本文選題：急性腹瀉 + 監(jiān)測��； 參考：《浙江大學》2016年碩士論文

【摘要】：研究背景及目的致瀉性大腸埃希菌(Diarrheagenic Escherichia coli, DEC)依其攜帶的毒力基因及致病性一般可分為腸致病性大腸埃希菌(EPEC)、腸出血性大腸埃希菌(EHEC)或產(chǎn)志賀毒素大腸埃希菌,STEC)、產(chǎn)腸毒素性大腸埃希菌(ETEC)、腸侵襲性大腸埃希菌(EIEC)、和腸集聚性大腸埃希菌(EAEC)以及彌散黏附性大腸埃希菌(DAEC)。目前,除DAEC外,其它各類的DEC在全球流行甚至暴發(fā)的事件多有報道。2011年3月至7月,德國暴發(fā)大規(guī)模的由DEC O104:H4引起胃腸炎和溶血性尿毒綜合征疫情,事件波及歐洲16個國家,共4125人發(fā)病,892例患者發(fā)生HUS,50人死亡,為迄今為止暴發(fā)的最嚴重的一次非0157 STEC疫情,此次疫情的罪魁禍首為stx基因編碼的Stx,可引起HUS,甚至死亡。文獻報道多種細菌可攜帶stx基因。因此本次疫情給全球一個強有力的警示。杭州獨特的地理位置、水域環(huán)境及旅游特色,歷來是腹瀉的高發(fā)城市,因此我們必須加強對DEC的監(jiān)測及防治研究,掌握其流行特征及應對措施,建立快速、準確的stx基因檢測方法,能及時向臨床醫(yī)生報告結(jié)果以便其指定后續(xù)治療方案,這對DEC或攜帶stx細菌疫情的預警及有效防止均有重要意義。方法1、收集2014年浙江大學附屬第一醫(yī)院急診及腸道門診的急性腹瀉糞便標本,分別采用常規(guī)微生物檢驗程序及RT-PCR等方法對主要腸道致病菌及病毒進行分離培養(yǎng)及檢測；2、采用多重PCR的方法對保存的大腸埃希菌進行鑒定及分型；3、統(tǒng)計分析2011-2014年在浙江大學附屬第一醫(yī)院急診及腸道門診收集的急性腹瀉患者資料及DEC檢測數(shù)據(jù)；4、建立快速檢測stx1、stx2單、雙重熒光定量PCR方法；5、采用熒光PCR方法對收集自腸道內(nèi)外的可疑臨床菌株及急性腹瀉標本進行檢測,陽性結(jié)果送出測序,進而進行序列比對及分析。結(jié)果1、對2014年735份急性腹瀉標本通過分離、培養(yǎng)、核酸檢測、免疫學檢測等方法展開病原體篩查,其中陽性標本(至少檢出1種細菌、真菌、病毒)338份,陽性檢出率為45.99%。其中致瀉性大腸埃希菌144份,檢出率為19.59%,列急性腹瀉病原首位,其次是諾如病毒和副溶血弧菌。2、在2014年735份急性腹瀉標本中,同時檢出兩種或兩種以上病原標本11份,其中有1份標本同時檢出3種腹瀉病原。3、采用多重PCR方法對2014年735例急性腹瀉標本進行DEC鑒定,共檢出144株DEC,檢出率為19.59%(144/735)。其中,EAEC 73株,檢出率為9.93%(73/735); ETEC 32株,檢出率為4.35%(32/735)；EPEC20株,其檢出率為2.72%(20/735)；分離到EIEC及STEC兩型DEC各2株,其檢出率均為0.27%(2/735)。此外,在分離到的DEC中,有15株菌同時攜帶EAEC、ETEC及EPEC的毒力基因。在分離到的DEC中,以EAEC為主,占50.69%(73/144),其次為分別為ETEC (22.22%)和EPEC (13.89%)。4、DEC一年四季均可分離到,主要以夏秋季為主,通過數(shù)據(jù)分析,2011、2012、2013年的檢出率存在季節(jié)差異(P分別為0.027、0.025和0.016),但是2014年的檢測數(shù)據(jù)顯示其四季檢出率無統(tǒng)計學差異,但其中EAEC/ETEC的檢出率夏秋季最高,存在季節(jié)性差異,P=0.025、0.001(P0.05),有統(tǒng)計學意義,其他各型DEC檢出率均無季節(jié)性差異。5、男性和女性患者的DEC檢出率分別為21.69%、17.87%,其差異在統(tǒng)計學上無意義。