DEC流行特征分析及stx基因快速检测方法的建立与应用

发布时间：2018-05-04 21:10

本文选题：急性腹泻 + 监测　；参考：《浙江大学》2016年硕士论文

【摘要】：研究背景及目的致泻性大肠埃希菌(Diarrheagenic Escherichia coli, DEC)依其携带的毒力基因及致病性一般可分为肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)或产志贺毒素大肠埃希菌,STEC)、产肠毒素性大肠埃希菌(ETEC)、肠侵袭性大肠埃希菌(EIEC)、和肠集聚性大肠埃希菌(EAEC)以及弥散黏附性大肠埃希菌(DAEC)。目前,除DAEC外,其它各类的DEC在全球流行甚至暴发的事件多有报道。2011年3月至7月,德国暴发大规模的由DEC O104:H4引起胃肠炎和溶血性尿毒综合征疫情,事件波及欧洲16个国家,共4125人发病,892例患者发生HUS,50人死亡,为迄今为止暴发的最严重的一次非0157 STEC疫情,此次疫情的罪魁祸首为stx基因编码的Stx,可引起HUS,甚至死亡。文献报道多种细菌可携带stx基因。因此本次疫情给全球一个强有力的警示。杭州独特的地理位置、水域环境及旅游特色,历来是腹泻的高发城市,因此我们必须加强对DEC的监测及防治研究,掌握其流行特征及应对措施,建立快速、准确的stx基因检测方法,能及时向临床医生报告结果以便其指定后续治疗方案,这对DEC或携带stx细菌疫情的预警及有效防止均有重要意义。方法1、收集2014年浙江大学附属第一医院急诊及肠道门诊的急性腹泻粪便标本,分别采用常规微生物检验程序及RT-PCR等方法对主要肠道致病菌及病毒进行分离培养及检测；2、采用多重PCR的方法对保存的大肠埃希菌进行鉴定及分型；3、统计分析2011-2014年在浙江大学附属第一医院急诊及肠道门诊收集的急性腹泻患者资料及DEC检测数据；4、建立快速检测stx1、stx2单、双重荧光定量PCR方法；5、采用荧光PCR方法对收集自肠道内外的可疑临床菌株及急性腹泻标本进行检测,阳性结果送出测序,进而进行序列比对及分析。结果1、对2014年735份急性腹泻标本通过分离、培养、核酸检测、免疫学检测等方法展开病原体筛查,其中阳性标本(至少检出1种细菌、真菌、病毒)338份,阳性检出率为45.99%。其中致泻性大肠埃希菌144份,检出率为19.59%,列急性腹泻病原首位,其次是诺如病毒和副溶血弧菌。2、在2014年735份急性腹泻标本中,同时检出两种或两种以上病原标本11份,其中有1份标本同时检出3种腹泻病原。3、采用多重PCR方法对2014年735例急性腹泻标本进行DEC鉴定,共检出144株DEC,检出率为19.59%(144/735)。其中,EAEC 73株,检出率为9.93%(73/735); ETEC 32株,检出率为4.35%(32/735)；EPEC20株,其检出率为2.72%(20/735)；分离到EIEC及STEC两型DEC各2株,其检出率均为0.27%(2/735)。此外,在分离到的DEC中,有15株菌同时携带EAEC、ETEC及EPEC的毒力基因。在分离到的DEC中,以EAEC为主,占50.69%(73/144),其次为分别为ETEC (22.22%)和EPEC (13.89%)。4、DEC一年四季均可分离到,主要以夏秋季为主,通过数据分析,2011、2012、2013年的检出率存在季节差异(P分别为0.027、0.025和0.016),但是2014年的检测数据显示其四季检出率无统计学差异,但其中EAEC/ETEC的检出率夏秋季最高,存在季节性差异,P=0.025、0.001(P0.05),有统计学意义,其他各型DEC检出率均无季节性差异。5、男性和女性患者的DEC检出率分别为21.69%、17.87%,其差异在统计学上无意义。其中ETEC/EAEC的男、女检出率分别为2.41%、0.50%,P=0.049(P0.05),具有统计学差异。此外,统计分析2011、2012及2013年监测数据,结果显示DEC及其各型在性别分布方面均无统计学差异。6、2014年期间DEC主要在19-60岁年龄段急性腹泻患者中检出,占77.78%(112/144),其次为60岁年龄段,占16.67%；但不同年龄段急性腹泻患者DEC及其各型DEC检出率并无统计学差异。与2014年一样,2011、2012、2013年杭州部分地区DEC也主要在19-60岁年龄段急性腹泻患者中检出,除2012年外,DEC及其各型检出率在不同年龄段不存在差异。2012年DEC检出率以60岁年龄段最高(20.43%),且存在统计学差异,P=0.024(P0.05)。7、2014年分离到DEC的急性腹泻患者多为水样便,占78.47%(113/144),其次为米泔样便(13.89%)；但DEC检出率最高的粪便性状为米泔样便混有粘液便及鲜血便,均为50%,其次为水样便加鲜血便,检出率为33.33%,但是DEC及各型检出率在各性状粪便间无显著差异。2011年DEC多在急性腹泻患者水样便中检出,占63.30%(69/109)；2012年主要在粘液样变和水样便中检出,分为占46.60%(48/103)、40.78%(42/103)；2013年则多在烂糊便和水样便中检出,各为28.81%(34/118)和21.19%(25/118)。且2011年DEC、EAEC、ETEC和EPEC检出率在各粪便性状组中存在统计学差异,2012年、2013年为DEC和EAEC检出率存在差异。此外,粪便中存有白细胞的各分组间DEC检出率有统计学差异,粪便中存在红细胞的各分组间EAEC检出率有统计学差异。8.2013、2014年检出的部分DEC菌株携带多型别毒力基因,分别为40株和15株,2013年40株DEC携带毒力基因情况如下,astA+estlb24株、astA+escV 6株、astA+elt+estlb2株,astA+elt、astA+elt+estIa、astA+elt+estlb+pic、 astA+estIb+escV、astA+estlb、astA+elt+estla+escV、invE+escV、elt+escV均为1株。2014年的15株DEC分别携带aggR+escV 1株、astA+escV 1株、pic+bfpB 1株、astA+elt 1株、astA+estla 2株、astA+estla 7株、astA+estlb+escV 1株、bfpB+stx21株。9.建立的快速检测stx基因的单、双重荧光PCR方法特异性较好,与肠道分离的其他细菌、真菌不存在交叉反应；检测范围为1×103～1×1012 copies/ml;在室温中的稳定性好,放置7天无明显影响；在重复性方面批间、批内变异系数均小于10%。10.用所建立的双重荧光PCR方法检测临床菌株,其中有4株大肠埃希菌six阳性,2株stx1阳性,2株stx2阳性。11.用所建立的双重荧光PCR方法直接检测急性腹泻标本,在抽取的55份标本中检出8份阳性。结论1、杭州地区急性腹泻患者DEC检出率高,列急性腹泻常见病原体首位,其中EAEC为本地区的主要流行类型,其次依次是ETEC、EPEC、EIEC和STEC。2、DEC在季节和粪便性状等因素上具有分布差异。3、通过特异性、灵敏度、稳定性、重复性等实验证明我们研究的单、双重荧光定量PCR方法可靠,可得以应用。4、通过对临床菌株的筛查,STEC的阳性率为0.54%。5、根据对急性腹泻粪便标本进行荧光PCR检测结果,我们建立的荧光PCR检测stx方法可实现对粪便标本直接检测,从而大大缩短检测周期。
[Abstract]:The virulence genes and pathogenicity carried by Diarrheagenic Escherichia coli (DEC) can be divided into intestinal pathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC) or Shiga toxin producing Escherichia coli, STEC, enterotoxigenic Escherichia coli (ETEC) and intestinal invasive large intestine. EIEC, enteroconcentrated Escherichia coli (EAEC) and diffuse adherent Escherichia coli (DAEC). At present, except DAEC, other various kinds of DEC are prevalent and even outbreaks of all kinds of events, from March to July. In Germany, the outbreak of gastroenteritis and hemolytic uremic syndrome caused by DEC O104: H4 caused the outbreak of the outbreak. 