【摘要】：一、研究背景和目的 近年來,隨著經(jīng)濟(jì)全球化進(jìn)程的加快,食品安全已成為當(dāng)今世界性公共衛(wèi)生熱點(diǎn)。食源性致病菌是引起食源性疾病的首要原因,對(duì)人類健康造成極大危害,是食品安全的重大隱患。隨著經(jīng)濟(jì)發(fā)展和技術(shù)進(jìn)步,食源性疾病并未減少或消失,世界范圍內(nèi)食品安全惡性事件反而接連發(fā)生,食品安全形勢(shì)嚴(yán)峻。目前,食源性致病菌的檢測(cè)存在特異性差、操作繁瑣、耗時(shí),以及不能現(xiàn)場(chǎng)應(yīng)用等問題。本研究旨在建立阪崎腸桿菌(Enterobacter Sakazakii)和痢疾志賀菌(Shigella dysenteriae)兩種常見食源性致病菌的快速檢測(cè)方法。 阪崎腸桿菌是一種重要的食源性致病菌,呈世界性分布,主要通過嬰幼兒配方奶粉經(jīng)消化道感染兒童,引起新生兒腦膜炎、敗血癥和壞死性結(jié)腸炎,甚或遺留神經(jīng)系統(tǒng)后遺癥或?qū)е滤劳觥Ｄ壳?引起阪崎腸桿菌感染事件的發(fā)生規(guī)模呈明顯上升趨勢(shì),已成為我國重要的食源性致病菌。國際食品微生物標(biāo)準(zhǔn)委員會(huì)于2002年將阪崎腸桿菌列為對(duì)特定人群產(chǎn)生嚴(yán)重的生命危害或產(chǎn)生慢性后遺癥的微生物。國家質(zhì)檢總局于2005年8月頒布了檢測(cè)阪崎腸桿菌的行業(yè)標(biāo)準(zhǔn)SN/T1632.1。2006年,針對(duì)嬰幼兒配方乳粉受阪崎腸桿菌污染的高風(fēng)險(xiǎn)性和污染后的巨大危害性,國家質(zhì)檢總局頒布了嬰幼兒配方乳粉中阪崎腸桿菌每批必檢的市場(chǎng)準(zhǔn)入要求。檢測(cè)阪崎腸桿菌的傳統(tǒng)方法步驟繁瑣,一般須耗時(shí)6d左右,近年來國內(nèi)外相繼建立了ELISA、PCR、核酸雜交等檢測(cè)技術(shù)。 痢疾志賀菌是人和動(dòng)物腸道內(nèi)寄生的革蘭陰性無芽胞桿菌,是一類具有較強(qiáng)傳染性、危害嚴(yán)重的革蘭陰性腸道致病菌,所致的細(xì)菌性痢疾(Shigellosis)是世界上尤其是發(fā)展中國家重要的傳染病之一,全球每年細(xì)菌性痢疾病例高達(dá)1.65億,其中1.63億發(fā)生在發(fā)展中國家,并導(dǎo)致110萬患者死亡,其中60%以上為5歲以下兒童。近年來,痢疾志賀菌食物中毒的發(fā)生規(guī)模呈明顯上升趨勢(shì),成為我國首要的食源性致病菌之一。目前,我國常用的志賀氏菌的檢驗(yàn)方法主要是國標(biāo)法(GB/T4789.5-2003)。整個(gè)檢測(cè)程序需要3-5d完成,檢測(cè)時(shí)間長,檢出限為104CFU/mL。免疫學(xué)方法簡(jiǎn)單,但靈敏度不高；PCR方法具有靈敏、快速等優(yōu)點(diǎn),但檢測(cè)成本較高,儀器昂貴,不適于基層單位推廣應(yīng)用。 綜上,建立準(zhǔn)確、靈敏、操作簡(jiǎn)便、不依賴昂貴儀器的檢測(cè)方法對(duì)于食源性致病菌感染的診斷、疾病控制和流行病學(xué)調(diào)查具有重要意義；發(fā)展快速準(zhǔn)確、操作簡(jiǎn)便的檢測(cè)與鑒定阪崎腸桿菌和痢疾志賀菌的方法成為相關(guān)研究的熱點(diǎn)。目前,常用快速檢測(cè)方法主要有ELISA、DNA探針、常規(guī)PCR、Real-time PCR等。環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)(loop-mediated isothermal amplification, LAMP)是一種新穎的核酸擴(kuò)增技術(shù),依賴于能夠識(shí)別靶DNA上6個(gè)特定區(qū)域的4條引物和一種具有鏈置換活性的DNA聚合酶,在恒溫條件下高效擴(kuò)增核酸,具有高特異性、快速、靈敏、操作簡(jiǎn)便等特點(diǎn)。