郑州地区人源隐孢子虫分离株种类/基因型/基因亚型研究
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摘要
本试验对郑州地区人隐孢子虫病进行了广泛的流行病学调查,获得2个人源隐孢子虫分离株,对2个分离株进行动物交叉感染实验,巢式PCR检测和PCR-RFLP分析;基于核糖体小亚基(18S rDNA)基因、70kDa热休克蛋白(HSP70)基因、肌动蛋白(actin)基因的种类与基因型分析和60kDa糖蛋白(GP60)基因的基因亚型分析,鉴定郑州地区人源隐孢子虫分离株种类、基因型和基因亚型,确定本地区人源隐孢子虫分离株和其它种类隐孢子虫之间的分子种系进化关系,推断人隐孢子虫病的感染来源和传播机制,为预防隐孢子虫病提供理论基础。
1、为了解人隐孢子虫病在我省的感染情况和流行特征,对河南省郑州市四个医院临床病人进行一年的隐孢子虫病感染情况调查。应用饱和蔗糖溶液漂浮法检测河南省郑州市四个医院病人,包括婴幼儿患者、肿瘤病人和其他疾病患者6720份粪便样品中的隐孢子虫,获得2个隐孢子虫分离株,均来自于婴幼儿,表明婴幼儿仍是隐孢子虫病的易感人群;为研究其生物学特性,对两个分离株进行动物交叉感染试验,用纯化的分离株卵囊感染免疫抑制小鼠,经过25天的小鼠粪便检查,未查到隐孢子虫卵囊。
2、本试验利用巢式PCR扩增2个隐孢子虫分离株18S rDNA基因部分片段,PCR产物经过连接转化,挑选阳性克隆进行测序,产物序列长度均为837bp;用限制性内切酶SspⅠ和VspⅠ对PCR产物进行消化酶切确定种类和基因型。SspⅠ酶切结果:2个隐孢子虫分离株酶切后产生108bp、251bp、485-495bp三个片段;VspⅠ酶切结果:2个隐孢子虫分离株酶切后产生100-110bp、590-600bp两个片段,根据酶切片段初步确定这2个隐孢子虫分离株为人隐孢子虫(C.hominis)。
3、为从种系发育关系确定郑州地区人源隐孢子分离株的种类或基因型,利用巢式PCR对2个隐孢子虫分离株进行18S rDNA基因、HSP70基因、actin基因特定片段扩增,并对扩增片段进行测序,测序后的序列用Blast或Fasta在NCBI、EMBL和DDBJ三大核酸序列数据库搜索同源序列,然后利用Clustal X1.81、Phylip3.64和DNAstar4.0等生物学软件对序列进行比对、构建分子进化树以及同源性分析。根据在18S rDNA基因位点、HSP70基因位点、actin基因位点种系进化关系分析,表明2个人源隐孢子虫分离株为人隐孢子虫C.hominis。
4、GP60基因,一种用于基因亚型分析的分子标记,利用巢式PCR扩增GP60基因的特定片段,通过序列分析,结果表明2个分离株为C.hominis Id基因亚型家族的基因亚型,进一步确定两个人源隐孢子虫分离株为C.hominis,与18SrRNA基因序列、HSP70基因序列和actin基因序列进行的种系发育分析结果一致。
基于PCR-RFLP分析和基因型与基因亚型的种系发育分析,确定本次试验获得的人源隐孢子虫分离株为C.hominis,推断本次试验中两名隐孢子虫病病人是通过直接或间接接触其他隐孢子虫病病人排泄在环境中的卵囊而感染发病,推断为人和人之间的循环传播方式.为下一步预防隐孢子虫病提供理论基
The experiment of epidemiological investigation of human cryptosporidiosis was carried out in Zhengzhou.The two Cryptosporidium isolates derived from children were made cross-transmission experiment,nested PCR-RFLP detection and analysis;genotypic analysis of the Small-Subunit rRNA(SSU rDNA) gene,70-kDa Heat Shock Protein(HSP70)gene and actin gene as well as subgenotypic analysis with GP60 gene were carried out to indentify species/ genotype/ subgenotype,determine molecular phylogenetic relationship between isolates drived from human of this area and other species,conclude human infection source and transmission mechanism in order to provide theoretical basis for prevention of human cryptosporidiosis.
