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PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit

A technique for Mycoplasma hyopneumoniae and Mycoplasma hyorhinosum, which is applied in the field of PCR, can solve the problems of cell contamination and low sensitivity, and achieve the effects of strong specificity, good sensitivity and pollution avoidance

Inactive Publication Date: 2017-01-11
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

; In addition, fluorescent staining is used to detect mycoplasma, but the sensitivity of this method is too low. When the detection is positive, the cells are often seriously polluted

Method used

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  • PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit
  • PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit
  • PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Primer Design and Synthesis

[0022] Referring to the gene sequence in GenBank, select the sequences of several representative mycoplasma-positive strains that are often easily contaminated in the laboratory, and select the conserved segments with partial fragment similarity rather than the conserved regions with a large number of similarities through comparison of nucleic acid sequences This can ensure that there are effective differences between different strains, and at the same time, the partial similarity can design common primers, so the differences in this segment can effectively distinguish different species of Mycoplasma fragments, and BLAST comparison It is interesting to find that this pair of sequences has homology to most species of mycoplasma except the representative mycoplasma. DNAStar software was used in the design, and a pair of primers were designed following the basic principles of primer design. The sequence of the upstream primer is: 5'-...

Embodiment 2

[0031] Construction of embodiment 2 positive control plasmid

[0032] (1) Extraction of nucleic acid: Take 200 μL each of the samples of positive strains, i.e. Mycoplasma hyorhina, Mycoplasma gallisynovii, and Mycoplasma hyopneumoniae, and extract the genome according to the instructions of the TaKaRa MiNiBEST Universal Genomic DNA Extraction KitVer.5.0 extraction kit. The extracted DNA Store at -20°C for subsequent PCR amplification reactions.

[0033] (2) Perform PCR amplification on the DNA of Mycoplasma hyorhina, Mycoplasma gallisynovii, and Mycoplasma hyopneumoniae respectively, and the PCR amplification reaction is carried out in a 25 μL system: 1 μL each of Primer1 / 2, 12.5 μL of PCR premix buffer, and 1 μL of genome template ,ddH 2 O was added to 25 μL; the PCR reaction program was as follows: 94°C for 5 min; 94°C for 30 s, 57°C for 1 min, 72°C for 1 min, 30 cycles; finally, 72°C for 5 min. Take 5 μL of the PCR amplification products of Mycoplasma hyorhina, 5 μL of th...

Embodiment 3

[0037] Example 3 Sensitivity Verification of PCR Detection Kit

[0038] The PCR detection kit used in this example includes: 500 μL PCR premix buffer, 1 mL ultrapure water, 50 μL Marker DL2000, 50 μL upstream primer with a concentration of 10 μmol / L and 50 μL downstream primer with a concentration of 10 μmol / L, 20 μL negative control, and 20 μL Positive control; the sequence of the upstream primer is: 5'-ACACCATGGGAGCTGGTAAT-3'; the sequence of the downstream primer is: 5'-GTTCATCGACTTTCAGACCCAAGGCAT-3'; the positive control includes the T vector plasmid containing Mycoplasma gallisovoida, Containing the T vector plasmid of Mycoplasma hyopneumoniae and the T vector plasmid containing Mycoplasma hyorhina; The negative control is ultrapure water; The PCR premix buffer is composed of 3mmol / μL of MgCl 2 , 500pmol / μL of dNTP, 25mmol / μL of Tris-HCl and 0.2μL of Taq enzyme, and its pH value is 8.3.

[0039] The operation steps of the sensitivity verification of the PCR detection kit...

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Abstract

The invention belongs to the technical field of PCR, and particularly relates to a PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of the PCR detection kit. The PCR detection kit comprises PCR premix buffering solution, superpure water, Marker DL2000, upstream primer, downstream primer, a negative control and a positive control. The sequence of the upstream primer is 5'-ACACCATGGGAGCTGGTAAT-3'; the sequence of the downstream primer is 5'-GTTCATCGACTTTCAGACCCAAGGCAT-3'. The PCR kit has the advantages of being low in detection limit, good in sensitivity, strong in specificity and the like, can rapidly detect and identify mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis.

Description

technical field [0001] The invention belongs to the technical field of PCR, and in particular relates to a PCR detection kit for rapidly differentiating Mycoplasma hyopneumoniae, Mycoplasma gallisynovium and Mycoplasma hyorhina and application thereof. Background technique [0002] Mycoplasma is pleomorphic between viruses and bacteria and can pass through a 0.22μm filter membrane. The production and inspection materials in the production and inspection of animal biological products are easily contaminated by Mycoplasma hyopneumoniae, Mycoplasma gallisynovium, and Mycoplasma hyorhina. Mycoplasma and chicken embryos may carry Mycoplasma gallisovoidum; (2) The positive control strains used in the test, such as Mycoplasma hyopneumoniae and Mycoplasma gallisynovium, are positive controls for mycoplasma test, and a large number of repeated uses in the test process may contaminate laboratory. [0003] Once the production materials are contaminated by the above-mentioned mycoplas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 陶醉徐高原尹争艳陈关平陈斌周明光金建云陈章表汤细彪
Owner WUHAN KEQIAN BIOLOGY CO LTD
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