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Publication Number
US-8778843-B1
Publication Date
2014-07-15
Expiration Date
Abstract
Disclosed are compositions, kits, and methods for detecting, extracting, visualizing, and identifying a pathogenic protozoan. Quantitative real time polymerase chain reaction in connection with specifically designed oligonucleotide probes are used to detect a variety of pathogenic protozoans in patient samples.
Core Innovation
The disclosure describes multiplex quantitative real time polymerase chain reaction (qPCR) for determining whether a sample contains or has an increased likelihood of containing a pathogenic protozoan. A reaction mixture in a vessel includes at least one forward primer comprising SEQ ID NO: 6, at least one reverse primer comprising SEQ ID NO: 7, a nucleic acid target from the sample, and a pool of oligonucleotide probes selected from groups defined by specific probe sequences. The reaction mixture is capable of amplifying a segment of the nucleic acid target to produce an amplicon that is primed by the forward and reverse primers.
After incubation under conditions allowing production of the amplicon if the sample contains the pathogenic protozoan, the sample is determined to contain the pathogenic protozoan, or to have an increased likelihood of containing it, based on detection of the amplicon. If the amplicon is not detected, the sample is determined not to contain the pathogenic protozoan, or not to have an increased likelihood of containing it. The probe pool is selected from sets comprising oligonucleotide probes with sequences of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16 and SEQ ID NO: 17.
The disclosure further supports identification using oligonucleotide probe fluorescence and sequence alignment of probe sequences to protozoal genomic sequence. Probe fluorescence is used for detection of the oligonucleotide probes in the reaction mixture, and identifying a pathogenic protozoan is performed by aligning the sequence of the oligonucleotide probes to a genomic sequence. Sequence identity thresholds are described as alignment criteria, including at least 60%, 70%, 80%, 90%, and 95% identity.
Claims Coverage
The document includes two independent claims: a method claim and a diagnostic kit claim. Across these independent claims, the coverage centers on multiplex quantitative real time qPCR using specified forward and reverse primers and a selectable pool of oligonucleotide probes, with probe fluorescence resulting in detection, and with kit-level inclusion of result indication and instructions.
Multiplex qPCR with defined primer and probe pool for pathogenic protozoan likelihood calling
A method that determines whether a sample contains or has an increased likelihood of containing a pathogenic protozoan by providing a vessel with a reaction mixture comprising at least one forward primer comprising SEQ ID NO: 6, at least one reverse primer comprising SEQ ID NO: 7, a nucleic acid target from the sample, and a pool of oligonucleotide probes selected from groups defined by SEQ ID NO: 4/8/9, SEQ ID NO: 10/12/15, SEQ ID NO: 11/13/14, or SEQ ID NO: 16/17; amplifying to produce an amplicon by multiplex quantitative real time qPCR; incubating to allow amplicon production if present; and determining presence or increased likelihood based on amplicon detection, and determining absence or decreased likelihood based on lack of amplicon detection.
Diagnostic kit for multiplex qPCR detection with defined primers, probe pool, result indication, and instructions
A diagnostic kit for determining whether a sample contains or has an increased likelihood of containing a pathogenic protozoan comprising at least one forward primer comprising SEQ ID NO: 6 and at least one reverse primer comprising SEQ ID NO: 7; a pool of oligonucleotide probes selected from groups defined by SEQ ID NO: 4/8/9, SEQ ID NO: 10/12/15, SEQ ID NO: 11/13/14, or SEQ ID NO: 16/17; wherein the oligonucleotide probes further comprise a fluorophore and/or a quencher; an indication of a result of the presence of a nucleic acid from a pathogenic protozoan; and instructions for using the kit, wherein the kit utilizes multiplex quantitative real time qPCR (qPCR).
The claim coverage is grounded in multiplex quantitative real time qPCR performed in a vessel or provided as a kit, using SEQ ID NO: 6 forward primers and SEQ ID NO: 7 reverse primers together with a selectable pool of oligonucleotide probes defined by the specified groups of SEQ ID NOs, with kit versions requiring probe fluorophore and/or quencher and including result indication and instructions.
Stated Advantages
Allows determination of whether a sample contains or has an increased likelihood of containing a pathogenic protozoan based on detection of an amplicon in multiplex quantitative real time qPCR.
Enables identification support by using probe fluorescence and aligning probe sequences to a genomic sequence, including use of sequence identity thresholds.
Provides a diagnostic kit including primers, selectable probe pool, probe fluorescence labeling (fluorophore and/or quencher), result indication, and instructions for use with multiplex qPCR.
Documented Applications
Clinical assays for detecting and identifying pathogenic protozoa in patient samples, including Protomyxzoa rheumatica, with Ct-based analysis and a workflow supporting protozoal detection.
Assay expansion for semi-pan and “pan” protozoal surveillance.
Microscopy support of biofilm-associated infections in ALS patients using fluorescent stains/microscopy described as part of the document context.
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