In order to understand the infected condition of Cryptosporidium and its epidemiological characteristics in Zhengzhou of Henan Provice,samples of fresh stool obtained from the patients including infant patients,tumour patients and common patients who were treating in 4 hospitals of Zhengzhou were detected for oocysts of Cryptosporidium by sugar centrifugal flotation method for one year.The results revealed that the total infection rate of Cryptosporidium was 0.0003% (2/6720).The two isolates were derived from infants.This suggests that the children are more vulnerable than adults.To study biological characteristics of cryptosporidium spp,the isolates were used to conduct cross-transmission experiment: Purified isolates oocysts were used to inoculate inmmunocomprise mice,mouse feces were detected for 25 days,but no cryptosporidiurn oocyst was detected from mice feces.
The Small-Subunit rRNA(SSU rRNA)specific fragments of isolates were amplified by nested PCR.The PCR products were digested by SspI restriction enzyme and VspI restriction enzyme respevtively to determine species and genotype. Fragments of 108bp,251bp and 485-495bp were got after digested with SspI and fragments of 100-110bp and 590-600bp were got after digested with VspI.Based on length of restriction fragments,the two isolates were initially considered to be C.hominis.
In order to determine species/genotype of cryptosporidium isolates from human in Zhengzhou,specific fragments of three genes including 18S rDNA gene,HSP70 gene,actin gene were amplified by nested PCR,cloned and sequenced.Then,Blast or Fasta methods was used to search homological sequences in NCBI,DDBJ and EMBL, after that,homological sequences were alignmented.Phylogenetic tree and homological analysis were made by some biological softwares such as ClustalⅩ1.81, and DNAstar 4.0.Based on the phylogenetic analysis of 18S rDNA gene,HSP70 gene and actin gene,the two isolates were idenfied as C.hominis.
As a molecular marker for subgenotyping,GP60 gene specific fragment were amplified by nested PCR,and sequenced,then,Blast or Fasta was used to search homological sequences in NCBI,DDBJ and EMBL.After that,homological sequences were alignmented.Phylogenetic tree and homological analysis were made by some biological softwares such as ClustalⅩ1.81,and DNAstar4.0.Based on the subgenotyping analysis,the two were identified as C.hominis Id allete,and were named C.hominis Id A31GS.
Based on PCR-RFLP,genotypic and subgenotypic analysis,the species/ genotype of isolates in this experiment were reached consensus and identified as C.hominis.it can be concluded that two human cryptosporidiosis patients of this experiment may be infected cryptosporidium through direct or indirect contact with oocysts which were excreted from cryptosporidiosis patients in environment and the transmission route of human cryptosporidiosis in this area is person to person cycle. pattern.
1、为了解人隐孢子虫病在我省的感染情况和流行特征,对河南省郑州市四个医院临床病人进行一年的隐孢子虫病感染情况调查。应用饱和蔗糖溶液漂浮法检测河南省郑州市四个医院病人,包括婴幼儿患者、肿瘤病人和其他疾病患者6720份粪便样品中的隐孢子虫,获得2个隐孢子虫分离株,均来自于婴幼儿,表明婴幼儿仍是隐孢子虫病的易感人群;为研究其生物学特性,对两个分离株进行动物交叉感染试验,用纯化的分离株卵囊感染免疫抑制小鼠,经过25天的小鼠粪便检查,未查到隐孢子虫卵囊。
2、本试验利用巢式PCR扩增2个隐孢子虫分离株18S rDNA基因部分片段,PCR产物经过连接转化,挑选阳性克隆进行测序,产物序列长度均为837bp;用限制性内切酶SspⅠ和VspⅠ对PCR产物进行消化酶切确定种类和基因型。SspⅠ酶切结果:2个隐孢子虫分离株酶切后产生108bp、251bp、485-495bp三个片段;VspⅠ酶切结果:2个隐孢子虫分离株酶切后产生100-110bp、590-600bp两个片段,根据酶切片段初步确定这2个隐孢子虫分离株为人隐孢子虫(C.hominis)。
3、为从种系发育关系确定郑州地区人源隐孢子分离株的种类或基因型,利用巢式PCR对2个隐孢子虫分离株进行18S rDNA基因、HSP70基因、actin基因特定片段扩增,并对扩增片段进行测序,测序后的序列用Blast或Fasta在NCBI、EMBL和DDBJ三大核酸序列数据库搜索同源序列,然后利用Clustal X1.81、Phylip3.64和DNAstar4.0等生物学软件对序列进行比对、构建分子进化树以及同源性分析。根据在18S rDNA基因位点、HSP70基因位点、actin基因位点种系进化关系分析,表明2个人源隐孢子虫分离株为人隐孢子虫C.hominis。
4、GP60基因,一种用于基因亚型分析的分子标记,利用巢式PCR扩增GP60基因的特定片段,通过序列分析,结果表明2个分离株为C.hominis Id基因亚型家族的基因亚型,进一步确定两个人源隐孢子虫分离株为C.hominis,与18SrRNA基因序列、HSP70基因序列和actin基因序列进行的种系发育分析结果一致。
