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SNP marker, detection primer pair and kit for identifying Chinese cervus elaphus and cervus elaphus and application of SNP marker and detection primer pair

A technology of primer pairs and kits, applied in the determination/testing of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the lack of effective identification of red deer and Chinese red deer product detection technology, red deer and Chinese red deer products Accurate identification of problems and other issues

Pending Publication Date: 2020-07-07
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, we lack the detection technology to effectively identify red deer and Chinese red deer products, and the accurate identification of red deer and Chinese red deer products is still a difficult problem in this field

Method used

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  • SNP marker, detection primer pair and kit for identifying Chinese cervus elaphus and cervus elaphus and application of SNP marker and detection primer pair
  • SNP marker, detection primer pair and kit for identifying Chinese cervus elaphus and cervus elaphus and application of SNP marker and detection primer pair
  • SNP marker, detection primer pair and kit for identifying Chinese cervus elaphus and cervus elaphus and application of SNP marker and detection primer pair

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Obtaining SNP markers for identifying Chinese red deer and red deer

[0030] 1. DNA extraction

[0031] Genomic DNA was extracted from white-lipped deer, sika deer, Altai red deer, Northeast red deer, Qingyuan red deer, Gansu red deer, Tahe red deer, duck, etc. For the operation steps, please refer to the instruction manual of Tiangen Blood Genome Kit (DP304).

[0032] 2. Screening of differential SNP sites between red deer and red deer in China

[0033]According to red deer, Chinese red deer (including Tahe red deer, Altai red deer, Gansu red deer, Qinghai red deer, Tianshan red deer, Northeast red deer, Alxa red deer, Tibetan red deer, Sichuan-Tibet red deer, high-yielding red deer king ) mtDNA full sequence, compared with Mega6.0, initially screened 123 differential SNP sites, and finally selected 7 specific SNP identification sites on the COX3 gene fragments of red deer and Chinese red deer (screening SNP sites mainly Consider from three aspects: one is...

Embodiment 2

[0036] Embodiment 2, establishment of the method for distinguishing Chinese red deer and red deer

[0037] According to the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 above, a PCR experiment was carried out to establish a method for identifying Chinese red deer and red deer. The theoretical value of the primer annealing temperature is 57°C, and a gradient is set every 2°C, within the range of 5°C above and below the theoretical value. That is, the annealing temperature was set to 53°C, 55°C, 57°C, 59°C, and 61°C at the same temperature to conduct experiments with three different sample DNA templates. The conditions are shown in Table 2.

[0038] Table 2 PCR detection reaction conditions

[0039]

[0040] Product electrophoresis detection results (see figure 2 ): When Tm is 61°C and 53°C, there is no electrophoresis band, indicating that too low or too high Tm will affect the combination of primers and templates. The Tm is 57°C, and there is a sample DNA that canno...

Embodiment 3

[0045] Embodiment 3 The inventive method specificity, stability, sensitivity experiment

[0046] 1. Specificity: Utilize the identification primers of the present invention to amplify the cervidae animals respectively: white-lipped deer, sika deer, red deer, Northeast red deer, Qingyuan red deer, Gansu red deer, Tahe red deer and duck, and perform PCR to determine the specificity of the primers. specificity.

[0047] The product electrophoresis test results are shown in Figure 4 , All deer family animals can amplify a single, bright band, and non-cervid family animals (ducks) have no band. It shows that the present invention has species specificity.

[0048] 2. Stability: Different operators operate at the same time or at different times (morning, noon, afternoon) or in different PCR instruments, and verify the stability by observing the presence or absence of electrophoretic bands.

[0049] Under different personnel, at different times and under different PCR instruments,...

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Abstract

The invention provides an SNP marker, detection primer pair and kit for identifying Chinese cervus elaphus and cervus elaphus and application of the SNP marker and the detection primer pair. The invention provides 5 specific SNP sites and a pair of identifying primers for accurately identifying the Chinese cervus elaphus and the cervus elaphus and establishes an accurate and reliable detection method. The method has very good specificity and stability and high sensitivity, and the detection limit is 0.25ng / ul. A molecular marker technique is applied to identification of the Chinese cervus elaphus and the cervus elaphus for the first time, so that economic loss caused due to difficult differentiation of appearances is effectively avoided. The method is used for detecting 100 pilos antlers and byproducts in the market, and the accuracy rate is 100%. Identifying procedures are simplified, and the method is easy in operation and grasping and strong in generalization performance. The deficiency that a detection technique for precisely identifying the Chinese cervus elaphus and the cervus elaphus is not reported in the field in the prior art is remedied, and precise identification of Chinese cervus elaphus products and cervus elaphus products is realized.

Description

technical field [0001] The invention relates to SNP markers, detection primers, kits and applications for identifying Chinese red deer and red deer, and belongs to the technical field of molecular biology detection. Background technique [0002] The red deer (Cervus elaphus) belongs to the order Artiodactyla, Cervidae, and the genus Cervus. There are 22 subspecies in the world, which are mainly divided into two groups: eastern red deer and western red deer. Eight subspecies are distributed in China (Tibetan subspecies, Northeast subspecies, Alxa subspecies, Altai subspecies, Sichuan subspecies, Tianshan subspecies, Tarim subspecies, Gansu subspecies). At present, on the basis of subspecies, my country has cultivated 3 cultivars (Tahe red deer, Qingyuan red deer, Tianshan red deer), 1 local breed (Northeast red deer), and 5 breeding populations (Tianshan population, Altai population, Gansu population, Alxa population and Qinghai population). [0003] Products such as deer an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 邢秀梅朱燕敏王天骄刘华淼董依萌
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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