Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes
© Saidac et al; licensee BioMed Central Ltd. 2009
Received: 15 December 2008
Accepted: 24 February 2009
Published: 24 February 2009
Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.
We show here that molecular beacons are effective, sensitive and specific probes for detecting and estimating wide-ranging numbers of B. burgdorferi in the presence of mouse DNA. In our assays, the spirochete recA and the mouse nidogen gene amplicons were detected simultaneously using molecular beacons labeled with different fluorophores. We further validated the application of these probes by quantifying the wild-type strain and bgp-defective mutant of B. burgdorferi. The bgp-defective mutant shows a ten-fold reduction in the level of spirochetes present in various tissues.
The high sensitivity and specificity of molecular beacons makes them superior probes for the detection of small numbers of B. burgdorferi. Furthermore, the use of molecular beacons can be expanded for the simultaneous detection and quantification of multiple pathogens in the infected hosts, including humans, and in the arthropod vectors.
Lyme disease, caused by the spirochete Borrelia burgdorferi, is a highly prevalent multisystemic illness that affects the heart, joints, skin, musculoskeletal and nervous system. Persistent infection with the spirochete results in potentially severe manifestations, such as, carditis, arthritis, acrodermatitis chronicum atrophicans and neuroborreliosis. The severity of the Lyme disease depends on several factors including; genotypes of both the host and the infecting Borrelia strain, age of the host, simultaneous infection with another tick-transmitted pathogen and the spirochete burden in the infected tissue [1–7].
The B. burgdorferi genome is relatively small (1.52 Mb) in size. Although the spirochete lacks major biosynthetic pathways, it contains a large number of surface proteins. Several of these are adhesins, which mediate attachment to various cell lines [8–13]. Each adhesin could contribute to the tissue specific colonization by the spirochetes. Alternatively, the presence of multiple adhesins exhibiting specificity for the same receptor can create a redundancy of function [9, 14]. In the latter case, a mutation in the gene encoding a particular B. burgdorferi adhesin can only moderately reduce the ability of the spirochete to colonize. Indeed, mutation in a specific spirochete gene has been shown to reduce the number of B. burgdorferi in the infected tissues [15, 16]. Therefore, although Bgp is not essential for infection it could contribute to tissue colonization by Lyme spirochetes. A sensitive detection system is critical to assess the burden of these mutant spirochetes in tissues and to determine the impact of mutation on a specific disease manifestation, and hence, could provide insight into the role of unique genes of B. burgdorferi in Lyme disease. Quantification of the spirochete burden in infected tissues by Real-time quantitative PCR (qPCR) using the fluorescent dye, SYBR Green I, is a commonly used method [5, 6, 17, 18]. However, this dye binds to the minor groove of the DNA double helix in a sequence-independent manner. Therefore, it is susceptible to detection of non-specific amplification products, including primer dimers.
Several types of fluorogenic hybridization probes have been described for the specific detection of PCR amplified products. The best characterized among these are the TaqMan probes. These probes are single stranded oligonucleotides labeled with a fluorophore-quencher pair that hybridize with the sequence present in the internal region of an amplified PCR product. When free in solution, TaqMan probes form random coils to bring fluorophore reporter and quencher in close proximity, enabling Fluorescence Resonance Energy Transfer (FRET) from the fluorophore to the quencher. This mechanism alleviates the fluorescence signal of the reporter. In the presence of the target, the TaqMan probe-target hybrid comes in contact with the Taq Polymerase during the extension phase of a PCR cycle. The inherent 5'exonuclease activity of the enzyme then cleaves the probe, releasing the fluorescent reporter from the probe. This prevents FRET and leads to an increase in the fluorescence intensity at each subsequent PCR cycle. Several researchers have employed this technique effectively to quantify B. burgdorferi in mammalian tissues and in ticks [15, 16, 19–26]. However, simultaneous quantification of spirochete and infected mammalian DNA has not been described.
The proximity of the fluorophore and the quencher in TaqMan probes in the free state depends on the formation of random coils and often results in only partial quenching of the fluorescence, and hence, can produce a high background . In contrast, molecular beacon probes are single-stranded oligonucleotides that form stem-loop structures with the recognition sequence mainly located in the loop region. A 5–7 base pair stem brings the fluorophore at the 5'end and non-fluorescent quencher at the 3'end together . This contact-dependent quenching mechanism is highly efficient and reduces the background fluorescence significantly when the probe is free in solution. The presence of the target sequence leads to the formation of a probe-target hybrid, which is longer and more stable than the stem. This spontaneous conformational reorganization forces dissociation of the fluorophore and the quencher resulting in a significant increase in fluorescence. Because of the specificity of the interaction between the probe region of the molecular beacon with the complementary target sequence within the PCR amplification product, the presence of the non-specific DNA does not interfere with the quantitative detection of the intended amplification product.
