Oligonucleotides and methods for detecting Borrelia burgdorferi

Abstract

The present invention provides methods and compositions for determining the presence and/or amount of Borrelia burgdorferi nucleic acids in a test sample related to Lyme disease. In particular, substantially purified oligonucleotide primers and probes are described that can be used for qualitatively and quantitatively detecting Borrelia burgdorferi nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for assessing internal quality control of the Borrelia burgdorferi assay.

1 . A method for detecting the presence or amount of  Borrelia burgdorferi  nucleic acids in a test sample, comprising: 
 (a) amplifying FlaA nucleic acids in  Borrelia burgdorferi  nucleic acid sequence if present in said sample using a pair of oligonucleotide primers having the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2;    (b) hybridizing said amplified FlaA nucleic acids with an oligonucleotide probe having the sequence set forth in SEQ ID NO:3, wherein said probe is conjugated to 6-carboxyflroresceine (FAM) and 6-carboxytetramethylrhodamine (TAMRA), in the presence of an enzyme that cleaves said probe when said probe hybridizes to said HBV nucleic acid; and    (c) detecting a signal from said probe, wherein said signal indicates the presence or amount of  Borrelia burgdorferi  nucleic acids in said test sample.    
     
     
         2 . The method of  claim 1 , wherein human placental nucleic acid are introduced into said test sample and amplified using the pair of oligonucleotide primers to produce human placental amplicons.  
     
     
         3 . The method of  claim 2 , wherein said human placental amplicons are hybridized to a control oligonucleotide probe having the sequence set forth in SEQ ID NO:6, wherein the control oligonucleotide probe is conjugated to 2′-chloro-5-fluoro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and 6-carboxytetramethylrhodamine (TAMRA).  
     
     
         4 . The method of  claim 1 , wherein said test sample is selected from the group consisting of serum, blood, plasma, cerebral spinal fluid, synovial fluid, and urine.  
     
     
         5 . The method of  claim 1 , wherein said  Borrelia burgdorferi  nucleic acids are purified from said sample prior to said amplifying step (a).  
     
     
         6 . The method of  claim 5 , wherein said human placental nucleic acid is introduced into said test sample prior to purifying said  Borrelia burgdorferi  nucleic acids from said sample.