New Immuno-PCR Assay for Detection of Low Concentrations of Shiga Toxin 2 and Its Variants

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

[1]  A. Mellmann,et al.  Shiga Toxin-Mediated Hemolytic Uremic Syndrome: Time to Change the Diagnostic Paradigm? , 2007, PloS one.

[2]  L. Beutin,et al.  Comparative evaluation of the Ridascreen® Verotoxin enzyme immunoassay for detection of Shiga‐toxin producing strains of Escherichia coli (STEC) from food and other sources , 2007, Journal of applied microbiology.

[3]  A. Mellmann,et al.  Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111. , 2007, International journal of medical microbiology : IJMM.

[4]  T. Whittam,et al.  Surveillance for Shiga Toxin–producing Escherichia coli, Michigan, 2001–2005 , 2007, Emerging infectious diseases.

[5]  Laboratory-confirmed non-O157 Shiga toxin-producing Escherichia coli--Connecticut, 2000-2005. , 2007, MMWR. Morbidity and mortality weekly report.

[6]  K. Omoe,et al.  A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins , 2007, Journal of Molecular Medicine.

[7]  C. Sears,et al.  The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. , 2006, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[8]  A. Friedrich,et al.  Shiga toxin activatable by intestinal mucus in Escherichia coli isolated from humans: predictor for a severe clinical outcome. , 2006, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[9]  A. Friedrich,et al.  Application of combined high-performance thin-layer chromatography immunostaining and nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry to the structural characterization of high- and low-affinity binding ligands of Shiga toxin 1. , 2005, Rapid communications in mass spectrometry : RCM.

[10]  A. Mellmann,et al.  Shiga Toxin 2e-Producing Escherichia coli Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells , 2005, Applied and Environmental Microbiology.

[11]  H. Karch,et al.  Pigeons as a possible reservoir of Shiga toxin 2f-producing Escherichia coli pathogenic to humans. , 2005, Berliner und Munchener tierarztliche Wochenschrift.

[12]  J. Wells,et al.  Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. , 2005, The Journal of infectious diseases.

[13]  H. Karch,et al.  Enterohaemorrhagic Escherichia coli in human medicine. , 2005, International journal of medical microbiology : IJMM.

[14]  A. Mellmann,et al.  Enterohemorrhagic Escherichia coli in human infection: in vivo evolution of a bacterial pathogen. , 2005, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[15]  Christof M Niemeyer,et al.  Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection. , 2005, Biochemical and biophysical research communications.

[16]  P. Tarr,et al.  Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome , 2005, The Lancet.

[17]  A. Friedrich,et al.  Transcriptional Analysis of Genes Encoding Shiga Toxin 2 and Its Variants in Escherichia coli , 2005, Applied and Environmental Microbiology.

[18]  A. Friedrich,et al.  A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli. , 2004, Molecular nutrition & food research.

[19]  D. Edelman,et al.  Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. , 2004, American journal of clinical pathology.

[20]  H. Karch,et al.  Phenotypic and Genotypic Analyses of Enterohemorrhagic Escherichia coli O145 Strains from Patients in Germany , 2004, Journal of Clinical Microbiology.

[21]  Hwan-Wun Liu,et al.  A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A. , 2004, Toxicon : official journal of the International Society on Toxinology.

[22]  F. Gunzer,et al.  Comparison of a Shiga Toxin Enzyme-Linked Immunosorbent Assay and Two Types of PCR for Detection of Shiga Toxin-Producing Escherichia coli in Human Stool Specimens , 2003, Journal of Clinical Microbiology.

[23]  Gilbert S Chen,et al.  Shiga toxin-producing Escherichia coli in Montana: bacterial genotypes and clinical profiles. , 2003, The Journal of infectious diseases.

[24]  Christof M Niemeyer,et al.  A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins. , 2003, Biochemical and biophysical research communications.

[25]  A. Edefonti,et al.  Shiga Toxin–Producing Escherichia coli Infections Associated with Hemolytic Uremic Syndrome, Italy, 1988–2000 , 2003, Emerging infectious diseases.

