Development of a PCR Diagnostic Kit for Cryptosporidium andersoni in Dairy Cow

Cryptosporidiosis is an important zoonosis caused by the Cryptosporidium species. To develop a PCR diagnostic kit for molecular detection and differential diagnosis of Cryptosporidium spp., a portion of ITS-1 sequence of Cryptosporidium. andersoni was chosen as the target DNA for designing the species-specific primers （ZRQF/ZR）. The kit components were determined after the PCR amplification conditions were serially optimized. A series of tests were conducted in the specificity, sensitivity, stability, reproducibility, and stored period of the kit, respectively. The results showed that only C. andersoni were amplified specific band of about 500 bp, while Cryptosporidium. parvum, Cryptosporidium. baileyi, Eimeria sp of dairy cow, Toxoplasma gondii, Eimeria sp of pig, Ascaris suum, Cyclospora sp, and E. coli could not be amplified. 254 oocysts of C. andersoni was the lowest number that could be detected using the kit. The kit worked well after being stored at room temperature, 4 and -20℃ for nine months. Fecal specimens, which were collected from a total of 243 calves on four different dairy farms in Guangdong Province, China, and one dairy farm in Henan Province, China, were examined using the kit; the positive rate of the kit was 2-13% higher than that of the routine methods. The results indicated that the kit can detect fecal samples faster, more sensitively, and conveniently, and can provide a useful tool for the identification and differentiation of C. andersoni from the other Cryptosporidium species; it also has implications for further studies on molecular epidemiology and differential diagnostics of cryptosporidiosis in animals.

Establishment of an Improved Loop-mediated Isothermal Amplification Technique for Visual Detection of Brucella

[Objectives]Loop-mediated isothermal amplification(LAMP)was developed and optimized for rapid and specific diagnosis of Brucella infection.[Methods]The conserved sequence of the brucellosis standard strain Omp25 gene fragment was selected,primers needed in the loop-mediated isothermal amplification system were designed,and nucleic acid of the standard strain was extracted.Primers,reaction system,reaction conditions and stain types were repeatedly optimized to establish an improved LAMP detection kit for brucellosis.Confirmatory tests were performed on blood samples from clinically confirmed cases and their sensitivity and specificity were confirmed.Then,the detection technology and kit were applied to the detection and screening of suspected B.melitensis in pastoral areas.Finally,SPSS21.0 was used to evaluate the consistency of LAMP and Q-PCR.[Results]LAMP system was optimized,and the optimal final concentrations of MgSO_(4) and betaine were 8 and 0.8 mmol/L,respectively.The sensitivity and specificity of the modified LAMP technique were verified by using human blood samples with confirmed brucellosis.In LAMP specific test,there was no nonspecific amplification,indicating good specificity.In the LAMP sensitivity test,the minimum detection line for B.melitensis,B.abortus,and B.suis was 10 copies/μL,which was an order of magnitude higher than that for Q-PCR.The consistency between modified LAMP and Q-PCR was 0.941.The established Brucella LAMP kit was used to detect the blood samples of 200 sheep suspected to be infected with Brucella.The LAMP kit detected 42 positive samples,with a positive rate of 21%and a detection sensitivity of 100%.Negative specimens were detected in 158 cases,with a specificity of 100%.There were 41 negative samples detected by PCR,with a positive rate of 20.5%.The detection specificity was consistent with that of LAMP kit.[Conclusions]The modified LAMP for Brucella has been successfully established.This technology is characterized by good sensitivity and specificity,low cost and high efficiency,and is conducive to the development in primary medical institutions.