食源性致病菌阪崎肠杆菌和痢疾志贺菌LAMP快速检测技术的建立与初步应用
发布时间：2018-09-04 20:21
【摘要】：一、研究背景和目的 近年来,随着经济全球化进程的加快,食品安全已成为当今世界性公共卫生热点。食源性致病菌是引起食源性疾病的首要原因,对人类健康造成极大危害,是食品安全的重大隐患。随着经济发展和技术进步,食源性疾病并未减少或消失,世界范围内食品安全恶性事件反而接连发生,食品安全形势严峻。目前,食源性致病菌的检测存在特异性差、操作繁琐、耗时,以及不能现场应用等问题。本研究旨在建立阪崎肠杆菌(Enterobacter Sakazakii)和痢疾志贺菌(Shigella dysenteriae)两种常见食源性致病菌的快速检测方法。 阪崎肠杆菌是一种重要的食源性致病菌,呈世界性分布,主要通过婴幼儿配方奶粉经消化道感染儿童,引起新生儿脑膜炎、败血症和坏死性结肠炎,甚或遗留神经系统后遗症或导致死亡。目前,引起阪崎肠杆菌感染事件的发生规模呈明显上升趋势,已成为我国重要的食源性致病菌。国际食品微生物标准委员会于2002年将阪崎肠杆菌列为对特定人群产生严重的生命危害或产生慢性后遗症的微生物。国家质检总局于2005年8月颁布了检测阪崎肠杆菌的行业标准SN/T1632.1。2006年,针对婴幼儿配方乳粉受阪崎肠杆菌污染的高风险性和污染后的巨大危害性,国家质检总局颁布了婴幼儿配方乳粉中阪崎肠杆菌每批必检的市场准入要求。检测阪崎肠杆菌的传统方法步骤繁琐,一般须耗时6d左右,近年来国内外相继建立了ELISA、PCR、核酸杂交等检测技术。 痢疾志贺菌是人和动物肠道内寄生的革兰阴性无芽胞杆菌,是一类具有较强传染性、危害严重的革兰阴性肠道致病菌,所致的细菌性痢疾(Shigellosis)是世界上尤其是发展中国家重要的传染病之一,全球每年细菌性痢疾病例高达1.65亿,其中1.63亿发生在发展中国家,并导致110万患者死亡,其中60%以上为5岁以下儿童。近年来,痢疾志贺菌食物中毒的发生规模呈明显上升趋势,成为我国首要的食源性致病菌之一。目前,我国常用的志贺氏菌的检验方法主要是国标法(GB/T4789.5-2003)。整个检测程序需要3-5d完成,检测时间长,检出限为104CFU/mL。免疫学方法简单,但灵敏度不高；PCR方法具有灵敏、快速等优点,但检测成本较高,仪器昂贵,不适于基层单位推广应用。 综上,建立准确、灵敏、操作简便、不依赖昂贵仪器的检测方法对于食源性致病菌感染的诊断、疾病控制和流行病学调查具有重要意义；发展快速准确、操作简便的检测与鉴定阪崎肠杆菌和痢疾志贺菌的方法成为相关研究的热点。目前,常用快速检测方法主要有ELISA、DNA探针、常规PCR、Real-time PCR等。环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)是一种新颖的核酸扩增技术,依赖于能够识别靶DNA上6个特定区域的4条引物和一种具有链置换活性的DNA聚合酶,在恒温条件下高效扩增核酸,具有高特异性、快速、灵敏、操作简便等特点。自从2000年以来,LAMP技术在临床疾病的诊断、病原性细菌或病毒的定性检测以及动物胚胎性别鉴定等方面应用广泛。LAMP技术在国内起步较晚,应用LAMP技术检测阪崎肠杆菌和痢疾志贺菌在国内外文献中报道较少。 本研究针对阪崎肠杆菌外膜蛋白OmpA基因和痢疾志贺茵ipaH基因,分别设计相应的LAMP引物,建立优化的反应体系,考察特异性和灵敏度,并应用于食品样品的直接检测,初步建立阪崎肠杆菌和痢疾志贺菌的LAMP检测方法。 二、研究方法 1.阪崎肠杆菌LAMP快速检测技术的建立与初步应用 (1)阪崎肠杆菌LAMP检测技术的建立 根据GenBank公布的阪崎肠杆菌外膜蛋白OmpA基因序列(Accession number:DQ000206)的保守区,利用LAMP专用软件Primer Explorer version4设计一套特异性引物,即外引物F3和B3、内引物BIP和FIP,以阪崎肠杆菌(ATCC29544)提取的DNA为模板,建立检测阪崎肠杆菌的LAMP检测技术体系,并对反应体系内各反应条件进行优化,最终摸索出优化后检测阪崎肠杆菌的LAMP反应体系。 (2)阪崎肠杆菌LAMP灵敏度、特异性实验和实际样品检测 利用建立的LAMP方法检测阪崎肠杆菌纯培养物,考察其灵敏度。将过夜培养的阪崎肠杆菌(ATCC29544)菌液10倍倍比稀释至10-1～10-8,取各稀释度菌液100ul进行平板计数。同时取各稀释度菌液1ml提取DNA,取2ul上清液作为模板进行LAMP扩增和PCR扩增,测试两种方法检测阪崎肠杆菌纯培养物的灵敏度。 选取3株阪崎肠杆菌标准菌株和大肠埃希菌、伤寒沙门菌、金黄色葡萄球菌等12株其它非阪崎肠杆菌致病菌进行特异性实验,检测LAMP方法的特异性。