Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

Crowder, Chris D.; Rounds, Megan A.; Phillipson, Curtis A.; Picuri, John M.; Matthews, Heather; Halverson, Justina; Schutzer, Steven E.; Ecker, David J.; Eshoo, Mark W.

doi:10.1093/jmedent/47.1.89

Cited by 61 publications

(41 citation statements)

References 25 publications

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“…Likewise, exclusive of the primer designing process which is somehow complicated and sensitive, other phases are simply applicable. It is noticeable that in all RT-based methods, RNA extraction is an unavoidable step, needing different protocol(s) followed by optimization (mostly is a time-consuming process) to acquire purified RNA stock [19,44], whereas IC-RT-LAMP can be easily performed with no attempt for RNA isolation. This method would lastly simplify the detection procedure and would result in saving of significant time which is needed for separation of the amplified products on the gel.…”

Section: Discussionmentioning

confidence: 99%

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

Almasi

Moradi

Nasiri

et al. 2012

Appl Biochem Biotechnol

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To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC-RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC-RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC-RT-LAMP products, both hydroxynaphthol blue and GeneFinder™ could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. Altogether, as IC-RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.

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Section: Discussionmentioning

confidence: 99%

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

Almasi

Moradi

Nasiri

et al. 2012

Appl Biochem Biotechnol

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“…Tick pools were homogenized for 25 s in 1.5 ml screw-cap tubes filled with zirconia/yttria-stabilized zirconium oxide beads (750 mg of 2.0 mm diameter and 150 mg of 0.1 mm diameter) (Glen Mills, Clifton, NJ, USA) (48), 650 µl of phosphate-buffered saline using a Mini-Beadbeater-16 (BioSpec, Bartlesville, OK, USA). Total nucleic acids were extracted from these homogenates in an automated MagNa Pure 96 extraction system (Roche Diagnostics, Risch-Rotkreuz, Switzerland) using the small volume DNA/viral RNA kits (Roche Diagnostics).…”

Section: Methodsmentioning

confidence: 99%

Molecular Detection of Tick-Borne Pathogen Diversities in Ticks from Livestock and Reptiles along the Shores and Adjacent Islands of Lake Victoria and Lake Baringo, Kenya

et al. 2017

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Although diverse tick-borne pathogens (TBPs) are endemic to East Africa, with recognized impact on human and livestock health, their diversity and specific interactions with tick and vertebrate host species remain poorly understood in the region. In particular, the role of reptiles in TBP epidemiology remains unknown, despite having been implicated with TBPs of livestock among exported tortoises and lizards. Understanding TBP ecologies, and the potential role of common reptiles, is critical for the development of targeted transmission control strategies for these neglected tropical disease agents. During the wet months (April–May; October–December) of 2012–2013, we surveyed TBP diversity among 4,126 ticks parasitizing livestock and reptiles at homesteads along the shores and islands of Lake Baringo and Lake Victoria in Kenya, regions endemic to diverse neglected tick-borne diseases. After morphological identification of 13 distinct Rhipicephalus, Amblyomma, and Hyalomma tick species, ticks were pooled (≤8 individuals) by species, host, sampling site, and collection date into 585 tick pools. By supplementing previously established molecular assays for TBP detection with high-resolution melting analysis of PCR products before sequencing, we identified high frequencies of potential disease agents of ehrlichiosis (12.48% Ehrlichia ruminantium, 9.06% Ehrlichia canis), anaplasmosis (6.32% Anaplasma ovis, 14.36% Anaplasma platys, and 3.08% Anaplasma bovis,), and rickettsiosis (6.15% Rickettsia africae, 2.22% Rickettsia aeschlimannii, 4.27% Rickettsia rhipicephali, and 4.95% Rickettsia spp.), as well as Paracoccus sp. and apicomplexan hemoparasites (0.51% Theileria sp., 2.56% Hepatozoon fitzsimonsi, and 1.37% Babesia caballi) among tick pools. Notably, we identified E. ruminantium in both Amblyomma and Rhipicephalus pools of ticks sampled from livestock in both study areas as well as in Amblyomma falsomarmoreum (66.7%) and Amblyomma nuttalli (100%) sampled from tortoises and Amblyomma sparsum (63.6%) sampled in both cattle and tortoises at Lake Baringo. Similarly, we identified E. canis in rhipicephaline ticks sampled from livestock and dogs in both regions and Amblyomma latum (75%) sampled from monitor lizards at Lake Victoria. These novel tick–host–pathogen interactions have implications on the risk of disease transmission to humans and domestic animals and highlight the complexity of TBP ecologies, which may include reptiles as reservoir species, in sub-Saharan Africa.

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“…Total DNA was extracted from 112 adult Black-legged Ticks, 49 from Thetford and 63 from Ascutney, using a proprietary extraction protocol (Total Nucleic Acid Extraction Tick or Tissue 002.8, Ibis Biosciences/Abbott Laboratories, Abbott Park, IL), previously published in modified form by Crowder et al (2010). Polymerase chain reaction (PCR) based B. burgdorferi tests were performed at Analytical Services, Inc. (ASI), Williston VT, in accordance with ASI SOP #385, which is based on procedures described by Marconi and Garon (1992) and Mouritsen, et al (1996).…”

Section: Methodsmentioning

confidence: 99%

Distribution of Ticks and Prevalence ofBorrelia burgdorferiin the Upper Connecticut River Valley of Vermont

Serra

Warden²,

Fricker³

et al. 2013

Northeastern Naturalist

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Ixodes scapularis (Black-legged Tick) has expanded its range in recent decades. To establish baseline data on the abundance of the Black-legged Tick and Borrelia burgdorferi (causative agent of Lyme disease) at the edge of a putative range expansion we collected 1398 ticks from five locations along the Connecticut River in Vermont. Collection locations were approximately evenly distributed between the villages of Ascutney and Guildhall. Relative abundance and distribution by species varied across sites. Black-legged Ticks dominated our collections (n = 1348, 96%), followed by Haemaphysalis leporispalustris (Rabbit Tick, n = 45, 3%) and Dermacentor variabilis (American Dog Tick, n = 5, <1%). Black-legged Tick abundance ranged from 6198 ticks per survey hectare (all life stages combined) at the Thetford site to zero at the Guildhall site. There was little to no overlap of tick species across sites. Phenology of Black-legged Ticks matched published information from other regions of the northeastern USA. Prevalence of B. burgdorferi in adult Black-legged Ticks was 8.9% (n = 112).

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Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

Cited by 61 publications

References 25 publications

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

Molecular Detection of Tick-Borne Pathogen Diversities in Ticks from Livestock and Reptiles along the Shores and Adjacent Islands of Lake Victoria and Lake Baringo, Kenya

Distribution of Ticks and Prevalence ofBorrelia burgdorferiin the Upper Connecticut River Valley of Vermont

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