检测肠出血性大肠埃希菌O157：H7及其它产志贺毒素大肠埃希菌的一种新的DNA扩增技术
摘要
肠出血性大肠埃希菌O157：H7和其它产志贺样毒素大肠埃希菌是引起人类疾病的重要食源性致病菌，它们主要通过食物链传播。引起严重的临床症状，包括出血性肠炎、溶血性尿毒综合征和血小板减少性紫癜。发病率和死亡率都很高，而且很低的感染剂量就可威胁生命。传统的检测方法如分离培养、血清学鉴定，缺乏特异性和灵敏度，需要几天才能出结果，费时又费力。而常用的分子生物学方法如PCR虽然具有高度的灵敏度和特异度，但需要特殊的仪器，而且易产生假阳性结果，标本中存在的抑制剂会抑制PCR扩增产生假阴性结果。因此急需建立一种快速、灵敏的检测方法，用于病人的早期诊断、食品的监测和疾病爆发的调查。
本实验介绍的网状分枝扩增技术是一种新奇的、恒温的DNA扩增方法，该方法利用环形探针和捕获探针分离和检测靶序列，当环形探针与靶基因结合时，在DNA连接酶的作用下以共价键连接成环状。然后，延着环形探针DNA聚合酶延伸连接的正向引物，置换下游链，产生多聚ssDNA，再以多聚ssDNA作为模板，反向引物与它杂交、延伸、置换下游链，产生大的分枝DNA复合物，导致指数级扩增，整个反应在等温条件下进行。
为了确定该方法检测志贺毒素基因的灵敏度和特异度，以及确定该方法检测从食品、动物粪便和临床标本中分离菌株的可行性，我们设计并合成了志贺毒素基因和检测靶基因的环形探针和捕获探针，结果表明RAM最
低能检测10个拷贝的志贺毒素基因，与PCR的灵敏度一致。我们又进一步检测不同血清型的产志贺样毒素大肠埃希菌O157：H7、O26：H11，O46：H38，O111：NM，O22：H8，以及3株非致病性大肠埃希菌和1株痢疾志贺杆菌，结果表明不同血清型产志贺样毒素大肠埃希菌和痢疾志贺杆菌志贺毒素均为阳性，而非致病性大肠埃希菌则为阴性，说明RAM具有高度的特异性。我们从食品、动物粪便和临床标本中分离127株大肠埃希菌，其中O157：H7为9株，来自牛粪、鸡粪、未消毒牛奶，而在临床病人标本中未检出O157：H7，用RAM和PCR同时要检测了这些菌株的志贺毒素基因，结果表明产志贺毒素大肠埃希菌为21株，占16.5%，而且两种方法检测结果一致。RAM除了具有高度的灵敏度和特异度，还具有以下特点。第一，由于闭合环形探针的形成是以环形探针与靶DNA特异性结合为前提，因此明显地提高了反应的特异性；第二，环形探针两端的连接与靶核酸的性质（DNA和RNA）无关，因而在检测RNA时无须进行逆转录，检测RNA 和DNA可以应用的一样的方法；第三，通过磁性分离方法废弃了以往、烦琐的人工DNA提取过程，使得临床实验室对大批量标本的检测成为可能；第四，由于没有对温度变化的要求，RAM反应可在最简单的水浴箱内进行，而无需价格昂贵的PCR仪器，因此RAM更适用于在基层医疗单位推广使用；第五，在RAM反应中，用于检测不同靶基因的环形探针可同时被一对引物扩增，消除了引物之间竞争，这样在一个反应中就可同时检测能引起腹泻的病原菌如志贺痢疾杆菌、伤寒沙门菌，STEC等。
虽然液相RAM检测简化了在实验室中检测大肠埃希菌O157及其它STEC的方法，提高了检出率，但它不适用于在实验室外监测食品和现场检测病人标本中的STEC。因此，我们研究了在固相支持物进行RAM反应，制成检测纸片，用于现场检测食品和病人标本。在本实验中，用抗O157多克隆抗体将菌株捕获在硝酸纤维素膜上，裂解细菌释放DNA，DNA变性后，
用环形探针与之杂交，在DNA连接酶的作用下环形探针闭合成环状，再在DNA 聚合酶作用下，扩增环形探针，用免疫学方法检测扩增产物并观察扩增结果。我们利用固相RAM检测来自不同国家地区分离的产志贺样毒素大肠埃希菌0157：H7，但该方法的灵敏度还有待进一步地提高。固相RAM操作不需复杂仪器，结果易于观察，便于推广，更适用于现场检测，这对早期诊断、病人治疗、追踪传染源、控制传播途径具有重要意义。
大肠埃希菌O157：H7和其它产志贺样毒素大肠埃希菌可通过食物链传播，牛是主要宿主，而且许多次疾病的暴发都是由于食用了污染牛肉引起的，牛肉馅中污染O157：H7的量是很少的，通常少于100CFU/g，但是极微量的O157：H7就可致病。PCR是目前比较理想的检测O157：H7和其它产贺样毒素大肠埃希菌的方法，但是标本中存在多种PCR抑制剂，如牛肉中血红蛋白、脂肪和有机化合物，就是PCR反应的潜在抑制剂，即使少量的抑制剂可能在很大程度上抑制PCR反应。在实验中，我们以牛肉馅为标本模型，在牛肉馅中加入不同浓度的大肠埃希菌O157：H7，通过离心、过滤回收牛肉馅中的菌株，去除牛肉馅中的食物颗粒和抑制剂，并且采用酶裂解和反复冻融使菌株彻底裂解，释放DNA。实验结果表明，100×g的离心速度是最适宜的，既可有效地去除食物颗粒又能高效地回收细菌。本实验在不增菌的条件下，采用酶裂解和冻融法制备模板，PCR在牛肉馅中最低难检出103CFU/g，而且重复性很好，仅需要6h。增菌过程明显地提高了检测的灵敏度，增菌6h后能检测1CFU/g，这样利用酶裂解和冻融法制备模板，检出1CFU/g，就需要12h，与分离方法相比明显地节省了时间。这种标本处理和制备方法简便易行，总消费与传统的增菌分离培养方法是基本相同的。利用该方法?
E.coli O157:H7 and other Shiga toxin-producing E.coli(STEC) are important human pathogens that are mainly transmitted through the food chain.They can result in severe clinical manifestations,including haemorrhagic colitis,haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura,symptoms associated with high morbidity and mortality.The pathogens have a low infectious dose and may cause life-threatening illness.Conventional methods for identification of these organisms such as culture isolation and serological test,lack specificity and sensitivity,and requires several days to generate results.They are laborious and consuming.Existing molecular methods such as PCR require sophisticated equipment and easy to cause false-positive result.The inhibitors in food and clinical speciments interferes with the reaction,therefore,leading to false-negative result.There is an urgent need for methods that can provide rapid,sensitive detection of the organism,in order to guide clinical management of patients,to monitor food processing and to investigate outbreaks.
