Molecular and Cellular Probes (1990) 4, 73-79
Detection of Borrelia burgdorferi DNA by the polymerase chain reaction
Siren L . Nielsen *J Karen K. Y . Young and Alan G . Barbourj' *Specialty Laboratories, Inc ., 2211 Michigan Avenue, Santa Monica, CA, U.S.A . 90404-3900 and tThe University of Texas, Departments of Microbiology and Medicine, Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7881, U .S .A . (Received 20 September 1989, Accepted 28 September 1989)
DNA amplification by the polymerase chain reaction (PCR) was used to detect DNA of the Lyme disease spirochaete Borrelia burgdorferi . Primers that specify the amplification of a 145 basepair DNA fragment of the OspA gene of B. burgdorferi were used . The amplification product was detected by gel electrophoresis and ethidium bromide staining or by hybridization to a radiolabelled oligonucleotide probe . The hybridization method was found to be more sensitive. As little as 50 fg of purified B . burgdorferi DNA could be detected by PCR . This corresponds to fewer than 50 spirochaetes . The specificity of PCR for B . burgdorferi was tested by using DNA from other organisms as templates for amplification . No cross-reactivity was found . The data shown provide useful information for the development of a PCR-based diagnostic test for Lyme disease .
KEYWORDS: Borrelia burgdorferi, PCR, DNA detection.
INTRODUCTION Lyme disease, the most common arthropod-borne disease in the United States and Europe, has been reported with increasing frequency during the last decade .' The disease is caused by the spirochaete Borrelia burgdorferi2-4 and transmitted by ticks, which account for the seasonal and geographic case clusters . The disease spectrum ranges from subclinical infections to severe skin, joint, cardiac and neurological manifestation S .3,4 Because of the variations in clinical picture and because the disease often mimics other neurological and rheumatological disorders, a definitive diagnosis based on clinical findings is difficult . Yet, early diagnosis is important because antibiotic treatment can prevent the development of neurological, cardiac and joint sequelae . s $ Author to whom correspondence should be sent.
0890-8508/90/010073+07 $03 .00/0
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© 1990 Academic Press Limited
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S . L. Nielsen et al .
Immunofluorescence assay, enzyme-linked immunosorbent assay and immunoblotting are currently used to detect B . burgdorferi-specific IgG and IgM antibodies in serum and cerebrospinal fluid . 6'' These tests, although useful in later stages of the disease, have only 50-60% sensitivity during the first 3-6 weeks after infection .' Lack of specificity is also a problem due to false-positive reactions in patients with other spirochaetal diseases such as syphilis and relapsing fever as well as in patients with rheumatic diseases . 6.8 Direct detection of the spirochaete by microscopic examination s or culture10 of blood, cerebrospinal fluid, synovial fluid and skin tissue are not practical for routine use and have low diagnostic sensitivities . A sensitive and specific test for the detection of B . burgdorferi is needed not only for early diagnosis of suspected Lyme disease, but also for therapeutic monitoring and studies of pathogenesis . In vitro amplification of DNA via the polymerase chain reaction (PCR) 11 has been shown to be valuable for the diagnosis of viral infections such as those involving human immunodeficiency virus and cytomegalovirus . 12,13 PCR has also been used for the detection of bacteria such as E. coli and Pseudomonas spp . 14,15 We tested the utility of the PCR technique for the detection of B . burgdorferi DNA . Our results show that a high degree of sensitivity and specificity were achieved with the PCR technique . This will provide useful information for the future development of a PCR-based diagnostic test for Lyme disease .
MATERIALS AND METHODS Organisms
Borrelia burgdorferi strain B31 (ATCC 35210) and Borrelia hermsii strain HS1 (ATCC 35209) were grown in BSK medium at 33 ° C . 16 The cells were harvested by centrifugation at 1500 X g for 30 minutes, washed in phosphate-buffered saline (PBS) and transferred to microcentrifuge tubes where the cells were pelleted again at 16,000 X g for 15 minutes . Treponema pallidum (ATCC 27087), Treponema denticola (ATCC 33521), Leptospirum interrogans (ATCC 43642) and Staphylococcus aureus (ATCC 6341) were obtained from ATCC either as frozen or freeze-dried cultures . The cells were resuspended and washed in PBS and pelleted as described above .
