US 6,087,097 AGrant

PCR Detection of Borrelia Burgdorferi

Issue Date:2000-07-11

•13 Claims

•22 Drawing Sheets

Abstract

A method for detecting in a biological sample the presence of Lyme-disease causing spirochetes using the polymerase chain reaction (PCR). This method includes the steps of: isolating DNA from the biological sample; amplifying the isolated DNA under hybridizing conditions with a primer pair that targets portions of extrachromosomal linear plasmid gene encoding outer surface protein A (OspA) or outer surface protein B (OspB) of the Lyme-disease causing spirochetes, probing the amplified DNA under hybridizing conditions with a labeled gene probe; and detecting the labeled gene probe that hybridized to the amplified DNA of the Lyme-disease causing spirochetes.

Metadata

Assignee

Mayo Foundation for Medical Education and Research

Inventor

David H. Persing

Application Information

Application Number:US 642807&

Filing Date:1996-05-03

Priority Date:1994-05-12

Art Unit:165

Classifications

IPC:

C12Q 168C12P 1934C07H 2102C07H 2104

Field of Search:

4355366;91.1;91.224.3;24.31;24.32;24.33

Patent Drawings (22 sheets)

Description

BACKGROUND OF THE INVENTION

Lyme borreliosis (Lyme disease), a systemic illness with a wide spectrum of clinical symptoms, was named for Lyme, Conn., where the disease was identified. Although subsets of the diverse clinical manifestations of Lyme disease were recorded in Europe early in this century, recognition of the disease as a distinct clinical entity did not occur until the mid-1970s. Today, Lyme disease is the most common tickborne zoonosis in the United States, with more than 6000 human infections reported each year.

Lyme disease is a multisystem disorder with dermatologic, neurologic and musculoskeletal components that is caused by the spirochete Borrelia burgdorferi. The risk of a human acquiring Lyme disease is dependent on an interplay of microbial, environmental, and demographic factors. Ultimately, transmission is effected by nymphal ticks of the Ixodes ricinus complex. Illness usually develops three to thirty days following the tick bite, and often begins with a primary skin lesion called erythema migrans, followed by cardiac, neurologic, or arthritic symptoms. These resulting symptoms vary in severity, are disease stage dependent, and often mimic other conditions. This multifaceted presentation often delays and confuses clinical diagnosis.

Currently bacterial culture and serologic methods are used in diagnosis. See, A. C. Steere, N. Engl. J. Med., 321, 568-596 (1989). In the early stages of Lyme disease, B. burgdorferi can be readily recovered by culture from biopsy specimens of the erythema migrans skin lesions. P. D. Mitchell et al., Am. J. Clin. Pathol., 99, 104 (1993). However, as the disease progresses, the organism becomes increasingly difficult to detect by culture. In addition, limited sensitivity and specificity and lack of test standardization between laboratories have hindered the interpretation of results.

The inadequacy of current diagnostic techniques is well-illustrated by the difficulties encountered in confirming diagnosis in patients with suspected Lyme arthritis, a late manifestation of Lyme disease that results in episodic synovial inflammation and swelling. In these patients, successful cultivation of spirochetes from synovial (joint) fluid specimens has been reported only twice. Lyme arthritis can usually be treated successfully with either a one-month course of doxycycline or amoxicillin or a two-week course of intravenous certriaxone or penicillin. In some patients, however, arthritis persists despite multiple courses of oral and intravenous antibiotic therapy. It has been unclear whether this treatment-resistant course results from persistent infection or from postinfective immune-mediated phenomena. The ability to demonstrate unequivocally the presence or absence of B. burgdorferi in the joint would improve the understanding of the pathogenesis of Lyme arthritis, and assist in identifying appropriate treatment protocols.

Detection of deoxyribonucleic acid (DNA) from B. burgdorferi in tissue and fluid specimens using the polymerase chain reaction (PCR) provides direct proof of continuing infection and is an important diagnostic tool. See, D. H. Persing, "Molecular detection of Borrelia burgdorferi," in S. Schutzer, ed. Lyme Disease: Molecular and Immunologic Approaches, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press, 299-315 (1993). To date, genomic and plasmid DNA sequences from B. burgdorferi have been used as targets with limited success. For example, specific DNA sequences encoding the outer surface protein A (OSPA) present on the 54-kb plasmid in B. burgdorferi B31, along with the genomic sequences encoding the flagellin and 16S rDNA genes have been used as targets. See, D. H. Persing et al., Science, 249, 1420-1423 (1990); D. H. Persing et al., J. Infect. Dis., 169, 668-672 (1994); S. L. Goodman et al., Infect. Immun., 59, 269-278 (1991); and P. A. Rosa et al., J. Infect. Dis., 160, 1018-1029 (1989). The range and quality of specimen types and collection and transport conditions have confounded attempts to design a single efficient standard processing technique; the varied physical characteristics and DNA content of the specimens, ranging from bacterial cultures, tick extracts, whole blood, serum, joint fluid, urine, and cerebrospinal fluid, require that protocols be developed for each specimen type to obtain a satisfactory yield of target DNA free from inhibitors that are often present. Moreover, genomic B. bergdorferi DNA is considerably more difficult than extrachromosomal DNA to detect in advanced cases of Lyme disease. D. H. Persing et al., J. Infect. Dis., 169, 668-672 (1994). Furthermore, existing methods for detecting B. burgdorferi DNA using PCR are strictly experimental and have no proven clinical value. Thus, what is needed is a highly selective, specific, sensitive, and practical method for detecting genetic evidence of the Lyme-disease causing spirochete in a variety of clinical specimens at varying stages of the disease.

