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Composition for detecting mycoplasma gallisepticum through microdroplet type digital PCR (Polymerase Chain Reaction) and application of composition

A technology of mycoplasma gallisepticum and composition, applied in the direction of microorganisms, methods based on microorganisms, measurement/testing of microorganisms, etc., can solve problems such as difficult diagnosis and difficult diagnosis, and achieve good specificity and repeatability

Pending Publication Date: 2022-07-05
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Symptoms are similar to those caused by other respiratory pathogens such as avian influenza and Newcastle disease, which brings difficulties to the diagnosis of MG, which needs to be distinguished by laboratory methods, but in the case of low viral load, conventional methods are also difficult For diagnosis, it is necessary to find a detection method for low copy

Method used

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  • Composition for detecting mycoplasma gallisepticum through microdroplet type digital PCR (Polymerase Chain Reaction) and application of composition
  • Composition for detecting mycoplasma gallisepticum through microdroplet type digital PCR (Polymerase Chain Reaction) and application of composition
  • Composition for detecting mycoplasma gallisepticum through microdroplet type digital PCR (Polymerase Chain Reaction) and application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Establishment and optimization of ddPCR reaction

[0060] 20μL reaction system: 2×Supermix for probe (No dUTP) 10μL, 5-40μmol / μL upstream and downstream primers 1μL each, 2.5-40μmol / μL probe 1μL, positive standard plasmid template 2μL, with ddH 2 O make up to 20 μL.

[0061] Droplet generation: Add 20μL of the above reaction solution and 70μL of droplet generation oil to the second and third rows of the droplet generation card DG8 respectively, cover with a special rubber pad, and place the droplet generator on the droplet generator to automatically generate droplets in the first row. .

[0062] Sealing film: suck the generated droplets into a 96-well plate, cover with an aluminum film, place it on a PX1 heat sealer, and seal the film at 180°C for 10s.

[0063] PCR amplification: The membrane-sealed 96-well plate was placed on a PCR amplicon for amplification. The reaction program was: 95°C for 10min; 94°C for 30s, set the annealing temperature to 50-60°C f...

Embodiment 2

[0084] Example 2, primer design and synthesis

[0085] According to the published conserved sequence of Mycoplasma gallisepticum strain MG mgpc gene (No.JX869132.1), use the online https: / / bioinfo.ut.ee / primer3-0.4.0 / software to design ddPCR primers and probes, and apply BLAST The designed primer and probe sequences were aligned and analyzed online, and the 5' end of the probe primer was marked with a FAM fluorophore, and the 3' end was marked with a BHQ1 quenching group. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The sequences of primers and probe oligonucleotides are shown in Table 1.

[0086] Table 1. Primer and probe oligonucleotide sequences

[0087]

Embodiment 3

[0088] Example 3. Construction of standard plasmids

[0089] 3.1. Extraction of viral nucleic acid

[0090] Referring to the instructions of the EasyPure Virual DNA / RNA extraction kit, extract the DNA / RNA of MG S6 and control strains, and store the extracted DNA / RNA at -80°C for later use.

[0091] 3.2. Preparation of standard templates

[0092] Using the DNA of the MG S6 strain extracted in 3.1 as a template, the MG mgpc gene was amplified with the designed primers MG-F and MG-R. The amplified PCR product was subjected to 15g / L agarose gel electrophoresis, and the target fragment was stained. The gel was excised, and the target fragment was recovered with a gel recovery kit to obtain a DNA molecule whose nucleotide sequence was shown in SEQ ID No. 1.

[0093] The obtained DNA molecule was ligated with PMD18-T vector (purchased from TaKaRa Biological Company, item number: 6011), and transfected into E. coli DH5α. The positive clones were screened out by the blue-white class...

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Abstract

The invention discloses a composition for detecting mycoplasma gallisepticum through microdroplet type digital PCR (polymerase chain reaction) and application of the composition, and belongs to the technical field of microorganism determination or detection methods. The composition comprises a mycoplasma gallisepticum specific primer probe, and the mycoplasma gallisepticum specific primer probe is composed of a primer and a probe, wherein the primer is used for specifically amplifying a mycoplasma gallisepticum genome DNA fragment containing a DNA fragment with the nucleotide sequence of SEQ ID No.1, and the probe is specifically combined with the DNA fragment with the nucleotide sequence of SEQ ID No.1. The ddPCR detection composition provided by the invention is high in sensitivity, strong in specificity and suitable for detection of low-copy samples.

Description

technical field [0001] The invention belongs to the technical field of determination or inspection methods of microorganisms, in particular to a method for detecting Mycoplasma gallisepticum by droplet digital PCR. Background technique [0002] Droplet digital PCR (droplet digital PCR, ddPCR) is a new generation of polymerase chain reaction (polymerase chain reaction, PCR) technology that appeared in recent years. The reaction solution is divided into tens of thousands of nano-sized droplets by the water-in-oil technology, and each droplet is an independent PCR reaction system. Then, according to the Poisson distribution principle in statistics, the number and proportion of positive droplets can be analyzed, and the initial copy number or concentration of the target molecule can be obtained, so as to realize the absolute quantification of nucleic acid molecules down to a single copy. . Compared with the traditional quantitative real-time PCR (qPCR) method, it does not rely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12R1/35
CPCC12Q1/689C12Q1/6851C12Q2600/166C12Q2600/158C12Q2531/113C12Q2563/159C12Q2545/113C12Q2563/107
Inventor 谢芝勋谢志勤张艳芳范晴谢丽基万丽军罗思思李孟
Owner GUANGXI VETERINARY RES INST
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