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Buffalo-specific primers, kits and their application in the identification of buffalo-derived components

A source-derived component and specific technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems that have not been studied in other species, and achieve low cost, wide applicability, highly specific effect

Active Publication Date: 2018-11-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific primers designed for this target sequence can only be used to identify buffalo, cattle and yak, and whether it is also applicable to other species has not been studied

Method used

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  • Buffalo-specific primers, kits and their application in the identification of buffalo-derived components
  • Buffalo-specific primers, kits and their application in the identification of buffalo-derived components
  • Buffalo-specific primers, kits and their application in the identification of buffalo-derived components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Specific detection of buffalo-derived components in the sample of Example 1

[0034] 1. Sample preparation and storage

[0035] 1.1 Sampling

[0036] Collect 1 g of buffalo meat samples and store them at -20°C for future use.

[0037] 1.2 DNA template preparation

[0038] The DNA template was prepared using the commonly used phenol-chloroform crude extraction method (Sambrook J, Fritsch E F, Mani Artis T. Molecular Cloning Experiment Guide [M]. 2nd Edition. Jin Dongyan, Li Mengfeng. Beijing: Science Publishing Society, 1999.465-467) or recognized other extraction methods with the same efficacy, these methods are the commonly used methods reported.

[0039] 2. Primer Design

[0040] The primer sequences of this embodiment are shown in Table 2 and SEQ ID NO: 2 and 3 in the sequence listing.

[0041] Table 2 PCR amplification primers designed by the present invention

[0042]

[0043]

[0044] The size of the expected amplified fragment is 231bp, and its nucleo...

Embodiment 2

[0066] Embodiment 2 sensitivity test

[0067] Using 0.1ng / μL, 1ng / μL, 10ng / μL, and 100ng / μL buffalo DNA as templates, the nucleotide-specific fragment of SEQ ID NO.1 was amplified according to the conditions of Example 1. The result is as figure 2 As shown, there is amplification at a template concentration of 1ng / μL, indicating that the fragment amplified by the specific primer has good sensitivity.

Embodiment 3

[0068] The assembly of embodiment 3 kits

[0069] The kit consists of:

[0070] Upstream primer (SEQ ID No.2);

[0071] Downstream primer (SEQ ID No.3);

[0072] 2×Taq DNA MasterMix;

[0073] Positive control DNA template (buffalo DNA template);

[0074] double distilled water.

[0075] Storage of this kit at -20°C for 12 months will not affect the use effect.

[0076] How to use the kit:

[0077] 1. Load the sample into a 200 μL EP tube according to the following PCR reaction system:

[0078]

[0079] 2×Taq DNA MasterMix (purchased from Beijing Aidelai Biotechnology Co., Ltd.) is the Taq enzyme, dNTP mixture, MgCl 2 And the reaction buffer is pre-configured into a 2-fold concentration mixture.

[0080] 2. Loading according to the PCR amplification conditions of Example 1

[0081] 3. After the reaction, take 5 μL of PCR amplification product, electrophoresis on 2.0% agarose gel (technical parameters: 2V / cm~5V / cm, electrophoresis 15min~30min), stain with ethidium br...

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Abstract

Belonging to the technical field of animal origin component molecular detection, the invention provides a buffalo specific primer, a kit and application thereof in buffalo origin component identification. In the invention, the buffalo specific primer is the primer pair SEQ ID No.2&3 able to specifically amplify the nucleotide sequence shown as SEQ ID No.1 or specific fragments of the sequence. The kit provided by the invention comprises the buffalo specific primer, a reaction reagent, positive control and blank control. The primer provided by the invention is employed to amplify sample DNA, and the PCR amplification product is analyzed to determine whether the sample contains buffalo origin components. The invention provides an effective, accurate and reliable means for detection of buffalo origin components in meat products, hides and feed, and the method and the kit have the characteristics of simplicity, fastness, comprehensiveness, strong specificity, low cost, and wide applicability.

Description

technical field [0001] The invention relates to the field of animal molecular biology, in particular to buffalo-specific primers, a kit and their application in the identification of buffalo-derived components. Background technique [0002] With the improvement of people's living standards and the increase of meat consumption, the adulteration of various meat products has also appeared in the market. In the import and export trade of meat products, due to the relatively backward inspection methods for meat quality in my country, false inspections and missed inspections occur from time to time. Shoddy, doping and adulteration have seriously affected fair trade, food safety, and even religious beliefs. Therefore, the safety of the source of meat products has become one of the focus issues that consumers care about, and the identification of the source of meat products and the establishment of convenient detection methods are the key to solving this problem. [0003] The iden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6888
Inventor 刘榜王文君刘建建张庆德
Owner HUAZHONG AGRI UNIV
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