SUMMARY OF THE INVENTION

The present invention is directed to methods based on the polymerase chain reaction (PCR) for the detection of Lyme-disease causing spirochetes in biological samples, and unique primers used therein. These methods are direct methods for the detection of spirochete DNA, whether the spirochete is active or inactive (inactivated by treatment, for example, with an antibiotic), in a variety of biological samples, e.g., samples from arthropods, domestic animals, humans, etc. The methods of the present invention can be used to detect spirochete DNA in, for example, fluid samples such as whole blood, blood serum, cerebrospinal fluid, urine, synovial fluid, etc., and in tissue samples such as brain tissue and other neurological tissues, cardiac tissue, skin, lymph nodes, etc. The methods of the present invention are particularly advantageous because they have proven clinical value. That is, they show greater than 90% sensitivity and greater than 90% specificity, and often greater than 95% sensitivity and 100% specificity, particularly when used to detect Lyme-arthritis causing spirochetes in synovial fluid.

One method of the present invention for detecting in a biological sample the presence of Lyme-disease causing spirochetes using the polymerase chain reaction (PCR) includes the steps of: isolating DNA from the biological sample; amplifying the isolated DNA under hybridizing conditions with a primer pair that targets portions of extrachromosomal linear plasmid gene encoding outer surface protein A (OspA) of the Lyme-disease causing spirochetes, wherein said primer pair is derived from highly conserved portions of said OspA gene; probing said amplified DNA under hybridizing conditions with a labeled gene probe; and detecting the labeled gene probe that hybridized to said amplified DNA of the Lyme-disease causing spirochetes. Another embodiment of the present invention uses a primer pair and probe that targets portions of extrachromosomal linear plasmid gene encoding outer surface protein B (OspB) of the Lyme-disease causing spirochetes.

These methods are useful for detecting a variety of Lyme-disease causing spirochetes, particularly those of the genus Borrelia. Any of a variety of strains of Borrelia spirochetes can be detected, including, for example, those within the species classifications Borrelia burgdorferi, Borrelia garnii, and Borrelia afzelii, as well as a variety of as yet unclassified strains. Specific examples of such strains that can be detected with the methods of the present invention include, for example, CDC strains 89-1421, 90-1246, 90-1654, 90-2246, 90-2810, 91-1226, 91-1828, 92-0889, 92-0953, as well as strains CA4, CA7, CA8, CA9, B172, B31, DN127, and N40. On the other hand, the methods described herein are specific for Lyme-disease causing spirochetes; they do not detect members of the genus Borrelia that are not associated with Lyme-disease.

The methods of the present invention use standard PCR techniques, preferably modified to include the use of isopsoralen, i.e., a three-ring compound activated by UV light, in the PCR master mix used for amplification of isolated DNA. An amount of isopsoralen is used that is effective to inactivate the amplified product, i.e., stop the polymerization process. This helps avoid false positives.

The OspA primers used in the PCR methods are derived from several highly conserved portions of the extracellular linear plasmid OspA gene. As used herein, "highly conserved" means that these portions of the gene have at least about 75% sequence identity among DNA of 14 spirochete strains, based on multiple sequence analysis and alignment of OspA genes. For example, one useful primer preferably contains 15-93 nucleotides, more preferably 18-30 nucleotides, and hybridizes under hybridizing conditions to nucleotides 135-227 in the Borrelia burgdorferi consensus sequence shown in FIG. 1. This primer preferably substantially corresponds to the nucleotide sequence of primer OspA149, shown below in Table 1. As used herein, "substantially corresponds to" means that the primer sequence of interest has at least about 50% sequence identity with the referenced primer sequence. Most preferably, this primer is the primer referred to herein as OspA149. This primer can be used with either of the two following primers: a primer containing 15-56 nucleotides, preferably 18-30 nucleotides, that hybridizes under hybridizing conditions to nucleotides 316-371 in the Borrelia burgdorferi consensus sequence shown in FIG. 1 (preferably this primer substantially corresponds to primer OspA319, and more preferably is primer OspA319); and a primer containing 15-37 nucleotides, preferably 18-30 nucleotides, that hybridizes under hybridizing conditions to nucleotides 457-493 in the Borrelia burgdorferi consensus sequence shown in FIG. 1 (preferably this primer substantially corresponds to primer OspA459, and more preferably is primer OspA459). Thus, two particularly preferred primer pairs are primers OspA149 and OspA319, and primers OspA149 and OspA459. The OspB primers used in the PCR methods of the present invention substantially correspond to the primers referred to in Table 1 as OspB1110 and OspB1411. Preferably, these primers are OspB1110 and OspB1411.