Isolation of nucleic acids Pellets of the microorganisms were resuspended in lysis buffer SET (25% sucrose ; 50 mm EDTA, 50 mm Tris, pH 8 . 0) and lysozyme was then added to a final concentration of 2 mg ml -1 . After incubation for 30 minutes at 37°C, proteinase K and sodium dodecyl sulphate were added to final concentrations of 0. 25 mg ml -1 and 1 . 2%, (w/v) respectively . The cells were incubated again at 37°C for 30 minutes with gentle shaking every 10 minutes . Nucleic acids were extracted twice with phenol and chloroform-isoamyl alcohol and once with chloroform-isoamyl alcohol alone . After adjusting the NaCl concentration to 0 . 2 M, the nucleic acids were precipitated with 2 volumes of 95% ethanol overnight at -20°C and then pelleted
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by centrifugation at 17,000 X g for 30 minutes . The pellet was then washed in cold 70% ethanol, lyophilized and resuspended in TE (10 mm Tris HCI, 1 mm EDTA, pH 8 . 0) . The nucleic acid suspension was then treated with DNase-free RNase . DNA concentration was estimated after electrophoresis on a 0 . 7% agarose gel by comparing the intensity of the ethidium bromide-stained DNA bands with DNA standards of known concentrations (Hind III digests of Lambda DNA, Bethesda Research Laboratories, Bethesda, MD) . Portions of the B. burgdorferi DNA were further purified to obtain a more accurate measurement of the DNA concentration . DNA was separated by electrophoresis through a 07% low-melt agarose (Bethesda Research Laboratories, Bethesda, MD) gel and then stained with ethidium bromide . The visible, highmolecular weight DNA was cut out of the gel and purified through a NACS Prepac mini-column (Bethesda Research Laboratories, Bethesda, MD), following the manufacturer's instructions . The eluted DNA was precipitated with ethanol and resuspended in TE . DNA concentration and purity were estimated by optical density at 260 nm/280 nm . Human DNA used in the study was obtained from peripheral blood mononuclear cells . The mononuclear cells were isolated from peripheral blood by banding on Ficoll paque (Pharmacia) following the manufacturer's instructions . The isolated cells was then lysed in lysis buffer (50 mm KCI, 10 mm Tris HCI, pH 8 . 3, 2 .5 MM MgCl 2, 0 . 5% (v/v) Tween 20, 0 . 5% (v/v) Triton X-100), and treated with proteinase K (120 gg ml -1 ) at 60° C for 1 hour. Proteinase K was then inactivated by a 10 minute incubation at 95°C . Aliquots of the lysate was used in the amplification reactions .
PCR Oligonucleotide primers SN1 and SN2 and oligonucleotide probe SN3 chosen from the sequence for the outer surface protein OspA gene of B . burgdorferi strain B31 17 were used throughout the study . The oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer and purified through oligonucleotide purification cartridges (Applied Biosystems) . Primer SN1 (5'-CGATCTAATTGCAACAGTAGACAAGC-3'), primer SN2 (5'-TTCAAGTGTGGTTTGACCTAGATCG-3') and probe SN3 (5'-CAATGGATCTGGAGTACTTGAAGGCGTAAAAGCTG-3') are located from nucleotide number 306-331, 450-426 and 360-394, respectively, of the published sequence for the OspA gene ." The primers specify the amplification of a 145 basepair (bp) DNA fragment and the probe hybridizes to a non-primer portion of the amplification product . Each PCR amplification was carried out in a 100 gl reaction volume containing 2 . 5 units of Taq DNA polymerase (Perkin Elmer Cetus or Strategene), each primer at 0 . 5 gm, each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, and dTTP) at 0 . 2 mm, 3 mm MgCl 2 , 10 mm Tris HCI (pH 8 . 3), 50 mm KCI and 0. 1 mg ml - ' gelatin . Before amplification, the reaction mixture was overlaid with 50 PI mineral oil and amplification was carried out for 45 cycles in a DNA Thermal Cycler (Perkin Elmer Cetus) using denaturing temperature of 95°C, annealing temperature of 56°C and extension temperature of 73°C . The amplification products were detected directly by gel electrophoresis or by oligomer hybridization in solution . For direct gel analysis, 25 pi of the amplification
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products were subjected to electrophoresis on a 3% Nusieve/1 % agarose gel . The DNA was visualized by ethidium-bromide staining . Presence of a 145 basepair DNA fragment indicated that specific amplification had occurred . For oligomer hybridization, part of the amplification products was hybridized in solution to probe SN3, end-labelled with gamma 32P-ATP as described previously . 18 After separation of the hybridization products from the unhybridized probe by electrophoresis through a 12 . 5% polyacrylamide gel, the gel was exposed to Kodak X-omat X-ray film at -70°C for 12-18 hours with one intensifying screen . Detection of a 145 by band on the autoradiograms indicates that amplification occurred . The size of amplification products was determined by comparison - with standards of known size .
RESULTS
Sensitivity The analytical sensitivity of the PCR method for detecting B. burgdorferi DNA (i .e . the smallest amount of B. burgdorferi DNA detectable) was determined by amplifying decreasing amounts of purified B. burgdorferi DNA (Fig . 1) . Using direct gel analysis to detect the amplification product, PCR can detect down to approximately 10 pg of B . burgdorferi DNA . A single 145 by band is seen on the ethidium bromide-stained gel . When the amplification product is detected by hybridization in solution to probe SN3, the analytical sensitivity is increased at least 100-fold . PCR amplification products can be detected from as little as 50 fg of template B. burgdorferi DNA . At the low levels of template B. burgdorferi DNA (50100 fg) only a 145 by band is seen on the autoradiogram after the hybridization analysis, whereas other bands are also present if PCR amplification is performed on higher levels of template B. burgdorferi DNA .
