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HRM primers and methods for differentiating Mycoplasma capricosum subsp.

A technology of mycoplasma and goats, applied in the field of rapid diagnosis of veterinary infectious diseases, can solve the problems of HRM primers and methods that have not yet been identified, and achieve rapid cost, strong specificity, and good repeatability

Active Publication Date: 2022-06-07
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After reviewing the existing literature, there is no HRM primer and method for the identification of Mycoplasma capricosum subsp.

Method used

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  • HRM primers and methods for differentiating Mycoplasma capricosum subsp.
  • HRM primers and methods for differentiating Mycoplasma capricosum subsp.
  • HRM primers and methods for differentiating Mycoplasma capricosum subsp.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Strain

[0022] Mycoplasma capricolum subspecies FJ-GT strain and Mycoplasma capricolum subsp. caprine pneumonia standard strain MccpF38 were all preserved by the Laboratory of Herbivore Diseases, Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0023] 2. Primer design and synthesis

[0024] Two pairs of specific primers were designed according to the sequence of Mmc 95010 strain MLC_0560 gene (GenBank accession number FQ377874.1) and the sequence of Mccp F38 strain MCCPF38_00984 gene (GenBank accession number LN515398.1):

[0025] Outer primer SEQ ID No. 5: MFW325: 5'-GAATGTTTAATCGATCAACTGCT-3',

[0026] SEQ ID No. 6: MRW325: 5'-TGCTAATAGTGGTCCTGCTAAGA-3'.

[0027] Inner primer SEQ ID No.7: 107F: 5'-CAATACATCCATTARMACTCTTGA-3',

[0028] SEQ ID No. 8: 107R: 5'-GAATWCAYTCAGGTTGATTATTAGGA-3'.

[0029] 3. The HRM method for identifying Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capricolum...

Embodiment 2

[0038] Example 2 Specificity Verification

[0039] Use the method of step (1) in Example 1 to extract the genomic DNAs of Mmc, Mccp, Mycoplasma ovine pneumonia (Movi), Orchitis Virus (ORFV), Escherichia coli, Salmonella, Pasteurella, and Mycoplasma arginine, respectively as templates , using HRM method for identification, to verify the specificity of the established HRM method for identification of Mycoplasma capricolum subspecies and Mycoplasma capricolum subspecies caprine pneumonia. see the results figure 1 . figure 1 The results show that the invention can accurately and rapidly identify Mycoplasma capricolum subspecies and Mycoplasma capricolum capitis subspecies, and can be used for clinical diagnosis and epidemiological investigation of Mycoplasma capricolum pneumonia and caprine infectious pleuropneumonia.

Embodiment 3

[0040] Example 3 Repeatability Verification

[0041] The Mmc and Mccp genomic DNAs extracted by Ezup column bacterial genomic DNA extraction kit were used as templates, and the extracted templates were amplified by PCR with primers MF-107 and MR-107, and the amplified products were recovered and purified after agarose gel electrophoresis. , connect the purified target gene to PMD-19T cloning vector, transform DH5α competent cells, use TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0 to extract the plasmid for PCR identification and enzyme digestion identification, measure its concentration and calculate the copy number as the HRM detection Standard. The standard was diluted 10-fold as a template for repeatable HRM detection. The intra-batch repeat test is to measure the samples of each concentration 3 times; the inter-batch repeat test is to measure 3 times of the standard substance of 3 dilutions, and the interval of each time is 3d. The HRM reaction system and cycle parameters...

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Abstract

The invention provides HRM primers and a method for distinguishing Mycoplasma capricum subsp. goat and Mycoplasma capricum subsp. goat pneumonia, and belongs to the technical field of rapid diagnosis of veterinary infectious diseases. The upstream primer of the HRM internal primer is 107F: CAATACATCCATTARMACTTCTTGA, and the downstream primer is 107R: GAATWCAYTCAGGTTGATTATTAGGA. This method uses the characteristic that the double-stranded DNA combined with a fluorescent dye will have a color reduction effect during the temperature rise process. By detecting the double-stranded DNA melting The characteristic melting curve formed by the fluorescent signal released during the process was used to identify the base difference in the nucleic acid sequences of the two mycoplasmas. Including DNA extraction, PCR amplification to obtain the target sequence and melting curve analysis. The identification method of the invention has strong specificity, high sensitivity, good repeatability and simple operation.

Description

technical field [0001] The invention belongs to the technical field of rapid diagnosis of veterinary infectious diseases, and in particular relates to an HRM primer and a method for rapidly identifying Mycoplasma capricolum subspecies and Mycoplasma capricolum capitis subspecies. Background technique [0002] Mycoplasma sheep pneumonia is a kind of pneumonia infectious disease in sheep, the incidence of the disease is 20% to 60%, and the mortality rate is 10% to 50%. Mycoplasma filamentosa goat subspecies ( Mycoplasma mycoides subsp. capri , Mmc) and Mycoplasma ovine pneumonia ( Mycopalsamovipneumonia, Movi) are the 2 main pathogens of mycoplasma pneumonia. goat infectious pleuropneumonia ( Contagious caprine pleuropneumonia, CCPP ) is a goat-specific acute or chronic high-contact infectious disease characterized by fibrinous pneumonia and pleurisy, with an incidence rate of 80-100% and a mortality rate of 60-80%. The disease is mainly prevalent in Africa, the Middle E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/35
CPCC12Q1/686C12Q1/689C12Q2527/107C12Q2563/107Y02A50/30
Inventor 胡奇林张靖鹏江锦秀林裕胜游伟
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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