| 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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