A 3 4 5 6 7
1
2
6
7
145 bp_
-145 bp
Fig. 1 . Analytical sensitivity of PCR . PCR amplification was performed on different amounts of purified B. burgdorferi DNA. Panel A shows the direct analysis of arrifcation products by gel electrophoresis and
ethidium bromide staining . Panel B shows the autoradiogram of the oligomer hybridization analysis of the same amplification products . Amplification was done on 1 ng (lane 1) ; 100 pg (lane 2) ; 10 pg (lane 3); 1 pg (lane 4); 100 fg (lane 5) ; 50 fg (lane 6); 10 fg (lane 7) of B. burgdorferi DNA . Lane 8 in Panel B is
negative control containing no DNA. Lane M in Panel A shows a 123 by DNA ladder as size markers (Bethesda Research Laboratories, Bethesda, MD). It should be noted that in lanes 2 and 3 in Panel A amplification products may be visible only in the original photograph .
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Specificity To determine the analytical specificity of the primers SN1 and SN2 and probe SN3 for B . burgdorferi, DNA from organisms other than B . burgdorferi was also tested by PCR (Fig . 2) . Thus, the closely related organisms Borrelia hermsii, Treponema pallidum, Treponema denticola and Leptospirum interrogans, as well as the common bacterium Staphylococcus aureus were tested for cross-reactivity . In contrast to the strong hybridization signal obtained with 1 ng of B . burgdorferi DNA, no amplification products were detected when 10-100 ng of DNA from each of the other organisms were used as templates for amplification by PCR . Likewise, 1 .tg of DNA from human peripheral blood mononuclear cells yielded no amplification products (Fig . 2) .
DISCUSSION The PCR method has successfully been applied in the diagnosis and study of infectious diseases .12-14•' 8•'9 Based on the principle of in vitro DNA amplification using oligonucleotide primers, the method is very sensitive and specific ." In this study we describe the use of PCR for the detection of B . burgdorferi DNA . The primers SN1 and SN2 used in the amplification of B . burgdorferi DNA by PCR were chosen from a region of the gene which codes for the outer surface protein OspA of B . burgdorferi strain B31 . 17 The outer surface protein OspA is demonstrable in almost all North American isolates of B . burgdorferi as assessed by monoclonal antibodies and apparent molecular weight in polyacrylamide gel . 20 We therefore anticipate that the OspA gene, which is located on a 49 kilobase plasmid 21 will be a useful target for amplification by PCR in tests of clinical specimens for the presence of B . burgdorferi DNA . However, variation in the DNA sequence of the OspA gene between different B . burgdorferi isolates may exist . Therefore it may be necessary to use more than one set of primers to optimize the detection of all North American isolates of B . burgdorferi .
p
Fig. 2.
Analytical specificity of PCR . PCR amplification was performed on 100 ng of DNA from
Leptospirum interrogans (lane 2) ; Borrelia hermsü (lane 4) ; Treponema denticola (lane 5) ; 50 ng DNA from Treponema pallidum (lane 3); 10 ng DNA from Staphylococcus aureus (lane 6); 1 gg human DNA (lane 7) and 1 ng of B. burgdorferi DNA (lane 1). Lane 8 is negative control containing no DNA . An
autoradiogram of the oligomer hybridization analysis of the amplification products is shown .
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Our studies on the sensitivity of the PCR for B. burgdorferi DNA show that PCR can be used to detect as little as 50 fg of B. burgdorferi DNA (Fig . 1) . This corresponds to fewer than 50 spirochaetes, based on a DNA content for B. burgdorferi of approximately 1100 kilobases22 and unpublished observations . The PCR is thus much more sensitive than dot blot hybridization for detection of B. burgdorferi which has a detection limit of 10,000 spirochaetes or 500 pg of purified plasmid DNA .", " Furthermore, PCR using the primers SN1 and SN2 is here shown to be very specific . No cross-reactivity was found with other bacteria tested or with human DNA (Fig . 2) . The detection of B. burgdorferi DNA by PCR is at least 100-fold more sensitive when hybridization with probe SN3 is used instead of direct gel analysis to detect the amplification products (Fig . 1) . This may be significant in the development of a diagnostic test using PCR, as the level of B . burgdorferi cells is expected to be very low in clinical specimens such as blood, synovial fluid and cerebrospinal fluid ." When analysing the amplification products by direct gel analysis a single band of 145 by was seen . In contrast, multiple bands were often seen when using hybridization in solution to analyse the products from amplifications . This is especially true when large amounts of template DNA are used (Fig. 1) . However, at low levels of template B. burgdorferi DNA (50-100 fg) a single, 145 by band was seen . The pattern of multiple bands, after hybridization analysis, was consistently found and was not affected by changing the number of amplification cycles or primer annealing temperature . No bands were seen when using DNA from other organisms as templates for amplification (Fig . 2) . Therefore, these bands are specific for B. burgdorferi DNA . The origin of the extraneous bands is not clear, but multiple bands have been observed in other PCR systems . 1S- 19 We have also observed multiple bands when using other primer sets specific for B. burgdorferi DNA (data now shown) . The difference in banding pattern between direct gel analysis and hybridization analysis is most likely due to the difference in the sensitivity of the two methods for the detection of amplification products . Our data demonstrate that the'detection of B. burgdorferi DNA using PCR is very sensitive and specific . The development of a PCR-based test for detection of B. burgdorferi DNA in clinical specimens promises improved methods for diagnosis and therapeutic monitoring of Lyme disease . The PCR should also be useful for studies of the pathogenesis of this disease .