16 European countries, a total of 4125 people, 892 patients with HUS, 50 people died, the most serious outbreak of a non 0157 STEC outbreak so far, the culprit is the STX gene encoded Stx, can cause HUS, even death. The literature reports that a variety of bacteria can carry the STX gene. Hangzhou's unique geographical location, water environment and tourism characteristics have always been the high incidence of diarrhea. Therefore, we must strengthen the monitoring and control research on DEC, grasp its epidemic characteristics and countermeasures, establish a fast and accurate method of STX gene detection, and report the results to clinicians in time so as to specify the follow-up treatment plan, This is of great significance for the early warning and effective prevention of the epidemic of DEC or STX bacteria. 1, a collection of acute diarrhoea specimens from the emergency and intestinal outpatients of the First Affiliated Hospital of Zhejiang University in 2014 was collected, and the main intestinal pathogenic bacteria and viruses were isolated and tested by routine microbiological test procedures and RT-PCR, respectively. 2, The multiple PCR method was used to identify and classify the preserved Escherichia coli. 3, the data of acute diarrhea patients and DEC data collected at the first hospital emergency and intestinal outpatient department of the Affiliated First Hospital of Zhejiang University were analyzed and analyzed by DEC. 4, the rapid detection of stx1, Stx2 single, double heavy fluorescence quantitative PCR method, 5, using fluorescence PCR method, Samples of suspected clinical strains and acute diarrhea from inside and outside of the intestine were collected, and the positive results were sequenced and sequence alignment and analysis were carried out. Results 1, 735 samples of acute diarrhoea in 2014 were screened by separation, culture, nucleic acid detection, immunological detection and so on. The positive specimens (at least 1 kinds of bacteria were detected, " The positive rate of 338 fungi was 45.99%., the positive rate of Escherichia coli was 144, the detection rate was 19.59%, the first for acute diarrhea, followed by norovirus and Vibrio parahaemolyticus.2. In the 735 acute diarrhoea specimens in 2014, two or more than two kinds of pathogenic specimens were detected in 11 cases, of which 1 specimens were detected at the same time in 3 abdomen simultaneously. .3, 735 cases of acute diarrhea in 2014 were identified by multiple PCR methods. A total of 144 strains of DEC were detected, and the detection rate was 19.59% (144/735). Among them, the detection rate of EAEC was 9.93% (73/735); ETEC 32 strains, the detection rate was 4.35% (32/735); EPEC20 strain was 2.72% (20/735), and 2 strains of EIEC and two types were detected, and their detection was detected. The yield was 0.27% (2/735). In addition, 15 strains carried EAEC, ETEC and EPEC virulence genes in the separated DEC. In the separated DEC, EAEC dominated 50.69% (73/144), followed by ETEC (22.22%) and EPEC (13.89%).4, DEC a year four Ji Junke, mainly in summer and autumn, through data analysis, There were seasonal differences in the detection rate of 013 years (P was 0.027,0.025 and 0.016 respectively), but the detection data in 2014 showed no statistical difference in the four seasons, but the detection rate of EAEC/ETEC was the highest in summer and autumn, there were seasonal differences, P=0.025,0.001 (P0.05), there were statistical significance, and there was no seasonal difference.5 in other types of DEC detection rates, male, male and female. The detection rates of DEC in sexual and female patients were 21.69% and 17.87% respectively, and the differences were statistically meaningless. The male and female detection rates of ETEC/EAEC were 2.41%, 0.50%, and P=0.049 (P0.05), with statistical differences. Moreover, statistical analysis of 20112012 and 2013 