Hostname: page-component-586b7cd67f-vdxz6 Total loading time: 0 Render date: 2024-11-22T16:20:58.710Z Has data issue: false hasContentIssue false
  • English
  • Français

Validation of droplet digital PCR for the detection and absolute quantification of Borrelia DNA in Ixodes scapularis ticks

Published online by Cambridge University Press:  03 November 2016

JENNY L. KING ,
ASHLEY D. SMITH ,
ELIZABETH A. MITCHELL  and
MICHAEL S. ALLEN
JENNY L. KING
Affiliation:
Department of Molecular and Medical Genetics, Center for Biosafety and Biosecurity, University of North Texas Health Science Center, Fort Worth, TX, USA
ASHLEY D. SMITH
Affiliation:
Department of Molecular and Medical Genetics, Center for Biosafety and Biosecurity, University of North Texas Health Science Center, Fort Worth, TX, USA
ELIZABETH A. MITCHELL
Affiliation:
Department of Molecular and Medical Genetics, Center for Biosafety and Biosecurity, University of North Texas Health Science Center, Fort Worth, TX, USA
MICHAEL S. ALLEN*
Affiliation:
Department of Molecular and Medical Genetics, Center for Biosafety and Biosecurity, University of North Texas Health Science Center, Fort Worth, TX, USA
*
*Corresponding author: University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2644, USA. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

We evaluated the QX200 Droplet Digital PCR (ddPCR™, Bio-Rad) system and protocols for the detection of the tick-borne pathogens Borrelia burgdorferi and Borrelia miyamotoi in Ixodes scapularis nymphs and adults collected from North Truro, Massachusetts. Preliminary screening by nested PCR determined positive infection levels of 60% for B. burgdorferi in these ticks. To investigate the utility of ddPCR as a screening tool and to calculate the absolute number of bacterial genome copies in an infected tick, we adapted previously reported TaqMan®-based qPCR assays for ddPCR. ddPCR proved to be a reliable means for detection and absolute quantification of control bacterial DNA with precision as low as ten spirochetes in an individual sample. Application of this method revealed the average carriage level of B. burgdorferi in infected I. scapularis nymphs to be 2291 spirochetes per nymph (range: 230–5268 spirochetes) and 51 179 spirochetes on average in infected adults (range: 5647–115 797). No ticks naturally infected with B. miyamotoi were detected. The ddPCR protocols were at least as sensitive to conventional qPCR assays but required fewer overall reactions and are potentially less subject to inhibition. Moreover, the approach can provide insight on carriage levels of parasites within vectors.

