Large-scale production of DNA sequencing templates by microtitre format PCR

Rosenthal, André; Coutelle, Oliver; Craxton, Molly

doi:10.1093/nar/21.1.173

Cited by 169 publications

(103 citation statements)

References 9 publications

(9 reference statements)

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“…Of each clone library, 20-30 clones were picked and sequenced. PCR products of clones were purified using an established protocol (45). Sequences are deposited in GenBank under the accession numbers JF510050-JF510160.…”

Section: Methodsmentioning

confidence: 99%

Microbial methane production in oxygenated water column of an oligotrophic lake

Grossart

Frindte

Dziallas

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

283

328

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The prevailing paradigm in aquatic science is that microbial methanogenesis happens primarily in anoxic environments. Here, we used multiple complementary approaches to show that microbial methane production could and did occur in the well-oxygenated water column of an oligotrophic lake (Lake Stechlin, Germany). Oversaturation of methane was repeatedly recorded in the well-oxygenated upper 10 m of the water column, and the methane maxima coincided with oxygen oversaturation at 6 m. Laboratory incubations of unamended epilimnetic lake water and inoculations of photoautotrophs with a lake-enrichment culture both led to methane production even in the presence of oxygen, and the production was not affected by the addition of inorganic phosphate or methylated compounds. Methane production was also detected by in-lake incubations of lake water, and the highest production rate was 1.8-2.4 nM·h −1 at 6 m, which could explain 33-44% of the observed ambient methane accumulation in the same month. Temporal and spatial uncoupling between methanogenesis and methanotrophy was supported by field and laboratory measurements, which also helped explain the oversaturation of methane in the upper water column. Potentially methanogenic Archaea were detected in situ in the oxygenated, methane-rich epilimnion, and their attachment to photoautotrophs might allow for anaerobic growth and direct transfer of substrates for methane production. Specific PCR on mRNA of the methyl coenzyme M reductase A gene revealed active methanogenesis. Microbial methane production in oxygenated water represents a hitherto overlooked source of methane and can be important for carbon cycling in the aquatic environments and water to air methane flux. epilimnic methane peak | methanogens A lthough methane makes up <2 parts per million by volume (ppmv) of the atmosphere, it accounts for 20% of the total radiative forcing among all long-lived greenhouse gases (1). In the aquatic environments, methane is also an important substrate for microbial production (2). The prevailing paradigm is that microbial methanogenesis occurs primarily in anoxic environments (3, 4). A commonly observed paradox is methane accumulation in well-oxygenated waters (2, 5), which is often assumed to be the result of physical transport from anoxic sediment and water (6-8) or in situ production within microanoxic zones (9-11). Two recent studies challenged this paradigm and suggested that microbes in oligotrophic ocean can metabolize methylated compounds and release methane even aerobically (12, 13). These claims are not without caveats, because the amounts of methylated compounds added [1-10 μM methylphosphonate (12) and 50 μM dimethyl sulfoniopropionate (13)] were far higher than their environmental concentrations, and therefore, the ecological relevance remains obscure. Moreover, dissolved oxygen (DO) was not monitored during the long incubation (5-6 d), and the possibility that the experimental setups had become anoxic before methane production could not be dismissed.* Despite the unce...

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Section: Methodsmentioning

confidence: 99%

Microbial methane production in oxygenated water column of an oligotrophic lake

Grossart

Frindte

Dziallas

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

283

328

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“…All amplification reactions were prepared in total volumes of 25 µl, consisting of 1x Colorless GoTaq Flexi Buffer (Promega, Mannheim, Germany), 1.5 mM MgCl 2 , 10 µM of each primer, 0. repens-specific primers were negative, the amplicons generated by using DIRD-F1 and DIDR-R1 primers were purified by polyethylene glycol precipitation (Rosenthal et al 1993) S65 and subjected to the sequencing in both directions. The obtained sequences were used to search for homologies using BLASTn in GenBank TM .…”

