Molecular assay development: detecting pathogenic Rickettsia in the tick vector Amblyomma americanum Open Access
Dugan, Ellen Jennifer (2013)
Abstract
Abstract
Molecular assay development: detecting pathogenic Rickettsia in the tick vector Amblyomma americanum
By Ellen Dugan
Purpose: Rickettsia amblyommii is a common and abundant member of the bacterial community of the tick vector Amblyomma americanum. While R. amblyommii is thought to cause only mild illness, if any, in humans, its presence makes the detection of rare and clinically more severe Rickettsia difficult to detect with available assays. A conventional PCR assay was developed to improve the detection of Rickettsia species of known or potential pathogenicity in the presence of R. amblyommii, in the tick vector Amblyomma americanum.
Methods: Homologous gene sequences from Rickettsia were aligned to detect polymorphisms unique to R. amblyommii. These polymorphisms were used to design 14 primer pairs. One primer pair was chosen for assay creation based on its ability to detect target Rickttsia DNA and to not detect R. amblyommii DNA. Further testing was conducted to validate the assay's sensitivity, specificity, and accuracy. Tests included annealing temperature optimization, determination of the LOD for Rickettsia, and testing of wild-caught tick samples with known and unknown Rickettsia infection.
Results: One hundred percent of 20 target Rickettsia species tested with the assay were detected and 0% of A. americanum DNA with known R. amblyommii infection detected R. amblyommii. Both results demonstrate assay specificity for the target Rickettsia species. Sensitivity testing indicated that R. parkeri could be detected in the presence of tick and R. amblyommii DNA in 92.8% of samples at a dilution as low as 1:6000 from the stock concentration. Rickettsia rhipicephali CWPP and Rickettsia 364D were detected in 100% of wild-caught naturally infected Dermacentor occidentalis ticks, indicating high accuracy of the assay to detect natural Rickettsia infection in ticks.
Conclusions: This simple, easily replicable, inexpensive, and rapid testing method detects Rickettsia other than R. amblyommii in ticks. It eliminates the need for sequencing to differentiate target Rickettsia from R. amblyommii, allowing for resources to be focused on less prevalent and abundant Rickettsia that cause more severe clinical disease.
Table of Contents
Table of Contents
Background......................................................................................................1
Amblyomma
americanum………………………………………………….............................…..…........1
Rickettsia species…………………………………………………………….........……...............................2
Rickettsia
amblyommii………………….........................…………………………….……..….…....…......2
Rickettsia amblyommii masking
effect…………………..….........................…..…………...........3
Rickettsia species identification and detection….......................……..………..…….............4
Relevance to environmental public
health…………………………......…................................…5
Methods………………………………....................................………….....…..………...6
Rickettsia samples…………………………………………..….…………..........................….....…....…..…6
Tick samples…………………………………………………..........................…..........………….....……..…7
Primer design and selection…………………………………..….…………….….................................…7
Conventional PCR screening and gel electrophoresis..………………................................….8
CFR performance measures applied to assay design
…..……………..…….........................….9
Results……………………………………………………….............................……………12
CFR performance measures applied to assay
results………..….........................………..…….12
Discussion…………………………………………….........................……..…..….………14
Discussion of the CFR performance measures applied to the assay
..........................…14
Conclusion……………………………………………………………..........................……16
References………………….........................………………………………………….……17
Tables………………………………………………..........................……….………………24
Figures…………………………………………...……………………………….....................................………25
Appendix…………………………………………………………………..............……..........................………32
.
.
List of Tables
Table 1. Prevalence of R. amblyommii in comparison to the prevalence of
other bacterial species in A. americanum
…………………..…………………................….….....….24
Table 2. Nucleotide sequences created as
potential candidates for the assay...............24
.
.
List of Figures
Figure 1. Distribution of tick samples across the
United States
…………….……........…..…..25
Figure 2. Assay sensitivity: Detection of R.
parkeri inoculated into wild
A. americanum at the LOD concentration of 1:6000…………………...….............………….…...26
Figure 3. Assay specificity: annealing temperature optimization for
A. americanum
DNA…………………………………………………….…………...............................…..…27
Figure 4a. Assay specificity: Spurious amplification
of bands in A. americanum
with known R. amblyommii infection ……………………………………......................……….….…...28
Figure 4b. Assay specificity: High resolution testing of spurious bands for
the detection of R.
amblyommii……………………………………….…......................…………..…....29
Figure 5. Assay accuracy: detection of 20
target Rickettsia species and the
absence of R. amblyommii and A. americanum detection……………….............………………...31
Figure 6. Assay accuracy: Detection of natural
Rickettsia infection in D.
occidentalis..…30
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