自從2000年以來,LAMP技術(shù)在臨床疾病的診斷、病原性細(xì)菌或病毒的定性檢測(cè)以及動(dòng)物胚胎性別鑒定等方面應(yīng)用廣泛。LAMP技術(shù)在國內(nèi)起步較晚,應(yīng)用LAMP技術(shù)檢測(cè)阪崎腸桿菌和痢疾志賀菌在國內(nèi)外文獻(xiàn)中報(bào)道較少。 本研究針對(duì)阪崎腸桿菌外膜蛋白OmpA基因和痢疾志賀茵ipaH基因,分別設(shè)計(jì)相應(yīng)的LAMP引物,建立優(yōu)化的反應(yīng)體系,考察特異性和靈敏度,并應(yīng)用于食品樣品的直接檢測(cè),初步建立阪崎腸桿菌和痢疾志賀菌的LAMP檢測(cè)方法。 二、研究方法 1.阪崎腸桿菌LAMP快速檢測(cè)技術(shù)的建立與初步應(yīng)用 (1)阪崎腸桿菌LAMP檢測(cè)技術(shù)的建立 根據(jù)GenBank公布的阪崎腸桿菌外膜蛋白OmpA基因序列(Accession number:DQ000206)的保守區(qū),利用LAMP專用軟件Primer Explorer version4設(shè)計(jì)一套特異性引物,即外引物F3和B3、內(nèi)引物BIP和FIP,以阪崎腸桿菌(ATCC29544)提取的DNA為模板,建立檢測(cè)阪崎腸桿菌的LAMP檢測(cè)技術(shù)體系,并對(duì)反應(yīng)體系內(nèi)各反應(yīng)條件進(jìn)行優(yōu)化,最終摸索出優(yōu)化后檢測(cè)阪崎腸桿菌的LAMP反應(yīng)體系。 (2)阪崎腸桿菌LAMP靈敏度、特異性實(shí)驗(yàn)和實(shí)際樣品檢測(cè) 利用建立的LAMP方法檢測(cè)阪崎腸桿菌純培養(yǎng)物,考察其靈敏度。將過夜培養(yǎng)的阪崎腸桿菌(ATCC29544)菌液10倍倍比稀釋至10-1～10-8,取各稀釋度菌液100ul進(jìn)行平板計(jì)數(shù)。同時(shí)取各稀釋度菌液1ml提取DNA,取2ul上清液作為模板進(jìn)行LAMP擴(kuò)增和PCR擴(kuò)增,測(cè)試兩種方法檢測(cè)阪崎腸桿菌純培養(yǎng)物的靈敏度。 選取3株阪崎腸桿菌標(biāo)準(zhǔn)菌株和大腸埃希菌、傷寒沙門菌、金黃色葡萄球菌等12株其它非阪崎腸桿菌致病菌進(jìn)行特異性實(shí)驗(yàn),檢測(cè)LAMP方法的特異性。依據(jù)本實(shí)驗(yàn)所建立的LAMP和PCR的條件和參數(shù),對(duì)36份奶粉樣品進(jìn)行檢測(cè),并與FDA檢測(cè)方法進(jìn)行比較。 2.痢疾志賀菌LAMP快速檢測(cè)技術(shù)的建立與初步應(yīng)用 (1)痢疾志賀菌LAMP檢測(cè)技術(shù)的建立 根據(jù)GenBank公布的痢疾志賀茵ipaH基因序列(Accession number:M76445)中的保守區(qū)作為靶序列,設(shè)計(jì)痢疾志賀菌特異性引物,包括外引物F3和B3、內(nèi)引物BIP和FIP、環(huán)引物L(fēng)F和LB。以痢疾志賀菌(CMCC48097)提取的DNA為模板,建立檢測(cè)痢疾志賀菌的LAMP檢測(cè)技術(shù),并對(duì)反應(yīng)體系內(nèi)各反應(yīng)條件進(jìn)行優(yōu)化。最終得出優(yōu)化后檢測(cè)痢疾志賀菌的LAMP反應(yīng)體系。 (2)痢疾志賀菌靈敏度、特異性實(shí)驗(yàn)和實(shí)際樣品檢測(cè) 采用建立的LAMP方法檢測(cè)痢疾志賀菌純培養(yǎng)液,考察其靈敏度。將過夜培養(yǎng)的痢疾志賀菌(CMCC48097)菌液10倍倍比稀釋至10-1～10-8,取各稀釋度菌液100ul進(jìn)行平板計(jì)數(shù)。同時(shí)取各稀釋度菌液1ml提取DNA,取2μl上清液作為模板進(jìn)行LAMP擴(kuò)增和PCR擴(kuò)增,測(cè)試兩種方法檢測(cè)痢疾志賀菌純培養(yǎng)物靈敏度。 