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Development of a PCR diagnostic kit for Cryptosporidium andersoni in Dairy Cow

Agricultural Sciences in China 2007年第4期6卷 493-498页

作者： ZHOU Rong-qiong LI Guo-qing XIAO Shu-min LI Wei-hua College of Veterinary Medicine South China Agricultural University Guangzhou 510642 P.R.China Department of Veterinary Medicine Rongchang Campus Southwest University Chongqing 402460 P.R.China

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.

关键词： Cryptosporidium andersoni ITS-1 diagnostic kit detection dairy cow

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Construction and expression of a synthetic gene encoding nonstructural glycoprotein NS1 of dengue 2 virus in Pichia pastoris

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Asian Pacific Journal of Tropical Biomedicine 2017年第8期7卷 689-693页

作者： Fernita Puspasari Riski Dwimalida Putri Aisyah Raden Roro Rika Damayanti Anita Yuwita Bachti Alisjahbana Sukwan Handali Ihsanawati Dessy Natalia Biochemistry Research Division Faculty of Mathematics and Natural SciencesInstitute of Technology Bandung Pakar Biomedika Indonesia Faculty of Medicine Universitas Padjadjaran

To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was *** recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid *** resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.

关键词： DENV 2 Dengue virus NS1 protein diagnostic kit Pichia pastoris

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

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Medicinal Plant 2022年第2期13卷 18-20,36页

作者： Sixi YANG Kexin ZHANG Jinxia LIU Experimental Center of Pathogenic Biology Chengde Medical University Chengde 067000 China

[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 ***,reaction system,reaction conditions and stain types were repeatedly optimized to establish an improved LAMP detection kit for *** tests were performed on blood samples from clinically confirmed cases and their sensitivity and specificity were ***,the detection technology and kit were applied to the detection and screening of suspected *** in pastoral ***,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,*** sensitivity and specificity of the modified LAMP technique were verified by using human blood samples with confirmed *** LAMP specific test,there was no nonspecific amplification,indicating good *** the LAMP sensitivity test,the minimum detection line for ***,***,and *** was 10 copies/μL,which was an order of magnitude higher than that for *** consistency between modified LAMP and Q-PCR was *** established Brucella LAMP kit was used to detect the blood samples of 200 sheep suspected to be infected with *** 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 *** technology is characterized by good sens

关键词： Brucella Loop-mediated isothermal amplification Omp25 diagnostic kit Development

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两种单克隆抗体—hCG、hLH的制备及其临床应用

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中华生殖与避孕杂志 1987年第1期 15-19页

作者：卢惟钊黄福全李吉祐上海市计划生育科学研究所加拿大温哥华大不列颠哥伦比亚大学妇产科

用半固体培养基筛选hCG和LH单克隆抗体。272株细胞克隆系分泌hCG抗体,hCGβ-4D6(亲和常数1.2×10m)和hCGβ-EO(亲和常数8.7×10M)选作制备hCG免疫测定药箱。43株细胞克隆系分泌LH抗体,LH(亲和常数6.7×10m)和LH(亲和常数5×10m)选作制... 详细信息

用半固体培养基筛选hCG和LH单克隆抗体。272株细胞克隆系分泌hCG抗体,hCGβ-4D6(亲和常数1.2×10m)和hCGβ-EO(亲和常数8.7×10M)选作制备hCG免疫测定药箱。43株细胞克隆系分泌LH抗体,LH(亲和常数6.7×10m)和LH(亲和常数5×10m)选作制备LH免疫测定药箱。设计放射免疫竞争测定法（RIA）,固相免疫放射测定法（IMRA）固相免疫酶标测定法等三种方法测定hCG和LH浓度。hCG和LH免疫测定药箱灵敏度分别为3mIU/ml和2mIU/ml。月经规则妇女的LH峰可用一种30分钟的程序从尿样中测出。为尽可能减低激素之间的交叉反应,hCG免疫测定药箱系统加入LH单克隆抗体,LH免疫测定药箱系统加入hCGβ-4D6。实验结果表示hCG和LH水平可用不同免疫测定药箱测定之,其免疫交叉反应极低。

关键词： hCG LH Monoclonal antibody diagnostic kit

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