本文選題：甲型副傷寒沙門氏菌　切入點：快速檢測　出處：《華中農(nóng)業(yè)大學》2008年碩士論文

【摘要】： 傷寒副傷寒是由傷寒桿菌(Salmonella typhi)和副傷寒桿菌甲、乙、丙(S.paratyphiA,B,C)引起的急性腸道傳染病,在全球分布很廣,估計每年傷寒發(fā)病1600萬～1700萬,死亡60萬,主要在發(fā)展中國家。以前我國傷寒發(fā)病占傷寒副傷寒總發(fā)病的90%以上,后來由于傷寒Vi疫苗的廣泛使用,20世紀90年代中期,流行菌株發(fā)生轉變,我國部分省份傷寒病例逐漸下降,而由甲型副傷寒沙門氏菌引起的副傷寒病例卻大幅上升,并呈現(xiàn)由散發(fā)到局部暴發(fā),由沿海省份向內(nèi)地省份推進的流行趨勢,近幾年來成為我國一些地區(qū)傳染病突發(fā)事件的主要病種,常發(fā)展成重大疫情,急需建立快速、準確的診斷方法。 實驗室常規(guī)檢測、診斷甲型副傷寒的主要方法為細菌培養(yǎng)、肥達氏凝集反應和傷寒血球快速診斷。這些方法費時費力,不利于早期診斷,給流行病學專家在追蹤傳染源、早期控制與臨床醫(yī)生早期診斷工作帶來了一定的難度。以酶聯(lián)免疫吸附試驗為原理建立起來的各種免疫學檢測方法,特異性好、靈敏度高、操作簡便、樣品處理量大,已經(jīng)廣泛應用于臨床和食品中病原菌的檢測。但是目前國內(nèi)外還很少有用ELISA方法檢測甲型副傷寒沙門氏菌的報道,本研究中,我們先制備抗甲型副傷寒沙門氏菌O2抗原的單克隆抗體和多克隆抗體,然后以此為基礎建立夾心ELISA方法,快速檢測該菌。 1.甲型副傷寒沙門氏菌單克隆抗體的制備與鑒定 甲型副傷寒沙門氏菌的菌體抗原包括O1、O2和O12三種,其中O2抗原為該菌所特有。我們選用甲型副傷寒沙門氏菌模式菌株50001,經(jīng)沸水浴2h后制備菌體抗原,免疫5只6-8周齡雌性Balb/C小鼠,經(jīng)過11次細胞融合篩選出兩株分泌特異性抗體的雜交瘤細胞株,2C6和4F10,其腹水效價分別為1:25600和1:12800,通過SDS-PAGE分析,兩株雜交瘤細胞所產(chǎn)生的單克隆抗體分子量均在180KD左右。2C6和4F10分泌的單克隆抗體不與傷寒等其它細菌發(fā)生交叉反應,為特異性針對甲型副傷寒沙門氏菌02抗原的抗體。 2.夾心ELISA檢測方法的建立 用相同抗原免疫兔子,制備多克隆抗體,選用單抗2C6和HRP標記的羊抗鼠酶標二抗,建立檢測甲型副傷寒沙門氏菌雙抗夾心ELISA方法。實驗結果表明,此ELISA方法不與屬內(nèi)外常見致病菌發(fā)生交叉反應,特異性較好;檢測限達到10~5CFU/mL,靈敏度高;整個檢測過程4-5h即可完成,為食品檢驗以及臨床醫(yī)生早期診斷提供一種快速、靈敏、有效的方法。 通過以上研究工作,初步確定了甲型副傷寒沙門氏菌的夾心ELISA檢測程序并優(yōu)化了反應條件。其方法的穩(wěn)定性和實際應用情況還有待進一步完善,以便為試劑盒的研制打下基礎。
[Abstract]:Typhoid paratyphoid is an acute enteric disease caused by Salmonella typhimurium (Salmonella typhimurium) and Bacillus paratyphiae A, B, C). It is widely distributed in the world. It is estimated that typhoid fever has an incidence of 16 million ~ 17 million per year and causes 600000 deaths. Mainly in developing countries. Previously, typhoid accounted for more than 90% of the total typhoid and paratyphoid fever in China. Later, due to the widespread use of typhoid Vi vaccine, epidemic strains changed in the mid-1990s. The number of typhoid cases in some provinces of China has gradually decreased, while the cases of paratyphoid fever caused by salmonella paratyphoid A have increased significantly, showing a trend of epidemic from sporadic to local outbreaks and from coastal provinces to inland provinces. In recent years, it has become the main type of infectious disease emergency in some regions of our country, and it often develops into a major epidemic situation. It is urgent to establish a rapid and accurate diagnostic method. Routine laboratory tests show that the main methods for diagnosis of paratyphoid A are bacterial culture, Widal's agglutination and rapid diagnosis of typhoid blood cells. These methods are time-consuming and laborious, not conducive to early diagnosis, giving epidemiologists the ability to track the source of infection. All kinds of immunological detection methods based on enzyme linked immunosorbent assay (Elisa) are characterized by good specificity, high sensitivity, simple operation and large amount of sample handling. It has been widely used in the detection of pathogenic bacteria in clinic and food. However, there are few reports about the detection of salmonella paratyphoid A by ELISA method at home and abroad. We first prepared monoclonal antibody and polyclonal antibody against O _ 2 antigen of Salmonella paratyphi A and then developed a sandwich ELISA method for rapid detection of the bacterium. 1. Preparation and identification of monoclonal antibody against salmonella paratyphoid A. The bacterial antigens of Salmonella paratyphi A include O _ 1O _ 2 and O _ 12, among which O _ 2 antigen is unique to this strain. We selected the model strain 50001 of Salmonella paratyphoid A and prepared the antigen after 2 hours of boiling water bath. Five 6-8 week-old female Balb/C mice were immunized. Two hybridoma cell lines 2C6 and 4F10 secreting specific antibodies were screened by cell fusion for 11 times. The ascites titers were 1: 25600 and 1: 12800, respectively. The ascites titers were analyzed by SDS-PAGE. The molecular weight of monoclonal antibodies produced by the two hybridoma cells were about .2C6 of 180KD and the monoclonal antibodies secreted by 4F10 did not cross react with other bacteria such as typhoid fever, so they were specific antibodies against the antigen of Salmonella paratyphi A 02. 2. Establishment of sandwich ELISA detection method. Rabbits were immunized with the same antigen and polyclonal antibodies were prepared. The double antibody sandwich ELISA method for detection of Salmonella paratyphi A was established by using the second antibody labeled by monoclonal antibody 2C6 and HRP. This ELISA method does not cross react with common pathogenic bacteria inside and outside of the family, and has good specificity, the detection limit is up to 105 CFU / mL, the sensitivity is high, the whole detection process can be completed 4-5 hours, which provides a fast and sensitive method for food testing and early diagnosis by clinicians. An effective method. Through the above research work, the sandwich ELISA detection procedure of Salmonella paratyphi A was preliminarily determined and the reaction conditions were optimized. The stability and practical application of the method need to be further improved. In order to lay a foundation for the development of the kit.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392;R446.6;R516.3

