Rapid Detection Of Enterohemorrhagic Escherichia Coli O157

Enterohemorrhagic Escherichia coli(EHEC)O157:H7 is a leading cause of foodborne illness. Infection with EHEC O157:H7 often leads to severe bloody diarrhea and abdominal cramps. In some persons the infection can also cause the life-threatening hemolytic uremic syndrome (HUS).EHEC O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea in USA. Now it spreads in many country include British, Canada, Japan, and so on. Recently, many cases of infection occur in China. An outbreak of EHEC O157:H7 infection in Jiangsu province in 1999 was reported. It indicated that EHEC O157 becomes threaten of public health in China. EHEC O157:H7 has been found in the intestines of healthy cattle, deer, goats, and sheep. Ruminants are principal reservoir. Because the organism lives in the intestines of healthy cattle, it would be difficult to eradicate this pathogenic bacterium thoroughly.Antibiotic therapy could increase releasing of Shiga Toxin and lead to severe consequence. Because there is no specific treatment of the disease currently available, there is an urgent need for effective preventive measures. Such measures will be dependent on the availability of rapid, sensitive, and simple procedures for the detection of the pathogen. Classical bacteriological separation method is gold standard, but it is labor and time consuming. Immunological methods and molecular biology methods for identification of EHEC O157:H7 are important more and more.Multiplex PCR method was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 and stx2), intimin (eaeA) and H7 flagellin antigen (fliC). Four pair's primers were synthesized. The size of amplified products is different, so it is easy to distinguish the bands on agarose gel. PCR products of these genes were generated with DNAs from EHEC O157:H7 simultaneously. The method includes enrichment before the PCR reaction. TSB medium was modified by adding antibiotics and bile salt to improve the sensibility of the Mul-PCR. The sensitivity was evaluated by detection of artificially contaminated samples. The results showed that this method is sensitive, specific and simple. Then a Mul-PCR kit was equipped and technology standard was formulated. The kit was used to detection of practical sample. EHEC O157 detection rate was 3%. This result exhibited 100% agreement with SMART II Rapid E. coli O157 Strip test.FQ-PCR assay was also used to quantify EHEC O157. Primers and TaqMan probes were designed to amplify and quantify the intimin gene (eae) of EHEC O157:H7. Specificity was confirmed with DNA from 3 EHEC O157:H7 strains and related strains. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity of EHEC O157:H7 DNA (correlation coefficient >0.998). A similar correlation was observed between the CT and number of CFU ml-1. A detection limit of approximately 3 CFU ml-1 of EHEC O157 based on plate counts was determined.For further improvement of detection methods, spleen cells collected from BALB/c mice immunized with the whole cell antigen of EHEC O157:H7 were fused with murine Sp2/0 myeloma cells. 3 hybridoma cell lines specific to EHEC O157 was established after screening and subcloning. One of them designated 3D6 has the best property. The monoclonal antibodies (McAb) 3D6 belonged to mouse IgG and reacted with all three EHEC O157:H7 strains but no other bacteria strains, including 7 strains of non-O157 E. coli and 11 non-E.coli strains. The titer of the purified antibody was 1:6.4×106. Affinity constant exceeded 6.14×10-11mol/L. Its speciesspecific epitope located on O157 LPS. It had been proved that this McAb process high degree of specificity by indirect fluorescent antibody test and coagglutination assay. The McAb should be a useful immunological reagent for EHEC O157 detection.In some cases, EHEC O157 may be present in low numbers and difficult to be identified among high numbers of other background bacteria. On the other hand, some materials in soil and feces samples could inhibit the reaction of PCR. In order to increase the sensitivity of PCR detection, an immunomagnetic separation (IMS) method was established. Magnetic beads were coated with McAb 3D6 which against surface antigan of O157 LPS. The pathogenic bacteria in artificially contaminated samples was separated by IMS and detected with Mul-PCR subsequently. It was recovered from initial inocula of 105 CFU g-1 soil by direct detection and 2×102~103 CFU g-1 by IMS. Sensitivity of detection increased more than 100 times for the artificially contaminated soil and fecal. After treated with IMS, quantification of EHEC O157 in soil and feces was possible when cell numbers were 102×103 CFU g-1 and 105 CFU g-1 respectively. These results indicated that the IMS can reduce the total test time, and improve the sensitivity for rapid detection of EHEC O157. With process of IMS method, the Mul-PCR and FQ-PCR offered a sensitive, efficient, and reliable approach to qualitative and quantitative detection of EHEC O157 in environment samples.Immunochromatographic (IC) strip for the rapid detection of Escherichia coli O157 in enriched samples was developed. McAb 3D6 was conjugated with colloidal gold particles by the citrate method. The specificity of the IC strip was determined using 21 pure-cultured bacteria, including 10 E. coli strains and 11 non-E. coli strains. All 3 EHEC O157 strains produced a positive signal, whereas the others did not. The sensitivity of the IC strip was determined using 10-fold diluted EHEC O157:H7, with a range of 5.6×107 to 56 CFU g-1 in raw beef after enrichment. These results indicated that the IC strip exhibited high specificity and sensitivity in the detection of EHEC O157, and this assay is rapid, economical, and simple, without requirement of complicated equipment.In this study, available Mul-PCR kit and other methods were established and improved for detection of EHEC O157. A set of systematical detection strategy was constructed, which can be used in routine clinical microbiological laboratories. These methods will be valuable in the rapid and accurate detection of EHEC O157 in environmental and clinical samples.