The labeled probes used in the methods of the present invention include any useful probe that hybridizes to the amplified products under hybridizing conditions. Such probes can be internal oligonucleotide probes, either synthetic or naturally occurring, an amplification product, or a plasmid or portion thereof containing an amplification product or a portion thereof. Preferred probes are those substantially corresponding to the probes listed in Table 1 below. More preferred probes are those listed in Table 1. The probes can be labeled with any standard detectable label, such as .sup.32 P, .sup.33 P, .sup.35 S, chemiluminescent labels, fluorescent labels such as fluorescene or rodamine, and enzymatic labels such as horseradish peroxidase or alkaline phosphatase.

The hybridizing conditions used in the present invention include an annealing temperature of about 45-60.degree. C., an extension temperature of about 70-75.degree. C., and a denaturation temperature of about 90-95.degree. C. for a total of about 30-50 cycles in a PCR mix containing a sufficient amount of buffer to maintain the pH at 8-8.5, and a sufficient amount of each of the following reagents to maintain: a final concentration of 50-200 .mu.M of each dNTP; a final concentration of 0.1-2 .mu.M primer; a final volume % of 5-15% glycerol; a final isopsoralen concentration of 25-150 .mu.g/ml; and about 0.1-1 Units of AmpliTaq per 50 .mu.l of the total volume of the reaction mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignment of published DNA sequences for OSPA gene for various strains of B. burgdorferi(SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32). For clarity sake, only the nucleotide sequences at variance with the consensus sequence are shown. Highly conserved regions (Region 1:135-227; Region 2:316-343; Region 3:457-493) are underlined. Primers (OSPA149:149-173; OSPA319:319-343; OSPA459:459-479) are overlined. Sequences were aligned using the Pileup utility made available by Genetics Computing Group, Madison, Wis. Strain B31 sequence numbering was used. Genbank Accession numbers are indicated in parentheses.

DETAILED DESCRIPTION OF THE INVENTION

A method is presented for detecting the presence of the Lyme-disease causative agent, particularly the spirochete Borrelia burgdorferi, in biological samples using the polymerase chain reaction (PCR). Samples (bacterial cultures, tick extracts, or clinical specimens) are carefully processed such that DNA is isolated free from nucleases and polymerase inhibitors. PCR primers OspA149, OspA319 and OspA459, hybridizing to the OspA gene located on the 54-Kb extracellular plasmid in B. burgdorferi, have been developed with reference to the consensus sequence of 14 different strains. These primers are highly sensitive and specific for B. burgdorferi. PCR primers OspB1110 and OspB1411, hybridizing to the OspB gene located on the same plasmid, were developed with reference to the OspB sequence of strain B31 and also exhibit high specificity and sensitivity for all tested strains of B. burgdorferi. Primers for the genomic sequences fla, encoding a flagellin protein (FLA107 and FLA337), and 16S rDNA (DD02 and DD04), were also used but showed lower sensitivity in clinical studies than the primers associated with the extracellular plasmid DNA.

PCR reactions incorporate the use of isopsoralen, which is highly effective in preventing the occurence of false positive results. The method shows high specificity and sensitivity in the detection of Lyme disease from dilute samples, particularly synovial fluid, and is shown to produce direct evidence of the spirochete in patients with advanced Lyme arthritis whose synovial fluid does culture for B. burgdorferi.

The following examples are offered to further illustrate the various specific and preferred embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the present invention.

EXPERIMENTAL EXAMPLES

Example 1

Sample Preparation and DNA Isolation

The diverse characteristics and DNA content of clinical samples require the use of different preparation methods. Specimens may contain nucleases (especially whole blood, serum, and urine) or Taq poLymerase inhibitors (especially blood and urine), which disallow direct use of the specimen for PCR. It is imperative that all clinical samples avoid contact with all sources of Borrelia DNA during collection, aliquot handling, and storage, or false-positive samples may occur. Avoid work areas where cultures, antigen preparations, or amplified DNAs have been manipulated. Samples are best sealed at the patient's bedside and sent directly to the PCR laboratory, where aliquots for serologic testing can be made.

A. Bacterial culture of B. burgdorferi. Borrelia isolates were grown in 7 ml of a modified BSK medium such as BSK H, a medium specifically developed for culture of Borrelia burgdorferi and available from Sigma Chemical, St. Louis, Mo., in screw-cap tubes (13 by 100 mm) at 32.degree. C. Cell pellets were harvested from 1 ml of BSK culture containing 1.times.10.sup.9 to 5.times.10.sup.9 organisms per ml. The pellet was resuspended in 50 .mu.l of TE buffer (TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM ethylene diamine tetraacetic acid (EDTA), both from Sigma Chemical Company, St. Louis, Mo.), and boiled for 10 minutes in a temperature block. A 10-fold dilution series was prepared in TE buffer, and PCR was performed directly on 1- to 5-.mu.l aliquots.