依据本实验所建立的LAMP和PCR的条件和参数,对36份奶粉样品进行检测,并与FDA检测方法进行比较。 2.痢疾志贺菌LAMP快速检测技术的建立与初步应用 (1)痢疾志贺菌LAMP检测技术的建立 根据GenBank公布的痢疾志贺茵ipaH基因序列(Accession number:M76445)中的保守区作为靶序列,设计痢疾志贺菌特异性引物,包括外引物F3和B3、内引物BIP和FIP、环引物LF和LB。以痢疾志贺菌(CMCC48097)提取的DNA为模板,建立检测痢疾志贺菌的LAMP检测技术,并对反应体系内各反应条件进行优化。最终得出优化后检测痢疾志贺菌的LAMP反应体系。 (2)痢疾志贺菌灵敏度、特异性实验和实际样品检测 采用建立的LAMP方法检测痢疾志贺菌纯培养液,考察其灵敏度。将过夜培养的痢疾志贺菌(CMCC48097)菌液10倍倍比稀释至10-1～10-8,取各稀释度菌液100ul进行平板计数。同时取各稀释度菌液1ml提取DNA,取2μl上清液作为模板进行LAMP扩增和PCR扩增,测试两种方法检测痢疾志贺菌纯培养物灵敏度。 同时选取4株志贺菌标准菌株和大肠埃希菌、伤寒沙门菌、金黄色葡萄球菌等其它非志贺菌进行特异性实验,检测LAMP方法的特异性,初步应用于人工污染猪肉样品痢疾志贺菌的检测,并与常规PCR方法比较。 三、结果 1.确定阪崎肠杆菌LAMP优化反应体系为：6mM MgCl2,0.6mMdNTPs,0.8M betaine,0.4μM外引物(F3和B3),1.6μM内引物(FIP和BIP),8U的Bst DNA聚合酶,2.5μl Thermopol buffer,2μL DNA模板,ddH2O补足体积至25μl；扩增温度58℃,反应60min,产物于2%琼脂糖凝胶电泳分析,得到典型的LAMP梯形条带,肉眼观察反应副产物焦磷酸镁白色沉淀。 LAMP法能检出所有的阪崎肠杆菌菌株,产生特异性扩增的梯形条带,直接用肉眼观察到焦磷酸镁白色沉淀,而其它肠道致病菌均未产生特异扩增的梯形条带。LAMP对阪崎肠杆菌纯菌液检测灵敏度为2.0×101cfu/mL,PCR检测灵敏度为2.0×102cfu/mL; LAMP检测的灵敏度是传统PCR灵敏度的10倍,检测时间缩短了2-3h。采用LAMP法检测36份奶粉样品,其中2份样品检出阪崎肠杆菌,阳性率为5.6%,与FDA方法检测结果一致。 2.确定痢疾志贺菌的LAMP反应体系为：5mM MgCl2,0.3mM dNTPs,0.6M betaine,0.4μM外引物(F3和B3),1.6μM内引物(FIP和BIP)与1.6μM环引物(LF和LB),8U Bst DNA聚合酶大片段,ThermoPol buffer2.5μl, DNA模板2μl, ddH2O补足体积至25μl；扩增温度63℃反应60min,产物于2.5%琼脂糖凝胶电泳分析,得到典型的LAMP梯形条带。 LAMP法能检出4株志贺菌,产生特异扩增的梯形条带,其余肠道致病菌均未产生特异扩增的梯形条带,表明选用的志贺菌ipaH基因引物的特异性强。LAMP法对痢疾志贺菌纯菌液检测灵敏度为5.3×101cfu/ml,对人工污染的猪肉样品的痢疾志贺菌检测限为6.8×101cfu/ml。PCR检测的灵敏度为5.3×102cfu/ml,猪肉样品为6.8×102cfu/mL, LAMP的灵敏度均比传统PCR灵敏度高10倍,整个检测过程可在2h内完成。 四、结论 本研究建立了乳制品中阪崎肠杆菌、肉制品中痢疾志贺菌的LAMP决速检测技术,并在实际样品检测中得到了初步应用。LAMP检测技术针对靶基因的6个区域设计4条(6条)特异引物,特异性强；LAMP检测限为101cfu/mL,是PCR法灵敏度的10倍。LAMP法能直接对奶粉、肉制品、乳制品等食品中的致病菌进行检测,实际样品一般在20～28h内完成检测,与传统的分离培养方法相比,大大缩短了检测时间,节省了人力、物力；使用恒温水浴装置即可完成反应,通过观察反应液浑浊度变化,或加入荧光染料SYBR Green I后观察反应液的颜色变化,直接用肉眼即可判读结果,不需要使用昂贵、精密的仪器设备,实现反应及产物检测一步完成,操作简便,检测成本低,适用于现场检测和大量样品高通量检测,尤其特别适合于病原微生物的现场快速检测、战时野外、小型实验室以及基层卫生单位的推广使用。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5
本文编号：2223228
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5
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