其中ETEC/EAEC的男、女檢出率分別為2.41%、0.50%,P=0.049(P0.05),具有統(tǒng)計學差異。此外,統(tǒng)計分析2011、2012及2013年監(jiān)測數(shù)據(jù),結(jié)果顯示DEC及其各型在性別分布方面均無統(tǒng)計學差異。6、2014年期間DEC主要在19-60歲年齡段急性腹瀉患者中檢出,占77.78%(112/144),其次為60歲年齡段,占16.67%；但不同年齡段急性腹瀉患者DEC及其各型DEC檢出率并無統(tǒng)計學差異。與2014年一樣,2011、2012、2013年杭州部分地區(qū)DEC也主要在19-60歲年齡段急性腹瀉患者中檢出,除2012年外,DEC及其各型檢出率在不同年齡段不存在差異。2012年DEC檢出率以60歲年齡段最高(20.43%),且存在統(tǒng)計學差異,P=0.024(P0.05)。7、2014年分離到DEC的急性腹瀉患者多為水樣便,占78.47%(113/144),其次為米泔樣便(13.89%)；但DEC檢出率最高的糞便性狀為米泔樣便混有粘液便及鮮血便,均為50%,其次為水樣便加鮮血便,檢出率為33.33%,但是DEC及各型檢出率在各性狀糞便間無顯著差異。2011年DEC多在急性腹瀉患者水樣便中檢出,占63.30%(69/109)；2012年主要在粘液樣變和水樣便中檢出,分為占46.60%(48/103)、40.78%(42/103)；2013年則多在爛糊便和水樣便中檢出,各為28.81%(34/118)和21.19%(25/118)。且2011年DEC、EAEC、ETEC和EPEC檢出率在各糞便性狀組中存在統(tǒng)計學差異,2012年、2013年為DEC和EAEC檢出率存在差異。此外,糞便中存有白細胞的各分組間DEC檢出率有統(tǒng)計學差異,糞便中存在紅細胞的各分組間EAEC檢出率有統(tǒng)計學差異。8.2013、2014年檢出的部分DEC菌株攜帶多型別毒力基因,分別為40株和15株,2013年40株DEC攜帶毒力基因情況如下,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均為1株。2014年的15株DEC分別攜帶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.建立的快速檢測stx基因的單、雙重熒光PCR方法特異性較好,與腸道分離的其他細菌、真菌不存在交叉反應；檢測范圍為1×103～1×1012 copies/ml;在室溫中的穩(wěn)定性好,放置7天無明顯影響；在重復性方面批間、批內(nèi)變異系數(shù)均小于10%。10.用所建立的雙重熒光PCR方法檢測臨床菌株,其中有4株大腸埃希菌six陽性,2株stx1陽性,2株stx2陽性。11.用所建立的雙重熒光PCR方法直接檢測急性腹瀉標本,在抽取的55份標本中檢出8份陽性。結(jié)論1、杭州地區(qū)急性腹瀉患者DEC檢出率高,列急性腹瀉常見病原體首位,其中EAEC為本地區(qū)的主要流行類型,其次依次是ETEC、EPEC、EIEC和STEC。2、DEC在季節(jié)和糞便性狀等因素上具有分布差異。3、通過特異性、靈敏度、穩(wěn)定性、重復性等實驗證明我們研究的單、雙重熒光定量PCR方法可靠,可得以應用。4、通過對臨床菌株的篩查,STEC的陽性率為0.54%。5、根據(jù)對急性腹瀉糞便標本進行熒光PCR檢測結(jié)果,我們建立的熒光PCR檢測stx方法可實現(xiàn)對糞便標本直接檢測,從而大大縮短檢測周期。
[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.

【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R446.5

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