B. Ticks. Ixodes ticks were identified. Live ticks are suitable for all test methods. Desiccation until testing was prevented. Dead ticks were stored in 70% alcohol prior to testing. Desiccated specimens could also be used. Whole ticks were processed as described in D. H. Persing et al., Science, 249, 1420-1423 (1990) and D. H. Persing et al., J. Clin. Micro., 28, 566-572 (1990), which are incorporated herein by reference. Briefly, ticks were removed from alcohol and air dried on filter paper disks for 5 minutes. Each whole tick was placed in a 1.5-ml microcentrifuge tube, covered with 20 .mu.l of K buffer (K Buffer: 10 mM Tris, pH 8.3, 50 mM KCl, 1.75 mM MgCl.sub.2, 0.01% bovine serum albumin (BSA, Fraction V), 0.45% Tween 20, 0.45% Nonidet P-40 (all from Sigma Chemical Company, St. Louis, Mo.)) and a small volume of BSA-coated glass beads (0.1-mm glass beads, BioSpec Products, Bartlesville, Okla.), and crushed with a disposable plastic pestle or pipette tip. The buffer becomes slightly turbid when the tick contents are released. Tubes were heated in a 95.degree. C. heating block for 10 minutes, and then immediately chilled on ice. PCR was used to test 5 .mu.l of supernatant. Additional buffer was be added to large specimens as necessary.

C. Urine. Sample sizes of 15 ml were used, preferably collected prior to antibiotic therapy. An equal volume of 95% ethanol was added prior to storage in some protocols. Solvent extraction was used to isolate the DNA as follows:

1. Urine (500 .mu.l) was added to a 1.5-ml microcentrifuge tube and centrifuged at 16,000.times. g for 5 minutes. The supernatant was removed and discarded, and the pellet was stored if needed for later processing at -20.degree. C.

2. The pellet was resuspended in 246.2 .mu.l of TE, 5 .mu.l of 1 M dithiothreitol (DTT, Sigma Chemical Company, St. Louis, Mo.), 20 .mu.l of 0.25 M EDTA (pH 8), 25 .mu.l of 10% sodium dodecyl sulfate (Amresco, Inc., Solon Ohio), and 3.8 .mu.l of 20-mg/ml proteinase K (VWR Scientific, Philadelphia, Pa.) and incubated 1 hour at 37.degree. C.

3. The digest (approximately 300 .mu.l) was transferred to pediatric serum separator tubes (Becton Dickinson, Bedford, Mass.). This product greatly simplifies solvent extraction, allowing multiple extractions to be performed in the same tube. Alternatively, the clinical specimen may be extracted directly by adding the specimen to the tube without digestion.

4. The sample was extracted with 150 .mu.l of phenol-chloroform-isoamyl alcohol (25:24:1) (Amresco, Solon Ohio). Mixing was accomplished by inversion or vortexing, then the mixture was centrifuged at 16,000.times. g for 30 seconds. The aqueous layer is above the inert plug and the organic solvent layer. A second extraction was performed in the same tube.

5. Next the sample was extracted with 150 .mu.l of chloroform-isoamyl alcohol (24:1) in the same tube.

6. The upper aqueous layer containing nucleic acids was transferred to a 1.5-ml microcentrifuge tube, and 30 .mu.l of 3 M sodium acetate and 300 .mu.l of isopropanol were added. The tube was gently inverted to mix.

7. The sample was centrifuged at 16,000.times. g for 15 minutes at 4.degree. C. The supernatant was carefully removed by using a fine-tip pipette without disturbing the pellet.

8. The pellet was washed with 500 .mu.l of 70% ethanol, then centrifuged at 16,000.times. g for 15 minutes at 4.degree. C. All but 20 .mu.l of ethanol was removed. The sample was evaporated to dryness in a Speed Vac centrifuge for 5 minutes. Air drying of the pellet is an alternative method.

9. The pellet was resuspended in 50 .mu.l of TE and stored overnight at 4.degree. C. to resolubilize the nucleic acid. Aliquots of 2.5 .mu.l were used in a 100-.mu.l PCR reaction.

D. Other body fluids.

1. Sample preparation.

Cerebrospinal fluid. Samples of 2 ml or more were used in sterile tubes. Aliquots were stored at -20.degree. C. until use. Multiple freeze-thaw cycles were avoided.

Joint fluid. Samples of 2 ml or more were collected in a red-top tube (no anticoagulant) or a purple-top tube (ethylene diamine tetraacetic acid (EDTA) additive). Heparin tubes (green-top) were not used, since they inhibit PCR (Venoject tubes, available from Terumo Medial, Elkton, Md.). Aliquots were stored at -20.degree. C. until use. Multiple freeze-thaw cycles were avoided.

Whole blood. Samples of 5 to 10 ml were collected in a purple-top tube (EDTA additive) and stored at 4.degree. C. They were processed or aliquoted and frozen as soon as possible after receipt.

Serum. Samples of 1 to 3 ml were stored in a sterile tube at -20.degree. C. until use. Multiple freeze-thaw cycles were avoided.

