Kit for identifying donkey hide, horse hide and mule hide on basis of MGB probe and detecting method thereof

A kit and mule skin technology, applied in the field of molecular biology, can solve the problems of high detection cost, false positive, and difficulty, and achieve the effect of good repeatability, low instrument requirements, and low cost

Pending Publication Date: 2016-11-16
济南诺贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For those skilled in the art, 5-fold multicolor fluorescent quantitative PCR is more difficult, mismatches between different primers and probes can easily lead to false positive results, and 5 molecular beacons lead to higher detection costs; on the other

Method used

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  • Kit for identifying donkey hide, horse hide and mule hide on basis of MGB probe and detecting method thereof
  • Kit for identifying donkey hide, horse hide and mule hide on basis of MGB probe and detecting method thereof
  • Kit for identifying donkey hide, horse hide and mule hide on basis of MGB probe and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 kit specificity test

[0050] A kit for identifying donkey hide, horse hide and mule hide based on the MGB probe method, the kit includes a PCR reaction solution for real-time fluorescent quantitative PCR detection.

[0051] Specifically, the PCR reaction solution includes: donkey and horse ckm gene general primer ckm-F / ckm-R, donkey MGB probe MGB-Lv, horse MGB probe MGB-Ma, quality control primers, quality control probes , Taq DNA polymerase, anhydrous D-(+)-trehalose; KCl; MgCl 2 ; dNTPs; glycerol; bovine serum albumin; ROX reference dye;

[0052] Wherein, donkey and horse ckm gene universal primer sequence is:

[0053] ckm-F: 5'-CAAGCTCTCCTAAACACCTATC-3'

[0054] ckm-R: 5'-CTGACCTAAAGCCTACGTA-3'

[0055] Each concentration is 250nM;

[0056] The MGB-Lv sequence is:

[0057] MGB-Lv: 5'-AGCCATTCATTACGCCT-3'

[0058] 5' modified FAM fluorescent group, 3' modified MGB group, the concentration is 500nM

[0059] The MGB-Ma sequence is:

[0060] MGB-Ma...

Embodiment 2

[0083] Use a commercially available animal genomic DNA extraction kit (D1700-50, Solarbio) to extract genomic DNA from donkey hide, horse hide, and mule hide, and dilute to 25 ng μL -1 As the original DNA solution, and serially diluted to 2.5ngμL -1 , 0.25ngμL -1 , 0.025ngμL -1 , 0.0025ngμL -1 , then the amount of genomic DNA in the PCR reaction solution configured according to the method described in Example 1 is 50ng, 5ng, 0.5ng, 0.05ng, 0.005ng in turn. PCR results such as Figure 1A , B shows that as the amount of genomic DNA decreases, the Ct value of the amplification curve decreases in turn. When the target DNA is as low as 0.05ng, the corresponding Ct value of MGB-LV is 35.31, and the corresponding Ct value of MGB-MA is 35.42 , close to the limit of positive results; when the genomic DNA continues to decrease to 0.005ng, the Ct value is above 38 or there is no obvious amplification, indicating that the detection limit of the kit for donkeys, horses, and mules is 0.0...

Embodiment 3

[0084] Reproducibility test in the laboratory of embodiment 3 kit

[0085] In order to verify the reproducibility of the kit when used by different personnel, three testers in the laboratory were invited to use the kit to test donkey skin, horse skin and mule skin respectively. Genomic DNA was extracted from donkey hide, horse hide and mule hide using Animal Genomic DNA Extraction Kit (D1700-50, Solarbio). Then the three inspectors respectively configured the PCR reaction amplification system according to the method described in Example 1, and set up a blank control with sterile water instead of DNA at the same time to carry out the PCR amplification reaction. The PCR amplification program was: 95°C for 30s; 95°C 5s, 60°C for 30s, collect fluorescence signal at 60°C, a total of 40 cycles. The results are shown in Table 2. The SD range of the standard deviation of the Ct values ​​obtained by the 3 testers is 0.72-0.86, and the RSD range is 2.76%-2.84%, indicating that the kit ...

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Abstract

The invention discloses a kit for identifying donkey hide, horse hide and mule hide on basis of a MGB probe and a detecting method thereof. The kit includes PCR (polymerase chain reaction) reaction liquid for real-time fluorescent quantitation PCR detection; the PCR reaction liquid includes a donkey and horse ckm gene general primer ckm-F/ckm-R, a donkey MGB probe MGB-Lv, a horse MGB probe MGB-Ma, a quality control primer, a quality control probe, Taq DNA (deoxyribonucleic acid) polymerase, anhydrous D-(+)-mycose, KCI, MgCl2, dNTPs, glycerinum, bovine serum albumin and ROX reference dye. The MGB probe technique is innovatively used; the MGB probe and template DNA can be completely combined, so that the stability is better; the result is more accurate. In addition, the kit has the advantages that the ingredients of the reaction liquid are optimized, so that the technical quality requirements on detection personnel are lowered; the practical operation steps are simplified; the practical application of colla corii asini production enterprises is convenient.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and more specifically relates to a kit for identifying donkey hide, horse hide and mule hide based on an MGB probe and a detection method thereof. Background technique [0002] "People's Republic of China Pharmacopoeia" (2010) stipulates that donkey-hide gelatin is made from the dry or fresh skin of the equine animal donkey (Equus asinus L.) after decoction and concentration. Donkey-hide gelatin has the functions of nourishing blood and stopping bleeding, nourishing yin and moistening dryness, etc. It is used as medicine and food. It is an important national intangible cultural heritage. It has been favored by doctors of all dynasties and the general public for thousands of years. However, in recent years, the number of donkeys in our country has been declining year by year, causing major donkey-hide gelatin companies to suffer from the shortage of donkey skin resources, and counterfeit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q1/6851C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101C12Q2563/107
Inventor 张广远
Owner 济南诺贝莱生物科技有限公司
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