同時(shí)選取4株志賀菌標(biāo)準(zhǔn)菌株和大腸埃希菌、傷寒沙門菌、金黃色葡萄球菌等其它非志賀菌進(jìn)行特異性實(shí)驗(yàn),檢測(cè)LAMP方法的特異性,初步應(yīng)用于人工污染豬肉樣品痢疾志賀菌的檢測(cè),并與常規(guī)PCR方法比較。 三、結(jié)果 1.確定阪崎腸桿菌LAMP優(yōu)化反應(yīng)體系為：6mM MgCl2,0.6mMdNTPs,0.8M betaine,0.4μM外引物(F3和B3),1.6μM內(nèi)引物(FIP和BIP),8U的Bst DNA聚合酶,2.5μl Thermopol buffer,2μL DNA模板,ddH2O補(bǔ)足體積至25μl；擴(kuò)增溫度58℃,反應(yīng)60min,產(chǎn)物于2%瓊脂糖凝膠電泳分析,得到典型的LAMP梯形條帶,肉眼觀察反應(yīng)副產(chǎn)物焦磷酸鎂白色沉淀。 LAMP法能檢出所有的阪崎腸桿菌菌株,產(chǎn)生特異性擴(kuò)增的梯形條帶,直接用肉眼觀察到焦磷酸鎂白色沉淀,而其它腸道致病菌均未產(chǎn)生特異擴(kuò)增的梯形條帶。LAMP對(duì)阪崎腸桿菌純菌液檢測(cè)靈敏度為2.0×101cfu/mL,PCR檢測(cè)靈敏度為2.0×102cfu/mL; LAMP檢測(cè)的靈敏度是傳統(tǒng)PCR靈敏度的10倍,檢測(cè)時(shí)間縮短了2-3h。采用LAMP法檢測(cè)36份奶粉樣品,其中2份樣品檢出阪崎腸桿菌,陽性率為5.6%,與FDA方法檢測(cè)結(jié)果一致。 2.確定痢疾志賀菌的LAMP反應(yīng)體系為：5mM MgCl2,0.3mM dNTPs,0.6M betaine,0.4μM外引物(F3和B3),1.6μM內(nèi)引物(FIP和BIP)與1.6μM環(huán)引物(LF和LB),8U Bst DNA聚合酶大片段,ThermoPol buffer2.5μl, DNA模板2μl, ddH2O補(bǔ)足體積至25μl；擴(kuò)增溫度63℃反應(yīng)60min,產(chǎn)物于2.5%瓊脂糖凝膠電泳分析,得到典型的LAMP梯形條帶。 LAMP法能檢出4株志賀菌,產(chǎn)生特異擴(kuò)增的梯形條帶,其余腸道致病菌均未產(chǎn)生特異擴(kuò)增的梯形條帶,表明選用的志賀菌ipaH基因引物的特異性強(qiáng)。LAMP法對(duì)痢疾志賀菌純菌液檢測(cè)靈敏度為5.3×101cfu/ml,對(duì)人工污染的豬肉樣品的痢疾志賀菌檢測(cè)限為6.8×101cfu/ml。PCR檢測(cè)的靈敏度為5.3×102cfu/ml,豬肉樣品為6.8×102cfu/mL, LAMP的靈敏度均比傳統(tǒng)PCR靈敏度高10倍,整個(gè)檢測(cè)過程可在2h內(nèi)完成。 四、結(jié)論 本研究建立了乳制品中阪崎腸桿菌、肉制品中痢疾志賀菌的LAMP決速檢測(cè)技術(shù),并在實(shí)際樣品檢測(cè)中得到了初步應(yīng)用。LAMP檢測(cè)技術(shù)針對(duì)靶基因的6個(gè)區(qū)域設(shè)計(jì)4條(6條)特異引物,特異性強(qiáng)；LAMP檢測(cè)限為101cfu/mL,是PCR法靈敏度的10倍。LAMP法能直接對(duì)奶粉、肉制品、乳制品等食品中的致病菌進(jìn)行檢測(cè),實(shí)際樣品一般在20～28h內(nèi)完成檢測(cè),與傳統(tǒng)的分離培養(yǎng)方法相比,大大縮短了檢測(cè)時(shí)間,節(jié)省了人力、物力；使用恒溫水浴裝置即可完成反應(yīng),通過觀察反應(yīng)液渾濁度變化,或加入熒光染料SYBR Green I后觀察反應(yīng)液的顏色變化,直接用肉眼即可判讀結(jié)果,不需要使用昂貴、精密的儀器設(shè)備,實(shí)現(xiàn)反應(yīng)及產(chǎn)物檢測(cè)一步完成,操作簡(jiǎn)便,檢測(cè)成本低,適用于現(xiàn)場(chǎng)檢測(cè)和大量樣品高通量檢測(cè),尤其特別適合于病原微生物的現(xiàn)場(chǎng)快速檢測(cè)、戰(zhàn)時(shí)野外、小型實(shí)驗(yàn)室以及基層衛(wèi)生單位的推廣使用。