2. DNA isolation via chaotropic lysis/solvent extraction.

The IsoQuick Nucleic Acid Extraction Kit (MicroProbe Corp., Bothell, Wash.) employs chaotropic lysis to disrupt cells and stabilize the nucleic acid contained in a variety of clinical samples, including bacteria and whole blood (MicroProbe Corp., IsoQuick Nucleic Acid Extraction Kit, Technical Insert, MicroProbe Corp., Concord, Calif. (1991)). This kit was used to successfully isolate DNA from 100- to 200-.mu.l volumes of joint fluid, CSF, plasma, serum, urine, and whole blood. The kit uses a modified solvent extraction method, which, in brief, includes guanidinium isothiocyanate lysis, partitioning of nucleic acid into an aqueous phase, and isopropanol precipitation of nucleic acid. The resulting nucleic acid was dissolved in 20 to 30 .mu.l of RNase-free water (sterile water for irrigation USP, Baxter Healthcare), and 5 .mu.l was used for PCR. Addition of 20 .mu.g of glycogen (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per sample during alcohol precipitation improved the recovery of small quantities of nucleic acid.

E. Tissue. A single standard 3- to 5-mm skin punch biopsy or equivalent-sized tissue specimen was stored in 70% ethanol at room temperature. Tissue (i.e., skin biopsy specimen, BB-sized to pea-sized tissue biopsy specimen) was immersed in 50 to 100 .mu.l of K buffer-proteinase K (K Buffer-proteinase K: K Buffer as described above in B. with 100 mg of proteinase K per ml (International Biotechnologies, Inc., New Haven, Conn.)) and incubated at 55.degree. C. in a temperature block until digestion was complete. The amount and type of tissue determine the required incubation time, which typically ranges from 1 to 24 hours. Briefly vortexing the sample several times during incubation speeds tissue digestion. Samples were heated at 95.degree. C. for 10 minutes to inactivate proteinase K and immediately chilled on ice. Excessive heating will decrease the DNA yield. Aliquots of 1 to 5 .mu.l of digest were tested by PCR. Processed samples were stored at -20.degree. C.

Example 2

PCR Amplification

1. DNA primers were synthesized on an oligonucleotide synthesizer (Applied Biosystems, Foster City, Calif.), desalted on an oligonucleotide-purification cartridge (Glen Research, Sterling, Va.), and used without further purification. Alternatively, they were purchased directly from Integrated DNA Technologies, Coralville, Iowa.

2. A PCR master mix was prepared with final concentrations of: 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl.sub.2, 0.01% BSA, 200 .mu.M of each deoxynucleoside triphosphate A, C, G, and T (dNTP) (Promega, Madison, Wis., or Boehringer Mannheim Biochemicals, Indianapolis, Ind.), 50 pmol of each primer per 50-ml reaction (1.0 mM), 10% glycerol, 100 .mu.g/ml of isopsoralen compound 10 (HRI Associates, Inc., Concord, Calif.), 0.025 U/.mu.l of AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.). The following substitutions were necessary: for OSPA149/OSPA319, substitute 2.5 mM MgCl.sub.2 for the 1.75 mM MgCl.sub.2 ; for DD02/DD06, substitute 62.5 .mu.M of each dNTP for the 200 mM of each dNTP, 50 .mu.g/ml of isopsoralen compound 10 instead of 100 .mu.g/ml, and 0.05 U/.mu.l of AmpliTaq polymerase instead of 0.025 U/.mu.l. In later experiments Ampliwax beads (Perkin-Elmer Cetus, Norwalk, Conn.) were added to prevent nonspecific hybridization at low temperatures. Isopsoralen was added to the reaction mixture for the second round but not the first round of the hemi-nested procedure for OSPA149/OSPA319, as described below. Processed DNA (5 .mu.l) and 1 or 2 drops of mineral oil was added to 20 .mu.l of the reaction mixture. All pipetting operations were performed with positive-displacement pipettors. It is useful to overestimate the volume of required master mix by one or two reactions to allow for pipetting inaccuracies and other volume losses during setup.

3. DNA was amplified in a thermal cycler by using a three-step protocol for the OSPA149/OSP319 target: denaturation at 94.degree. C. for 45 seconds, annealing at 55.degree. C. for 45 seconds, and extension at 72.degree. C. for 45 seconds for a total of 50 cycles. A similar procedure was used for the OSPB1110/OSPB1411 target and the FLA107/FLA335 target: denaturation at 94.degree. C. for 45 seconds, annealing at 50.degree. C. for 45 seconds, and extension at 72.degree. C. for 45 seconds for a total of 50 cycles. For these two targets the thermal cycling was preceded by a 4 minutes incubation at 94.degree. C. and followed by a 7 minute extension at 72.degree. C. For the DD02/DD06 target a two-step protocol was used: denaturation at 94.degree. C. for 25 seconds and annealing at 60.degree. C. for 25 seconds for a total of 50 cycles, and then heated to 72.degree. C. for a 5-minute final extension. For the OSPA149/OSPA459 target a hemi-nested procedure was used: the first round of amplification utilized the primer pair OSPA149/OSPA459 and consisted of initial heating at 94.degree. C. for 4 minutes, followed by 25 cycles of denaturation at 94.degree. C. for 45 seconds, annealing at 50.degree. C. and extension at 72.degree. C., then a final 7 minute extension at 72.degree. C. Upon completion, the following solution (75 .mu.l) was added to the reaction mixture: 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl.sub.2, 0.01% BSA, 200 mM of each dNTP A, C, G, and T, 50 pmol of each primer OSPA149 and OSPA319, 10% glycerol, isopsoralen compound 10 (100 .mu.g/ml, HRI Associates, Inc., Concord, Calif.), 2.5 units of AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.). The same cycling procedure was performed a second time.

