【作者】 郗宁；

【导师】 韩俭；

【作者基本信息】 兰州大学 ， 病原生物学， 2008， 硕士

【摘要】 目的:调查兰州市区腹泻患者中志贺菌的感染率和感染型别;确定兰州市区分离志贺菌株的毒力基因型;测定不同毒力基因型志贺菌的毒力,以探讨毒力与毒力基因型的关系。方法:选择兰州市区三家三甲医院门、急诊急性腹泻患者,采用分离培养、生化反应和血清学鉴别确定兰州市区腹泻患者中志贺菌的感染率和感染型别;设计引物,应用聚合酶链反应(PCR)方法,检测志贺菌肠毒素1基因(setlA和setlB)、志贺菌肠毒素2基因(sen)、侵袭性质粒抗原H基因(ipaH)、侵袭相关位点基因(ial)以及多重调控基因virA,确定兰州市区分离志贺菌株的毒力基因型;以腹腔注射为感染途径,测定不同毒力基因型的志贺菌对昆明种小鼠的半数致死量(LD50)。结果:1.从80例急性腹泻患者粪便中分离出志贺菌16株,阳性率为20.0%。其中福氏志贺菌9株,占56.2%,宋内志贺菌6株,占37.5%,痢疾志贺菌1株,占6.2%。同一菌群的不同志贺菌间在生化反应和抗原性方面存在差异。2.兰州市区的志贺菌毒力基因virA、setlA阳性率最高,为100%;ipaH次之,阳性率为93.7%;ial、sen阳性率分别为56.2%、62.5%;setlB阳性率最低,仅为25.0%。除virA、ipaH、setlA外,宋内志贺菌sen阳性率较高,为50.0%;ial检出率较低,为33.3%,6株宋内志贺菌均未检出setlB。福氏志贺菌ial、setlB、sen阳性率分别为66.7%、44.4%、66.7%均高于宋内志贺菌。3.7株不同毒力基因型(virA+ipaH+setlA,virA+ial+ipaH+setlA+setlB+sen,virA+setlA,virA+ial+ipaH+setlA+sen,virA+ipaH+setlA+sen,virA+ipaH+setlA+setlB,virA+ial+ipaH+setlA+setlB)志贺菌LD50测定的结果分别为:1.422×10~9cfu/ml、6.653×10~7 cfu/ml、阴性、6.653×10~8 cfu/ml、1.422×10~9 cfu/ml、1.422×10~9cfu/ml、6.653×10~8 cfu/ml;对照宋内志贺菌51334的LD50为3.062×10~9 cfu/ml。结论:1.兰州市区腹泻患者中志贺菌的感染率为20.0%左右,感染菌群以福氏志贺菌为主,其次为宋内志贺菌。宋内志贺菌感染的比例相对较高,占37.5%。2.兰州市区志贺菌中存在virA、ial、ipaH、setlA、setlB、sen毒力基因,以virA,setlA,ipaH较稳定。志贺菌相关毒力基因检测中,福氏志贺菌检出率高于宋内志贺菌。宋内志贺菌携带setlA。3兰州市区志贺菌的毒力与毒力基因型密切相关,以virA、ial、ipaH、setlA、setlB和sen均阳性的福氏志贺菌Ⅳ型毒力最强。4.virA,setlA,ipaH基因可作为靶基因用于兰州市区志贺菌的检验。setlB可用于筛选福氏志贺菌Ⅳ型菌株。更多还原

【Abstract】 Objective:To investigate the rate and the type of Shigella spp infection among the patients suffered with the diarrhea in Lanzhou city.To detect the virulent genotype of the Shigella spp separated from the patients lived in Lanzhou city.To measure the virulence of Shigella with different virulent genes in order to investigate their relation among virulent genotype and virulence.Methods:The ordinary and acute diarrheal patients came to the three third A grade hospitals in Lanzhou city were enrolled in the study.The isolated culture, biochemical reaction and serological method were used to identify the rate and the type of Shigella spp infection among the patients in the area.The virulent genes of SHET1(setlA、setlB),SHET2(sen),ipaH,ial and virA of Shigella spp were detected by PCR.The Shigella with different virulent genes was chosen to detect its virulence. The healthy Kunming mouse were chosen as laboratory animal to detect the median lethal dose(LD50)by intraperitoneal injection.Results:1.16 Shigella strains were isolated from 80 diarrheal patients.The positive rate of Shigella was 20%.All the isolated Shigella strains were composed with 9 S.flexneri strains(56.3%),6 S.sonnei strains(37.5%)and 1 S.dysenteriae strain(6.3%).There exit difference in biochemical reaction and antigenicity in different strain among the same Shigella group.2.The virulent genes of virA and setlA were presented in all the Shigella strains. The positive rate of ipaH,ial and sen genes were 93.7%,56.2%and 62.5% respectively.The setlB only presented in some S.flexneri strains and the positive rate were 25%.Except for virA,ipaH and setlA genes,the positive rate of sen in S.sonnei strains were 50.0%.The positive rate of ial were 33.3%.The setlB were not found in the 6 S.sonnei strains.The positive rate of ial,setlB and sen in S.flexneri strains were 66.7%,44.4%and 66.7%respectively.The positive rate of ial,setlB and sen in S. flexneri strains were higher than that in S.sonnei strains.3.The LD50 of the seven Shiglla strains with the different virulent genotype in virA+ipaH+setlA,virA+ial+ipaH+setlA+setlB+sen,virA+setlA,virA+ial+ipaH+ setlA+sen,virA+ipaH+setlA+sen,virA+ipaH+setlA+setlB and virA+ial+ipaH+setlA +setlB were 1.422×10~9 cfu/ml,6.653×10~7 cfu/ml,-,6.653×10~8 cfu/ml,1.422×10~9 cfu/ml,1.422×10~9 cfu/ml and 6.653×10~8 cfu/ml respectively.The LD50 of the quality control S.sonnei 51334 was 3.062×10~9 cfu/ml.Conclusions:1.There were about 20%diarrheal patients infected by Shigella spp.The predominant prevalent species was S.flexneri and the second was S.sonnei.The S. sonnei strain infection was higher in Lanzhou city comparatively with the other place.2.There present virulent genes of virA,ial,ipaH,setlA,setlB and sen among the Shigella strains in Lanzhou city.The virA,setlA and ipaH present stably in the Shigella strain in the area.The positive rate of virulent genes of the S.flexneri strains was higher than that in the S.sonnei strains.There exists setlA in the S.sonnei strains in the area.3.There has good correlation between Shigella virulence and virulent genotype.S. flexneri typeⅣwith virA,ial,ipaH,setlA,setlB and sen have the strongest virulence in all genotype strains.4.The virA,setlA and ipaH can be used as the target genes to detect the Shigella in Lanzhou city.SetlB can be used to select the S.flexneri typeⅣin the area.更多还原