Primer group for detecting mycoplasma hyopneumoniae, application of primer group and real-time fluorescent quantitative PCR detection method
A real-time fluorescence quantitative technology for Mycoplasma hyopneumoniae, applied in the biological field, can solve problems such as fluctuation of results, identification, counting errors, time-consuming and labor-intensive, etc., and achieve rapid detection, accurate quantification, and good repeatability
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[0058] Example 1
[0059] 1Primer and probe design and synthesis
[0060] Aiming at the highly conserved region of the 16S rRNA gene, which is specific and is the main target gene for bacterial classification and identification, as the amplification region, the first primer pair realtime F / R for Real Time PCR and the second primer pair clone for constructing standard plasmids were 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'-GCGGATCATTAACGCGTTAGC-3' (SEQ ID NO. 5).
[0066] The product of primer pair realtime F / R is located within the sequence amplified by primer pair clone F / R. The 5' end of the Real Time PCR probe is bound with a fluorescent reporter group (FAM), and the...
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[0092] Example 2
[0093] Comparison of the results of detection of Mycoplasma hyopneumoniae by CCU method and fluorescence quantitative PCR method
[0094] 1 Culture of Mycoplasma hyopneumoniae
[0095] Take out the production seeds from the strain bank, dissolve them in the modified Friis liquid medium, subculture them in the modified Friis liquid medium at a ratio of 1:10, and put them into a constant temperature incubator at a temperature of 36-38°C for 48-72 hours. When the color of the bacterial liquid changes, and the pH value drops to 6.70-6.80, the bacterial liquid is collected and used as the first-class seed liquid for purity inspection, and it should be pure. Propagation and identification of secondary seeds and tertiary seeds were carried out according to the above methods; the tertiary seed liquid was added to the improved Friis liquid medium at a ratio of 1:10, the mouth of the fermenter was sealed, and sterile air was introduced, and the pressure in the tank r...
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