4. The reaction tubes were removed from the DNA cycler and exposed to 20 mW/cm.sup.2 of 300- to 400-nm UV light for 15 minutes at 4.degree. C. in an HRI-100 UV Photochemical Reaction Chamber for post-PCR sterilization of amplicon (HRI Associates, Inc., Concord, Calif.). Postamplification inactivation of amplified DNA is optional; however, routine use significantly reduces the risk of laboratory contamination with amplified DNA.

5. A 4% agarose gel (3% NuSieve and 1% SeaPlaque or 1% SeaKem (FMC Bioproducts, Portland, Me.) agarose gel was prepared in 1.times. TBE buffer (20.times. TBE buffer: 216 g of Tris base (Sigma), 110 g of boric acid (Sigma), 80 ml of 0.5 M EDTA (pH 8.0; Sigma), H.sub.2 O to 1 liter). The PCR products were electrophoresed, 5 .mu.l of product to 2 .mu.l of gel-loading buffer (27 ml of glycerol, 3 ml of 10.times. TBE, 1 ml of 10% sodium dodecyl sulfate (SDS), 10 ml of 0.5 M EDTA (pH 8.0), 10 mg of bromphenol blue, H.sub.2 O to 100 ml) until the bromphenol blue indicator migrated at least two-thirds the length of the gel (35 to 100 V for 1 to 3 hours).

5. The amplification products were visualized after staining with ethidium bromide (Sigma Chemical Company, St. Louis, Mo.). Use of isopsoralen increased the apparent molecular weight of amplification products 10 to 25%.

6. The gel was denatured and neutralized by soaking in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 45 minutes followed by neutralization buffer (0.5 M Tris (pH 7.5; Sigma), 1.5 M NaCl) for 15 minutes. The reaction products were then transferred to nylon membrane (Hybond N; Amersham Corp., Arlington Heights, Ill.) by Southern blot using 10.times. SSC transfer buffer (10.times. SSC: 1.5 M NaCl plus 0.15 M sodium citrate).

7. Positive amplification and sensitivity control was carried out as follows. DNA (60-100 pg) was prepared from a boiled B. burgdorferi B31 (ATCC 35210) bacterial culture. Use of both high-positive and low-positive reactions provided useful quality control information. DNA concentrations were determined as follows. A 10-fold dilution series of B. burgdorferi DNA was prepared, and 5-.mu.l aliquots of each dilution were amplified. Two of the last DNA dilutions yielding positive amplification reactions were chosen for routine controls. The specificity controls were 1 ng each of Borrelia hermsii (ATCC 35209) and Treponema pallidum (ATCC 27087). The negative amplification control was prepared by adding 5 .mu.l of water directly to the PCR master mix (triplicate).

Example 3

DNA Probe Hybridization and Detection of Amplification Products

1. Internal oligonucleotide probes (180 ng per hybridization) were synthesized on an oligonucleotide synthesizer (Applied Biosystems, Foster City, Calif.) and desalted on an oligonucleotide-purification cartridge (Glen Research, Sterling, Va.). Alternatively, they were purchased from Integrated DNA Technologies (Coralville, Iowa). The oligonucleotides were .sup.32 P-end-labeled. As an example, probe OspA6 was labeled by adding to a 1.5 .mu.l microcentrifuge tube, the following: 12.3 .mu.l water, 1.7 .mu.l 10.times. T4 buffer, 1.0 .mu.l probe, 1.0 .mu.l T4 polynucleotide kinase, and 1.0 .mu.l .gamma..sup.32 P ATP. This mixture was incubated for 45-60 minutes in a 37.degree. C. water bath. The labeled probe was then heated at 95.degree. C. for 6 minutes and cooled to room temperature to inactivate the kinase. In later experiments, amplification products were detected with a chemiluminescent internal hybridization probe constructed by amplification of internal sequences or by direct coupling of the amplification product itself with horseradish peroxidase as directed by the manufacturer for the ECL kit (Amersham Laboratories, Arlington Heights, Ill.). Briefly a 142 nucleotide probe was produced using primers OspA175 (GGT CTA ATA TTA GCC TTA ATA GC) (SEQ ID NO:10) and OspA316 (CAA TTA GAT CGT ACT TGC) (SEQ ID NO:11) and an annealing temperature of 46.degree. C. with 72.degree. C. for 72 minutes final extension. For full-length probes, the presence of PCR primer sequences in nonspecific amplification products did not result in lower hybridization specificity. For a detailed description of detection of PCR amplification products by means of a chemiluminescent probe, see D. H. Persing et al., J. Clin. Microbiol., 30, 2097-2103 (1992), incorporated herein by reference.