Section: Pcr To Detect and Discriminate Between Canine Microfilariaementioning

confidence: 99%

Diagnosis of Imported Canine Filarial Infections in Germany 2008 – 2010

et al. 2011

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“…There are conflicting results between the relation of giardiosis and diarrhoea [19]. However, most of the studies found a positive correlation between the occurrence of diarrhoea and Giardia infection in calves [38,39]. A prevalence study with molecular characterization of G. duodenalis isolates in cattle has not yet been reported from Turkey.…”

Section: Discussionmentioning

confidence: 99%

“…For this purpose, the DNA copies obtained from PCR were purified using polyethylene glycol 4000 [39]. Sequencing was accomplished by using Big Dye--terminator chemistry 3 following the instructions of the manufacturer.…”

Section: Dna Sequence Analysis Of Nested Pcr Positive Samplesmentioning

confidence: 99%

Prevalence and Molecular Characterization of Giardia duodenalis in Calves in Turkey

Gültekin

Ural

Aysul

et al. 2017

Acta Scientiae. Vet.

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Background: Giardia duodenalis (G. duodenalis) is an ubiquitous, flagellated intestinal protozoan with major public health significance worldwide. Limited data are available on the epidemiology of G. duodenalis in dairy cattle from Turkey. Determining the zoonotic potential of the Giardia infection requires molecular characterization. The aim of the present study was to investigate the prevalence and to molecularly characterize G. duodenalis in calves less than three months of age in Aydin, Aegean region of Turkey. Materials, Methods & Results: The study was conducted on different dairy farms in the south-western part of the Turkey, Aegean Region, Aydin. A total of 198 Holstein Friesian calves less than three months of age, of both sexes were enrolled into the study. Faecal samples from each calf were collected manually from the rectum using a disposable latex glove. The consistency of collected samples was recorded as diarrhoeic or non-diarrhoeic. Diagnosis of G. duodenalis infection was made microscopically by detection of cysts in the faecal samples. One hundred and sixteen (58.5%) of the 198 faecal samples were diarrheic. Giardia cysts were found in 27 (23.28%) of the diarrheic samples and in 8 (9.76%) of nondiarrheic samples (P < 0.05). The overall prevalence of giardiosis in calves was determined as 17.67%. The prevalence of Giardia genotypes was identified by DNA sequence analysis of the beta-giardin gene for every PCR positive sample. The beta-giardin nested PCR assay was revealed assemblage A and sub-genotype A3 was detected in all of 35 samples (100%). Discussion: The highest prevalence of Giardia infection in calves is reported at the age between 1 and 6 months, and the prevalence shows decreased rate from the age of 6 months. The present study was conducted in Aydin, a province of south-western Turkey in the Aegean Region, and the overall prevalence from a total of 198 dairy calves was 17.67%. The prevalence rate in calves with diarrhoea was higher and reached up to 23.28%, whereas it was 9.76% in non-diarrhoeic calves. A prevalence study with molecular characterization of G. duodenalis isolates in cattle has not yet been reported from Turkey. Molecular studies have shown that mostly assemblage E predominates in cattle, but recent studies denoted that assemblage A is increasingly being detected and might be more widespread than expected before. In the present study, Giardia positive samples identified with a beta-giardin nested PCR assay. The sub-genotype A3 was identified in all samples. The same sub-genotype was identified in human and dog samples from different countries. Furthermore, sub-genotype A3 was found in humans and dogs from Turkey. In this context, results of the present study suggested an important role of calves as potential reservoirs of human infections in Turkey. In conclusion, epidemiological data revealed that G. duodenalis infection is frequent in calves with diarrhoea in Aydin, Turkey. The presence of the potentially zoonotic sub-genotype A3 and high prevalence of Giardi...

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Large-scale production of DNA sequencing templates by microtitre format PCR

Cited by 169 publications

References 9 publications

Microbial methane production in oxygenated water column of an oligotrophic lake

Microbial methane production in oxygenated water column of an oligotrophic lake

Diagnosis of Imported Canine Filarial Infections in Germany 2008 – 2010

Prevalence and Molecular Characterization of Giardia duodenalis in Calves in Turkey

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