Oligonucleotides and methods for detecting Borrelia burgdorferi

Details Oligonucleotides and methods for detecting Borrelia burgdorferi Oligonucleotides and methods for detecting Borrelia burgdorferi

2001-12-04

2004-02-03

Fredman, Jeffrey (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06686162

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to compositions and methods for detecting nucleic acids for the organism
Borrelia burgdorferi
in a test sample.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Lyme disease, also known as Lyme borreliosis, is a tick-transmitted, spirochetal, inflammatory disorder causing a rash (erythema [chronicum] migrans) that may be followed weeks to months later by neurologic, cardiac, or joint abnormalities. Lyme disease was recognized in 1975 because of close clustering of cases in Lyme, Conn. It has since been reported in many states in USA and numerous foreign countries.
Lyme disease has been the most commonly reported tick-borne illness in the USA. It is caused by a spirochete,
Borrelia burgdorferi
, transmitted primarily by minute ticks of the
ixodes ricinus
complex. Once attached to the skin, they continue to engorge on blood for days. Transmission of
Borrelia burgdorferi
does not usually occur until the infected tick has been in place for at least 36 to 48 hrs; thus, screening for ticks after potential exposure and removing them can help prevent infection.
Borrelia burgdorferi
enters the skin at the site of the tick bite. It may spread in lymph, producing regional adenopathy, or disseminate in blood to organs or other skin sites. The relative paucity of organism in the involved tissue suggests that most manifestations of infection are due to host immune response rather than to the destructive properties of the organisms.
Patients with Lyme disease suffer from a variety of chronic and acute syndromes caused by
Borrelia burgdorferi
. The signs and symptoms of Lyme disease vary over time and between individuals. The characteristic clinical manifestation following the bite of an infected tick is a distinctive skin rash, erythema migrans, which often occurs in conjunction with mild constitutional symptoms. Later stages of Lyme disease may include severe arthritic, neurological, and cardiac manifestations.
The diagnosis of Lyme disease is made by clinical examination combined with evidence of tick bite or exposure in endemic areas, and this usually coincides with evidence of seroreactivity to the organism. Diagnosis of early Lyme disease in a patient with typical erythema migrans in an endemic area does not require laboratory confirmation. Titers of specific antispirochetal antibodies (first IgM, then IgG) can be determined by ELISA or by indirect immunofluorescence, but are not useful before the patient has made antibodies. Confirmation of positive titers by Western blot is also needed. In addition, false- positive results can be high. Thus, testing is best reserved for patients in whom suspicion is high. Moreover, serological testing does not recognize the presence of the spirochete itself, but rather the host's immunological response to the organism following a recent or past infection. Culture of
Borrelia burgdorferi
from blood and other body tissues is possible, but the recovery rate is low, and it may require many weeks before growth of the organism is evident.
The polymerase chain reaction (PCR) provides a sensitive and specific means of detecting the presence of
Borrelia burgdorferi
in clinical specimens, and it has been used successfully to detect
Borrelia burgdorferi
in body fluids including blood. See, e.g., (Benach et al., N. Engl. J. Med. 308: 740-742, 1983); serum (Liebling et al., Arth Rheum 36: 665-675, 1993); cerebral spinal fluid and synovial fluid (Liebling et al., Arth Rheum 36: 665-675, 1993); and in urine (Schmidt et al., Diagn. Microbiol. Infect. Dis. 21: 121-128, 1995). A diagnosis of
Borrelia burgdorferi
infections using a real-time PCR assay is also reported (Schwaiger et al.,
Clin. Microbiol. Infect.
7(9): 461-9, 2001).
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for determining the presence and/or amount of
Borrelia burgdorferi
nucleic acids in a test sample. In particular, substantially purified oligonucleotides for qualitatively and/or quantitatively detecting
Borrelia burgdorferi
nucleic acids in a test sample by amplification methods are described herein. The present invention can provide a specific, sensitive method that can exhibit a broad dynamic range of detection of
Borrelia burgdorferi
nucleic acids.
In various embodiments of the present invention, oligonucleotide primers and probes are used in the methods described herein to provide the
Borrelia burgdorferi
assay. Thus, in certain embodiments, the invention relates to primer sequences that can be used to amplify FlaA gene in the
Borrelia burgdorferi
gene sequence present in a sample. The FlaA gene in the
Borrelia burgdorferi
gene sequence encodes the flagellin protein. In addition, primers can also be used to amplify one or more control nucleic acid sequences. Control amplification primers may contain only control-specific sequences, or may be hybrid primers that can amplify the control sequence(s) while simultaneously introducing FlaA gene sequences into the control amplicon produced. By introducing FlaA gene sequences into the control amplicon, the control can be introduced into test samples and amplified by the same primers used to amplify the target FlaA gene in the
Borrelia burgdorferi
gene sequences, providing a convenient positive control.
In additional embodiments, the invention relates in part to probe nucleic acids that can be conjugated to a detectable label, preferably, a fluorescent dye, and most preferably a dye pair located at the 5′ and 3′ end of the oligonucleotides. Certain labeled oligonucleotides are described that hybridize to amplified FlaA gene in the
Borrelia burgdorferi
gene sequences, if present, in the sample. Similarly, certain labeled oligonucleotides are described that hybridize to a control amplicon that may have been introduced into the test sample as a positive control.
In a first aspect, the invention relates to one or more substantially purified oligonucleotides having sequences selected from the following group:
5′-TTG CAA ATC TTT TCT CTG GTG-3′ (SEQ ID NO:1), a Fla A gene sequence;
5′-AGA ATT AAC TCC GCC TTG AGA-3′ (SEQ ID NO:2), a Fla A gene sequence;
5′-CCT TCC TGT TGA ACA CCC TCT TGA AC-3′ (SEQ ID NO:3), a probe hybridizing to Fla A gene sequence;
5′-CTT GTA CCA GTT GTA CGG TCC-3′ (SEQ ID NO:4), a human placental DNA sequence suitable for use as a primer for internal positive control amplification;
5′-GGT AGC AGC GGT AGA GTT GTA-3′ (SEQ ID NO:5)), a human placental DNA sequence suitable for use as a primer for internal positive control amplification; and
5′-ATC ATG ATG TTC AAG TTG TGT TTT GC-3′ (SEQ ID NO:6), a human placental DNA sequence suitable for use as a probe for hybridizing to human placental DNA.
In preferred embodiments, one or more of the selected oligonucleotides can be conjugated to a detectable label, preferably a fluorescent dye, and most preferably a dye pair. Particularly preferred oligonucleotide dye conjugates are 5′-[6-carboxyfluorescein(FAM)]-CCT TCC TGT TGA ACA CCC TCT TGA AC-[6-carboxytetramethylrhodamine (TAMRA)]-3′ (SEQ ID NO:7); and 5′[2′-Chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC)]-ATC ATG ATG TTC AAG TTG TGT TTT GC-[6-carboxytetramethylrhodamine (TAMRA)]3′ (SEQ ID NO:8). These may be used as probes for FlaA gene and human placental DNA, respectively, in methods to detect the presence or amount of specific nucleic acids present in a test sample.
In another aspect, the present invention relates in part to methods that use primers to a sequence unrelated to
Borrelia burgdorferi
, e.g., human placental nucleic acid primers, to produce control amplicons as a positive assay control. In preferred embodiments, oligonucleotides having

Affiliated with

Also associated with

Rating

Oligonucleotides and methods for detecting Borrelia burgdorferi does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Oligonucleotides and methods for detecting Borrelia burgdorferi, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oligonucleotides and methods for detecting Borrelia burgdorferi will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3310601