Detection of Shiga toxin- or Shiga-like toxin-producing organisms
First Claim
Patent Images
1. A method for detecting the presence or absence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in a biological sample from an individual, said method comprising:
- performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of stx1 primers to produce an amplification product if a nucleic acid molecule encoding a Shiga toxin- or Shiga-like toxin is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of stx1 probes, wherein the members of said pair of stx1 probes hybridize to said amplification product within no more than five nucleotides of each other, wherein a first stx1 probe of said pair of stx1 probes is labeled with a donor fluorescent moiety and wherein a second stx1 probe of said pair of stx1 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first stx1 probe and said acceptor fluorescent moiety of said second stx1 probe, wherein the presence of FRET is indicative of the presence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in said biological sample, and wherein the absence of FRET is indicative of the absence of a Shiga toxin- or Shiga-like toxin-producing organism in said biological sample.
0 Assignments
0 Petitions
Accused Products
Abstract
The invention provides methods to detect Shiga toxin- or Shiga-like toxin-producing organisms, particularly Shiga-like toxin-producing E. coli organisms, in biological samples using real-time PCR. Primers and probes for the detection of Shiga toxin- or Shiga-like toxin-producing organisms are provided by the invention. Articles of manufacture containing such primers and probes for detecting Shiga toxin- or Shiga-like toxin-producing organisms are further provided by the invention.
-
Citations
36 Claims
-
1. A method for detecting the presence or absence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in a biological sample from an individual, said method comprising:
-
performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of stx1 primers to produce an amplification product if a nucleic acid molecule encoding a Shiga toxin- or Shiga-like toxin is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of stx1 probes, wherein the members of said pair of stx1 probes hybridize to said amplification product within no more than five nucleotides of each other, wherein a first stx1 probe of said pair of stx1 probes is labeled with a donor fluorescent moiety and wherein a second stx1 probe of said pair of stx1 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first stx1 probe and said acceptor fluorescent moiety of said second stx1 probe, wherein the presence of FRET is indicative of the presence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in said biological sample, and wherein the absence of FRET is indicative of the absence of a Shiga toxin- or Shiga-like toxin-producing organism in said biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 27)
-
-
24. A method for detecting the presence or absence of one or more Shiga-like toxin-producing E. coli organisms in a biological sample from an individual, said method comprising:
-
performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of stx2 primers to produce a stx2 amplification product if an E. coli Shiga-like toxin stx2 nucleic acid molecule is present in said sample, wherein said hybridizing step comprises contacting said sample with a pair of stx2 probes, wherein the members of said pair of stx2 probes hybridize to said amplification product within no more than five nucleotides of each other, wherein a first stx2 probe of said pair of stx2 probes is labeled with a donor fluorescent moiety and wherein a second stx2 probe of said pair of stx2 probes is labeled with a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first stx2 probe and said acceptor fluorescent moiety of said second stx2 probe, wherein the presence of FRET is indicative of the presence of one or more Shiga-like toxin-producing E. coli organisms in said biological sample, and wherein the absence of FRET is indicative of the absence of a Shiga-like toxin-producing E. coli organism in said biological sample. - View Dependent Claims (25, 26)
-
-
28. A method for detecting the presence or absence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in a biological sample from an individual, said method comprising:
-
performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of stx1 primers to produce an amplification product if a nucleic acid molecule encoding Shiga toxin or Shiga-like toxin is present in said sample, wherein said hybridizing step comprises contacting said sample with a stx1 probe, wherein said stx1 probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and
detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety and said acceptor fluorescent moiety of said stx1 probe, wherein the presence or absence of FRET is indicative of the presence or absence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in said sample. - View Dependent Claims (29, 30, 31, 32, 33)
-
-
34. A method for detecting the presence or absence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in a biological sample from an individual, said method comprising:
-
performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step, wherein said amplifying step comprises contacting said sample with a pair of stx1 primers to produce an amplification product if a nucleic acid molecule encoding Shiga toxin or Shiga-like toxin is present in said sample, wherein said dye-binding step comprises contacting said amplification product with a double-stranded nucleic acid binding dye; and
detecting the presence or absence of binding of said double-stranded nucleic acid binding dye to said amplification product, wherein the presence of binding is indicative of the presence of one or more Shiga toxin- or Shiga-like toxin-producing organisms in said sample, and wherein the absence of binding is indicative of the absence of a Shiga toxin- or Shiga-like toxin-producing organism in said sample. - View Dependent Claims (35, 36)
-
Specification