基于PCR-RFLP分析和基因型与基因亚型的种系发育分析,确定本次试验获得的人源隐孢子虫分离株为C.hominis,推断本次试验中两名隐孢子虫病病人是通过直接或间接接触其他隐孢子虫病病人排泄在环境中的卵囊而感染发病,推断为人和人之间的循环传播方式.为下一步预防隐孢子虫病提供理论基
The experiment of epidemiological investigation of human cryptosporidiosis was carried out in Zhengzhou.The two Cryptosporidium isolates derived from children were made cross-transmission experiment,nested PCR-RFLP detection and analysis;genotypic analysis of the Small-Subunit rRNA(SSU rDNA) gene,70-kDa Heat Shock Protein(HSP70)gene and actin gene as well as subgenotypic analysis with GP60 gene were carried out to indentify species/ genotype/ subgenotype,determine molecular phylogenetic relationship between isolates drived from human of this area and other species,conclude human infection source and transmission mechanism in order to provide theoretical basis for prevention of human cryptosporidiosis.
In order to understand the infected condition of Cryptosporidium and its epidemiological characteristics in Zhengzhou of Henan Provice,samples of fresh stool obtained from the patients including infant patients,tumour patients and common patients who were treating in 4 hospitals of Zhengzhou were detected for oocysts of Cryptosporidium by sugar centrifugal flotation method for one year.The results revealed that the total infection rate of Cryptosporidium was 0.0003% (2/6720).The two isolates were derived from infants.This suggests that the children are more vulnerable than adults.To study biological characteristics of cryptosporidium spp,the isolates were used to conduct cross-transmission experiment: Purified isolates oocysts were used to inoculate inmmunocomprise mice,mouse feces were detected for 25 days,but no cryptosporidiurn oocyst was detected from mice feces.
The Small-Subunit rRNA(SSU rRNA)specific fragments of isolates were amplified by nested PCR.The PCR products were digested by SspI restriction enzyme and VspI restriction enzyme respevtively to determine species and genotype. Fragments of 108bp,251bp and 485-495bp were got after digested with SspI and fragments of 100-110bp and 590-600bp were got after digested with VspI.Based on length of restriction fragments,the two isolates were initially considered to be C.hominis.
In order to determine species/genotype of cryptosporidium isolates from human in Zhengzhou,specific fragments of three genes including 18S rDNA gene,HSP70 gene,actin gene were amplified by nested PCR,cloned and sequenced.Then,Blast or Fasta methods was used to search homological sequences in NCBI,DDBJ and EMBL, after that,homological sequences were alignmented.Phylogenetic tree and homological analysis were made by some biological softwares such as ClustalⅩ1.81, and DNAstar 4.0.Based on the phylogenetic analysis of 18S rDNA gene,HSP70 gene and actin gene,the two isolates were idenfied as C.hominis.
As a molecular marker for subgenotyping,GP60 gene specific fragment were amplified by nested PCR,and sequenced,then,Blast or Fasta was used to search homological sequences in NCBI,DDBJ and EMBL.After that,homological sequences were alignmented.Phylogenetic tree and homological analysis were made by some biological softwares such as ClustalⅩ1.81,and DNAstar4.0.Based on the subgenotyping analysis,the two were identified as C.hominis Id allete,and were named C.hominis Id A31GS.
Based on PCR-RFLP,genotypic and subgenotypic analysis,the species/ genotype of isolates in this experiment were reached consensus and identified as C.hominis.it can be concluded that two human cryptosporidiosis patients of this experiment may be infected cryptosporidium through direct or indirect contact with oocysts which were excreted from cryptosporidiosis patients in environment and the transmission route of human cryptosporidiosis in this area is person to person cycle. pattern.
引文
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