Due to their potential superiority , we used molecular beacons for PCR-based quantification of B. burgdorferi in this study and assessed their efficiency, sensitivity and specificity relative to the SYBR Green I based detection system. Furthermore, the molecular beacons were used to detect B. burgdorferi, including the bgp mutant, in infected mouse tissues effectively.
Analysis of molecular beacon probes for qPCR detection of recA gene of B. burgdorferi and nidogen gene of mouse
Sequence of primers for PCR, molecular beacon probes and their specific targets
PCR Primers, Probes and Targets
5' GTG GAT CTA TTG TAT TAG ATG AGG CTC TCG 3'
5' GCC AAA GTT CTG CAA CAT TAA CAC CTA AAG 3'
5' CCA GCC ACA GAA TAC CAT CC 3'
5' GGA CAT ACT CTG CTG CCA TC 3'
5' CGG CGC ACC CAG CTT CGG CTC AGT AGC GCC G 3'
5' ta GGC GCT ACT GAG CCG AAG CTG GGT G at 3'
5' CCC GCG CGT CTG GCA AGA CTA CTT TAA CTC TTC GCG GG 3'
5' ta GAA GAG TTA AAG TAG TCT TGC CAG ACG at 3'
5' CGCGAG TCG TCT GGC AAG ACT ACT TTA A CTCGCG 3'
5' ttG AGT TAA AGT AGT CTT GCC AGA CGA CTC tt 3'
5' CTG GCG GAT ATC CTA GGG GG CGC CAG 3'
5' ttG CGC CCC CTA GGA TAT CCG CCt t 3'
B. burgdorferi and mouse DNA can be quantified simultaneously using molecular beacons in multiplex system
Since molecular beacons are specific hybridization probes for particular PCR products, simultaneous detection of pathogen and host PCR products is possible using molecular beacons tagged with different fluorophores. Therefore, normalization of the host DNA in different tissue samples is more convenient and accurate. To test this premise, a ten-fold serial dilution of genomic DNA of B. burgdorferi strain N40 spiked in the same concentration of the uninfected mouse tissue DNA, i.e., 105 nidogen copies per reaction, were used as template for the PCR assays. The "threshold cycle" (Ct) is the PCR cycle at which specific fluorescence rises significantly above the fluorescence background. In this assay, the threshold was set at twenty times the standard deviation of the noise in the background fluorescence of each PCR assay (recorded between the third and 20th thermal cycle).
Sensitivity and specificity of detection of qPCR amplicons is not affected by multiplex analysis
Molecular beacons can be used effectively to quantify B. burgdorferi in the infected tissues
Quantitative PCR is a widely used method for determining the burden of pathogens, including the Lyme disease-causing spirochetes, present in infected tissues. The fluorescent dye SYBR Green I, which binds non-specifically to double stranded DNA, has mainly been used to detect the qPCR product obtained for the recA or fla genes of B. burgdorferi for quantification. However, sensitivity of this detection system is poor when the number of spirochetes present in the tissues is low [8, 29]. To overcome the background fluorescence obtained by binding of SYBR Green to the non-specific amplified products, such as primer dimers , a higher temperature (80°C) is needed for the detection of the amplicon. This could also contribute to the low sensitivity of this detection system when a small spirochete population and high primer dimer concentrations are present. Clinical Lyme disease manifestations are not always dependent on high B. burgdorferi burden. Furthermore, qPCR of a mouse gene, such as nidogen, using specific primers needs to be conducted separately to normalize the quantity of mouse tissue in the sample when SYBR Green is used. Hence, it is important to explore newer, more specific probes, which remain sensitive even when less than one hundred spirochetes are present in the PCR sample.