ACKNOWLEDGEMENTS We thank Dr. James B . Peter, Specialty Laboratories, Inc ., for helpful discussions .
REFERENCES 1 . Ciesielski, C. A ., Markowitz, L . E ., Horsley, R., Hightower, A . W ., Russell, H . & Broome, C . V . (1988) . The geographic distribution of Lyme disease in the United States . In Annals of the New York Academy of Sciences : Lyme Disease and Related Disorders . ( Benach, J . L . & Bosler, E . M ., eds) pp . 283-8 . New York : New York Academy of Sciences . 2 . Burgdorfer, W., Barbour, A. G ., Hayes, S . F ., Benach, J . L ., Grunwaldt, E . & Davis, J . P . (1982) . Lyme disease-a tick-borne spirochetosis? Science 216, 1317-9. 3 . Benach, J . L ., Bosler, E. M ., Hanrahan, J . P. et al. (1983) . Spirochetes isolated from the blood of two patients with Lyme disease . New England Journal of Medicine 308, 740-2 .
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burgdorferi DNA
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4. Steere, A . C., Grodzicki, R. L., Kornblatt, A . N . et al. (1983). The spirochetal etiology of Lyme disease . New England Journal of Medicine 308, 733-40 . 5 . Steere, A . C ., Hutchinson, G . J ., Rahn, D. W. et al . (1983). Treatment of the early manifestations of Lyme disease. Annals of Internal Medicine 99, 22-6 . 6. Craft, J . E ., Grodzicki, R. L. & Steere, A. C. (1984) . Antibody response in Lyme disease : evaluation of diagnostic tests. journal of Infectious Diseases 149, 789-95. 7. Grodzicki, R . L., & Steere, A. C. (1988) . Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease . journal of Infectious Diseases 157, 790-7 . 8 . Magnarelli, L . A., Anderson, J . F ., & Johnson, R . C . (1987) . Cross-reactivity in serological tests for Lyme disease and other spirochetal infections, journal of Infectious Diseases 156, 183-8 . 9. de Koning, J ., Bosma, R . B . & Hoogkamp-Korstanje, J . A . A. (1987) . Demonstration of spirochaetes in patients with Lyme disease with a modified silver stain . Journal of Medical Microbiology 23, 261-7. 10 . Steere, A . C ., Grodzicki, R. L ., Craft, J . E ., Shrestha, M ., Kornblatt, A. M. & Malawista, S . E . (1984) . Recovery of Lyme disease spirochetes from patients . Yale Journal of Biology and Medicine 57, 55760. 11 . Saiki, R . K., Gelfand, D . H ., Stoffel, S . et al. (1988) . Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 12. Ou, C .-Y ., Kwok, S ., Mitchell, S . W . et al. (1988). DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells . Science 239, 295-7. 13 . Demmler, G . J ., Buffone, G. J ., Schimbor, C . M . & May, R. A. (1988) . Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. Journal of Infectious Diseases 158, 1177-1184 . 14 . Olive, D. M. (1989) . Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase . Journal of Clinical Microbiology 27, 261-5 . 15 . Steffan, R . J . & Atlas, R. M. (1988) . DNA amplification to enhance detection of genetically engineered bacteria in environmental samples . Applied and Environmental Microbiology 54, 2185-91 . 16. Barbour, A . G . (1984). Isolation and cultivation of Lyme disease spirochetes . Yale Journal of Biological and Medicine 57, 521-5 . 17 . Bergstrom S ., Bundoc, V . G . & Barbour, A . G . (1989) . Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi . Molecular Microbiology 3, 479-86 . 18 . Abbott, M . A ., Poiesz, B . J ., Byrne, B . C ., Kowk S ., Sninsky, J . J . & Ehrlich, G . D . (1988) . Enzymatic gene amplification : qualitative and quantitative methods for detecting proviral DNA amplified in vitro . journal of Infectious Diseases 158, 1158-69 . 19. Kaneko, S ., Miller, R . H ., Feinstone, S . M . et al. (1989) . Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay . Proceedings of the National Academy of Sciences, USA 86, 312-6. 20. Barbour, A . G ., Heiland, R. A . & Howe, T . R. (1985) . Heterogeneity of major proteins in Lyme disease borrelia : a molecular analysis of North America and European isolates . Journal of Infectious Diseases 152, 478-84 . 21 . Barbour, A . G . & Garon, C. F. (1987). Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends . Science 237, 409-11 . 22 . Ferdows, M. S . & Barbour, A . G . (1989' . Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent . Proceedings of the National Academy of Sciences, USA 86, 5969-73 . 23 . Schwan, T . G . & Barbour, A . G . (1988) . Efficacy of nucleic acid hybridization probes for the detection and identification of Borrelia burgdorferi. In Annals of the New York Academy of Sciences : Lyme Disease and Related Disorders . (Benach, J . L. & Bosler, E . M., eds) pp . 419-21 . 24 . Schwan, T . G ., Simpson, W . J ., Schrumpf, M . E . & Karstens, R . H . (1989) . Identification of Borrelia burgdorferi and B. hermsii using DNA hybridization probes . Journal of Clinical Microbiology 27, 1734-8 .