[Abstract]:I. background and purpose
In recent years, with the acceleration of economic globalization, food safety has become a worldwide public health hotspot. Food borne pathogens are the primary cause of foodborne diseases, causing great harm to human health and a major hidden danger to food safety. With the development of economic and technological progress, foodborne diseases have not decreased or disappeared. At present, the detection of food-borne pathogenic bacteria has some problems, such as poor specificity, tedious operation, time-consuming, and unable to be used in the field. This study aimed to establish two common strains, Enterobacter Sakazakii and Shigella dysenteriae. Rapid detection of foodborne pathogens.
Enterobacter sakazakii is an important foodborne pathogen, which is distributed worldwide. It mainly infects children through the digestive tract of infant formula milk powder, causing meningitis, sepsis and necrotizing colitis in newborn infants, and even the sequelae of the nervous system or death. At present, the incidence of Enterobacter sakazakii infection is obvious. Enterobacter sakazakii was listed as a serious life-threatening or chronic sequelae by the International Committee on Food Microbiological Standards in 2002. The General Administration of Quality Supervision and Inspection issued the industry standard SN/T1632.1.2006 for the detection of Enterobacter sakazakii in August 2005. In 1997, the State Administration of Quality Supervision and Inspection (AQSIQ) promulgated the requirements for market access for each batch of Enterobacter sakazakii in infant formula milk powder, aiming at the high risk of contamination by Enterobacter sakazakii and the great harm after contamination. ELISA, PCR, nucleic acid hybridization and other detection techniques were established.