2. Blots were prehybridized for 1 hour at 55.degree. C. in 10 ml of hybridization solution (5.times. Denhardt's solution [1.times. Denhardt's solution is 0.02 percent Ficoll, 0.02 percent polyvinylpyrrolidone, and 0.02 percent BSA], 5.times. SSPE (Sigma Chemical Company 20.times. stock: 0.2 M phosphate, 2.98 M NaCl, 0.02 M EDTA), 0.5% SDS, 100 mg/mL of denatured salmon sperm DNA (Sigma Chemical, St. Louis, Mo.). Probe was added and hybridized for 3 hours at 55.degree. C. All prehybridization, hybridization, and wash steps were performed in glass hybridization bottles and incubated in a hybridization oven (both from Hybaid Limited, Middlesex, UK).

3. The probe solution was decanted and the blots washed twice for 10 minutes with 150 ml of 2.times. SSC, 0.1% SDS; a third high-stringency wash was performed for 30 minutes at 55.degree. C. with 150 ml of prewarmed 1.times. SSC, 0.1% SDS. Increased stringency may be achieved by repeating wash 3 or by adding a 15-minute wash with 150 ml of hybridization wash 2 containing 2% sodium pyrophosphate.

4. This Saran-wrapped blot was exposed to Kodak X-Omat film or Kodak XAR-5 film in an exposure cassette for up to 48 hours at -70.degree. C. as necessary for adequate visualization.

Example 4

Multiple Target Detection of B. Burgdorferi-Specific Plasmid and Genomic Targets in Synovial Fluid Specimens Using DNA Primer Pairs OspA149/OspA319, OspB1110/OspB1411, DD02/DD06 and Fla107/Fla335

DNA primer pairs OspA149/OspA319 (Table 1), OspB1110/OspB1411 (Table 1), DD02/DD06 (DD02 sequence: CCC TCA CTA AAC ATA CCT (SEQ ID NO:12); DD06 sequence: ATC TGT TAC CAG CAT GTA AT) (SEQ ID NO:13) and Fla107/Fla335 (Table 1) were synthesized as described in Example 2. PCR experiments were conducted as described in Examples 2 and 3 on synovial fluid DNA from nineteen patients with chronic Lyme disease. The samples were aliquoted soon after collection in a biosafety hood kept free of contaminating B. burgdorferi DNA and then frozen at -70.degree. C. or -20.degree. C. until further analysis. The patients met Centers for Disease Control and Prevention case definition criteria for Lyme disease as set out in the Morbidity and Mortality Weekly Report. All patients had intermittent episodes of arthritis, lived in areas endemic for Lyme disease, and had elevated antibody titers for B. burgdorferi by ELISA (enzyme-linked immunosorbant assay). Eleven control synovial fluid specimens were collected from patients with non-Lyme arthritic disorders and were processed and sorted the same way as those from patients with Lyme arthritis.

As shown in Table 2, all 19 synovial fluid specimens from the Lyme arthritis patients reacted with the extracellular OspA149/OspA319 primer pair and the extracellular OspB1110/OspB1411 primer pair, while only 8 or the 19 samples of the same volume reacted with the genomic 16S rDNA primer pair (DD02/DD06) and only 9 of 17 samples were positive for the flagellin target (FLA107/FLA35).

Example 5

Detection of B. Burgdorferi-Specific Plasmid and Genomic Targets in Synovial Fluid Specimens Using DNA Primer Pairs OspA149/OspA319, OspA2/OspA4 and DD02/DD06

Synovial fluid was collected from Lyme arthritis patients treated in the Lyme disease clinics at Yale-New Haven Hospital (1975-1987) or New England Medical Center (1987-1992) over a 17-year period. Synovial fluid was also collected from control patients with other forms of arthritis during the same period. Primer pairs OspA149/OspA319, OspA2/OspA4 and DD02/DD06, and related oligonucleotide probes as described in Table 3 were synthesized and end-labeled as described in Examples 2 and 3. Samples were processed, PCR reactions conducted, and amplification products detected as described in Examples 2 and 3.

As shown in Table 4 below, B. burgdorferi DNA was detected with at least one of the primer-probe sets in 75 out of the 88 patients with Lyme arthritis. The three OspA primer sets, which probe for extrachromosomal DNA, each detected B. burgdorferi DNA in 75 to 89% of the 75 patients with positive test results. Primer set 4, which detected genomic DNA, was less sensitive; only 56 percent of the 75 patients had positive results with this set. No B. burgdorferi DNA was found in any of the 69 control patients or in the blank control samples. Importantly, B. bergdorferi DNA was detected in 70 of 73 (96%) Lyme-disease patients who had not received antibiotic treatment or were tested prior to treatment.

