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...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.展开更多
Coagulation test, in principle, is an immunodiagnostics technique, in which immunoglobuline G of antibody is bound to protein A from Staphylococcus aureus. The aim of study is to develop a rapid test kit for detecting...Coagulation test, in principle, is an immunodiagnostics technique, in which immunoglobuline G of antibody is bound to protein A from Staphylococcus aureus. The aim of study is to develop a rapid test kit for detecting iridovirus infection in fish. Method was summarized as follows： （1） vaccine of iridovirus was injected to rabbit four times with a dosage as 0.5 mL, 1 mL, 2mL, 3 mL each week. Serum was collected at the fifth week as a coagglutination test kit; （2） through the positif polymerase chain reaction （PCR） test, the kidney and spleen sample infected with iridovirus are homogenized by using the phosphate buffered saline （PBS） solution of pH 7.2 with ratio 1：2 （WN）; （3） the supernatant material is collected after centrifugation at 8,000 rpm for 15 min; （4） filtrate/supernatant from sample was dropped on a slide an added with coagglutination test kit with the same volume （l：l）; （5） the agglutination observation is done after the 30, 60 and 90 min incubate at room temperature. The coagglutination test gave positive result in 25% of the test samples.展开更多
A cDNA fragment of about 860 bp corresponding to the c33-c gene in the non-structural region 3 (NS3) of HCV genome was obtained from one plasma derived from a Chinese HCV carrier who came from Tai'an of Shandong P...A cDNA fragment of about 860 bp corresponding to the c33-c gene in the non-structural region 3 (NS3) of HCV genome was obtained from one plasma derived from a Chinese HCV carrier who came from Tai'an of Shandong Province, China by the application of reverse transcription (RT) and polymerase chain reaction (PCR) techniques. After the sequence of the cDNA fragment was determined and compared with the equivalent region of. the HCV-I (HCV-US) and HCV-II (HCV-BK) genomes, the nucleotide/ amino acid sequence homologies were found to be 79. 2%/91. 3% and 91. 3%/93. 9%, respectively. The prokaryotic expression vector pBV220 was employed for the overproduction of c33-c native recombinant protein in E. coli cells. The expression products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting with antisera of chronic hepatitis C patients, and a molecular weight 31 kD of c33-c viral protein was shown to account for 14% of the total cellular soluble proteins. This product was extracted from the bacterial lysate by lysozyme, Triton X-100 and urea treatment, and purified through ion exchange chromatography. The purified c33-c protein combined with a branch peptide MAP-C-19 representing immunodominant epitopes on the nucleocapsid region of HCV genome was used to develop a Chinese HCV EIA 2nd-generation diagnostic kit for the detection of anti-HCV antibodies. Its specifity, sensitivity and re-producibility were all in keeping with the indexes of the national standard for the quality control of the HCV diagnostic kit. The agreement rate between our kit and Abbott company's HCV EIA second-generation diagnostic kit was 99. 33% , and the identified rate of positive anti-HCV of our kit was 2% more than that of the Abbott company's kit.展开更多
目的评价结核分枝杆菌特异性蛋白（tuberculosis specific antigen, TB-SA）抗体检测试剂在结核病中的应用价值。方法以天津市和平区结核病控制中心、山东省烟台芝罘区肺科医院和河南省南阳市卧龙区结核病防治中心为研究现场，连续纳入2...目的评价结核分枝杆菌特异性蛋白（tuberculosis specific antigen, TB-SA）抗体检测试剂在结核病中的应用价值。方法以天津市和平区结核病控制中心、山东省烟台芝罘区肺科医院和河南省南阳市卧龙区结核病防治中心为研究现场，连续纳入2012年4月至10月门诊就诊的所有肺结核疑似患者944例，同时纳入110名健康志愿者作为对照。对所有纳入患者及健康志愿者进行痰涂片、培养、结核菌素试验（PPD）和胸部X线检查，签署知情同意书后留取血清进行TB-SA抗体检测。痰涂片采用萋-尼（Ziehl-Neelsen）染色法，培养采用接种酸性罗氏固体培养基培养。现场数据由双人录入，在国家结核病参比实验室统一整理分析。结果755例确诊的活动性结核病患者，TB-SA抗体检测法阳性率为74．8％（565／755，95％CI=71．7％-77．9％），远高于涂片阳性率25．6％（193／755）（X^2=366．59，P〈0．0001）和培养阳性率39．6％（21）9／755）（X^2=190．12，P〈0．0001）。在培养阳性结核病患者中，TB-SA抗体检查敏感度为88．5％（255／288，95％CI=84．8％～92．2％）。在菌阴活动性结核病患者中，TB-SA抗体检查敏感度为68．0％（310／456，95％CI=63．7％-72．3％）。TB-SA抗体检测法在活动性结核病患者中的阳性预测值为92．3％（565／612，95％CI=90．2％～94．4％），在健康人群中检测特异度为97．3％（95％CI=94．3％～100．3％）。结论TB-SA抗体检测法在结核病患者中阳性检出率显著高于涂片和培养，适用于结核病，特别是菌阴结核病的诊断；TB-SA抗体检测法诊断结核病的特异度高，可用于人群健康体检。展开更多
文摘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.
