Primer pair, kit and method for rapidly detecting horse-derived components in horse hide and mule hide

A detection reagent and horse-derived technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. On-site detection methods of horse-derived components and other issues

Active Publication Date: 2021-04-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent (CN201610716622.3) discloses the quantitative identification of donkey hide, horse hide, and mule hide based on MGB probes using fluorescence; Horsehide and mule hide; but the above methods require expensive instruments and equipment, are time-consuming, and cannot achieve on-site detection; therefore, it is necessary to establish a rapid on-site detection method for horse-derived components in horse hide and mule hide
[0005] However, the current RPA-based on-site detection method for horse-derived components in horsehide and mule hide has no special software for designing RPA primers, and the requirements for primer design are relatively high. Primer dimers cannot be formed, so the primer design of RPA is more difficult, so there is a lack of on-site detection methods for horse-derived components in horse hide and mule hide

Method used

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  • Primer pair, kit and method for rapidly detecting horse-derived components in horse hide and mule hide
  • Primer pair, kit and method for rapidly detecting horse-derived components in horse hide and mule hide
  • Primer pair, kit and method for rapidly detecting horse-derived components in horse hide and mule hide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Embodiment 1 specific detection

[0037] 1. RPA amplification primer design

[0038] Because the detection method of RPA amplification combined with test strips has high requirements for primer design, it is necessary to ensure that it is specific among species, conserved within species, and cannot form primer dimers, otherwise it will directly affect the RPA amplification speed and detection sensitivity. Thus affecting the judgment of on-site inspection results. The present invention screens horse-specific sequences from the aspects of intra-species consistency, stability, and inter-species specificity, and compares the horse genome sequence with the whole genome of other mammals (cattle, buffalo, sheep, goat, pig, donkey) through local BLAST. Sequences were compared, and finally the horse LOC102147906 gene sequence was selected as the detection target; according to the design principles of RPA primers, the primer length was 28-35bp, and several specific RPA primers we...

Embodiment 2

[0056] Embodiment 2 sensitivity test

[0057] The horsehide DNA concentration was quantified to 10ng / μL with Nanodrop, and then 10-fold serial dilution (10 1 、10 0 、10 -1 、10 -2 ), with the concentration of horsehide genomic DNA as 10 1 ng / μL, 10 0 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL was used as a template, and RPA amplification was performed according to the conditions of Example 1. The amplified products were detected by test strips, and the results were as follows: image 3 As shown, when the template concentration is as low as 1ng / μL, both the quality control line and the detection line are colored, that is, adding 1ng of template DNA to the reaction system can be detected. Since the detection method of the present invention is established for donkey hide, horse hide and mule hide, the result of the sensitivity test can meet the detection requirements.

[0058] Embodiment 3 test strip

[0059] The invention provides a colloidal gold test strip for detecting RPA nucle...

Embodiment 4

[0060] Example 4 Application of test strips for rapid detection of horse-derived components in horse hide and mule hide

[0061] Two horsehide samples, two mule hide samples and one donkey hide sample were respectively used to extract the genomic DNA of the samples with a DNA genome extraction kit, and the extracted DNA samples were used as templates, and RPA amplification was carried out according to the conditions of Example 1 . The amplified products were detected by test strips, and the results were as follows: Figure 4 shown. Number 1 is distilled water as a blank control, 2 is a donkey hide DNA sample, 3-4 are two horse hide DNA samples, 5-6 are two mule hide DNA samples, the results are shown in the test strips in 3-6 samples Bands appeared on both T and C lines, and only bands appeared on the C line in the test strip in the 2 samples, indicating that the sequences of SEQ ID NO.1-SEQ ID NO.2 can be used for the detection of real samples.

[0062] For the detection o...

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Abstract

The invention provides an RPA labeled primer pair, a kit and a method for quickly detecting horse-derived components in horse hide and mule hide, and belongs to the technical field of biology. The RPA labeled primer pair has sequences as shown in SEQ ID No. 1 and SEQ ID No. 2. The method includes: on the basis of naked eye preliminary identification, extracting DNA from DNA horse hide and mule hide that are suspected to be donkey hide, and amplifying by adopting the RPA labeled primer pair; detecting the RPA amplification product by using a detection test strip and judging a result. Whether a sample is horse hide or mule hide is identified by judging whether the sample contains horse-derived components or not, so that authenticity of the donkey hide is judged. The method for detecting the horse-derived components in horse hide and mule hide does not need special equipment, only needs 40 minutes of reaction time, has strong specificity, has advantages of simplicity, convenience, quickness and the like, provides an effective, accurate and reliable means for detecting the horse-derived components in the horse hide and mule hide, and is very suitable for detecting authenticity of donkey hide on site.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer pair, a kit and a method for rapid detection of horse-derived components in horse hide and mule hide. Background technique [0002] Donkey-hide gelatin is a solid gelatin obtained by decocting and concentrating the dry or fresh skin of an equine animal donkey (Equus asinus L.). Meet the needs of production. The shortage of donkey hides has led some lawbreakers to use horse hides and mule hides similar to donkey hides (mule hides are derived from mules, and mules are produced by crossbreeding horses and donkeys) to sell as donkey hides. The ingredients of horsehide and mule skin are mixed in, which changes the efficacy of donkey-hide gelatin, disrupts the market order, and damages the reputation of businesses and the rights and interests of consumers. Therefore, there is an urgent need for detection methods for the identification of horse-derived components in d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
Inventor 刘榜谌阳付明周翔王文君
Owner HUAZHONG AGRI UNIV
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