In this study,we introduced a noval,isothemal DNA
amplification technology,termed ramification amplification method(RAM),which utilizes a circular probe and a capture probe for target seperation and detection.When the C-probe hybridizes to a target,a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand,generating a multimeric single-stranded DNA(ssDNA).Then this ssDNA serve as a template for multiple reverse primers to hybridize,extend and displace downstream DNA,generating a large ramified DNA complex,and resulting in an exponential amplification under an isothermal condition.
In order to determine the analytical sensitivity and specificity of RAM to detect Shiga toxin and to determine the feasibility to detect E.coli O157:H7 and other STEC isolated from food and clinical specimens,we designed and synthesized Shiga toxin target ,C-probe and capture probe.The result showed that as few as 10 copies of synthesized target can be detected by RAM,which indicated that RAM assay is as sensitive as conventional PCR.We further test different serotypes which contain stx genes,including a E.coli O157:H7,a E.coli O26：H11，a E.coli O46：H38，a E.coli O111：NM，a E.coli O22：H8,and a Shigalla as well as 3 nonpathogenic E.coli by RAM.The results showed that different serotypes of strains were all positive for Shiga toxin gene,and Shigalla was also positive,
while nonpathogenic E.coli were negative.We isolated 127 strains of E.coli from food,animal feces and clinical specimens,among of which 9 strains are O157:H7,isolated from cattle fecal,chicken
fecal and raw milk.O157:H7 couldn’t be detected in clinical specimens.We further detected shiga toxin gene from these strains by PCR and RAM.21 Shiga toxin-producing isolates were detected.The result of RAM is similar to that of PCR.Besides of high sensitivity and specificity,RAM also offers several other unique features.
First,the formation of closed C-probe requires target-specific ligation of the C-probe,therefore significantly increasing assay specificity.Second,both ends of the probe can be ligated regardless of the nature of target(DNA or RNA)eliminating the need for reverse transcription for detecting RNA and creating a uniform assay format for both RNA and DNA detection.Third, the use of magnetic isolation eliminates complicated, laborious manual DNA extraction procedures and enables the high-throughput processing of a large number of specimens in a clinical laboratory setting.Forth,since no temperature cycling is required,the reaction can be carried out in a simple water bath,obviating the use of an expensive thermocycler.
