采用3对特异性引物,对中国近海常见3种原甲藻的18S rDNA的部分序列进行PCR扩增,运用变性梯度凝胶电泳技术(Denaturing Gradient Gel Electrophoresis,DGGE)对PCR产物进行了差异性分析。结果表明,各对引物具有不同的区分效果,选择包含较...采用3对特异性引物,对中国近海常见3种原甲藻的18S rDNA的部分序列进行PCR扩增,运用变性梯度凝胶电泳技术(Denaturing Gradient Gel Electrophoresis,DGGE)对PCR产物进行了差异性分析。结果表明,各对引物具有不同的区分效果,选择包含较多差异碱基的短片段进行扩增,可以对藻种进行更好的区分;PCR-DGGE技术具有较高的灵敏性,不同藻种间即使1个碱基的差异也可以区分开;PCR-DGGE技术可以对3种原甲藻进行区分。展开更多
河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病。根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内容物寄生虫DNA,采用PCR方法检测。结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染。所建立的PCR方法具有...河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病。根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内容物寄生虫DNA,采用PCR方法检测。结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染。所建立的PCR方法具有灵敏、特异、快速等优点,不仅能用于鸡组织滴虫病临床诊断,还能用于开展流行病学研究。展开更多
为建立一种快速、准确检测猫等孢球虫的方法,本研究采用GenBank登录的猫等孢球虫18s r RNA基因序列(L76471.1)保守区设计并合成1对特异引物,从经显微镜检测为猫等孢球虫阳性的粪便样品中选取一份提取猫等孢球虫DNA,以其为模板经PCR扩增...为建立一种快速、准确检测猫等孢球虫的方法,本研究采用GenBank登录的猫等孢球虫18s r RNA基因序列(L76471.1)保守区设计并合成1对特异引物,从经显微镜检测为猫等孢球虫阳性的粪便样品中选取一份提取猫等孢球虫DNA,以其为模板经PCR扩增猫等孢球虫18s r RNA基因片段,构建重组质粒标准品p UCm-T-IF1并经PCR及测序鉴定正确后作为质粒标准品。通过优化反应条件,建立了猫等孢球虫的荧光定量PCR检测方法。特异性试验结果显示,该方法可以特异性的检测猫等孢球虫DNA,对猫贾第虫、猫滴虫、猫细小病毒、猫冠状病毒DNA/cDNA的检测结果均为阴性;对重组质粒标准品的检测限可达6.26×10^(2)拷贝/μL。批内和批间重复性试验的变异系数分别是0.61%~2.06%和0.32%~0.50%。表明本研究建立的方法特异性强、敏感性高、重复性好。利用该方法对23份临床疑似猫等孢球虫感染的样品进行检测,荧光定量PCR的检出率为78.26%(18/23),明显高于漂浮法检测的阳性率43.48%(10/23),并且经漂浮法检测为阳性的样品采用该方法检测均为阳性。结果表明,本研究建立的荧光定量PCR方法能够更准确的检测猫等孢球虫的感染,为开展猫等孢球虫病和流行病学调查提供更准确的检测手段。展开更多
Objective:To investigate the prevalence of Cryptosporidium spp.in goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen stain...Objective:To investigate the prevalence of Cryptosporidium spp.in goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen staining and nested PCR approach to identify and characterize the Cryptosporidium sp.from diarrhoeic feces of goat kids.A total of 100 diarrhoeic feces samples were collected from Chittagong region in Southern Bangladesh.For nested PCR analysis,specific primers for amplification of 581 base pair fragments of 18 S rRNA gene were used.Results:A total of 15%and 3%samples were found positive in microscopic study and in nested PCR analysis respectively.Phylogenetic analysis of sequence data showed similarity with that of Cryptosporidium xiaoi recorded from sheep and goat.Conclusions:To our knowledge,this is the first report of Cryptosporidium xiaoi responsible for diarrhoea in goat kids in Bangladesh.Further study can highlight their zoonotic significance along with genetic diversity in other host species inside the country.展开更多
Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-bas...Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.展开更多
文摘采用3对特异性引物,对中国近海常见3种原甲藻的18S rDNA的部分序列进行PCR扩增,运用变性梯度凝胶电泳技术(Denaturing Gradient Gel Electrophoresis,DGGE)对PCR产物进行了差异性分析。结果表明,各对引物具有不同的区分效果,选择包含较多差异碱基的短片段进行扩增,可以对藻种进行更好的区分;PCR-DGGE技术具有较高的灵敏性,不同藻种间即使1个碱基的差异也可以区分开;PCR-DGGE技术可以对3种原甲藻进行区分。
文摘河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病。根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内容物寄生虫DNA,采用PCR方法检测。结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染。所建立的PCR方法具有灵敏、特异、快速等优点,不仅能用于鸡组织滴虫病临床诊断,还能用于开展流行病学研究。
文摘为建立一种快速、准确检测猫等孢球虫的方法,本研究采用GenBank登录的猫等孢球虫18s r RNA基因序列(L76471.1)保守区设计并合成1对特异引物,从经显微镜检测为猫等孢球虫阳性的粪便样品中选取一份提取猫等孢球虫DNA,以其为模板经PCR扩增猫等孢球虫18s r RNA基因片段,构建重组质粒标准品p UCm-T-IF1并经PCR及测序鉴定正确后作为质粒标准品。通过优化反应条件,建立了猫等孢球虫的荧光定量PCR检测方法。特异性试验结果显示,该方法可以特异性的检测猫等孢球虫DNA,对猫贾第虫、猫滴虫、猫细小病毒、猫冠状病毒DNA/cDNA的检测结果均为阴性;对重组质粒标准品的检测限可达6.26×10^(2)拷贝/μL。批内和批间重复性试验的变异系数分别是0.61%~2.06%和0.32%~0.50%。表明本研究建立的方法特异性强、敏感性高、重复性好。利用该方法对23份临床疑似猫等孢球虫感染的样品进行检测,荧光定量PCR的检出率为78.26%(18/23),明显高于漂浮法检测的阳性率43.48%(10/23),并且经漂浮法检测为阳性的样品采用该方法检测均为阳性。结果表明,本研究建立的荧光定量PCR方法能够更准确的检测猫等孢球虫的感染,为开展猫等孢球虫病和流行病学调查提供更准确的检测手段。
基金Supported by two grants(No.-09-148 RG/BIO/AS_G and No.-10-087 RG/ITC/AS_C)from the Academy of Science for the developing World(TWAS)
文摘Objective:To investigate the prevalence of Cryptosporidium spp.in goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen staining and nested PCR approach to identify and characterize the Cryptosporidium sp.from diarrhoeic feces of goat kids.A total of 100 diarrhoeic feces samples were collected from Chittagong region in Southern Bangladesh.For nested PCR analysis,specific primers for amplification of 581 base pair fragments of 18 S rRNA gene were used.Results:A total of 15%and 3%samples were found positive in microscopic study and in nested PCR analysis respectively.Phylogenetic analysis of sequence data showed similarity with that of Cryptosporidium xiaoi recorded from sheep and goat.Conclusions:To our knowledge,this is the first report of Cryptosporidium xiaoi responsible for diarrhoea in goat kids in Bangladesh.Further study can highlight their zoonotic significance along with genetic diversity in other host species inside the country.
基金Supported by the National Natural Science Foundation of China(No.41076085)
文摘Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.
