design soft Primer Express3 was used to assist in the design and analysis of primers. After the specificity was tested by NCBI Blast, the primers were s...

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 100ng / μL with TE solution, and stored at -20°C for later use (the DNA concentration of canned abalone i...

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Abstract

The invention provides a method for detecting and identifying haliotis discus hannai ino through fluorogenic quantitative PCR, and belongs to the technical field of species resource identification. The method comprises the step of identifying molecular characteristics of the haliotis discus hannai ino by applying a specific primer and a probe. Through the adoption of the method, the haliotis discus hannai ino can be quickly and accurately detected and identified, the method has the advantages of high specificity, high sensitivity, short consumed time, and accurate and convenient determinationof results, PCR aftertreatment is not needed, and false positive and cross contamination can be effectively avoided. A new technological means is provided for quickly and accurately identifying the haliotis discus hannai ino in entry and exit species resource inspection, 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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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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