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 produ

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
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Example Embodiment

[0029] Example 1. Obtaining of SNP markers for the identification of Chinese red deer and red deer

[0030] 1. DNA extraction

[0031] Extract the genomic DNA of white-lipped deer, sika deer, Altai red deer, Northeast red deer, Qingyuan red deer, Gansu red deer, Tahe red deer, ducks, etc. See the instructions of Tiangen Blood Genome Kit (DP304) for the operation steps.

[0032] 2. Screening of different SNP loci between Chinese red deer and red deer

[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, Alashan red deer, Tibet red deer, Sichuan-Tibet red deer, high-yield deer king) ), compared with Mega6.0, 123 differential SNP sites were initially screened, and finally 7 specific SNP identification sites on the COX3 gene fragments of red deer and Chinese red deer were selected (the main SNP sites for screening were Consider from three aspects: one is to find the unique SNP of...

Example Embodiment

[0036] Example 2. Establishment of a method to identify Chinese red deer and red deer

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

[0038] Table 2 PCR detection reaction conditions

[0039]

[0040] Product electrophoresis test results (see figure 2 ): When Tm is 61°C and 53°C, there is no electrophoresis band, indicating that Tm is too low or too high to affect the combination of primer and template. Tm is 57°C, and there is a sample DNA that cannot be amplified, indicating that prim...

Example Embodiment

[0045] Example 3 Specificity, stability, and sensitivity experiments of the method of the present invention

[0046] 1. Specificity: Using the identification primers of the present invention to amplify deer animals are: white-lipped deer, sika deer, red deer, northeastern red deer, Qingyuan red deer, Gansu red deer, Tahe red deer and ducks. PCR was performed to determine the primer Specificity.

[0047] See the product electrophoresis test results Figure 4 , Deer animals can amplify single, bright bands, non-deer animals (duck) have no bands. 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 on different PCR instruments, and verify the stability by observing the presence or absence of bands in the electropherogram.

[0049] Using the method of the present invention to detect the DNA of red deer products under different personnel, different times and unde...

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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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