【參考文獻】

相關期刊論文 前10條

1 陳瓊;孔繁德;張長弓;彭海濱;徐淑菲;;沙門氏菌快速檢測試紙條的研制與應用[J];福建畜牧獸醫(yī);2006年05期

2 郭愛玲;鄭華英;馬愛民;余功保;呂均;秦巧玲;王震;;沙門氏菌全細胞的熱裂解氣相色譜-質譜分析[J];分析化學;2007年05期

3 魏承毓;我國甲型副傷寒的流行趨勢及對防控對策之探討[J];國外醫(yī)學.流行病學傳染病學分冊;2005年02期

4 黃勁;王和;;用PCR擴增16SrRNA基因鑒定甲型副傷寒沙門菌細胞壁缺陷突變株[J];貴陽醫(yī)學院學報;2006年04期

5 游旅,張玉瓊;貴州省傷寒和甲型副傷寒沙門氏菌流行株的耐藥性變遷及意義[J];貴州醫(yī)藥;2004年04期

6 鄭友限;食源性致病菌中沙門氏菌檢測方法的探討[J];河南預防醫(yī)學雜志;2005年02期

7 邵軍軍;�；菔|;;膠體金免疫層析試紙條在病原體檢測及醫(yī)學診斷中的應用[J];實用診斷與治療雜志;2008年01期

8 田克誠,闞飆,呂太富,游旅,張玉瓊;貴州省2000年甲型副傷寒沙門菌分離株16SrRNA基因型分析[J];疾病監(jiān)測;2001年12期

9 閆梅英,梁未麗,李偉,闞飆;1995-2004年全國傷寒副傷寒的流行分析[J];疾病監(jiān)測;2005年08期

10 孫洋,王云翔,柳增善;吖啶橙免疫熒光菌團培養(yǎng)法對沙門氏菌的快速檢測[J];吉林畜牧獸醫(yī);1994年06期

，

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