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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: Michael.Allen@unthsc.edu
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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

Information

Type
Research Article
Information
Parasitology , Volume 144 , Issue 4 , April 2017 , pp. 359 - 367
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Copyright
Copyright © Cambridge University Press 2016 

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