monitoring data showed that DEC and its types were not statistically different in gender distribution. In the period of.62014 years, DEC was detected mainly in 19-60 years old patients with acute diarrhea, accounting for 77.78% (112/144), followed by 60 years of age, accounting for 16.67%. However, there was no statistical difference in the detection rate of DEC and its various types of DEC in patients with acute diarrhoea in different ages. As in 2014, DEC in parts of Hangzhou was also mainly at the age of 19-60 years. In the patients with acute diarrhea, except 2012, the detection rates of DEC and their types were not different in different age groups, and the DEC detection rate was the highest (20.43%) at the age of 60 years (20.43%), and there were statistical differences. The acute diarrhea patients who were separated to DEC in P=0.024 (P0.05).72014 were mostly water samples, 78.47% (113/144), and then rice hogwash (13.89%). But the fecal traits with the highest detection rate of DEC were mixed with mucous stool and blood stool, 50%, followed by water and fresh blood, and the detection rate was 33.33%, but the detection rate of DEC and the detection rates were no significant difference between the feces of each character in.2011 years and 63.30% (69/109) in acute diarrhea patients, which was mainly in 2012. 46.60% (48/103) and 40.78% (42/103) were detected in fluid like change and water sample, and 28.81% (34/118) and 21.19% (25/118) were detected in 2013, and the detection rates of DEC, EAEC, ETEC and EPEC in every fecal trait group were different in 2011, and in 2012, the detection rates of DEC and EAEC were different. Besides, in 2013, the detection rates of DEC and EAEC were different. There were statistical differences in the detection rates of DEC between the various groups of white blood cells in the feces, and the EAEC detection rates of red cells in the feces were statistically different, and some of the DEC strains detected in.8.20132014 years were 40 and 15, respectively, and 40 strains of DEC carrying virulence genes in 2013 were as follows, astA+estlb24 strain, astA+es CV 6, astA+elt+estlb2, astA+elt, astA+elt+estIa, astA+elt+estlb+pic, astA+estIb+escV, astA+estlb, astA+elt+estla+escV, invE+escV, elt+escV were all 15 strains of DEC, 1, 1, 1, 2, 7, 1, respectively. The rapid detection of STX gene was established by strain.9., and the dual fluorescence PCR method had better specificity. There was no cross reaction with other bacteria isolated from the intestinal tract; the detection range was 1 x 103~1 x 1012 copies/ml; the stability was good at room temperature, and there was no obvious effect on the placement for 7 days; the intra batch variation coefficient was less than 10%.10. in refolding. The clinical strains were detected by the dual fluorescence PCR method, including 4 Escherichia coli six positive, 2 stx1 positive and 2 Stx2 positive.11. using double fluorescent PCR to detect acute diarrhoea specimens directly. 8 positive were detected in 55 samples. Conclusion 1, acute diarrhea in Hangzhou region has high DEC detection rate and acute acute diarrhea. EAEC is the main epidemic type of common pathogens in diarrhoea, in which the main epidemic types in the region are ETEC, EPEC, EIEC and STEC.2, and DEC has the distribution difference of.3 in seasonal and fecal traits. It is confirmed by the specificity, sensitivity, stability, repeatability and other experiments. The double fluorescence quantitative PCR method is reliable and can be applied. .4, through the screening of clinical strains, the positive rate of STEC is 0.54%.5. According to the results of fluorescence PCR detection for the stool specimens of acute diarrhea, our fluorescence PCR detection method can be used to detect the fecal specimens directly, thus greatly shortening the detection cycle.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5

【参考文献】