[26]  D. Samuel,et al.  A quantitative immuno-PCR assay for the detection of mumps-specific IgG. , 2002, Journal of immunological methods.

[27]  J. Wells,et al.  Shiga toxin-producing Escherichia coli in children with diarrhea: a prospective point-of-care study. , 2002, The Journal of pediatrics.

[28]  H. Karch,et al.  Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli , 2002, Journal of Clinical Microbiology.

[29]  H. Karch,et al.  Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans , 2002, Journal of Clinical Microbiology.

[30]  A. Friedrich,et al.  Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. , 2002, The Journal of infectious diseases.

[31]  E. Elliott,et al.  Nationwide study of haemolytic uraemic syndrome: clinical, microbiological, and epidemiological features , 2001, Archives of disease in childhood.

[32]  J. H. Green,et al.  The United States National Prospective Hemolytic Uremic Syndrome Study: microbiologic, serologic, clinical, and epidemiologic findings. , 2001, The Journal of infectious diseases.

[33]  F. Gunzer,et al.  Rapid Detection of Enterohemorrhagic Escherichia coliby Real-Time PCR with Fluorescent Hybridization Probes , 2001, Journal of Clinical Microbiology.

[34]  H. Karch,et al.  Characterization of a Shiga Toxin 2e-Converting Bacteriophage from an Escherichia coli Strain of Human Origin , 2000, Infection and Immunity.

[35]  A. Kater,et al.  Toxin‐mediated haemolytic uraemic syndrome without diarrhoea , 2000, Journal of internal medicine.

[36]  A. Caprioli,et al.  A New Shiga Toxin 2 Variant (Stx2f) fromEscherichia coli Isolated from Pigeons , 2000, Applied and Environmental Microbiology.

[37]  D. Kobayashi,et al.  Detection of human serum tumor necrosis factor-alpha in healthy donors, using a highly sensitive immuno-PCR assay. , 1999, Clinical chemistry.

[38]  M. Petric,et al.  Evaluation of a Microplate Latex Agglutination Method (Verotox-F Assay) for Detecting and Characterizing Verotoxins (Shiga Toxins) in Escherichia coli , 1999, Journal of Clinical Microbiology.

[39]  G. Remuzzi,et al.  The hemolytic uremic syndrome. , 2005, Kidney international. Supplement.

[40]  S. Lauwers,et al.  Identification of New Verocytotoxin Type 2 Variant B-Subunit Genes in Human and Animal Escherichia coliIsolates , 1998, Journal of Clinical Microbiology.

[41]  K. Yuen,et al.  A unique strain of Escherichia coli O157:H7 that produces low verocytotoxin levels not detected by use of a commercial enzyme immunoassay kit. , 1998, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[42]  T. D. de Koning-Ward,et al.  Hemolytic-uremic syndrome following urinary tract infection with enterohemorrhagic Escherichia coli: case report and review. , 1998, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[43]  A. Stapleton,et al.  Hemolytic-uremic syndrome in a six-year-old girl after a urinary tract infection with Shiga-toxin-producing Escherichia coli O103:H2. , 1996, The New England journal of medicine.

[44]  A. O’Brien,et al.  Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice , 1996, Infection and immunity.

[45]  R. Joerger,et al.  High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. , 1995, Nucleic acids research.

[46]  M. McKee,et al.  Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escherichia coli strains are responsible for the antigenic heterogeneity of the O157:H- strain E32511 , 1991, Infection and immunity.

[47]  H. Karch,et al.  Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction , 1989, Journal of clinical microbiology.

[48]  H. Karch,et al.  Evaluation of oligonucleotide probes for identification of shiga-like-toxin-producing Escherichia coli , 1989, Journal of clinical microbiology.

[49]  H. Karch,et al.  Growth of Escherichia coli in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay , 1986 .

[50]  M. Bennish,et al.  Enzyme-linked immunosorbent assay for shigella toxin , 1986, Journal of clinical microbiology.

[51]  R. Holmes,et al.  Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities , 1986, Infection and immunity.

[52]  H. Lior,et al.  The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. , 1985, The Journal of infectious diseases.

[53]  S. Cutillo.  [Hemolytic-uremic syndrome]. , 1973, La Pediatria.