Isolation,Identification And Establish Detection Method Of Candida In Fresh Milk

Dairy products are important consumer goods in people's lives.The quality of fresh milk affects the quality of dairy products to a large extent.Pathogen contamination is an important aspect of the quality of fresh milk.In recent years,the incidence of fungal mastitis in dairy cows has increased significantly,and reports on the contamination of fresh milk by Candida are increasing.The pathogenic Candida in fresh milk not only destroys the nutritional quality of the dairy,but also risks the infection of canines in humans and animals.Therefore,the establishment of specific,sensitive and rapid detection methods based on the isolation and identificatio of Candida in fresh milk is of great significance for the diagnosis and treatment of dairy cow candidia mastitis and dairy safety.In this study,96 milk samples were randomLy collected from dairy farms in Southern Hebei Province.First,15 suspected Candida strains were isolated by plate culture and morphological observation,and then rDNA-ITS gene sequence analysis and morphology were used.12 strains of Candida were identified by the combination method:8 strains of Candida albicans,2 strains of Candida parapsilosis,2 strains of Candida glabrata,and the other 3 strains were non-Candida yeast fungi.The homology between Candida isolates and Candida three species in GenBank ranged from 97.6%to 99.8%.In this study,the extracellular hydrolase test was carried out using aspartic protease,extracellular phospholipase and extracellular lipase as indicators to understand its main virulence factors and strength.The results showed that Candida albicans and Candida parapsilosis could produce aspartic protease,extracellular phospholipase and extracellular lipase,Candida glabrata mainly produced extracellular phospholipase;through three kinds of Candida extracellular hydrolase Compared with the activity,Candida albicans is the most toxic.The survival experiment of mice with Candida albicans,Candida parapsilosis and Candida glabrata,which had the strongest extracellular hydrolase activity,was selected.The results showed that the survival rate of mice injected with Candida albicans was 10%;The survival rate of mice was 80%;the survival rate of mice injected with Candida glabrata was 60%.It was further verified that Candida albicans was the most toxic.It is known from the formation and sterilization test of chlamydospores in milk that Candida glabrata does not produce chlamydospores in milk at room temperature,and Candida albicans and Candida parapsilosis are prone to thick chlamymate in milk at room temperature,although It can be killed by high temperature sterilization,but there will still be some residue after the pasteurization.The results of pasteurization showed that the survival rate of Candida albicans is 9.69%~24.23%,and the survival rate of Candida albicans is 16.17%~16.38%.Three pairs of specific primers for Candida albicans,Candida parapsilosis and Candida glabrata were designed for GenBank's published benAfum gene sequence,and SYBR Green I fluorescence quantitative PCR method for detecting three Candida species in fresh milk was established.The results showed that the method can simultaneously amplify the specific target fragments of three Candida species,without primer dimer,and the Tm value difference between the strains can be distinguished by 1°C or more;the detection of Candida albicans,near smoothing beads The sensitivity of bacteria and Candida glabrata reached 1.63?10~1 cfu/mL,1.61?10~1 cfu/mL,and 1.65?10~1 cfu/mL,respectively,which was 100 times higher than that of conventional PCR technology.The fluorescence curve and melting curve of the repeatability test tended to be consistent;the detection speed was fast.It can be completed in 2 hours.