Compositions and methods for detecting
Disclosed are oligonucleotides useful in methods for determining whether a sample contains Borrelia afzelii , a spirochete which is a causative agent of Lyme disease in humans. These oligonucleotides, which have nucleotide sequences derived from a coding segment of the gene encoding the p24 gene for the outer surface protein of Borrelia afzelii , are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as templates, and as probes for detecting any resultant amplicon. Detection of an amplicon indicates the sample contains Borrelia afzelii . Real-time PCR and detection using florescence resonance energy transfer is disclosed.
1. A method of detecting the presence of Borrelia afzelii in a biological sample, comprising:
(a) mixing
(i) DNA extracted from said biological sample,
(ii) a primer pair comprising a forward primer and a reverse primer that target a p24 gene of Borrelia afzelii , and
(iii) a hybridization probe, in a PCR vessel, wherein said hybridization probe comprises a fluorescent moiety;
(b) amplifying said mixture of step (a), in a real-time PCR reaction, under conditions to allow production of an amplicon; and
(c) detecting a presence or absence of Borrelia afzelii , by detecting a presence or absence of a fluorescent signal resulting from the formation of said amplicon, wherein the presence of a fluorescent signal is indicative of the presence of Borrelia afzelii,
wherein said forward primer consists of nucleotide sequence 453-474 set forth in SEQ ID NO: 1, said reverse primer consists of nucleotide sequence 74-95 set forth in SEQ ID NO: 2, and said hybridization probe consists of nucleotide sequence 492-521 of SEQ ID NO: 1.
2. A kit for PCR detection of Borrelia afzelii , comprising:
(a) a forward primer that consists of nucleotides 453-474 of SEQ ID NO: 1;
(b) a reverse primer that consists of nucleotides 74-95 of SEQ ID NO: 2; and
(c) a hybridization probe that consists of nucleotides 492-521 of SEQ ID NO: 1.