Shigellosis is one of the most important infectious diseases in the world, especially in developing countries. There are 165 million cases of bacillary dysentery in the world every year. In recent years, Shigella dysenteriae food poisoning has become one of the most important food-borne pathogens in China. At present, the commonly used method for testing Shigella in China is the national standard method (GB/T4789.5-20). 03). The whole detection procedure needs 3-5 days to complete, the detection time is long, the detection limit is 104CFU/mL. Immunoassay method is simple, but the sensitivity is not high; PCR method has the advantages of sensitivity and speed, but the detection cost is high, the instrument is expensive, and is not suitable for grass-roots units to promote the application.
To sum up, it is of great significance to establish accurate, sensitive, easy-to-operate, non-expensive instrument-based detection methods for the diagnosis, disease control and epidemiological investigation of foodborne pathogenic bacterial infections; to develop rapid, accurate and easy-to-operate methods for the detection and identification of Enterobacter sakazakii and Shigella dysenteriae have become a hot spot of related research. Rapid detection methods include ELISA, DNA probes, conventional PCR, Real-time PCR and so on. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique, which relies on four primers capable of identifying six specific regions of the target DNA and a DNA polymerase with chain substitution activity at constant temperature. Since 2000, LAMP technology has been widely used in the diagnosis of clinical diseases, the qualitative detection of pathogenic bacteria or viruses and the sex identification of animal embryos. Shigella dysentery is rarely reported in domestic and foreign literature.
In this study, the LAMP primers were designed for the outer membrane protein OmpA gene of Enterobacter sakazakii and the ipaH gene of Shigella dysenteriae, and the optimized reaction system was established. The specificity and sensitivity of the LAMP primers were investigated. The LAMP method for the detection of Enterobacter sakazakii and Shigella dysenteriae was established.
Two, research methods.
Establishment and preliminary application of 1. LAMP rapid detection technology for Enterobacter sakazakii
(1) establishment of LAMP detection technology for Enterobacter sakazakii
According to the conserved region of Enterobacter sakazakii outer membrane protein OmpA gene (Accession number: DQ000206) published by GenBank, a set of specific primers, namely external primers F3 and B3, internal primers BIP and FIP, were designed with LAMP software Primer Explorer version 4. DNA extracted from Enterobacter sakazakii (ATCCre4) was used as template to establish a method for detection of Enterobacter sakazakii. LAMP detection technology system, and optimize the reaction conditions in the reaction system, and finally find out the optimized LAMP reaction system for detection of Enterobacter sakazakii.
(2) sensitivity, specific and practical detection of Enterobacter sakazakii LAMP
The LAMP method was used to detect the pure culture of Enterobacter sakazakii, and its sensitivity was investigated. The overnight culture of Enterobacter sakazakii (ATCC29544) was diluted 10 times to 10-1-10-8, and the dilutions of 100 UL were counted on a plate. Two methods were used to detect the sensitivity of pure cultures of Enterobacter sakazakii.
Three standard strains of Enterobacter sakazakii and 12 other non-Enterobacter sakazakii pathogenic bacteria such as Escherichia coli, Salmonella typhi and Staphylococcus aureus were selected to test the specificity of LAMP method. According to the conditions and parameters of LAMP and PCR established in this experiment, 36 milk powder samples were detected and improved with FDA method. Comparison.
Establishment and preliminary application of LAMP rapid detection technology for Shigella dysentery 2.
(1) establishment of LAMP detection technology for Shigella dysentery
Based on the conserved region of the accession number (M76445) of Shigella dysenteriae ipaH gene published by GenBank, specific primers for Shigella dysenteriae were designed, including external primers F3 and B3, internal primers BIP and FIP, cyclic primers LF and LB. Finally, the LAMP reaction system for detecting Shigella dysenteriae was obtained.
(2) sensitivity, specificity and practical detection of Shigella dysentery.
