本文選題：甲型副傷寒沙門(mén)氏菌　切入點(diǎn)：快速檢測(cè)　出處：《華中農(nóng)業(yè)大學(xué)》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(jì)每年傷寒發(fā)病1600萬(wàn)～1700萬(wàn),死亡60萬(wàn),主要在發(fā)展中國(guó)家。以前我國(guó)傷寒發(fā)病占傷寒副傷寒總發(fā)病的90%以上,后來(lái)由于傷寒Vi疫苗的廣泛使用,20世紀(jì)90年代中期,流行菌株發(fā)生轉(zhuǎn)變,我國(guó)部分省份傷寒病例逐漸下降,而由甲型副傷寒沙門(mén)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(jìn)的流行趨勢(shì),近幾年來(lái)成為我國(guó)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(zhǔn)確的診斷方法。 實(shí)驗(yàn)室常規(guī)檢測(cè)、診斷甲型副傷寒的主要方法為細(xì)菌培養(yǎng)、肥達(dá)氏凝集反應(yīng)和傷寒血球快速診斷。這些方法費(fèi)時(shí)費(fèi)力,不利于早期診斷,給流行病學(xué)專(zhuān)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(lái)了一定的難度。以酶聯(lián)免疫吸附試驗(yàn)為原理建立起來(lái)的各種免疫學(xué)檢測(cè)方法,特異性好、靈敏度高、操作簡(jiǎn)便、樣品處理量大,已經(jīng)廣泛應(yīng)用于臨床和食品中病原菌的檢測(cè)。但是目前國(guó)內(nèi)外還很少有用ELISA方法檢測(cè)甲型副傷寒沙門(mén)氏菌的報(bào)道,本研究中,我們先制備抗甲型副傷寒沙門(mén)氏菌O2抗原的單克隆抗體和多克隆抗體,然后以此為基礎(chǔ)建立夾心ELISA方法,快速檢測(cè)該菌。 1.甲型副傷寒沙門(mén)氏菌單克隆抗體的制備與鑒定 甲型副傷寒沙門(mén)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(mén)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(guò)11次細(xì)胞融合篩選出兩株分泌特異性抗體的雜交瘤細(xì)胞株,2C6和4F10,其腹水效價(jià)分別為1:25600和1:12800,通過(guò)SDS-PAGE分析,兩株雜交瘤細(xì)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(xì)菌發(fā)生交叉反應(yīng),為特異性針對(duì)甲型副傷寒沙門(mén)氏菌02抗原的抗體。 2.夾心ELISA檢測(cè)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(biāo)記的羊抗鼠酶標(biāo)二抗,建立檢測(cè)甲型副傷寒沙門(mén)氏菌雙抗夾心ELISA方法。實(shí)驗(yàn)結(jié)果表明,此ELISA方法不與屬內(nèi)外常見(jiàn)致病菌發(fā)生交叉反應(yīng),特異性較好;檢測(cè)限達(dá)到10~5CFU/mL,靈敏度高;整個(gè)檢測(cè)過(guò)程4-5h即可完成,為食品檢驗(yàn)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(guò)以上研究工作,初步確定了甲型副傷寒沙門(mén)氏菌的夾心ELISA檢測(cè)程序并優(yōu)化了反應(yīng)條件。其方法的穩(wěn)定性和實(shí)際應(yīng)用情況還有待進(jìn)一步完善,以便為試劑盒的研制打下基礎(chǔ)。
[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(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R446.6;R516.3

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