Fifth,different C-probes specific for different targets can be amplified with the same set of primers,eliminating competition among primers.This offers great adva ntage for the detection of most common food contaminants that can cause diarrhea(i.e. STEC,
Shigella,Salminella,etc) in a single reaction.
The RAM assay will
本实验介绍的网状分枝扩增技术是一种新奇的、恒温的DNA扩增方法，该方法利用环形探针和捕获探针分离和检测靶序列，当环形探针与靶基因结合时，在DNA连接酶的作用下以共价键连接成环状。然后，延着环形探针DNA聚合酶延伸连接的正向引物，置换下游链，产生多聚ssDNA，再以多聚ssDNA作为模板，反向引物与它杂交、延伸、置换下游链，产生大的分枝DNA复合物，导致指数级扩增，整个反应在等温条件下进行。
为了确定该方法检测志贺毒素基因的灵敏度和特异度，以及确定该方法检测从食品、动物粪便和临床标本中分离菌株的可行性，我们设计并合成了志贺毒素基因和检测靶基因的环形探针和捕获探针，结果表明RAM最
低能检测10个拷贝的志贺毒素基因，与PCR的灵敏度一致。我们又进一步检测不同血清型的产志贺样毒素大肠埃希菌O157：H7、O26：H11，O46：H38，O111：NM，O22：H8，以及3株非致病性大肠埃希菌和1株痢疾志贺杆菌，结果表明不同血清型产志贺样毒素大肠埃希菌和痢疾志贺杆菌志贺毒素均为阳性，而非致病性大肠埃希菌则为阴性，说明RAM具有高度的特异性。我们从食品、动物粪便和临床标本中分离127株大肠埃希菌，其中O157：H7为9株，来自牛粪、鸡粪、未消毒牛奶，而在临床病人标本中未检出O157：H7，用RAM和PCR同时要检测了这些菌株的志贺毒素基因，结果表明产志贺毒素大肠埃希菌为21株，占16.5%，而且两种方法检测结果一致。RAM除了具有高度的灵敏度和特异度，还具有以下特点。第一，由于闭合环形探针的形成是以环形探针与靶DNA特异性结合为前提，因此明显地提高了反应的特异性；第二，环形探针两端的连接与靶核酸的性质（DNA和RNA）无关，因而在检测RNA时无须进行逆转录，检测RNA 和DNA可以应用的一样的方法；第三，通过磁性分离方法废弃了以往、烦琐的人工DNA提取过程，使得临床实验室对大批量标本的检测成为可能；第四，由于没有对温度变化的要求，RAM反应可在最简单的水浴箱内进行，而无需价格昂贵的PCR仪器，因此RAM更适用于在基层医疗单位推广使用；第五，在RAM反应中，用于检测不同靶基因的环形探针可同时被一对引物扩增，消除了引物之间竞争，这样在一个反应中就可同时检测能引起腹泻的病原菌如志贺痢疾杆菌、伤寒沙门菌，STEC等。
虽然液相RAM检测简化了在实验室中检测大肠埃希菌O157及其它STEC的方法，提高了检出率，但它不适用于在实验室外监测食品和现场检测病人标本中的STEC。因此，我们研究了在固相支持物进行RAM反应，制成检测纸片，用于现场检测食品和病人标本。在本实验中，用抗O157多克隆抗体将菌株捕获在硝酸纤维素膜上，裂解细菌释放DNA，DNA变性后，
用环形探针与之杂交，在DNA连接酶的作用下环形探针闭合成环状，再在DNA 聚合酶作用下，扩增环形探针，用免疫学方法检测扩增产物并观察扩增结果。我们利用固相RAM检测来自不同国家地区分离的产志贺样毒素大肠埃希菌0157：H7，但该方法的灵敏度还有待进一步地提高。固相RAM操作不需复杂仪器，结果易于观察，便于推广，更适用于现场检测，这对早期诊断、病人治疗、追踪传染源、控制传播途径具有重要意义。
大肠埃希菌O157：H7和其它产志贺样毒素大肠埃希菌可通过食物链传播，牛是主要宿主，而且许多次疾病的暴发都是由于食用了污染牛肉引起的，牛肉馅中污染O157：H7的量是很少的，通常少于100CFU/g，但是极微量的O157：H7就可致病。PCR是目前比较理想的检测O157：H7和其它产贺样毒素大肠埃希菌的方法，但是标本中存在多种PCR抑制剂，如牛肉中血红蛋白、脂肪和有机化合物，就是PCR反应的潜在抑制剂，即使少量的抑制剂可能在很大程度上抑制PCR反应。在实验中，我们以牛肉馅为标本模型，在牛肉馅中加入不同浓度的大肠埃希菌O157：H7，通过离心、过滤回收牛肉馅中的菌株，去除牛肉馅中的食物颗粒和抑制剂，并且采用酶裂解和反复冻融使菌株彻底裂解，释放DNA。实验结果表明，100×g的离心速度是最适宜的，既可有效地去除食物颗粒又能高效地回收细菌。本实验在不增菌的条件下，采用酶裂解和冻融法制备模板，PCR在牛肉馅中最低难检出103CFU/g，而且重复性很好，仅需要6h。增菌过程明显地提高了检测的灵敏度，增菌6h后能检测1CFU/g，这样利用酶裂解和冻融法制备模板，检出1CFU/g，就需要12h，与分离方法相比明显地节省了时间。这种标本处理和制备方法简便易行，总消费与传统的增菌分离培养方法是基本相同的。利用该方法?
