Abstract
Real-time PCR assays have recently been implemented in diagnostics for many bacterial pathogens, allowing rapid and accurate detection, which ultimately results in improved clinical intervention. Here, we describe a sensitive method of detection for three common tick-borne pathogens Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti since coinfections with these pathogens have started occurring with increasing frequency over the last several years in both North America and Europe. A shared geographic region, the same tick vectors, and similar transmission cycle all favor simultaneous transmission of these three tick-borne pathogens. Furthermore, early symptoms of the diseases are often similar and somewhat nonspecific leading to poor clinical identification. The multiplex real-time PCR assay we describe here utilizes gene-specific primers, molecular beacon probes tagged with different fluorophores, and optimized PCR conditions to detect even small amounts of specific pathogen DNA without interference. Application of this detection method will offer better diagnostics for acute and persistent infection compared to the two-tier serological tests that are currently approved in North America and Europe, which do not necessarily detect active infection.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Marques AR (2010) Lyme disease: a review. Curr Allergy Asthma Rep 10:13–20
Wright WF, Riedel DJ, Talwani R, Gilliam BL (2012) Diagnosis and management of Lyme disease. Am Fam Physician 85:1086–1093
Steere AC (2001) Lyme disease. N Engl J Med 345:115–125
Dantas-Torres F, Chomel BB, Otranto D (2012) Ticks and tick-borne diseases: a one health perspective. Trends Parasitol 28:437–446
Ginsberg HS (2008) Potential effects of mixed infections in ticks on transmission dynamics of pathogens: comparative analysis of published records. Exp Appl Acarol 46:29–41
Belongia EA (2002) Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis 2:265–273
Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, Krause PJ, Bakken JS, Strle F, Stanek G, Bockenstedt L, Fish D, Dumler JS, Nadelman RB (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the infectious diseases society of America. Clin Infect Dis 43:1089–1134
Maurin M (2012) Real-time PCR as a diagnostic tool for bacterial diseases. Expert Rev Mol Diagn 12:731–754
Wilson ML (2011) Recent advances in the laboratory detection of mycobacterium tuberculosis complex and drug resistance. Clin Infect Dis 52:1350–1355
Lee JH, Uhl JR, Cockerill FR 3rd, Weaver AL, Orvidas LJ (2008) Real-time PCR vs standard culture detection of group a beta-hemolytic streptococci at various anatomic sites in tonsillectomy patients. Arch Otolaryngol Head Neck Surg 134:1177–1181
Green BN, Johnson CD, Egan JT, Rosenthal M, Griffith EA, Evans MW (2012) Methicillin-resistant Staphylococcus aureus: an overview for manual therapists(). J Chiropr Med 11:64–76
Selva L, Esteva C, Gene A, de Sevilla MF, Hernandez-Bou S, Munoz-Almagro C (2010) Direct detection of Streptococcus Pneumoniae in positive blood cultures by real-time polymerase chain reaction. Diagn Microbiol Infect Dis 66:204–206
Colborn JM, Kosoy MY, Motin VL, Telepnev MV, Valbuena G, Myint KS, Fofanov Y, Putonti C, Feng C, Peruski L (2010) Improved detection of bartonella DNA in mammalian hosts and arthropod vectors by real-time PCR using the NADH dehydrogenase gamma subunit (nuoG). J Clin Microbiol 48:4630–4633
Riehm JM, Rahalison L, Scholz HC, Thoma B, Pfeffer M, Razanakoto LM, Al Dahouk S, Neubauer H, Tomaso H (2011) Detection of Yersinia Pestis using real-time PCR in patients with suspected bubonic plague. Mol Cell Probes 25:8–12
Saidac DS, Marras SA, Parveen N (2009) Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes. BMC Microbiol 9:43
Chan K, Marras SA, Parveen N (2013) Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti. BMC Microbiol 13:295
Marras SAE, Russell Kramer F, Tyagi S (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal 14:151–156
Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16:49–53
Marras SA, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30:e122
Liveris D, Schwartz I, McKenna D, Nowakowski J, Nadelman R, Demarco J, Iyer R, Bittker S, Cooper D, Holmgren D, Wormser GP (2012) Comparison of five diagnostic modalities for direct detection of Borrelia burgdorferi in patients with early Lyme disease. Diagn Microbiol Infect Dis 73:243–245
Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP (2005) Diagnosis of Lyme borreliosis. Clin Microbiol Rev 18:484–509
Schotthoefer AM, Frost HM (2015) Ecology and epidemiology of Lyme borreliosis. Clin Lab Med 35:723–743
Herwaldt BL, Linden JV, Bosserman E, Young C, Olkowska D, Wilson M (2011) Transfusion-associated babesiosis in the United States: a description of cases. Ann Intern Med 155:509–519
Acknowledgment
This work was supported by NIH grant AI089921, and New Jersey Health Foundation and The Office of Research & Economic Development grants to NP.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Schlachter, S., Chan, K., Marras, S.A.E., Parveen, N. (2017). Detection and Differentiation of Lyme Spirochetes and Other Tick-Borne Pathogens from Blood Using Real-Time PCR with Molecular Beacons. In: Bishop-Lilly, K. (eds) Diagnostic Bacteriology. Methods in Molecular Biology, vol 1616. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7037-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7037-7_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7035-3
Online ISBN: 978-1-4939-7037-7
eBook Packages: Springer Protocols