Example 6

Sequence Alignment and Identification of Highly Conserved Regions and Primer Pairs for OspA Gene

Sequences for the gene OspA from fourteen strains of B. burgdorferi were aligned with the Pileup utility of the GCG Sequence Analysis Package (Genetics Computing Group, Madison, Wis.). See FIG. 1. The numbering system used in FIG. 1 is that of the B31 strain, strictly for ease of reference. Included in the alignment set were two newly identified isolates, Dn127 and 25015 (GenBank accession number S88693) that exhibited sequences that differed substantially from the previously known sequences. Comparison of the aligned sequences permitted generation of a consensus sequence (a textual string of nucleotides representing the most commonly found base at each site on the gene) and identification of the most highly conserved regions among all the strains. These regions, labeled Region 1 (135 through and including 227), Region 2 (316 through and including 371) and Region 3 (457 through and including 493) were utilized as the basis for oligonucleotide primer generation. The program OLIGO (OLIGO Primer Analysis Sorgware version 4.0 for the Macintosh, National Biosciences, Inc. Plymouth, Minn.) was used to generate preliminary compatible primer sets, which upon visual inspection suggested primers OspA149 (nucleotides 149-173), OspA319 (nucleotides 319-343) and OspA459 (nucleotides 459-479) as likely candidates for successful PCR reactions, based upon their melting points, interaction energies and predicted secondary structure. These primers were synthesized and tested as described in Examples 2 and 3.

The foregoing detailed descriptions and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

Certificate of Correction present

The claims shown below may not include correction changes. Use the corrected PDF for authoritative text.

What is claimed is:

1. A method of detecting in a biological sample the presence of Lyme-disease causing spirochetes comprising: (a) amplifying DNA isolated from the biological sample with a primer pair that targets portions of extrachromosomal linear plasmid gene encoding outer surface protein A of the Lyme-disease causing spirochetes; wherein a first primer of said primer pair hybridizes to the complement of Borrelia burgdorferi consensus sequence having SEQ ID NO:32 and a second primer of said primer pair hybridizes to the Borrelia burgdorferi consensus sequence having SEQ ID NO:32, wherein the first primer of said primer pair comprises nucleotides 135-227 of SEQ ID NO:32 or a portion thereof, and wherein the second primer of said primer pair comprises the complement of nucleotides 316-371 of SEQ ID NO:32 or a portion thereof or comprises the complement of nucleotides 457-493 of SEQ ID NO:3 or a portion thereof, and wherein the primers do not hybridize to Borrelia hermsii DNA or Treponema pallidum DNA; (b) probing said amplified DNA with a labeled gene probe so as to form a complex comprising said amplified DNA and labeled gene probe; and (c) detecting the presence or absence of the complex, wherein the presence of the complex is indicative of the presence of Lyme-disease causing spirochetes in the biological sample.

2. A method of detecting in a biological sample the presence of Lyme-disease causing spirochetes comprising: (a) amplifying DNA isolated from the biological sample with a primer pair that targets portions of extrachromosomal linear plasmid gene encoding outer surface protein A of the Lyme-disease causing spirochetes; wherein one primer of said primer pair hybridizes to the complement of nucleotides 135-227 of the Borrelia burgdorferi consensus sequence having SEQ ID NO:32, and wherein the other of said primer pair hybridizes to nucleotides 316-371 or nucleotides 457-493 of the Borrelia burgdorferi consensus sequence having SEQ ID NO:32, wherein the primers do not hybridize to Borrelia hermsii DNA or Treponema pallidum DNA; (b) probing said amplified DNA with a labeled gene probe so as to form a complex comprising said amplified DNA and labeled gene probe; and (c) detecting the presence or absence of the complex, wherein the presence of the complex is indicative of the presence of Lyme-disease causing spirochetes in the biological sample.

3. The method of claim 1 or 2 wherein the Lyme-disease causing spirochetes are of the genus Borrelia.

4. The method of claim 3 wherein the Lyme-disease causing spirochetes are Borrelia burgdorferi spirochetes.

5. The method of claim 1 or 2 wherein the biological sample is a fluid or tissue sample from a human patient.

6. The method of claim 5 wherein the biological sample is synovial fluid.

7. The method of claim 1 or 2 wherein the step of amplifying the isolated DNA comprises using a polymerase chain reaction mix containing an amount of isopsoralen effective to inactivate the amplified product.

8. The method of claim 1 wherein one primer is primer OspA149 (SEQ ID NO:1).

9. The method of claim 1 wherein one primer is primer OspA319 (SEQ ID NO:2).

10. (Third amendment) The method of claim 1 wherein one primer is primer OspA459 (SEQ ID NO:3).

11. The method of claim 1 wherein the primer pair consists of primers OspA149 (SEQ ID NO:1) and OspA319 (SEQ ID NO:2).

12. The method of claim 1 wherein the primer pair consisting of primers OspA149 (SEQ ID NO: 1) and OspA459 (SEQ ID NO:3).

13. The method of claim 1 or 2 wherein portions of the labeled probe consist of sequences comprising the Borrelia burgdorferi consensus sequence having SEQ ID NO:32 or a portion thereof.

Patent Citations (5)

Patent	Date	Inventor
US5279938	1994-01-01	Rosa
US5530103	1996-06-01	Livey et al.
US5582990	1996-12-01	Bergstrom et al.
CAX2001328	1991-04-01
WOX93/08306	1993-01-01

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Abstract

Description