Thermal profile with alternately raised and lowered annealing temperature improves the PCR amplification using highly degenerate primers.

Sachadyn, Paweł; Sobiewska, Gabriela; Kur, Józef

doi:10.18388/abp.1998_4262

Cited by 5 publications

(2 citation statements)

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“…For colony PCR, a loopful of freshly grown bacteria was suspended in 200μl of water and 1 μl of the suspension was directly used (without prior boiling) as template with HotStarTaq (Qiagen). Degenerate PCR and chromosomal walking were performed as previously described [36,37]. Sequencing was carried out on duplicate samples with BigDye Terminator mix (Applied Biosystems) according to the manufacturer's instructions, the sequences analyzed using an ABI 3700 DNA sequencer (Applied Biosystems).…”

Section: Pcr and Dna Sequencingmentioning

confidence: 99%

γ-Glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

Barnes

Bagnall

Browning

et al. 2007

Microbial Pathogenesis

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The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.

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Section: Pcr and Dna Sequencingmentioning

confidence: 99%

γ-Glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

Barnes

Bagnall

Browning

et al. 2007

Microbial Pathogenesis

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“…During the design process, this mainly concerns the annealing temperature for each primer. The annealing temperature of a primer depends on the GC content of the primer sequence and the length of the primer itself (Sachadyn, Sobiewska and Kur, 1998). The G (guanine) and C (cytosine)…”

Section: Sampling Protocolmentioning

confidence: 99%

Characterising the foraging ecology and mercury exposure of the Nationally Critical Whenua Hou diving petrel

Tocker¹

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<p>With seabird species in decline globally, significant research has gone into characterising their key prey species and foraging areas that need protection. Knowledge on the diet of a species has important implications for the development of conservation programmes. The sand dune system on Whenua Hou is home to the endemic Whenua Hou diving petrel (Pelecanoides whenuahouensis; hereafter WHDP) and a population of common diving petrels (Pelecanoides urinatrix; hereafter CDP). The WHDP is considered ‘Nationally Critical’ due to its small population size (~200 individuals) and restricted breeding range (0.018 km2) on Whenua Hou. The foraging ecology of the WHDP is relatively unknown, as is its exposure to sources of marine pollution. This thesis aimed to characterise the foraging ecology of the WHDP, the potential interspecific competition with the sympatric CDPs, the prey present in the diets of each species, and their resulting exposure to mercury from the environment. In chapter 2, I used stable isotope analysis to infer the trophic dynamics of the WHDP. By sampling and analysing both blood and feathers, I was able to investigate potential differences in WHDP foraging ecology between the breeding and non-breeding seasons. I found a difference between the foraging ecology of male and female WHDPs, with results indicating females forage further out to sea than males and on prey of lower trophic value. I found that WHDPs forage an entire trophic level higher during the breeding season than the non-breeding season. As my sampling effort spanned three consecutive breeding seasons (2017-2019), I was able to detect interannual variation in the foraging ecology of WHDPs. The results revealed that WHDPs foraged at a higher trophic level during the breeding season of 2018 compared to that of 2017 or 2019. By characterising the isotopic niches of both the WHDPs and CDPs over the three years, I was able to demonstrate a degree of trophic segregation between the two species during the breeding season. In chapter 3, I designed and went through the initial development stages for a novel multiplex-PCR assay to identify the prey species present in the diets of WHDPs and CDPs. The obstacles faced in the development of this protocol highlighted the suitability of DNA metabarcoding as an alternative method. In chapter 4, I analysed the mercury concentration in the same blood and feather samples used for stable isotope analysis. I demonstrated that male WHDPs had higher concentrations of mercury in their tissues than females, correlating with their foraging at a higher trophic level. The interannual variation in mercury concentration did not correlate with the trophic variation of WHDPs among years, indicating that the environmental fluctuations in mercury levels had a stronger effect on mercury exposure than diet. WHDP tissues consistently had higher concentrations of mercury than CDPs, correlating with their isotopic niche segregation and highlighting a potential threat to individual survival and reproductive success in WHDPs. Overall, my results describe patterns in the foraging ecology of the WHDP, as well as highlighting the potential threat from mercury exposure. This research can be used as a baseline for future investigations into the key prey species for the endangered WHDP and the impacts mercury exposure may be having on the population growth of this species. </p>

