宁波市腹泻患者隐孢子虫分子鉴定及基因特征分析

Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City

目的通过了解宁波市某哨点医院临床腹泻患者隐孢子虫感染情况及基因特征分析,为临床探究腹泻病因提供新思路。方法2019年11月—2021年7月,收集宁波市某哨点医院临床腹泻患者粪样650份,提取粪样中基因组DNA,巢式PCR扩增隐孢子虫核糖体小亚基RNA (SSU rRNA)基因,产物经1.5%琼脂糖凝胶电泳检测。扩增出条带的阳性粪样DNA进行60-kDa糖蛋白(gp60)基因的巢氏PCR扩增,产物经1.5%琼脂糖凝胶电泳检测。将与目的条带大小相近的PCR扩增产物进行双向测序,根据GenBank公共数据库资源行BLAST分析,使用Clustal X 2.1软件比对和校准,根据序列分析结果进行隐孢子虫虫种鉴定和亚型分型。运用Mega 11.0软件,以neighbour-joining (N-J)法绘制系统进化树。结果收集的粪样中男性腹泻患者369份(56.8%),女性腹泻患者281份(43.2%);年龄分布为1~99岁。经SSU rRNA基因扩增,仅1份DNA样品在830 bp左右出现特异性条带,呈隐孢子虫阳性。患者为53岁的男性,农民,水样便。经核苷酸序列分析,鉴定为火鸡隐孢子虫感染,与上海市人源分离株、黑龙江省奶牛源分离株、云南省鸡源分离株等的序列同源性为100%,系统进化树均落入同一分支。经gp60基因扩增,在955 bp左右出现特异性条带,鉴定为Ⅲb亚型家族,与人源ⅢbA24G1R1亚型的同源性最高(99.8%);经序列比对,为火鸡隐孢子虫新亚型ⅢbA26G1 (GenBank登录号:OQ032548)。结论宁波地区临床腹泻患者中鉴定到人兽共患的火鸡隐孢子虫,提示应加强腹泻人群隐孢子虫的监测和流调溯源。

Objective To analyze the Cryptosporidium infection and genetic characterization of outpatients with diarrhea in a sentinel hospital in Ningbo,to provide new ideas for clinical investigation on etiology of diarrhea.Methods A total of 650 fecal samples were collected from clinical diarrhea patients in NingBo City from November 2019 to July 2021.The genomic DNA from these samples was extracted,and the small subunit ribosomal ribonucleic acid gene(SSU rRNA)of Cryptosporidium was amplified by nested PCR.The products were analyzed by 1.5%agarose gel electrophoresis.The DNA from above positive sample was used as the template to amplify the 60-kDa glycoprotein(gp60)gene by nested PCR,of which the product was analyzed by 1.5%agarose gel electrophoresis.PCR amplification products of similar size to the target bands were sequenced in both directions,and blasted to identify the species or genotype according to the GenBank public database resources using Clustalx 2.1 software.Use Mega 11.0 software to draw the phylogenetic tree by the neighbour-joining(N-J)method.Results Among the fecal samples collected,369(56.8%)were from male diarrhea cases,and 281(43.2%)female cases;of them the ages ranged from 1 to 99 years.After amplifying of the SSU rRNA gene,only one DNA sample showed specific bands at around 830 bp in electrophoresis,indicating Cryptosporidium spp.positive.The patient was a 53-year-old male,farmer with watery stool.According to the analysis of DNA sequence,it was identified as C.meleagridis,which has 100%homology with those of a human-derived isolate from Shanghai,a dairy-derived isolate from Heilongjiang Province,a chicken-derived isolate from Yunnan Province.The phylogenetic tree showed that these isolates locate in the same branch.The product of gp60 gene amplification displayed a specific band at around 955 bp,which was identified as theⅢb subtype family,with the highest homology(99.8%)to the human derivedⅢbA24G1R1 subtype,sequence alignment indicate it is a new subtypeⅢbA26G1 of C.meleagridis(GenBank accession No.OQ032548).Conclusion Zoonotic C.meleagridis was identified from a clinical diarrhea case in Ningbo area,suggesting that surveillance on Cryptosporidium infection in people with diarrhea and epidemiological tracing for infection source should be strengthened.