Detection of Borrelia burgdorferi DNA by the polymerase chain reaction
Siren L . Nielsen *J Karen K. Y . Young and Alan G . Barbourj' *Specialty Laboratories, Inc ., 2211 Michigan Avenue, Santa Monica, CA, U.S.A . 90404-3900 and tThe University of Texas, Departments of Microbiology and Medicine, Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7881, U .S .A . (Received 20 September 1989, Accepted 28 September 1989)
DNA amplification by the polymerase chain reaction (PCR) was used to detect DNA of the Lyme disease spirochaete Borrelia burgdorferi . Primers that specify the amplification of a 145 basepair DNA fragment of the OspA gene of B. burgdorferi were used . The amplification product was detected by gel electrophoresis and ethidium bromide staining or by hybridization to a radiolabelled oligonucleotide probe . The hybridization method was found to be more sensitive. As little as 50 fg of purified B . burgdorferi DNA could be detected by PCR . This corresponds to fewer than 50 spirochaetes . The specificity of PCR for B . burgdorferi was tested by using DNA from other organisms as templates for amplification . No cross-reactivity was found . The data shown provide useful information for the development of a PCR-based diagnostic test for Lyme disease .
KEYWORDS: Borrelia burgdorferi, PCR, DNA detection.
INTRODUCTION Lyme disease, the most common arthropod-borne disease in the United States and Europe, has been reported with increasing frequency during the last decade .' The disease is caused by the spirochaete Borrelia burgdorferi2-4 and transmitted by ticks, which account for the seasonal and geographic case clusters . The disease spectrum ranges from subclinical infections to severe skin, joint, cardiac and neurological manifestation S .3,4 Because of the variations in clinical picture and because the disease often mimics other neurological and rheumatological disorders, a definitive diagnosis based on clinical findings is difficult . Yet, early diagnosis is important because antibiotic treatment can prevent the development of neurological, cardiac and joint sequelae . s $ Author to whom correspondence should be sent.
0890-8508/90/010073+07 $03 .00/0
73
© 1990 Academic Press Limited
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S . L. Nielsen et al .
Immunofluorescence assay, enzyme-linked immunosorbent assay and immunoblotting are currently used to detect B . burgdorferi-specific IgG and IgM antibodies in serum and cerebrospinal fluid . 6'' These tests, although useful in later stages of the disease, have only 50-60% sensitivity during the first 3-6 weeks after infection .' Lack of specificity is also a problem due to false-positive reactions in patients with other spirochaetal diseases such as syphilis and relapsing fever as well as in patients with rheumatic diseases . 6.8 Direct detection of the spirochaete by microscopic examination s or culture10 of blood, cerebrospinal fluid, synovial fluid and skin tissue are not practical for routine use and have low diagnostic sensitivities . A sensitive and specific test for the detection of B . burgdorferi is needed not only for early diagnosis of suspected Lyme disease, but also for therapeutic monitoring and studies of pathogenesis . In vitro amplification of DNA via the polymerase chain reaction (PCR) 11 has been shown to be valuable for the diagnosis of viral infections such as those involving human immunodeficiency virus and cytomegalovirus . 12,13 PCR has also been used for the detection of bacteria such as E. coli and Pseudomonas spp . 14,15 We tested the utility of the PCR technique for the detection of B . burgdorferi DNA . Our results show that a high degree of sensitivity and specificity were achieved with the PCR technique . This will provide useful information for the future development of a PCR-based diagnostic test for Lyme disease .
MATERIALS AND METHODS Organisms
Borrelia burgdorferi strain B31 (ATCC 35210) and Borrelia hermsii strain HS1 (ATCC 35209) were grown in BSK medium at 33 ° C . 16 The cells were harvested by centrifugation at 1500 X g for 30 minutes, washed in phosphate-buffered saline (PBS) and transferred to microcentrifuge tubes where the cells were pelleted again at 16,000 X g for 15 minutes . Treponema pallidum (ATCC 27087), Treponema denticola (ATCC 33521), Leptospirum interrogans (ATCC 43642) and Staphylococcus aureus (ATCC 6341) were obtained from ATCC either as frozen or freeze-dried cultures . The cells were resuspended and washed in PBS and pelleted as described above .