文摘Coagulation test, in principle, is an immunodiagnostics technique, in which immunoglobuline G of antibody is bound to protein A from Staphylococcus aureus. The aim of study is to develop a rapid test kit for detecting iridovirus infection in fish. Method was summarized as follows： （1） vaccine of iridovirus was injected to rabbit four times with a dosage as 0.5 mL, 1 mL, 2mL, 3 mL each week. Serum was collected at the fifth week as a coagglutination test kit; （2） through the positif polymerase chain reaction （PCR） test, the kidney and spleen sample infected with iridovirus are homogenized by using the phosphate buffered saline （PBS） solution of pH 7.2 with ratio 1：2 （WN）; （3） the supernatant material is collected after centrifugation at 8,000 rpm for 15 min; （4） filtrate/supernatant from sample was dropped on a slide an added with coagglutination test kit with the same volume （l：l）; （5） the agglutination observation is done after the 30, 60 and 90 min incubate at room temperature. The coagglutination test gave positive result in 25% of the test samples.
文摘A cDNA fragment of about 860 bp corresponding to the c33-c gene in the non-structural region 3 (NS3) of HCV genome was obtained from one plasma derived from a Chinese HCV carrier who came from Tai'an of Shandong Province, China by the application of reverse transcription (RT) and polymerase chain reaction (PCR) techniques. After the sequence of the cDNA fragment was determined and compared with the equivalent region of. the HCV-I (HCV-US) and HCV-II (HCV-BK) genomes, the nucleotide/ amino acid sequence homologies were found to be 79. 2%/91. 3% and 91. 3%/93. 9%, respectively. The prokaryotic expression vector pBV220 was employed for the overproduction of c33-c native recombinant protein in E. coli cells. The expression products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting with antisera of chronic hepatitis C patients, and a molecular weight 31 kD of c33-c viral protein was shown to account for 14% of the total cellular soluble proteins. This product was extracted from the bacterial lysate by lysozyme, Triton X-100 and urea treatment, and purified through ion exchange chromatography. The purified c33-c protein combined with a branch peptide MAP-C-19 representing immunodominant epitopes on the nucleocapsid region of HCV genome was used to develop a Chinese HCV EIA 2nd-generation diagnostic kit for the detection of anti-HCV antibodies. Its specifity, sensitivity and re-producibility were all in keeping with the indexes of the national standard for the quality control of the HCV diagnostic kit. The agreement rate between our kit and Abbott company's HCV EIA second-generation diagnostic kit was 99. 33% , and the identified rate of positive anti-HCV of our kit was 2% more than that of the Abbott company's kit.
文摘目的评价结核分枝杆菌特异性蛋白（tuberculosis specific antigen, TB-SA）抗体检测试剂在结核病中的应用价值。方法以天津市和平区结核病控制中心、山东省烟台芝罘区肺科医院和河南省南阳市卧龙区结核病防治中心为研究现场，连续纳入2012年4月至10月门诊就诊的所有肺结核疑似患者944例，同时纳入110名健康志愿者作为对照。对所有纳入患者及健康志愿者进行痰涂片、培养、结核菌素试验（PPD）和胸部X线检查，签署知情同意书后留取血清进行TB-SA抗体检测。痰涂片采用萋-尼（Ziehl-Neelsen）染色法，培养采用接种酸性罗氏固体培养基培养。现场数据由双人录入，在国家结核病参比实验室统一整理分析。结果755例确诊的活动性结核病患者，TB-SA抗体检测法阳性率为74．8％（565／755，95％CI=71．7％-77．9％），远高于涂片阳性率25．6％（193／755）（X^2=366．59，P〈0．0001）和培养阳性率39．6％（21）9／755）（X^2=190．12，P〈0．0001）。在培养阳性结核病患者中，TB-SA抗体检查敏感度为88．5％（255／288，95％CI=84．8％～92．2％）。在菌阴活动性结核病患者中，TB-SA抗体检查敏感度为68．0％（310／456，95％CI=63．7％-72．3％）。TB-SA抗体检测法在活动性结核病患者中的阳性预测值为92．3％（565／612，95％CI=90．2％～94．4％），在健康人群中检测特异度为97．3％（95％CI=94．3％～100．3％）。结论TB-SA抗体检测法在结核病患者中阳性检出率显著高于涂片和培养，适用于结核病，特别是菌阴结核病的诊断；TB-SA抗体检测法诊断结核病的特异度高，可用于人群健康体检。
