| Streptococcus is one of the most common pathogenic bacterium in bovine mastitis, which can lead to a serious harm to mammary gland without apparent symptoms, causing great loss of production and poor quality of milk. Meanwhile, inflammatory factor, pathogenic bacteria and bacteria toxins in the milk are injurious to the health of public. So a rapid, accurate pathogen diagnosis is quite crucial and necessary to take effective treatment measures, only that can help daily control mastitis correctly. Except for decreasing economic loss, the benefits of pathogenic diagnosis can also included:avoiding antibiotic abuse validly, reducing the odds of bacterial drug resistance and ensure the lower residual amount of drug in milk. In addition, antibiotics, like penicillin, ampicillin, vacomycin, can be chosen to give if a Streptococcus mastitis develops because Streptococcus are very sensitive to those antiibiotics. Therefore, developing a rapid and convenient method to diagnose Streptococcus will have a high utility value for mastitis control as well as protection of public health in clinical.Traditional detection methods require lengthy culture enrichment steps and several days to detect bacterial pathogens present in samples at low levels. The PCR methods provide powerful tools for rapid, specific, and sensitive detection of pathogens. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common. Loop-mediated isothermal amplification (LAMP) is a novel method which can provide a simpler and more sensitive, specific, cost-effective test in detecting microbes and viruses, especially in the field.In this experiment, we designed two pairs of primers based on elongation factor tuf gene which was highly conserved in Streptococcus spp.. After optimization of reaction system, we finally confirmed a25uL system as the best choice:inner primer (FIP/BIP)1.2μmol/L, outer primer (F3/B3)0.2μmol/L, Mg2+8mmol/L, dNTP1.2mmol/L, betaine0.8mol/L, Calcein25μmol/L, MnCl20.5mmol/L,8U Bst DNA polymerase,2.5μL10×Bst Buffer,2ul template, the rest filled up with ultrapure water to25uL. The mixture was incubated at63℃for60min and then heated to80℃for5min to terminate the reaction. We can obtain the results of detection when either the white precipitate or green fluorescent light was observed by high speed centrifugation or UV light.According to our reaction system and LAMP primers, we detected several common specieses of mastitis pathogens, which including Streptococcus agalactiae, Streptococcus dysgalactiaes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Staphylococcus epidermidis and Salmonella. The positive results only in Streptococcus indicated that LAMP possessed a high specificity. Sensitivity of Streptococcus agalactiae, Streptococcus dysgalactiaes and Streptococcus uberis was52.6fg/μL,48.9fg/μL and55fg/μL, respectively. In artificial samples, we obtained positive testing result with both pyrolysis and commercial kit that implied an undemanding quality of DNA templates. Sensitivity of the method in Streptococcus detection was100%.Compared with traditional PCR, the specificity ratio of LAMP and PCR was90.74%and96.08%, coincidence rate was94.25%,97.70%,respectively. Adding Calcein/MnCl2in LAMP reaction can not only avoid aerosol contamination caused by uncapped operation but also observed an obvious result in naked eyes.The rapid LAMP method is an easy and convenient testing method with characteristics of high sensitivity, high specificity, simple operation and cheap requirement of equipments, which affords a new orientation for detection of mastitis pathogens. It will be readily applied for rapid diagnosis of mastitis pathogenic streptococcus, demand of the amount of testing samples in the field as well as an reliable tool for mastitis monitoring in dairies which has a profound influence in yield increase, loss reduction and food safety improvement. |
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