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Publication Number
US-10227660-B2
Publication Date
2019-03-12
Expiration Date
Abstract
A method for detecting toxigenic Clostridium difficile (C. difficile) by strand-invasion based DNA amplification is provided, together with oligonucleotides, compositions and kits suitable for use in this method.
Core Innovation
The invention describes a method for detecting a target nucleic acid sequence of toxigenic C. difficile in a sample using strand-invasion amplification. The method contacts the sample with at least one upstream primer, at least one downstream primer, and at least one strand invasion oligonucleotide under conditions suitable for amplification by strand invasion of the target nucleic acid sequence, and each primer and the strand invasion oligonucleotide comprises a region complementary to the target nucleic acid sequence.
A strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of the upstream primer and the downstream primer. The method includes specific sequence constraints on the oligonucleotides used for strand invasion and amplification, including upstream primer length and specified sequences from SEQ ID references, downstream primer length and specified sequences from SEQ ID references, and a specified strand invasion oligonucleotide sequence.
For detection, the method assays for amplification of the target nucleic acid sequence and uses the amplification as indicative of the presence of the target nucleic acid sequence of toxigenic C. difficile in the sample. The disclosed approach is aligned with specific toxin-associated targets, including tcdA and tcdB, and supports detection performance characterized in the document, including specificity and sensitivity across relevant toxinotypes.
Claims Coverage
The document includes two independent method claims, each defining a strand-invasion-based nucleic acid detection workflow for toxigenic C. difficile with specific primer and oligonucleotide sequence constraints. Across the independent claims, there are 3 main inventive elements: primer and oligonucleotide complementarity, strand invasion rendering a target region single-stranded for primer binding, and assaying amplification as indicative of target presence; each independent claim further specifies particular SEQ ID content and length or identity constraints for the primers and the strand invasion oligonucleotide.
Strand-invasion primer set to detect toxigenic C. difficile target nucleic acid
Contacts the sample with at least one upstream primer, at least one downstream primer, and at least one strand invasion oligonucleotide under conditions suitable for amplification by strand invasion of the target nucleic acid sequence, wherein each primer and the strand invasion oligonucleotide comprises a region complementary to the target nucleic acid sequence.
Strand invasion oligonucleotide makes target single-stranded for primer binding
Renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of the upstream primer and the downstream primer, enabling amplification to be indicative of the presence of the target nucleic acid sequence of toxigenic C. difficile in the sample.
Specific primer length and SEQ ID constraints for upstream, downstream, and invasion oligonucleotide
Requires the upstream primer to be an oligonucleotide of less than 30 nucleotides comprising the sequence of SEQ ID NO: 2 or a variant thereof having at least 70% sequence identity to the sequence of SEQ ID NO: 2, requires the downstream primer to be an oligonucleotide of less than 30 nucleotides comprising the sequence of SEQ ID NO: 3 or a variant thereof having at least 70% sequence identity to the sequence of SEQ ID NO: 3, and requires the strand invasion oligonucleotide to be of the sequence of SEQ ID NO: 5.
Specific primer length and SEQ ID constraints for upstream, downstream, and invasion oligonucleotide
Requires the upstream primer to be an oligonucleotide of less than 30 nucleotides comprising the sequence of SEQ ID NO: 7 or a variant thereof having at least 70% sequence identity to the sequence of SEQ ID NO: 7, requires the downstream primer to be an oligonucleotide of less than 30 nucleotides comprising the sequence of SEQ ID NO: 8 or a variant thereof having at least 70% sequence identity to the sequence of SEQ ID NO: 8, and requires the strand invasion oligonucleotide to be of the sequence of SEQ ID NO: 10.
The claim coverage centers on strand-invasion amplification for detecting toxigenic C. difficile nucleic acid, using complementary upstream and downstream primers and a strand invasion oligonucleotide that renders a target region single-stranded for primer binding, with detection based on amplification being indicative of target presence. Independent claims additionally fix particular primer and invasion sequence identities and length constraints via specified SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5, or SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10.
Stated Advantages
High specificity and sensitivity for detecting tcdA and tcdB targets.
Inclusivity across C. difficile toxinotypes.
Documented Applications
Medical diagnostic use for diagnosis of C. difficile infection, including gastrointestinal illness (diarrhea), based on detecting tcdA/tcdB.
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