The Separation And Development Of Detection Method By Insulated Isothermal Fluorescence PCR Of Mycoplasma Bovis

Mycoplasma is an important pathogen causing respiratory diseases in cattle,It has been proved that Mycoplasma mycoides subsp.Mycoides SC,MmmSC,Mycoplasma bovis,Mycoplasma bovoculi,Mycoplasma dispar,Mycoplasma bovirhinis and other mycoplasma are pathogenic to cattle.Among them,Mycoplasma bovis is the most common cause of bovine respiratory diseases.A variety of Mycoplasma bovis were reported in China,but the infection of other mycoplasma is still not clear.In this study,we investigated the infections of Mycoplasma bovis,Mycoplasma bovoculi,Mycoplasma dispar,and Mycoplasma bovirhinis in cattle with respiratory diseases,and established a Insulated Isothermal Fluorescence PCR of Mycoplasma bovis to facilitate the use of a portable convenience.and detection in field.The results acquired are as follows:1.Investigation of mycoplasma infection in bovine respiratory diseases54 nasal cavity sample of beef cattle and 16 nasal cavity sample of yak nasal swabs were collected from beef cattle and yak with respiratory diseases.were collected.The samples were tested to investigate the infection of mycoplasma using the specific primers for Mycoplasma bovis,Mycoplasma bovoculi,Mycoplasma dispar,and Mycoplasma bovirhinis.Additional,The 1,021-bp in length of 16S rRNA were amplified for phylogenetic analysis by primers for Mycoplasma genus.The results of the four species of mycoplasma showed that the infection rates of Mycoplasma bovis in the samples of beef cattle and yak were 57.30%?29/54?and 68.75%?11/16?,respectively.The infection rates of Mycoplasma dispar in beef cattle and yak were 29.63%?16/54?and 56.20%?9/16?,the detection rate of Mycoplasma bovoculi in beef cattle and yak samples were20.37%?11/54?and 37.50%?6/16?,respectively,and the detection rates of Mycoplasma bovirhinis in beef cattle and yak were 16.67%?9/54?and 25.00%?8/16?,respectively.Among the two mycoplasma mixed infections,the relatively high detection rate was a mixed infection of Mycoplasma bovis and Mycoplasma dispar.The detection rates in beef cattle and yak samples were 16.67%?9/54?and 50.00%?8/16?,respectively.The situation was found in beef cattle and yak,and the mixed infection of the four mycoplasma cases was only detected in yak samples.The results of this study show that the infection of Mycoplasma in cattle with respiratory diseases is common in China,and a variety of species belong to Mycoplasma were detected in beef cattle and yak.The detection rate of Mycoplasma bovis is the highest.Notably,the first detection of Mycoplasma bovoculi in China has enriched the pathogenic spectrum of cattle respiratory diseases2.Isolation,identification and drug sensitivity test of Mycoplasma bovis15 positive samples of Mycoplasma bovis were isolated using a commercial Mycoplasma basal medium.After the sample was prepared,the liquid medium was inoculated and cultured at 37°C and 5%CO₂.The medium was discolored and inoculated on a solid medium,and cultured at 37°C in a 5%CO₂ incubator.After 3 days of culture,"omelette-like"colonies appeared.After 3 purifications,10 pure cultures of Mycoplasma bovis were obtained.Drug susceptibility test results showed that isolated M.bovis was sensitive to tylosin,ofloxacin,oxytetracycline,gentamycin,cefquinox,and florfenicol,and to enrofloxacin and ceftiofur.Produced resistance.The results of this study provide a reference for the prevention and treatment of bovine mycoplasma disease.3.Establishment of iiPCR method for Mycoplasma bovisThe advantage of Insulated Isothermal Fluorescence PCR?iiPCR?detection is rapid.In this study,the primers and probes for detecting Mycoplasma bovis reported by Sachse K et al.were used.After optimization of the reaction system,a thermostatically isolated fluorescent PCR method for M.bovis was established for the first time.This method has good specificity and stability.It can be detected for both Mycoplasma bovis reference strain?CD-2?and 10 strains of Mycoplasma bovis isolates.Hemolytic Mansfield,Streptococcus,A.galactiae,BVDV,BCoV,etc.were not detected;the sensitivity of this method was high with aminimum detection limit 44.40 copies/?L-1.With the Pet NAD nucleic acid extraction kit and POCKIT handheld nucleic acid analyzer,the field detection of Mycoplasma bovis was constituted.It only takes one hour from the extraction of the nucleic acid to the results,providing a reliable method for field detection of Mycoplasma bovis.