Semi-pan-protozoal by quantitative PCR

摘要

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.

主权项

1. A method for determining whether a sample contains or has an increased likelihood of containing a pathogenic protozoan comprising:
a) providing a vessel containing a reaction mixture, wherein the reaction mixture comprises 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 the group consisting of:
i) oligonucleotide probes comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 9;ii) oligonucleotide probes comprising the sequences of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 15;iii) oligonucleotide probes comprising the sequences of SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14; andiv) oligonucleotide probes comprising the sequences of SEQ ID NO: 16 and SEQ ID NO: 17;wherein the reaction mixture is capable of amplifying, by a multiplex quantitative real time polymerase chain reaction (qPCR), a segment of the nucleic acid target to produce an amplicon; and wherein production of the amplicon is primed by the at least one forward primer and the at least one reverse primer;
b) incubating the vessel under conditions allowing production of the amplicon if the sample contains the pathogenic protozoan; and c) determining that the sample contains the pathogenic protozoan or that the sample has an increased likelihood of containing the pathogenic protozoan if the amplicon is detected, or determining that the sample does not contain the pathogenic protozoan or that the sample does not have an increased likelihood of containing the pathogenic protozoan if the amplicon is not detected.