Primer pair for detecting Metschnikowia bicuspidate and application of primer pair

A technology of bicuspid plums and primer pairs, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as low sensitivity, low specificity, and complex processes of bicuspid yeast, Achieve the effect of good sensitivity, good sensitivity, and simple detection method

Active Publication Date: 2021-01-12
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, the present invention provides a pair of primers for detecting Metschia bicuspidis and its application, so as to solve the problems of complex process, low sensitivity and specificity in the prior art.

Method used

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  • Primer pair for detecting Metschnikowia bicuspidate and application of primer pair
  • Primer pair for detecting Metschnikowia bicuspidate and application of primer pair
  • Primer pair for detecting Metschnikowia bicuspidate and application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the design of the specific primers that are used to detect Metschnischiana bitipum of the present invention

[0037] 1. Materials and methods

[0038] 1.1 Sample plasmids and strains

[0039] The PMD19T vector was purchased from Baoriyi Biotechnology Co., Ltd.; Escherichia coli DH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd.; the positive sample of Metchiella bimini was collected from Panjin, Liaoning, and then obtained by laboratory separation and purification.

[0040] 1.2 Samples and reagents

[0041]DNA extraction kits were purchased from Tiangen (Beijing) Biochemical Technology Co., Ltd., PCR Mix and PCR product recovery kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid extraction kits were purchased from Dingguo Biotechnology Co., Ltd.

[0042] 1.3 DNA extraction and purification of target gene fragments

[0043] Extract, recover, purify, and extract plasmids according to the instructions of each...

Embodiment 2

[0048] Example 2, the annealing temperature of the first specific primer PCR amplification of Metchis bicuspii

[0049] Using the nucleic acid of Metchiella bitipina as a template, PCR amplification was carried out, and the reaction system was: 13 μL of 2×PCR Mix, 1 μL of specific primer pair (nmol / L), and 1 μL of DNA template of Metchi bimini.

[0050] Determine the annealing temperature of the primers, and select 45°C, 50°C, and 55°C for PCR amplification according to the reference temperature after primer synthesis. The PCR amplification program is as follows: pre-denaturation at 94°C, 10min; denaturation at 94°C, 1min; annealing at 45°C / 50°C / 55°C, 45s; extension at 72°C, 1min; final extension at 72°C, 10min; and extended for 30 loops. After the PCR reaction was completed, 5 μL of the PCR product was taken for 1.5% agarose gel electrophoresis analysis.

[0051] Test results: Set different annealing temperatures. After the PCR reaction, take 5 μL of the PCR product for 1.5...

Embodiment 3

[0052] Example 3, Sensitivity Identification of Metschia bicuspii-specific Primer Pairs

[0053] Plasmids were extracted from the PCR products recovered by gel cutting in Example 1, and the concentration was measured. The copy number of each sample was calculated according to the DNA copy number calculation formula. The results are shown in Table 2.

[0054] Table 2

[0055]

[0056] Copy number = (6.02×10 14 ×concentration (ng / μL) / (DNA length×660)

[0057] Select Metchimos bicuspii DNA template 3 for dilution, and dilute the copy number to about 8.62×10 9 , 8.62×10 8 , 8.62×10 7 , 8.62×10 6 , 8.62×10 5 , 8.62×10 4 , 8.62×10 3 , 8.62×10 2 , 8.62×10 1 . Take 1 μL of them as a template, and use the first specific primer pair P1 / P2 primers of the present invention to carry out PCR amplification. The reaction conditions are the same as those in Example 2, and the annealing temperature is 50°C. After the end, 5 μL of PCR products were taken for 1.5% agarose gel elect...

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Abstract

The invention discloses a primer pair for detecting Metschnikowia bicuspidate and application of the primer pair. Nucleotide sequences of the specific primer pair for detecting the Metschnikowia bicuspidate are shown as SEQ ID NO.1 and SEQ ID NO.2. According to the primer pair for detecting the Metschnikowia bicuspidate and application of the primer pair, specificity experiment results show that the primer pair only has specific fragments in the Metschnikowia bicuspidate, has no cross reaction with other common saccharomycetes and bacteria, and has good specificity; sensitivity experiments show that the minimum limit of the primer pair for detecting the Metschnikowia bicuspidate is 8.62 * 10 / micro L, and the primer pair has good sensitivity; and the primer pair provides an effective technical means for the detection or diagnosis of the Metschnikowia bicuspidate disease, by a method, the detection method of the Metschnikowia bicuspidate is simpler and more convenient, the diagnosis ismore accurate, and the method has an important significance in prevention and control of the Metschnikowia bicuspidate disease of economically cultured species such as Eriocheir sinensis, Macrobrachium rosenbergii, Chenook salmon and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a pair of primers for detecting Metschniella bitipina and its application. Background technique [0002] Metchiella bitipina is a kind of pathogenic yeast in aquatic animals, which can infect a variety of economically farmed species, such as Chinese mitten crab, giant giant rosenbergii, and chinook salmon. Metchiella bitipina can reproduce and develop in large numbers in various tissues of aquatic animals, and cause degenerative lesions in these tissues, mainly necrosis, resulting in swelling, erosion, and necrosis of the body cells, and in severe cases, it will lead to death. . Seriously threaten the healthy farming of economical aquatic animals, and have a great impact on its breeding industry. [0003] At present, it is difficult to confirm the infection of aquatic animals through clinical diagnosis. At present, the conventional detection method is morphologica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2565/125Y02A50/30
Inventor 陈启军姜宏波包杰李晓东申洪彬
Owner SHENYANG AGRI UNIV
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