Multiplex-polymerase chain reaction (PCR) kit for rapidly detecting deer product and preparation method thereof

A technology of deer products and kits, applied in the field of multiplex PCR kits

Inactive Publication Date: 2012-07-25
邢秀梅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention provides a multiplex PCR kit for rapid detection of deer products, which solves the problems of sika deer, red deer, Tarim red deer, reindeer and red deer as well as pigs, cattle and sheep. Problems with rapid detection of common counterfeit mixed products such as , chicken, etc.

Method used

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  • Multiplex-polymerase chain reaction (PCR) kit for rapidly detecting deer product and preparation method thereof
  • Multiplex-polymerase chain reaction (PCR) kit for rapidly detecting deer product and preparation method thereof
  • Multiplex-polymerase chain reaction (PCR) kit for rapidly detecting deer product and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1: Composition and preparation of kit

[0098] 1) Preparation of DNA extraction solution

[0099] (1) Low-salt buffer (pH 7.6): Dissolve 1.21g Tris base (trishydroxymethylaminomethane) and 3.38g Na in 800ml distilled water 2 EDTA (disodium ethylenediaminetetraacetic acid), 0.76g MgCl 2 , 0.47gNaCl. Add concentrated hydrochloric acid to adjust the pH to the desired value, add distilled water to make up to 1L.

[0100] (2) Cell lysate: Add 1g SDS (sodium dodecyl sulfate) and 1ml Triton X-100 (polyethylene glycol p-isooctylphenyl ether) to 200ml low-salt buffer.

[0101] (3) Proteinase K: Dissolve 100 mg proteinase K in 5 ml sterile deionized water and store at -20°C.

[0102] Extraction method: Add 200ul anticoagulant blood and 400ul low-salt buffer solution into a 1.5ml centrifuge tube, mix well, centrifuge at 1000RPM for 10min, discard the supernatant; add 500ul low-salt buffer solution, mix well, and centrifuge at 1000RPM for 5min at room temperature...

Embodiment 2

[0139] Embodiment 2: the specificity test of kit

[0140] 1. The interspecies specificity and intraspecies generality of each primer pair in deer-derived multiplex PCR reaction.

[0141] (1) To prove that each primer pair in the deer-derived multiplex PCR reaction has cross-species specificity.

[0142] ① Genomic DNA of sika deer, red deer, Tarim red deer, red deer, reindeer, sambar, eld deer, white-lipped deer, elk, fallow deer, pig, cattle, sheep and chicken were extracted from the blood according to the method in Example 1. The extracted DNA was used to prepare the following reaction systems, and sterile water was used as a negative control.

[0143] ②Preparation of the reaction system: step ① Add 1.0ul of the extracted DNA to the deer-derived multiplex PCR reaction solution in Example 1.

[0144] ③According to the program: 94°C for 5 minutes; 94°C for 30s, 56°C for 30s, 67°C for 1min; 67°C for 5min, amplify for 30 cycles. Perform PCR amplification.

[0145] ④ Results...

Embodiment 3

[0156] Example 3: Sensitivity of multiplex PCR.

[0157] 1. To evaluate the sensitivity of deer-derived multiplex PCR reaction.

[0158] (1) According to the extraction method in Example 1, blood genome DNA of sika deer, red deer, Tarim red deer, red deer and reindeer were extracted respectively. Carry out serial dilution respectively, and the dilution conditions are as follows: 10, 5, 1, 0.5, 0.2, 0.1, 0.05, 0.02 ng.

[0159] (2) Preparation of reaction systems: Step (1) 1.0 ul of extracted DNA was added to the deer-derived multiplex PCR reaction solution in Example 1.

[0160] (3) According to the program: 94°C for 5 minutes; 94°C for 30s, 56°C for 30s, 67°C for 1min, 67°C for 5min, amplify for 30 cycles. Perform PCR amplification.

[0161] (4) Results: The minimum detection limits of genomic DNA samples of sika deer, red deer, Tarim red deer, red deer and reindeer were 0.05, 0.05, 0.1, 0.5 and 0.02 ng, respectively.

[0162] 2. Evaluate the sensitivity of multiple PCR...

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Abstract

The invention discloses a multiplex-polymerase chain reaction (PCR) kit for rapidly detecting a deer product, which is used for rapidly and accurately distinguishing the deer product. The invention also discloses the preparation method of the kit. A multiplex-PCR technology is adopted to amplify the specific molecular fragments of mitochondrial DNAs of sika deer, red deer, Tarim red deer, wapiti, reindeer, pig, cow, sheep and chicken, so that the kit is prepared and used for detecting a detected sample contains the components of the animal components and detecting a mixed sample; and the kit and the method have the characteristics that is operation is simple, sensitivity is high and speed is rapid.

Description

technical field : [0001] The invention discloses a multiplex PCR kit for rapidly detecting deer products, which is used for quickly and accurately identifying deer products, and also discloses a preparation method of the kit, which belongs to the technical field of biological detection kits. Background technique: [0002] Deer products are some tissues or organs of deer, mainly including: deer antler, deer heart, deer kidney, deer fetus, deer tendon, deer whip, deer tail, venison meat and deer blood, etc. Since most deer products are precious Chinese medicinal materials and are expensive, it is common to see fake and inferior products pretending to be valuable deer products in the market. [0003] At present, the identification of deer products mostly adopts traditional methods, mainly including trait identification, microscopic identification and physical and chemical identification. Although the traditional identification method is simple, fast, and low in cost, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邢秀梅杨福合査代明荣敏苏伟林吴琼
Owner 邢秀梅
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