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Identification of Genetic Evidence for Dobrava Virus Spillover in Rodents by Nested Reverse Transcription (RT)-PCR and TaqMan RT-PCR

et al. 2005

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A survey of 158 rodents caught in the Czech Republic identified Dobrava virus sequences closely related to that of the Dobrava virus type strain in Apodemus sylvaticus and Mus musculus rodents. The identity of A. sylvaticus was unequivocally confirmed by random amplified polymorphic DNA analysis. The data seem to indicate hantavirus spillover from Apodemus flavicollis to other rodents.Hantaviruses are subdivided into the Eurasian and the American groups. The distinction manifests itself in serological and genetic differences as well as in the clinical picture of the diseases elicited in humans. The up to 20 individual hantavirus types have been observed to be strictly coevolving with the distinct rodent species that carry them. Prominent hantaviruses in western and central Europe are Dobrava virus (DOBV), Puumala virus (PUUV), and Tula virus (TULV) (6,16,21).DOBV was originally isolated in Slovenia from the rodent Apodemus flavicollis (DOBV-Af) (3) and is a Eurasian hantavirus that predominantly causes disease in humans in southeastern Europe. DOBV-Af has also been isolated in Greece, Albania, and Bosnia-Herzegovina (1,10,14). Serological studies have indicated the presence of DOBV-Af beyond the geographical limits of the Balkans in Slovakia (22) and recently in the Czech Republic (15,25). The isolation of a Dobrava-like virus from the rodent Apodemus agrarius (DOBV-Aa) was reported in Estonia (11), Russia (17), and Slovakia (23), supplemented by a report on genetic evidence of this DOBV-Aa type from rodent samples in Hungary (20). Whether this new type is a type of its own, as comparative serological and genetic analyses suggest, is still a matter of debate (7,12,18). The distribution of the two rodent host species overlaps in Europe, but rodent carriers of both DOBV types coexist only in the border region of southeastern and central Europe (2, 23). In order to investigate the occurrence of hantaviruses in a rodent population from the Czech Republic, we analyzed the kidneys, lungs, and spleens of 157 rodents and 1 shrew (A. flavicollis, n ϭ 77; A. sylvaticus, n ϭ 34; Mus musculus, n ϭ 2; Clethrionomys glareolus, n ϭ 41; Microtus arvalis, n ϭ 2; Microtus agrestis, n ϭ 1; Sorex araneus, n ϭ 1) caught at the military training area of Boletice, close to Ceské Budejovice in the south of the Czech Republic, from May to November 1999. MATERIALS AND METHODSA total of 10 to 50 mg of tissue of each organ was homogenized in a FastPrep machine with the FastRNA Green kit (Qbiogene, Hilden, Germany) to perform total RNA extraction. This closed system avoids the generation of contaminating aerosols. We screened the extracted RNA for DOBV-Af RNA by a one-step TaqMan reverse transcription (RT)-PCR and for hantavirus RNA with the degenerate primers for nested PCR published by Scharninghausen et al. (20). For the DOBV TaqMan RT-PCR, we used 500 nM concentrations of primers DOBFP (5Ј-TGGCTTGACCTCCCGTG-3Ј) and DOBRP (5Ј-CAAGCGCTCCT TGTCTTTGA-3Ј) and 200 nM probe DOBP (5Ј-ATCTCCAACGTCTTTGAC CAAAGGCCC-3Ј) tagged with 6-carboxyfluores...

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Thermal profile with alternately raised and lowered annealing temperature improves the PCR amplification using highly degenerate primers.

Cited by 5 publications

References 1 publication

γ-Glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

γ-Glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

Characterising the foraging ecology and mercury exposure of the Nationally Critical Whenua Hou diving petrel

Identification of Genetic Evidence for Dobrava Virus Spillover in Rodents by Nested Reverse Transcription (RT)-PCR and TaqMan RT-PCR

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