Isolation of nucleic acids Pellets of the microorganisms were resuspended in lysis buffer SET (25% sucrose ; 50 mm EDTA, 50 mm Tris, pH 8 . 0) and lysozyme was then added to a final concentration of 2 mg ml -1 . After incubation for 30 minutes at 37°C, proteinase K and sodium dodecyl sulphate were added to final concentrations of 0. 25 mg ml -1 and 1 . 2%, (w/v) respectively . The cells were incubated again at 37°C for 30 minutes with gentle shaking every 10 minutes . Nucleic acids were extracted twice with phenol and chloroform-isoamyl alcohol and once with chloroform-isoamyl alcohol alone . After adjusting the NaCl concentration to 0 . 2 M, the nucleic acids were precipitated with 2 volumes of 95% ethanol overnight at -20°C and then pelleted
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by centrifugation at 17,000 X g for 30 minutes . The pellet was then washed in cold 70% ethanol, lyophilized and resuspended in TE (10 mm Tris HCI, 1 mm EDTA, pH 8 . 0) . The nucleic acid suspension was then treated with DNase-free RNase . DNA concentration was estimated after electrophoresis on a 0 . 7% agarose gel by comparing the intensity of the ethidium bromide-stained DNA bands with DNA standards of known concentrations (Hind III digests of Lambda DNA, Bethesda Research Laboratories, Bethesda, MD) . Portions of the B. burgdorferi DNA were further purified to obtain a more accurate measurement of the DNA concentration . DNA was separated by electrophoresis through a 07% low-melt agarose (Bethesda Research Laboratories, Bethesda, MD) gel and then stained with ethidium bromide . The visible, highmolecular weight DNA was cut out of the gel and purified through a NACS Prepac mini-column (Bethesda Research Laboratories, Bethesda, MD), following the manufacturer's instructions . The eluted DNA was precipitated with ethanol and resuspended in TE . DNA concentration and purity were estimated by optical density at 260 nm/280 nm . Human DNA used in the study was obtained from peripheral blood mononuclear cells . The mononuclear cells were isolated from peripheral blood by banding on Ficoll paque (Pharmacia) following the manufacturer's instructions . The isolated cells was then lysed in lysis buffer (50 mm KCI, 10 mm Tris HCI, pH 8 . 3, 2 .5 MM MgCl 2, 0 . 5% (v/v) Tween 20, 0 . 5% (v/v) Triton X-100), and treated with proteinase K (120 gg ml -1 ) at 60° C for 1 hour. Proteinase K was then inactivated by a 10 minute incubation at 95°C . Aliquots of the lysate was used in the amplification reactions .
PCR Oligonucleotide primers SN1 and SN2 and oligonucleotide probe SN3 chosen from the sequence for the outer surface protein OspA gene of B . burgdorferi strain B31 17 were used throughout the study . The oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer and purified through oligonucleotide purification cartridges (Applied Biosystems) . Primer SN1 (5'-CGATCTAATTGCAACAGTAGACAAGC-3'), primer SN2 (5'-TTCAAGTGTGGTTTGACCTAGATCG-3') and probe SN3 (5'-CAATGGATCTGGAGTACTTGAAGGCGTAAAAGCTG-3') are located from nucleotide number 306-331, 450-426 and 360-394, respectively, of the published sequence for the OspA gene ." The primers specify the amplification of a 145 basepair (bp) DNA fragment and the probe hybridizes to a non-primer portion of the amplification product . Each PCR amplification was carried out in a 100 gl reaction volume containing 2 . 5 units of Taq DNA polymerase (Perkin Elmer Cetus or Strategene), each primer at 0 . 5 gm, each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, and dTTP) at 0 . 2 mm, 3 mm MgCl 2 , 10 mm Tris HCI (pH 8 . 3), 50 mm KCI and 0. 1 mg ml - ' gelatin . Before amplification, the reaction mixture was overlaid with 50 PI mineral oil and amplification was carried out for 45 cycles in a DNA Thermal Cycler (Perkin Elmer Cetus) using denaturing temperature of 95°C, annealing temperature of 56°C and extension temperature of 73°C . The amplification products were detected directly by gel electrophoresis or by oligomer hybridization in solution . For direct gel analysis, 25 pi of the amplification
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products were subjected to electrophoresis on a 3% Nusieve/1 % agarose gel . The DNA was visualized by ethidium-bromide staining . Presence of a 145 basepair DNA fragment indicated that specific amplification had occurred . For oligomer hybridization, part of the amplification products was hybridized in solution to probe SN3, end-labelled with gamma 32P-ATP as described previously . 18 After separation of the hybridization products from the unhybridized probe by electrophoresis through a 12 . 5% polyacrylamide gel, the gel was exposed to Kodak X-omat X-ray film at -70°C for 12-18 hours with one intensifying screen . Detection of a 145 by band on the autoradiograms indicates that amplification occurred . The size of amplification products was determined by comparison - with standards of known size .
RESULTS
Sensitivity The analytical sensitivity of the PCR method for detecting B. burgdorferi DNA (i .e . the smallest amount of B. burgdorferi DNA detectable) was determined by amplifying decreasing amounts of purified B. burgdorferi DNA (Fig . 1) . Using direct gel analysis to detect the amplification product, PCR can detect down to approximately 10 pg of B . burgdorferi DNA . A single 145 by band is seen on the ethidium bromide-stained gel . When the amplification product is detected by hybridization in solution to probe SN3, the analytical sensitivity is increased at least 100-fold . PCR amplification products can be detected from as little as 50 fg of template B. burgdorferi DNA . At the low levels of template B. burgdorferi DNA (50100 fg) only a 145 by band is seen on the autoradiogram after the hybridization analysis, whereas other bands are also present if PCR amplification is performed on higher levels of template B. burgdorferi DNA .
