Real-time fluorescent quantitative PCR method for detecting canine mycoplasmas

Abstract

The invention provides a specific primer and real-time fluorescent quantitative PCR detection method for detecting canine mycoplasmas, and belongs to the technical field of biology. According to the method, the specific primer is designed according to the sequence of a conserved region of a sialidase gene of the canine mycoplasmas published by Genebank, a target fragment is amplified and connectedto a pMD-18T vector, a recombinant standard plasmid is constructed and subjected to 10-fold dilution, a Ct value and a copy number have a good linear relationship, and the correlation coefficient is0.9947. The method is high in sensitivity, the minimum detection limit is 1.85*10<4> copy / microliter, and the sensitivity is improved by 100 times compared with that of common PCR detection; the specificity is good, and there is no specific amplification curve for three common mycoplasmas and common canine pathogens; the repeating stability is good, and the between-group and within-group variancecoefficients are both smaller than 0.5%. The method overcomes the defects that the identification time of traditional isolated culture is long, and a serological method easily causes a cross reactionand a conventional PCR false positive result, can be used for early diagnosis and detection of the canine mycoplasmas, and has important significance for the prevention and control of canine mycoplasmosis.

Description

technical field [0001] The invention relates to a detection method of canine pathogenic bacteria, in particular to a real-time fluorescent quantitative PCR detection method of canine mycoplasma, belonging to the field of biotechnology. Background technique [0002] With the improvement of people's living standards, more and more people start to keep pets. According to the "2017 White Paper on China's Pet Industry" released by Goumin.com, pet dogs accounted for about 61.74%, about 150 million. A pet dog costs about 6,000 yuan a year, and pet medical treatment accounts for about 30% of the total cost. [0003] Mycoplasma belongs to the kingdom Bacteria, Firmicutes, Mollicula, Mycoplasma, Mycoplasma, and Mycoplasma, the smallest known prokaryotic microorganisms that lack cell walls and can survive independently. Mycoplasma can infect many animals, including dogs, poultry, pigs, ruminants, humans, and reptiles. The genome of mycoplasma is extremely small, and the cells can re...

AI Technical Summary

Problems solved by technology

The most reliable method for detecting Mycoplasma canis is to isolate Mycoplasma from samples, but the isolation of Mycoplasma canis is time-consuming, poor in sensitivity, extremely demanding on nutrition, slow in vitro culture, takes a long time, and is easily contaminated by other pathogenic microorganisms. Therefore, the traditional isolation and culture method cannot meet the needs of clinical diagnosis.

Cross-reactivity between serological assays and other mycoplasma complicates differential diagnosis of M. canis

The sensitivity of conventional PCR detection technology is not high, it is easy to be polluted by the environment, and it is impossible to monitor the PCR process in real time

At present, domestic research on Mycoplasma canis is still blank, and it is urgent to establish a rapid, accurate and sensitive detection method

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