The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 wa...The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 was constructed by inserting CP15/60 gene intopcDNA3 (+) in XhoⅠand EcoRⅠ. A vaccination protocol was the adult pregnant goatsinoculated intranasally with the pcDNA3-15/60 plasmid and their offspring were infectedwith C.parvum oocysts. The results showed that the pcDNA3-15/60 plasmid can induce theimmune response of goats and the vaccinated goats can transfer the immunity to offspringconferring protection against C.parvum infection. These suggested that the recombinantplasmid could be a DNA vaccine candidate.展开更多
With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, many diagnostic techniques were quickly implemented such as microscopic visualization, immunofluorescence and PCR...With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, many diagnostic techniques were quickly implemented such as microscopic visualization, immunofluorescence and PCR. Currently, there is no effective drug treatment and none of the current diagnostic methods is 100% accurate. In this study, a BALB/C mouse was subcutaneously immunized with Cryptosporidium parvum oocysts extract. The spleen was removed and the splenocytes were fused with SP2/0 myeloma cells in order to obtain hybridoma cells secreting antibodies specific to C. parvum antigens. Human and cattle fecal samples previously characterized by microscopy [Ziehl-Neelsen staining (ZN) and Lugol] and PCR for the presence of C. parvum and Giardia duodenalis, were analyzed by indirect immunofluorescence, using the developed hybridomas supernatants. The study shows that the selected hybridomas supernatants identify C. parvum oocysts in fecal samples in correlation with C. parvum oocysts identified using ZN/PCR. No false positive results were obtained and the two best supernatants gave 20% -30% of false negative results. No cross reaction with G. duodenalis was observed. By comparing our results with those obtained with an immunofluorescence commercial kit, it suggests the potential use of the monoclonal antibodies present in two of the hybridomas supernatants as a detection tool of C. parvum. With a reliability of 80.8% and 73.1% versus ZN and PCR methods for IFI, compared with a reliability of 76.9% and 92.3% versus ZN and PCR for commercial DIF kit, the supernatant 4.1D5 seems to be the most promising subject to further study its usefulness for C. parvum detection.展开更多
Background:Access to clean and safe drinking water that is free from pathogenic protozoan parasites,especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans,is still an issue...Background:Access to clean and safe drinking water that is free from pathogenic protozoan parasites,especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans,is still an issue in Southeast Asia(SEA).This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA,using real-time polymerase chain reaction(qPCR)assays.Methods:A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia(53),Thailand(120),the Philippines(33),and Vietnam(15).A physicochemical analysis was conducted.The water samples were processed in accordance with the US Environmental Protection Agency’s methods 1622/1623.1,microscopically observed and subsequently screened using qPCR assays.Results:Cryptosporidium oocysts were detected in treated water samples from the Philippines(1/10),with a concentration of 0.06±0.19 oocyst/L,and untreated water samples from Thailand(25/93),Malaysia(17/44),and the Philippines(11/23),with concentrations ranging from 0.13±0.18 to 0.57±1.41 oocyst/L.Giardia cysts were found in treated water samples from the Philippines(1/10),with a concentration of 0.02±0.06 cyst/L,and in untreated water samples from Thailand(20/93),Vietnam(5/10),Malaysia(22/44),and the Philippines(16/23),with concentrations ranging from 0.12±0.3 to 8.90±19.65 cyst/L.The pathogens C.parvum and G.lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene,respectively.C.parvum was detected in untreated water samples from the Philippines(1/23)and Malaysia(2/44),whilst,G.lamblia detected was detected in treated water samples from the Philippines(1/10)and in untreated water samples from Thailand(21/93),Malaysia(12/44),and the Philippines(17/23).Nitrate concentration was found to have a high positive correlation with(oo)cyst(0.993).Conclusion:The presence of(oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries.Detection using qPCR is feasible for quantifying both pathogenic C.parvum and G.lamblia in large water samples.展开更多
目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性...目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性和80℃酶失活过程,其最佳反应温度和时间是63℃和60min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8mol/L、8mmol/L、0.8mmol/L、8U/25μL,内外引物浓度分别为1.2μmol/L、0.2μmol/L,添加环引物后体系反应时间缩短为20min。对贾第虫、肠阿米巴、柔嫩艾美耳球虫、类圆线虫检测为阴性。该方法简便、快速,无需特殊设备。结论和常规PCR方法比较,LAMP检测隐孢子虫的灵敏度提高了100倍。该方法的建立为野外和临床隐孢子虫的快速检测提供技术手段。展开更多
基金supported by Natural Science Foundation of Jilin Province for Outstanding Young scientists Award,China(2002).