More recently, TaqMan probes have also been employed for detection of the PCR products from both B. burgdorferi and host/vector genes [16, 19–26]. Although TaqMan probes have been reported to be a sensitive detection system for PCR of B. burgdorferi amplicon by several laboratories [19–22, 24, 25], high background fluorescence of the unhybridized probe, i.e., low signal-to-noise ratio, and lower sensitivity due to incomplete enzymatic hydrolysis has been observed with these probes [19, 20, 27]. In addition, compatibility of the fluorophore and quencher due to the requirement for sufficient spectral overlap remains a significant issue due to the requirement of FRET in TaqMan probes. This limits its application in the multiplex analysis to some extent. To the best of our knowledge, simultaneous detection of mouse and spirochete DNA using TaqMan probes in multiplex analysis has not been reported. In contrast to TaqMan probes, quenching due to a direct interaction between fluorophore and quencher in molecular beacons is much more efficient. It also offers a choice of a variety of fluorophores with quenchers. Indeed, the efficiency of molecular beacons is not affected significantly by the choice of different fluorophores-quencher combinations 
Denaturation profiles of the Nidogen molecular probe as well as three different RecA molecular beacons, and detection of B. burgdorferi by PCR assays indicate that RecA3 emits most fluorescence and shows the highest sensitivity of detection. RecA3 has a high GC content, and thereby, forms the most stable probe-target hybrid and hairpin structures. Furthermore, its detection step temperature is most compatible with that of the Nidogen molecular beacon (Table 1). This also makes RecA3 most suitable for multiplex analyses. The ABI7700 sequence detector software from Applied Biosystems can distinguish the emission of a particular fluorescence signal (from FAM or TET fluorophores) associated with each molecular beacon in PCR assays. Lower background signal facilitated the efficient detection of B. burgdorferi at seven different dilutions, and a high co-efficient of correlation between Ct values and spirochete number (r2 = 0.996) was obtained. In addition, sensitivity of detection of B. burgdorferi DNA was not affected by the presence of mouse DNA and remained comparable in monoplex versus multiplex analyses. These results, as well as a high correlation (R2 = 0.998) between threshold cycle number and the amount of mouse DNA, made quantification of the spirochetes burden in different infected mouse tissues convenient and accurate since a single PCR tube per sample was used for the analysis of both B. burgdorferi and mouse amplicons. This could be of great importance if this system is employed for detection of B. burgdorferi, as well as other pathogens, in patient tissues or fluids, where quantities of samples are often limiting.
Efficient amplification by PCR due to the small size of the amplicons and high signal to noise ratio obtained by the use of molecular beacon probes resulted in high sensitivity of this assay. However, a strong TET signal from the Nidogen molecular beacon sometimes hampered the sensitivity of detection of approximately one spirochete in the sample in multiplex systems (unpublished observation). This can be overcome by synthesizing molecular beacons with a combination of red (such as Texas red) and green (TET or FAM) fluorophore for use in multiplex analyses. This will be especially useful when the pathogen is present in very small numbers in the infected tissues.
Simultaneous infection by several pathogens often creates difficulty in identifying the causative agent for a particular disease manifestation. Multiplex analysis using molecular beacons allows detection of a pathogen and the host tissue by PCR. Furthermore, additional pathogen(s) can be detected by including the appropriate molecular beacon in the assay. This is possible for up to seven molecular beacons, each labeled with different fluorophores, which can be combined in one reaction to detect different amplicons, as long as PCR conditions are compatible. This is of great importance especially for the detection of multiple vector-borne bacterial illnesses in humans such as Lyme disease and human granulocytic anaplasmosis (HGA), caused by Anaplasma phagocytophila. Both of these organisms, along with several viruses, can be transmitted together to humans by Ixodes ticks, often complicating the diagnosis of Lyme disease. This study is focused on quantification specifically of B. burgdorferi, and not other Lyme spirochetes, in the mouse tissues. We anticipate that in the future, after slight modifications of the primers and molecular beacon, we will be able to distinguish the presence of different Lyme spirochetes in clinical samples. An appropriate human gene region will also be selected for amplification and a specific molecular beacon designed for diagnostic purposes. In addition, we will be able to detect Lyme spirochetes in combination with other organisms in clinical samples after an infected tick bite using the specific primers and different fluorophore-tagged molecular beacons. This will help to identify the actual causative agent, facilitate proper treatment strategy and offer a better clinical outcome for the patient. Furthermore, it will be possible to adapt this system to detect microbes in other systems, such as in the infected plants.
In conclusion, molecular beacons have several advantages over other fluorescent probes for qPCR or Real-Time PCR including; (1) specificity of interaction with a particular amplicon, (2) possibility to select a variety of compatible fluorophores and quenchers, which show minimum interference, (3) high signal-to-noise ratio resulting in sensitivity of detection of the amplicons at low copy number, and (4) multiplex analysis (potentially of up to seven different amplicons simultaneously), which may make it feasible to detect multiple pathogens in infected tissue. Hence, molecular beacon probes will be very useful for the detection of various microbial pathogens in patients in the future.
Bacterial strains and mouse infection
N40, clone D10/E9, is an infectious B. burgdorferi (sensu stricto) isolate. We generated bgp-defective mutant of this strain, NP1.3, by disruption of the gene with a kanamycin resistance cassette . Both B. burgdorferi strains were grown at 33°C in BSKII medium containing 6% rabbit serum. To conduct the infection studies, immunocompetent C3H/HeN mice were injected subcutaneously at a dose of 5 × 104 spirochetes per mouse. Mice were euthanized after two weeks of infection and tissues harvested for qPCR. UMDNJ-New Jersey Medical School is accredited (Accreditation number 000534) by the International Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC International), and the animal protocol used was approved by the Institutional Animal Care and Use Committee (IACUC) at UMDNJ.