A 3 4 5 6 7
1
2
6
7
145 bp_
-145 bp
Fig. 1 . Analytical sensitivity of PCR . PCR amplification was performed on different amounts of purified B. burgdorferi DNA. Panel A shows the direct analysis of arrifcation products by gel electrophoresis and
ethidium bromide staining . Panel B shows the autoradiogram of the oligomer hybridization analysis of the same amplification products . Amplification was done on 1 ng (lane 1) ; 100 pg (lane 2) ; 10 pg (lane 3); 1 pg (lane 4); 100 fg (lane 5) ; 50 fg (lane 6); 10 fg (lane 7) of B. burgdorferi DNA . Lane 8 in Panel B is
negative control containing no DNA. Lane M in Panel A shows a 123 by DNA ladder as size markers (Bethesda Research Laboratories, Bethesda, MD). It should be noted that in lanes 2 and 3 in Panel A amplification products may be visible only in the original photograph .
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Specificity To determine the analytical specificity of the primers SN1 and SN2 and probe SN3 for B . burgdorferi, DNA from organisms other than B . burgdorferi was also tested by PCR (Fig . 2) . Thus, the closely related organisms Borrelia hermsii, Treponema pallidum, Treponema denticola and Leptospirum interrogans, as well as the common bacterium Staphylococcus aureus were tested for cross-reactivity . In contrast to the strong hybridization signal obtained with 1 ng of B . burgdorferi DNA, no amplification products were detected when 10-100 ng of DNA from each of the other organisms were used as templates for amplification by PCR . Likewise, 1 .tg of DNA from human peripheral blood mononuclear cells yielded no amplification products (Fig . 2) .
DISCUSSION The PCR method has successfully been applied in the diagnosis and study of infectious diseases .12-14•' 8•'9 Based on the principle of in vitro DNA amplification using oligonucleotide primers, the method is very sensitive and specific ." In this study we describe the use of PCR for the detection of B . burgdorferi DNA . The primers SN1 and SN2 used in the amplification of B . burgdorferi DNA by PCR were chosen from a region of the gene which codes for the outer surface protein OspA of B . burgdorferi strain B31 . 17 The outer surface protein OspA is demonstrable in almost all North American isolates of B . burgdorferi as assessed by monoclonal antibodies and apparent molecular weight in polyacrylamide gel . 20 We therefore anticipate that the OspA gene, which is located on a 49 kilobase plasmid 21 will be a useful target for amplification by PCR in tests of clinical specimens for the presence of B . burgdorferi DNA . However, variation in the DNA sequence of the OspA gene between different B . burgdorferi isolates may exist . Therefore it may be necessary to use more than one set of primers to optimize the detection of all North American isolates of B . burgdorferi .
p
Fig. 2.
Analytical specificity of PCR . PCR amplification was performed on 100 ng of DNA from
Leptospirum interrogans (lane 2) ; Borrelia hermsü (lane 4) ; Treponema denticola (lane 5) ; 50 ng DNA from Treponema pallidum (lane 3); 10 ng DNA from Staphylococcus aureus (lane 6); 1 gg human DNA (lane 7) and 1 ng of B. burgdorferi DNA (lane 1). Lane 8 is negative control containing no DNA . An
autoradiogram of the oligomer hybridization analysis of the amplification products is shown .
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S. L. Nielsen et al.
Our studies on the sensitivity of the PCR for B. burgdorferi DNA show that PCR can be used to detect as little as 50 fg of B. burgdorferi DNA (Fig . 1) . This corresponds to fewer than 50 spirochaetes, based on a DNA content for B. burgdorferi of approximately 1100 kilobases22 and unpublished observations . The PCR is thus much more sensitive than dot blot hybridization for detection of B. burgdorferi which has a detection limit of 10,000 spirochaetes or 500 pg of purified plasmid DNA .", " Furthermore, PCR using the primers SN1 and SN2 is here shown to be very specific . No cross-reactivity was found with other bacteria tested or with human DNA (Fig . 2) . The detection of B. burgdorferi DNA by PCR is at least 100-fold more sensitive when hybridization with probe SN3 is used instead of direct gel analysis to detect the amplification products (Fig . 1) . This may be significant in the development of a diagnostic test using PCR, as the level of B . burgdorferi cells is expected to be very low in clinical specimens such as blood, synovial fluid and cerebrospinal fluid ." When analysing the amplification products by direct gel analysis a single band of 145 by was seen . In contrast, multiple bands were often seen when using hybridization in solution to analyse the products from amplifications . This is especially true when large amounts of template DNA are used (Fig. 1) . However, at low levels of template B. burgdorferi DNA (50-100 fg) a single, 145 by band was seen . The pattern of multiple bands, after hybridization analysis, was consistently found and was not affected by changing the number of amplification cycles or primer annealing temperature . No bands were seen when using DNA from other organisms as templates for amplification (Fig . 2) . Therefore, these bands are specific for B. burgdorferi DNA . The origin of the extraneous bands is not clear, but multiple bands have been observed in other PCR systems . 1S- 19 We have also observed multiple bands when using other primer sets specific for B. burgdorferi DNA (data now shown) . The difference in banding pattern between direct gel analysis and hybridization analysis is most likely due to the difference in the sensitivity of the two methods for the detection of amplification products . Our data demonstrate that the'detection of B. burgdorferi DNA using PCR is very sensitive and specific . The development of a PCR-based test for detection of B. burgdorferi DNA in clinical specimens promises improved methods for diagnosis and therapeutic monitoring of Lyme disease . The PCR should also be useful for studies of the pathogenesis of this disease .
