Multiplex PCR (polymerase chain reaction) primers and method for identifying Panthera pardus, and application thereof in identifying Panthera pardus

A golden leopard, multiple technology, applied in the fields of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of time-consuming and labor-intensive, and achieve the reduction of sampling difficulty, economical and practical identification, and simplicity. The effect of identification

Inactive Publication Date: 2014-09-24
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In view of the above-mentioned prior art, the purpose of the present invention is to overcome the limitation of determining the species of leopards by morphological characteristics in the prior art, or the time-consuming and labor-intensive method of determining the species by determining the DNA in feces by conventional methods And cost a lot of problems, provide a simple, quick and economical multiplex PCR primers for identifying leopards and a method for identifying leopards, and their application in identifying leopards

Method used

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  • Multiplex PCR (polymerase chain reaction) primers and method for identifying Panthera pardus, and application thereof in identifying Panthera pardus
  • Multiplex PCR (polymerase chain reaction) primers and method for identifying Panthera pardus, and application thereof in identifying Panthera pardus
  • Multiplex PCR (polymerase chain reaction) primers and method for identifying Panthera pardus, and application thereof in identifying Panthera pardus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Put 0.5 g of the sample to be tested numbered 5 in Table 1 into a centrifuge tube and cut the sample to be tested in the centrifuge tube with sterilized scissors, then add 500 μL of lysate and 30 μL of 10% ten Sodium dialkyl sulfate and 3 μL of 20 mg / ml proteinase K were thoroughly mixed and then digested in a water bath at 56°C for 12 hours until the liquid in the tube was clear. Add 500 μL of Tris-balanced phenol to the above-mentioned digested mixture, shake slightly for 5 minutes, then place it in a centrifuge at 11000 r / min for 10 minutes, and take the supernatant after centrifugation. Repeat the above centrifugation step twice. Add 1,000 μL of frozen absolute ethanol to the supernatant obtained after repeated centrifugation, place it at -20°C for 1 hour, then centrifuge it in a centrifuge at 12,000 r / min for 13 minutes, discard the supernatant, and add 800 μL of 70% ethanol was shaken slightly for 0.5 min, then placed in a centrifuge at 13000 r / min for 13 min, an...

Embodiment 2

[0039] Operate according to the method of Example 1, the difference is that the sample number to be tested is 12 (in Table 1), the consumption of each primer is 0.75 μ L, the consumption of the total DNA is 45 ng, and the annealing temperature is 55°C, the annealing time is 40s, the electrophoresis results are as follows figure 1 Shown (lane number is 12).

Embodiment 3

[0041] According to the method of Example 1, the amount of each primer used was 1.5 μL, the amount of the total DNA used was 55 ng, the annealing temperature was 60° C., and the annealing time was 25 s. The electrophoresis results were consistent with those in Example 1. same.

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Abstract

The invention discloses multiplex PCR primers for identifying Panthera pardus, which comprise a primer pair 1 disclosed as SEQ ID NO:1 and SEQ ID NO:2 and a primer pair 2 disclosed as SEQ ID NO:3 and SEQ ID NO:4. The invention also discloses a method for identifying Panthera pardus by utilizing multiplex PCR, which comprises the following steps: extracting total DNA (deoxyribonucleic acid) of a sample to be detected, and carrying out PCR amplification on the total DNA; and carrying out agarose gel electrophoresis detection on the amplified product, wherein the multiplex PCR primers are the multiplex PCR primers in the invention, and the sample to be detected is judged to be from Panthera pardus if two strips appear in the lane. The invention also discloses application of the multiplex PCR primers or identification method in identifying Panthera pardus. The two pairs of primers are designed to achieve the goal of identifying Panthera pardus in a simple, feasible, economical and practical way.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a multiple PCR primer for identifying leopards, a method for identifying leopards using the multiple PCR primers, and their application in identifying leopards. Background technique [0002] Accurate species identification based on taxonomy is a necessary prerequisite for human cognition of nature and sustainable development. It provides a knowledge and theoretical basis for the conservation of biodiversity, the sustainable use of species resources, and the development of new products from biological sources. For the identification of species, traditional methods mainly use morphological evidence. However, some shortcomings of this method have also caused limitations in the protection of endangered animals, such as phenotypic plasticity and genetic variability, inability to distinguish cryptic taxa, and restrictions by biological sex and developmental stage. In the practice ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2537/143C12Q2565/125
Inventor 晏龙晏鹏耿章珍吴孝兵
Owner ANHUI NORMAL UNIV
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