Purification of B. burgdorferi and mouse genomic DNA
Total genomic DNA was isolated from the low passage B. burgdorferi strain N40 grown to a density of 108spirochetes/ml using the protocol we described previously . DNA from mouse tissues was isolated using the previously described protocol  with two modifications. Firstly, PLG-containing tubes (Qiagen Sciences, MD) were used for phenol and chloroform extraction, since they allow clean separation of the top aqueous layer by decantation after centrifugation. Secondly, a final step of passing the DNA through DNA-Easy kit columns was included to obtain good quality DNA for qPCR.
A 222-bp amplicon from recA gene of B. burgdorferi using RecF and RecR primers and a 154-bp amplicon from mouse nidogen gene using NidoF and NidoR primers (Table 1) were amplified by PCR in 0.2 ml optical tubes, as previously described , using an ABI7700 sequence detector (Applied Biosystems, NJ). Data was processed using the software from the manufacturer. Amplification was performed in 25 μl reaction mixture containing Amplitaq PCR reaction buffer supplemented with 3 mM MgCl2, 500 ng/μl of bovine serum albumin, 250 μM of each deoxynucleoside triphosphate (dNTP), 0.5 μM of each set of primers and 2.5 U of Amplitaq polymerase. Previous work has shown that a single copy of recA and two copies of nidogen gene are present per B. burgdorferi and mouse genomes respectively . Since genome sizes of B. burgdorferi and mouse are 1.5 Mb and 2.5 Gb respectively, 2 ng of B. burgdorferi genomic DNA contains approximately 106 copies of recA gene, while 200 ng of mouse genomic DNA contains approximately 105 copies of nidogen gene. For each amplification reaction, 5 μl of the sample was used to minimize the variation due to pipetting error.
Molecular beacons design
Molecular beacons probes were designed to hybridize to the recA and the nidogen gene amplicons using the previously described strategies . The lengths of the probe sequences were chosen so that they would form a stable hybrid with the target at 5 to 10°C above the annealing temperature (60°C) of the PCR assay. The 5' and 3' arm sequences of the molecular beacons were designed to form a stable hybrid at 5 to 10°C above the annealing temperature of the PCR assay. After selecting three slightly different probe and arm sequences, the molecular beacon for recA amplicon were optimized. These probes were labeled with a Fluorescein (FAM) reporter molecule at their 5' terminals and Black Hole Quencher 1 (BHQ-1) or dabcyl at their 3' terminals. Using similar parameters, a nidogen specific molecular beacon was also designed. The Nidogen molecular beacon was labeled with a 5' Tetrachlorofluorescein (TET) reporter molecule and a 3' BHQ-1 quencher. The fluorophores and quenchers were chosen based on the specifications of the spectrofluorometric thermal cycler platform on which the assays were carried out. The sequences of the molecular beacons used in this study are listed in Table 1. A detailed protocol for the synthesis and purification of molecular beacons can be found at http://www.molecular-beacons.org.
PCR products were detected by fluorescence measurement by including SYBR Green I dye (Molecular Probes, OR) or molecular beacons in the assays. The amplification program for SYBR Green I consisted of heating at 95°C for 2 minutes, followed by 50 cycles of heating at 95°C for 15 s, annealing at 60°C for 30 s, polymerization at 72°C for 20 s and fluorescence detection at 80°C for 10 s. Mouse nidogen was amplified similarly except that fluorescence was detected at 82°C instead of 80°C. For PCR assays using molecular beacon probes, 200 nM of RecA or Nidogen molecular beacon were included per reaction. The amplification program consisted of heating at 95°C for 2 minutes, followed by 50 cycles of heating at 95°C for 15 s, annealing and fluorescence detection at 60°C for 30 s, and polymerization at 72°C for 20 s.
Determination of thermal denaturation profiles
In order to determine the melting temperatures of the molecular beacon stem and the molecular beacon probe-target hybrid, a denaturation profile analysis was carried out. For each probe, three tubes containing 200 nM molecular beacon, 3 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 8.0), in a 50-μl volume were prepared. A two-fold molar excess of an oligonucleotide that is complementary to the molecular beacon probe sequence, a two-fold excess of an oligonucleotide unrelated to the probe sequence, or only buffer were added in these tubes. The fluorescence of each solution was determined as a function of temperature. The thermal cycler was programmed to decrease the temperature of the solutions from 80°C to 30°C in 1°C steps, with each step lasting 1 min, while monitoring fluorescence during each step.
We thank John M. Leong, Sanjay Tyagi and Diana Palmeri for careful review of the manuscript. This work was supported by National Research Foundation for Tick-borne Diseases grant to NP. Arthritis Foundation (New Jersey) grant to NP paid for the open access publication charges for this article.
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