Keywords

droplet digital PCRBorrelia miyamotoiBorrelia burgdorferipathogen detection
Type
Research Article
Information
Parasitology , Volume 144 , Issue 4 , April 2017 , pp. 359 - 367
Check for updates
Copyright
Copyright © Cambridge University Press 2016 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Bio-Rad Laboratories, I. (2014). qPCR/Real-Time PCR. Vol. 2014 Bio-Rad Laboratories, Inc. http://www.bio-rad.com/en-us/applications-technologies/qpcr-real-time-pcr Google Scholar
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A. and Turnbaugh, P. J. (2011). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America 108.Google Scholar
CDC (2013). B. miyamotoi. In Ticks, Vol. 2014 Center for Disease Control and Prevention. http://www.cdc.gov/ticks/miyamotoi.html Google Scholar
CDC (2014 a). Interactive Lyme disease map. In Lyme Disease, Vol. 2014 pp. Map of Reported Cases of Lyme Disease in the United States in 2013. Centers for Disease Control and Prevention. http://www.cdc.gov/lyme/stats/maps/interactivemaps.html Google Scholar
CDC (2014 b). Lyme Disease. Vol. 2014 Center for Disease Control and Prevention. http://www.cdc.gov/lyme/ Google Scholar
CDC (2015). How many people get Lyme disease? Vol. 2015. Center for Disease Control and Prevention. http://www.cdc.gov/lyme/stats/humancases.html Google Scholar
Dietrich, F., Schmidgen, T., Maggi, R. G., Richter, D., Matuschka, F.-R., Vonthein, R., Breitschwerdt, E. B. and Kempf, V. A. J. (2010). Prevalence of Bartonella henselae and Borrelia burgdorferi sensu lato DNA in Ixodes ricinus Ticks in Europe. Applied and Environmental Microbiology 76, 1395–1398.CrossRefGoogle ScholarPubMed
Fraser, C. M., Casjens, S., Huang, W. M., Sutton, G. G., Clayton, R., Lathigra, R., White, O., Ketchum, K. A., Dodson, R., Hickey, E. K., Gwinn, M., Dougherty, B., Tomb, J. F., Fleischmann, R. D., Richardson, D., Peterson, J., Kerlavage, A. R., Quackenbush, J., Salzberg, S., Hanson, M., van Vugt, R., Palmer, N., Adams, M. D., Gocayne, J., Weidman, J., Utterback, T., Watthey, L., McDonald, L., Artiach, P., Bowman, C. et al. (1997). Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi . Nature 390, 580–586.CrossRefGoogle ScholarPubMed
Gugliotta, J. L., Goethert, H. K., Berardi, V. P. and Telford, S. R. (2013). Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient. New England Journal of Medicine 368, 240–245.CrossRefGoogle Scholar
Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., Bright, I. J., Lucero, M. Y., Hiddessen, A. L., Legler, T. C., Kitano, T. K., Hodel, M. R., Petersen, J. F., Wyatt, P. W., Steenblock, E. R., Shah, P. H., Bousse, L. J., Troup, C. B., Mellen, J. C., Wittmann, D. K., Erndt, N. G., Cauley, T. H., Koehler, R. T., So, A. P., Dube, S., Rose, K. A., Montesclaros, L., Wang, S., Stumbo, D. P., Hodges, S. P. et al. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical Chemistry 83, 8604–8610.CrossRefGoogle ScholarPubMed
Hue, F., Ghalyanchi Langeroudi, A. and Barbour, A. G. (2013). Chromosome sequence of Borrelia miyamotoi, an uncultivable tick-borne agent of human infection. Genome Announcements 1.CrossRefGoogle ScholarPubMed
Kibbe, W. A. (2007). OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Research 35, W43–W46.CrossRefGoogle ScholarPubMed
Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X. and Wilson, A. C. (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America 86, 6196–6200.CrossRefGoogle ScholarPubMed
Krause, P. J., Narasimhan, S., Wormser, G. P., Rollend, L., Fikrig, E., Lepore, T., Barbour, A. and Fish, D. (2013). Human Borrelia miyamotoi Infection in the United States. New England Journal of Medicine 368, 291–293.CrossRefGoogle ScholarPubMed
Miller, S. C., Porcella, S. F., Raffel, S. J., Schwan, T. G. and Barbour, A. G. (2013). Large linear plasmids of Borrelia species that cause relapsing fever. Journal of Bacteriology 195, 3629–3639.CrossRefGoogle ScholarPubMed
Mitchell, E. A., Williamson, P. C., Billingsley, P. M., Seals, J. P., Ferguson, E. E. and Allen, M. S. (2016). Frequency and distribution of Rickettsiae, Borreliae, and Ehrlichiae detected in human-parasitizing ticks in Texas. Emerging and Infectious Diseases 22(2).CrossRefGoogle ScholarPubMed
Padgett, K., Bonilla, D., Kjemtrup, A., Vilcins, I. M., Yoshimizu, M. H., Hui, L., Sola, M., Quintana, M. and Kramer, V. (2014). Large scale spatial risk and comparative prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus . PLoS ONE 9, e110853.CrossRefGoogle ScholarPubMed
Piesman, J., Oliver, J. R. and Sinsky, R. J. (1990). Growth kinetics of the Lyme disease spirochete (Borrelia burgdorferi) in vector ticks (Ixodes dammini). American Journal of Tropical Medicine and Hygiene 42, 352–357.CrossRefGoogle ScholarPubMed
Staff, B. (2012). A Simple Method for Obtaining Absolute Quantification of DNA Molecules Using the Innovative Droplet Digital PCR Technology. Vol. 2014 Bio-Rad Laboratories, Inc. http://www.bioradiations.com/focus-on-technology/783-ddpcr/1340-simpleddpcr Google Scholar
Strain, M. C., Lada, S. M., Luong, T., Rought, S. E., Gianella, S., Terry, V. H., Spina, C. A., Woelk, C. H. and Richman, D. D. (2013). Highly precise measurement of HIV DNA by droplet digital PCR. PLoS ONE 8, e55943.CrossRefGoogle ScholarPubMed
Sze, M. A., Abbasi, M., Hogg, J. C. and Sin, D. D. (2014). A Comparison between droplet digital and quantitative PCR in the analysis of bacterial 16S load in lung tissue samples from control and COPD GOLD 2. PLoS ONE 9, e110351.CrossRefGoogle ScholarPubMed
Tilly, K., Rosa, P. A. and Stewart, P. E. (2008). Biology of infection with Borrelia burgdorferi . Infectious Disease Clinics of North America 22, 217–234.CrossRefGoogle ScholarPubMed
Ullmann, A. J., Gabitzsch, E. S., Schulze, T. L., Zeidner, N. S. and Piesman, J. (2005). Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected Ixodes nymphs in North America. Journal of Medical Entomology 42, 1057–1062.CrossRefGoogle ScholarPubMed
Vayssier-Taussat, M., Moutailler, S., Michelet, L., Devillers, E., Bonnet, S., Cheval, J., Hébert, C. and Eloit, M. (2013). Next generation sequencing uncovers unexpected bacterial pathogens in ticks in western Europe. PLoS ONE 8, e81439.CrossRefGoogle ScholarPubMed
Williamson, P. C., Billingsley, P. M., Teltow, G. J., Seals, J. P., Turnbough, M. A. and Atkinson, S. F. (2010). Borrelia, Ehrlichia, and Rickettsia spp. in ticks removed from persons, Texas, USA. Emerging Infectious Diseases 16, 441–446.CrossRefGoogle ScholarPubMed