文摘The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 was constructed by inserting CP15/60 gene intopcDNA3 (+) in XhoⅠand EcoRⅠ. A vaccination protocol was the adult pregnant goatsinoculated intranasally with the pcDNA3-15/60 plasmid and their offspring were infectedwith C.parvum oocysts. The results showed that the pcDNA3-15/60 plasmid can induce theimmune response of goats and the vaccinated goats can transfer the immunity to offspringconferring protection against C.parvum infection. These suggested that the recombinantplasmid could be a DNA vaccine candidate.
文摘With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, many diagnostic techniques were quickly implemented such as microscopic visualization, immunofluorescence and PCR. Currently, there is no effective drug treatment and none of the current diagnostic methods is 100% accurate. In this study, a BALB/C mouse was subcutaneously immunized with Cryptosporidium parvum oocysts extract. The spleen was removed and the splenocytes were fused with SP2/0 myeloma cells in order to obtain hybridoma cells secreting antibodies specific to C. parvum antigens. Human and cattle fecal samples previously characterized by microscopy [Ziehl-Neelsen staining (ZN) and Lugol] and PCR for the presence of C. parvum and Giardia duodenalis, were analyzed by indirect immunofluorescence, using the developed hybridomas supernatants. The study shows that the selected hybridomas supernatants identify C. parvum oocysts in fecal samples in correlation with C. parvum oocysts identified using ZN/PCR. No false positive results were obtained and the two best supernatants gave 20% -30% of false negative results. No cross reaction with G. duodenalis was observed. By comparing our results with those obtained with an immunofluorescence commercial kit, it suggests the potential use of the monoclonal antibodies present in two of the hybridomas supernatants as a detection tool of C. parvum. With a reliability of 80.8% and 73.1% versus ZN and PCR methods for IFI, compared with a reliability of 76.9% and 92.3% versus ZN and PCR for commercial DIF kit, the supernatant 4.1D5 seems to be the most promising subject to further study its usefulness for C. parvum detection.
基金study was supported by the University of Malaya High Impact Research Grant(UM.C/625/1/HIR/MoHE/MED/23 and UM.0000041/HIR.C3)from the Ministry of Higher Education,Malaysia+1 种基金postgraduate research grants(PV 049/2011B and PV 014/2012A)University Malaya Research Grants(UMRG 544/14HTM and UMRG 362/15AFR).
文摘Background:Access to clean and safe drinking water that is free from pathogenic protozoan parasites,especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans,is still an issue in Southeast Asia(SEA).This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA,using real-time polymerase chain reaction(qPCR)assays.Methods:A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia(53),Thailand(120),the Philippines(33),and Vietnam(15).A physicochemical analysis was conducted.The water samples were processed in accordance with the US Environmental Protection Agency’s methods 1622/1623.1,microscopically observed and subsequently screened using qPCR assays.Results:Cryptosporidium oocysts were detected in treated water samples from the Philippines(1/10),with a concentration of 0.06±0.19 oocyst/L,and untreated water samples from Thailand(25/93),Malaysia(17/44),and the Philippines(11/23),with concentrations ranging from 0.13±0.18 to 0.57±1.41 oocyst/L.Giardia cysts were found in treated water samples from the Philippines(1/10),with a concentration of 0.02±0.06 cyst/L,and in untreated water samples from Thailand(20/93),Vietnam(5/10),Malaysia(22/44),and the Philippines(16/23),with concentrations ranging from 0.12±0.3 to 8.90±19.65 cyst/L.The pathogens C.parvum and G.lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene,respectively.C.parvum was detected in untreated water samples from the Philippines(1/23)and Malaysia(2/44),whilst,G.lamblia detected was detected in treated water samples from the Philippines(1/10)and in untreated water samples from Thailand(21/93),Malaysia(12/44),and the Philippines(17/23).Nitrate concentration was found to have a high positive correlation with(oo)cyst(0.993).Conclusion:The presence of(oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries.Detection using qPCR is feasible for quantifying both pathogenic C.parvum and G.lamblia in large water samples.
文摘目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性和80℃酶失活过程,其最佳反应温度和时间是63℃和60min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8mol/L、8mmol/L、0.8mmol/L、8U/25μL,内外引物浓度分别为1.2μmol/L、0.2μmol/L,添加环引物后体系反应时间缩短为20min。对贾第虫、肠阿米巴、柔嫩艾美耳球虫、类圆线虫检测为阴性。该方法简便、快速,无需特殊设备。结论和常规PCR方法比较,LAMP检测隐孢子虫的灵敏度提高了100倍。该方法的建立为野外和临床隐孢子虫的快速检测提供技术手段。