E.coli O157:H7 and other Shiga toxin-producing E.coli(STEC) are important human pathogens that are mainly transmitted through the food chain.They can result in severe clinical manifestations,including haemorrhagic colitis,haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura,symptoms associated with high morbidity and mortality.The pathogens have a low infectious dose and may cause life-threatening illness.Conventional methods for identification of these organisms such as culture isolation and serological test,lack specificity and sensitivity,and requires several days to generate results.They are laborious and consuming.Existing molecular methods such as PCR require sophisticated equipment and easy to cause false-positive result.The inhibitors in food and clinical speciments interferes with the reaction,therefore,leading to false-negative result.There is an urgent need for methods that can provide rapid,sensitive detection of the organism,in order to guide clinical management of patients,to monitor food processing and to investigate outbreaks.
In this study,we introduced a noval,isothemal DNA
amplification technology,termed ramification amplification method(RAM),which utilizes a circular probe and a capture probe for target seperation and detection.When the C-probe hybridizes to a target,a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand,generating a multimeric single-stranded DNA(ssDNA).Then this ssDNA serve as a template for multiple reverse primers to hybridize,extend and displace downstream DNA,generating a large ramified DNA complex,and resulting in an exponential amplification under an isothermal condition.
In order to determine the analytical sensitivity and specificity of RAM to detect Shiga toxin and to determine the feasibility to detect E.coli O157:H7 and other STEC isolated from food and clinical specimens,we designed and synthesized Shiga toxin target ,C-probe and capture probe.The result showed that as few as 10 copies of synthesized target can be detected by RAM,which indicated that RAM assay is as sensitive as conventional PCR.We further test different serotypes which contain stx genes,including a E.coli O157:H7,a E.coli O26：H11，a E.coli O46：H38，a E.coli O111：NM，a E.coli O22：H8,and a Shigalla as well as 3 nonpathogenic E.coli by RAM.The results showed that different serotypes of strains were all positive for Shiga toxin gene,and Shigalla was also positive,
while nonpathogenic E.coli were negative.We isolated 127 strains of E.coli from food,animal feces and clinical specimens,among of which 9 strains are O157:H7,isolated from cattle fecal,chicken
fecal and raw milk.O157:H7 couldn’t be detected in clinical specimens.We further detected shiga toxin gene from these strains by PCR and RAM.21 Shiga toxin-producing isolates were detected.The result of RAM is similar to that of PCR.Besides of high sensitivity and specificity,RAM also offers several other unique features.
First,the formation of closed C-probe requires target-specific ligation of the C-probe,therefore significantly increasing assay specificity.Second,both ends of the probe can be ligated regardless of the nature of target(DNA or RNA)eliminating the need for reverse transcription for detecting RNA and creating a uniform assay format for both RNA and DNA detection.Third, the use of magnetic isolation eliminates complicated, laborious manual DNA extraction procedures and enables the high-throughput processing of a large number of specimens in a clinical laboratory setting.Forth,since no temperature cycling is required,the reaction can be carried out in a simple water bath,obviating the use of an expensive thermocycler.
Fifth,different C-probes specific for different targets can be amplified with the same set of primers,eliminating competition among primers.This offers great adva ntage for the detection of most common food contaminants that can cause diarrhea(i.e. STEC,
Shigella,Salminella,etc) in a single reaction.
The RAM assay will
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