Primer group for detecting mycoplasma hyopneumoniae, application of primer group and real-time fluorescent quantitative PCR detection method

Abstract

The invention provides a primer group for detecting mycoplasma hyopneumoniae, application of the primer group and a real-time fluorescent quantitative PCR detection method, and relates to the technical field of biology. The primer group for detecting the mycoplasma hyopneumoniae comprises a first primer pair and a probe, has the characteristics of strong specificity and high sensitivity, and can be used for detecting the mycoplasma hyopneumoniae. The real-time fluorescent quantitative PCR detection method for the mycoplasma hyopneumoniae adopts the primer group, and comprises the following steps: firstly, performing real-time fluorescent quantitative PCR on a mycoplasma hyopneumoniae standard substance with a known initial copy number to obtain a relationship between the copy number and a Ct value; then extracting DNA of a to-be-detected sample for real-time fluorescent quantitative PCR, and obtaining the target gene copy number in the to-be-detected sample through calculation. The detection method is high in specificity, good in repeatability, high in sensitivity, rapid in detection and accurate in quantification and has great significance in rapid quantitative detection of mycoplasma hyopneumoniae cultures, vaccine semi-finished products and the like.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a primer set for detecting mycoplasma hyopneumoniae, an application thereof, and a real-time fluorescent quantitative PCR detection method. Background technique

[0002] Mycoplasma pneumonia, also known as swine endemic pneumonia (Swine enzootic hyopneumoniae), commonly known as swine panting disease, is a chronic, contact, respiratory infectious disease caused by Mycoplasma hyopneumoniae. The main clinical symptoms are cough and wheezing, often secondary infection by other bacteria (such as Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, etc.). The disease is widely distributed all over the world and occurs in pig farms in many areas of our country. Due to the existence of pigs with bacteria-carrying disease, the production performance of the pig herd will usually be significantly reduced, the daily gain of the infected pig herd will be reduced, the fe...

Examples

Embodiment 1

[0059] 1 Primer and probe design and synthesis

[0060] Aiming at the highly conserved region of the 16S rRNA gene genus, which is specific and the main target gene for bacterial classification and identification, as the amplification region, the first primer pair realtime F / R for Real Time PCR was designed, and the second primer pair clone for constructing a standard plasmid was designed F / R, the sequences of the primers and probes are:

[0061] realtime F: 5'-AACTGGTCATATATTGACACTAAGGG-3' (SEQ ID NO.1);

[0062] realtime R: 5'-TGTGTTAGTGACTTTTGCCACCAACT-3' (SEQ ID NO. 2).

[0063] Probe: 5'-CAGGATTAGATACCCTGGTAGTCCAC-3' (SEQ ID NO.3);

[0064] clone F: 5'-GAGCCTTCAAGCTTCACCAAGA-3' (SEQ ID NO.4);

[0065] clone R: 5'-GCGGATCATTTAACGCGTTAGC-3' (SEQ ID NO. 5).

[0066] The product of the primer pair realtime F / R is located within the sequence amplified by the primer pair clone F / R. The 5' end of the Real Time PCR probe is bound with a fluorescent reporter group (FAM), and ...

Embodiment 2

[0093] Comparison of the results of detection of Mycoplasma hyopneumoniae by CCU method and fluorescent quantitative PCR method

[0094] 1 Culture of Mycoplasma hyopneumoniae

[0095] Take out the production seeds from the strain bank, dissolve them with the improved Friis liquid medium, and subculture in the improved Friis liquid medium at a ratio of 1:10, put them into a constant temperature incubator at a temperature of 36-38°C and cultivate them for 48-72 hours, and wait for The color of the bacterial liquid changes, and when the pH value drops to 6.70-6.80, the bacterial liquid is collected and used as a first-class seed liquid for pure inspection. It should be pure. Propagate and identify the secondary and tertiary seeds according to the above method; add the tertiary seed liquid into the improved Friis liquid medium at a ratio of 1:10, seal the mouth of the fermenter, feed sterile air, and the pressure in the tank reaches 0.08MPa , close all the inlets and outlets on t...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a primer set for detecting mycoplasma hyopneumoniae, an application thereof, and a real-time fluorescent quantitative PCR detection method. Background technique

[0002] Mycoplasma pneumonia, also known as swine endemic pneumonia (Swine enzootic hyopneumoniae), commonly known as swine panting disease, is a chronic, contact, respiratory infectious disease caused by Mycoplasma hyopneumoniae. The main clinical symptoms are cough and wheezing, often secondary infection by other bacteria (such as Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, etc.). The disease is widely distributed all over the world and occurs in pig farms in many areas of our country. Due to the existence of pigs with bacteria-carrying disease, the production performance of the pig herd will usually be significantly reduced, the daily gain of the infected pig herd will be reduced, the fe...

Examples

Embodiment 1

[0059] 1 Primer and probe design and synthesis

[0060] Aiming at the highly conserved region of the 16S rRNA gene genus, which is specific and the main target gene for bacterial classification and identification, as the amplification region, the first primer pair realtime F / R for Real Time PCR was designed, and the second primer pair clone for constructing a standard plasmid was designed F / R, the sequences of the primers and probes are:

[0061] realtime F: 5'-AACTGGTCATATATTGACACTAAGGG-3' (SEQ ID NO.1);

[0062] realtime R: 5'-TGTGTTAGTGACTTTTGCCACCAACT-3' (SEQ ID NO. 2).

[0063] Probe: 5'-CAGGATTAGATACCCTGGTAGTCCAC-3' (SEQ ID NO.3);

[0064] clone F: 5'-GAGCCTTCAAGCTTCACCAAGA-3' (SEQ ID NO.4);

[0065] clone R: 5'-GCGGATCATTTAACGCGTTAGC-3' (SEQ ID NO. 5).

[0066] The product of the primer pair realtime F / R is located within the sequence amplified by the primer pair clone F / R. The 5' end of the Real Time PCR probe is bound with a fluorescent reporter group (FAM), and ...

Embodiment 2

[0093] Comparison of the results of detection of Mycoplasma hyopneumoniae by CCU method and fluorescent quantitative PCR method

[0094] 1 Culture of Mycoplasma hyopneumoniae

[0095] Take out the production seeds from the strain bank, dissolve them with the improved Friis liquid medium, and subculture in the improved Friis liquid medium at a ratio of 1:10, put them into a constant temperature incubator at a temperature of 36-38°C and cultivate them for 48-72 hours, and wait for The color of the bacterial liquid changes, and when the pH value drops to 6.70-6.80, the bacterial liquid is collected and used as a first-class seed liquid for pure inspection. It should be pure. Propagate and identify the secondary and tertiary seeds according to the above method; add the tertiary seed liquid into the improved Friis liquid medium at a ratio of 1:10, seal the mouth of the fermenter, feed sterile air, and the pressure in the tank reaches 0.08MPa , close all the inlets and outlets on t...

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