ACKNOWLEDGEMENTS We thank Dr. James B . Peter, Specialty Laboratories, Inc ., for helpful discussions .
REFERENCES 1 . Ciesielski, C. A ., Markowitz, L . E ., Horsley, R., Hightower, A . W ., Russell, H . & Broome, C . V . (1988) . The geographic distribution of Lyme disease in the United States . In Annals of the New York Academy of Sciences : Lyme Disease and Related Disorders . ( Benach, J . L . & Bosler, E . M ., eds) pp . 283-8 . New York : New York Academy of Sciences . 2 . Burgdorfer, W., Barbour, A. G ., Hayes, S . F ., Benach, J . L ., Grunwaldt, E . & Davis, J . P . (1982) . Lyme disease-a tick-borne spirochetosis? Science 216, 1317-9. 3 . Benach, J . L ., Bosler, E. M ., Hanrahan, J . P. et al. (1983) . Spirochetes isolated from the blood of two patients with Lyme disease . New England Journal of Medicine 308, 740-2 .
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4. Steere, A . C., Grodzicki, R. L., Kornblatt, A . N . et al. (1983). The spirochetal etiology of Lyme disease . New England Journal of Medicine 308, 733-40 . 5 . Steere, A . C ., Hutchinson, G . J ., Rahn, D. W. et al . (1983). Treatment of the early manifestations of Lyme disease. Annals of Internal Medicine 99, 22-6 . 6. Craft, J . E ., Grodzicki, R. L. & Steere, A. C. (1984) . Antibody response in Lyme disease : evaluation of diagnostic tests. journal of Infectious Diseases 149, 789-95. 7. Grodzicki, R . L., & Steere, A. C. (1988) . Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease . journal of Infectious Diseases 157, 790-7 . 8 . Magnarelli, L . A., Anderson, J . F ., & Johnson, R . C . (1987) . Cross-reactivity in serological tests for Lyme disease and other spirochetal infections, journal of Infectious Diseases 156, 183-8 . 9. de Koning, J ., Bosma, R . B . & Hoogkamp-Korstanje, J . A . A. (1987) . Demonstration of spirochaetes in patients with Lyme disease with a modified silver stain . Journal of Medical Microbiology 23, 261-7. 10 . Steere, A . C ., Grodzicki, R. L ., Craft, J . E ., Shrestha, M ., Kornblatt, A. M. & Malawista, S . E . (1984) . Recovery of Lyme disease spirochetes from patients . Yale Journal of Biology and Medicine 57, 55760. 11 . Saiki, R . K., Gelfand, D . H ., Stoffel, S . et al. (1988) . Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 12. Ou, C .-Y ., Kwok, S ., Mitchell, S . W . et al. (1988). DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells . Science 239, 295-7. 13 . Demmler, G . J ., Buffone, G. J ., Schimbor, C . M . & May, R. A. (1988) . Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. Journal of Infectious Diseases 158, 1177-1184 . 14 . Olive, D. M. (1989) . Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase . Journal of Clinical Microbiology 27, 261-5 . 15 . Steffan, R . J . & Atlas, R. M. (1988) . DNA amplification to enhance detection of genetically engineered bacteria in environmental samples . Applied and Environmental Microbiology 54, 2185-91 . 16. Barbour, A . G . (1984). Isolation and cultivation of Lyme disease spirochetes . Yale Journal of Biological and Medicine 57, 521-5 . 17 . Bergstrom S ., Bundoc, V . G . & Barbour, A . G . (1989) . Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi . Molecular Microbiology 3, 479-86 . 18 . Abbott, M . A ., Poiesz, B . J ., Byrne, B . C ., Kowk S ., Sninsky, J . J . & Ehrlich, G . D . (1988) . Enzymatic gene amplification : qualitative and quantitative methods for detecting proviral DNA amplified in vitro . journal of Infectious Diseases 158, 1158-69 . 19. Kaneko, S ., Miller, R . H ., Feinstone, S . M . et al. (1989) . Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay . Proceedings of the National Academy of Sciences, USA 86, 312-6. 20. Barbour, A . G ., Heiland, R. A . & Howe, T . R. (1985) . Heterogeneity of major proteins in Lyme disease borrelia : a molecular analysis of North America and European isolates . Journal of Infectious Diseases 152, 478-84 . 21 . Barbour, A . G . & Garon, C. F. (1987). Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends . Science 237, 409-11 . 22 . Ferdows, M. S . & Barbour, A . G . (1989' . Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent . Proceedings of the National Academy of Sciences, USA 86, 5969-73 . 23 . Schwan, T . G . & Barbour, A . G . (1988) . Efficacy of nucleic acid hybridization probes for the detection and identification of Borrelia burgdorferi. In Annals of the New York Academy of Sciences : Lyme Disease and Related Disorders . (Benach, J . L. & Bosler, E . M., eds) pp . 419-21 . 24 . Schwan, T . G ., Simpson, W . J ., Schrumpf, M . E . & Karstens, R . H . (1989) . Identification of Borrelia burgdorferi and B. hermsii using DNA hybridization probes . Journal of Clinical Microbiology 27, 1734-8 .