The sensitivity of the established LAMP method for the detection of Shigella dysenteriae was investigated. The overnight cultured Shigella dysenteriae (CMCC48097) was diluted 10 times to 10-1-10-8, and the diluted bacterial liquid 100 UL was counted on the plate. The DNA was extracted from the diluted bacterial liquid 1 ml, and the supernatant 2 ml was used as the template for LAMP amplification and PCR amplification. Increase, test two methods to detect the sensitivity of dysentery Shika Sonojun culture.
Four standard strains of Shigella and other non-Shigella strains such as Escherichia coli, Salmonella typhi, Staphylococcus aureus were selected for specificity test to detect the specificity of LAMP method. The method was preliminarily applied to detect Shigella dysenteriae in artificially contaminated pork samples and compared with conventional PCR method.
Three, the result
1. The optimized LAMP reaction system of Enterobacter sakazakii was determined as follows: 6mMgCl2, 0.6mmdNTPs, 0.8mbetaine, 0.4mu external primers (F3 and B3), 1.6mu internal primers (FIP and BIP), 8U Bst DNA polymerase, 2.5mu L Thermopol buffer, 2mu L DNA template, ddH2O replenishment Volume 25 mu; amplification temperature 58 C, reaction time 60 min, product was obtained by 2% agarose gel electrophoresis analysis. To the typical LAMP trapezoidal strip, the reaction by-product magnesium pyrophosphate and white precipitates were observed.
All Enterobacter sakazakii strains could be detected by LAMP, producing specific amplified trapezoidal bands. Magnesium pyrophosphate white precipitation was observed directly with the naked eye, while other enteropathogenic bacteria did not produce specific amplified trapezoidal bands. The sensitivity of AMP was 10 times higher than that of conventional PCR, and the detection time was shortened by 2-3 hours. The positive rate of Enterobacter sakazakii was 5.6% in 2 of 36 milk powder samples detected by LAMP, which was consistent with that of FDA.
2. The LAMP reaction system of Shigella dysenteriae was determined as follows: 5mM MgCl2, 0.3mdNTPs, 0.6M betaine, 0.4um external primers (F3 and B3), 1.6um internal primers (FIP and BIP) and 1.6um ring primers (LF and LB), 8U Bst DNA polymerase large fragments, Thermo Polbuffer 2.5ml, DNA template 2ml, ddH_2O replenishment volume to 25 mul; amplification temperature 63 C, reaction 60min, product was 2.5 mu_ ml. The typical LAMP trapezoid strip was obtained by agarose gel electrophoresis.
Four strains of Shigella were detected by LAMP method, producing specific amplified trapezoidal bands, while none of the other intestinal pathogenic bacteria produced specific amplified trapezoidal bands, indicating the specificity of the selected primers for the ipaH gene of Shigella. The sensitivity of LAMP method for detection of Shigella dysenteriae was 5.3 *101 cfu/ml, and it was suitable for detection of Shigella dysenteriae from artificially contaminated pork samples. The sensitivity of PCR was 5.3
Four. Conclusion
In this study, a LAMP rapid detection method for Enterobacter sakazakii in dairy products and Shigella dysenteriae in meat products was established, and was applied to the detection of real samples. Four (6) specific primers were designed for six regions of the target gene, and the detection limit of LAMP was 101 cfu/mL, which was 10 times the sensitivity of PCR. Direct detection of pathogenic bacteria in milk powder, meat products, dairy products and other foods, the actual sample is usually completed within 20-28 hours, compared with the traditional isolation and culture method, greatly shorten the detection time, save manpower and material resources; the use of a constant temperature water bath device can complete the reaction, by observing the changes in the turbidity of the reaction solution, or adding fluorescence. Light dye SYBR Green I can be used to observe the color change of the reaction solution. The results can be read directly by naked eyes. It is not necessary to use expensive and precise instruments to complete the reaction and product detection in one step. It is easy to operate and low cost. It is suitable for on-site detection and high-throughput detection of a large number of samples, especially for pathogenic microorganisms. Rapid detection, promotion and use in wartime field, small laboratories and primary health units.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5

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