Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Schabereiter-Gurtner, Claudia; Lubitz, Werner; Rölleke, Sabine

doi:10.1016/s0167-7012(02)00186-0

Cited by 101 publications

(87 citation statements)

References 26 publications

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“…One sequence was identified as 18S rDNA of I. ricinus. This artefactual amplification has also been shown in a previous similar study and is supposed to be related to the complete annealing of the reverse primer to the I. ricinus 18S rDNA [18]. Nevertheless, this aspecific amplification did not limit amplification of bacterial DNA.…”

Section: Discussionsupporting

confidence: 75%

“…An automated standardized reading process would improve sensitivity and accuracy. Moreover, preferential amplification of 16S rDNA of some bacterial taxons [18] could also explain these biases. Indeed, the number of B. burgdorferi sl is known to be low in questing nymphs [19].…”

Section: Discussionmentioning

confidence: 99%

“…We also found sequences that have already been described in ticks such as the sequences related to environmental bacteria such as Rhodococcus sp. or arthropod symbiotic bacteria (Coxiellaceae) [18]. One sequence was identified as 18S rDNA of I. ricinus.…”

Section: Discussionmentioning

confidence: 99%

“…Temporal Temperature Gradient gel Electrophoresis (TTGE) is one technique that allows the sequencespecific separation on the basis of the GC content of amplicons by using the denaturing conditions of an increasing temperature [3,23]. This technique, as well as the very similar DGGE (Denaturing Gradient Gel Electrophoresis), are commonly used to determine the bacterial profile of different biotopes such as soil and lakes [3,15] and have also been successfully applied to study the microflora of Ixodes ricinus ticks [18]. Although they have been proposed to detect fish-borne pathogenic bacteria [10], these techniques have not yet been used for the detection of bacterial pathogens in ticks.…”

Section: Introductionmentioning

confidence: 99%

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Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Halos

Mavris

Vourc’H³

et al. 2006

Vet. Res.

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-Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.Ixodes ricinus / Borrelia burgdorferi sensu lato / Anaplasma phagocytophilum / Rickettsia spp. / bacterial diversity

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Halos

Mavris

Vourc’H³

et al. 2006

Vet. Res.

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“…These include members of the Rickettsiaceae and Anaplasmataceae (7) that are not known to be human pathogens, bacteria belonging to the Wolbachia persica group (21,23,32,38), and a variety of other bacteria detected by direct isolation or observation (15,19). In related ticks, denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments has been used to assess bacterial populations (27).…”

mentioning

confidence: 99%

Intracellular Symbionts and Other Bacteria Associated with Deer Ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts

Benson

Gawronski

Eveleigh

et al. 2004

Appl Environ Microbiol

121

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The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group. Several strains of members of the Sphingomonadaceae were also detected in all but one tick. The results provide a view of the diversity of bacteria associated with I. scapularis ticks in the field.

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Analyzing Arthropods for the Presence of Bacteria

Andrews

2013

CP Microbiology

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Bacteria within arthropods can be identified using culture‐independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis. Curr. Protoc. Microbiol. 28:1E.6.1‐1E.6.14. © 2013 by John Wiley & Sons, Inc.

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Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Cited by 101 publications

References 26 publications

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Intracellular Symbionts and Other Bacteria Associated with Deer Ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts

Analyzing Arthropods for the Presence of Bacteria

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