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Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis

A technology of mycoplasma hyorhinosum and primer pairs, applied in the biological field, can solve the problems of long detection time and achieve the effects of short quarantine period, easy promotion and strong specificity

Active Publication Date: 2014-06-04
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defect that the detection time of Mycoplasma hyorhina is obviously long in the existing separation and culture-based detection, and to provide a nested PCR detection method for Mycoplasma hyorhina that can be detected quickly, sensitively and accurately

Method used

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  • Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis
  • Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis
  • Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis

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Effect test

Embodiment 1

[0033] Embodiment 1, the nested PCR detection method of Mycoplasma hyorhinus of the present invention mainly comprises the following steps:

[0034] 1) Extract the DNA of Mycoplasma hyorrhinosus samples and store at low temperature for future use;

[0035] The DNA of the Mycoplasma hyorrhinos sample used in the present invention can be extracted by conventional methods, and the specific method is to carry out the following steps after extracting the swine lung tissue containing Mycoplasma hyorrhinosus:

[0036] 1) Add 100 mg of tissue to 600 μl of protein lysate, then add 3 μl of proteinase K (20 mg / ml), 3 μl of RNase inhibitor (10 mg / ml), mix well, and water bath at 55°C for 2-4 hours, shaking several times during this period.

[0037] 2) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), shake gently for 10min, and centrifuge at 1,2000rpm for 5min.

[0038] 3) Take the supernatant (about 300 μl), add 600 μl of chloroform, and centrifuge at 1,2000 rpm for 5 ...

Embodiment 2

[0071] Example 2, in order to verify the specificity of Mycoplasma hyorrhea, the electrophoresis detection method can be used to detect Mycoplasma flocculum, Mycoplasma hyos synovium, Mycoplasma hyopneumoniae, Mycoplasma gallisepticum, Actinobacillus pleuropneumoniae, swine fever virus, swine fever virus, respectively. Influenza virus, porcine circovirus and Mycoplasma hyorrhinos negative and positive controls were tested. The gel electrophoresis results of the amplified products of each strain are shown in figure 1 , the results showed that Mycoplasma hyorhinosus could amplify a 346bp band, while the negative control and other strains did not amplify a specific band.

Embodiment 3

[0072] Embodiment 3, the sensitivity test of common double primer PCR

[0073] 1. Through the determination of the discoloration unit of Mycoplasma hyorrhinosus, the content of Mycoplasma hyorrhinosus was measured to be 10 9 CCU / mL;

[0074] 2. Dilute the above-mentioned Mycoplasma hyorrhea cell stock solution as: 10 7 CCU / mL, 10 6 CCU / mL, 10 5 CCU / mL, 10 4 CCU / mL, 10 3 CCU / mL, 10 2 CCU / mL, extract the DNA of each bacterial body, take 1 μl

[0075] As a template, only one PCR amplification was performed with specific primers Mhr P01 / P02, and the electrophoresis results after amplification were shown in figure 2 . The same results were obtained by repeating the amplification three times.

[0076] figure 2 , M is a 2000bp DNA marker; 1-6 represent DNA templates of 10 7 CCU / mL, 10 6 CCU / mL, 10 5 CCU / mL, 10 4 CCU / mL, 10 3 CCU / mL, 10 2 A PCR amplification product of Mycoplasma hyorhinosa at CCU / mL. figure 2 showed that the minimum detectable pathogens were 10 fo...

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Abstract

The invention relates to a nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis causing polyserositis, arthritis, otitis and the like of swines. The method includes the main steps: firstly, extracting DNA (deoxyribonucleic acid) from a mycoplasma hyorhinis sample, and keeping the DNA at a low temperature for later use; secondly, performing primary PCR amplification for the mycoplasma hyorhinis sample by the aid of a pair of specific primers of Mhr P01 and Mhr P02; thirdly, performing secondary PCR amplification for a primary PCR product of the mycoplasma hyorhinis sample by the aid of another pair of specific primers of Mhr P03 and Mhr P04; and fourthly, performing electrophoresis detection for an amplified product, and if a specific band of 346bp exists, determining the detected pathogenic bacteria as the mycoplasma hyorhinis. By the method, the mycoplasma hyorhinis can be accurately detected in the presence of trace pathogenic bacteria, and can be detected only in the presence of 102CCU / mL of the pathogenic bacteria. The nested PCR detection method for the mycoplasma hyorhinis has the advantages of high detecting speed, high specificity, high sensitivity and easiness to popularize.

Description

technical field [0001] The invention relates to a detection method of Mycoplasma hyorrhinosus which causes multiple serositis, arthritis, otitis and the like of pigs, in particular to a nested PCR detection method of Mycoplasma hyorrhinosus, which belongs to the field of biotechnology. Background technique [0002] Mycoplasma hyorrhea ( Mycoplasma hyohinis ) is a pathogenic mycoplasma that can cause porcine polyserositis, arthritis, otitis, pneumonia and other diseases. As a common pathogen, M. hyorrhinosus is usually transmitted from sows or pigs to piglets. Ross and Spear (1993) demonstrated that the mycoplasma could be isolated from the nasal secretions of 10% of sows and 30% to 40% of weaned piglets. Once infected, the mycoplasma spreads rapidly in the upper respiratory tract and can be isolated from the lungs and nasopharyngeal tube of infected pigs. [0003] The presence of M. hyorrhinos can be detected in more than 50% of chronic swine pneumonia cases in the UK and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/35
Inventor 白方方邵国青武昱孜刘茂军冯志新熊祺琰
Owner JIANGSU ACAD OF AGRI SCI
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