An improved primer set and PCR amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region using the SymVar primer pair v1 (protocols.io.n8edhte)

protocols.io ◽  
2018 ◽  
Author(s):  
Benjamin Hume ◽  
Maren Ziegler ◽  
Christian Voolstra
Keyword(s):  
Pcr Amplification ◽  
Its2 Region ◽  
Specificity And Sensitivity ◽  
Amplification Protocol

scholarly journals An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4816 ◽  
Author(s):  
Benjamin C.C. Hume ◽  
Maren Ziegler ◽  
Julie Poulain ◽  
Xavier Pochon ◽  
Sarah Romac ◽  
...  
Keyword(s):  
Large Scale ◽  
Marine Invertebrates ◽  
Temperature Cycle ◽  
Its2 Region ◽  
Rrna Gene ◽  
Surrounding Water ◽  
Cycle Number ◽  
Specificity And Sensitivity ◽  
Amplification Protocol ◽  
Generation Sequencing

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

scholarly journals Detection of zoonotic and livestock-specific assemblages of Giardia duodenalis in free-living wild lizards

2017 ◽  
Vol 26 (3) ◽  
pp. 395-399 ◽  
Author(s):  
Aurora Reboredo-Fernández ◽  
Elvira Ares-Mazás ◽  
Pedro Galán ◽  
Simone Mario Cacciò ◽  
Hipólito Gómez-Couso
Keyword(s):  
Giardia Duodenalis ◽  
Pcr Amplification ◽  
Protozoan Parasite ◽  
Its2 Region ◽  
Free Living ◽  
Wide Range ◽  
Two Samples ◽  
Zoonotic Parasite ◽  
Rock Lizard ◽  
Podarcis Bocagei

Abstract Giardia duodenalis is a zoonotic parasite that infects the gut of a wide range of vertebrates, including numerous wildlife species. However, little is known about this protozoan parasite in reptiles. Fecal samples from 31 wild lizards were collected in Galicia (northwest Spain) and screened for the presence of Giardia by PCR amplification and sequencing of the ITS1-5.8S-ITS2 region in the ribosomal unit. This allowed detection of the parasite in 5 samples (16.1%), and enabled identification of G. duodenalis assemblage A2 in two samples of Iberian rock lizard (Iberolacerta monticola), G. duodenalis assemblage B in other two samples of I. monticola, and G. duodenalis assemblage E in one sample of Bocage’s wall lizard (Podarcis bocagei). The results obtained after PCR amplification and sequencing of the SSU-rDNA gene confirmed the presence of G. duodenalis assemblage A in two samples of I. monticola. This is the first report of G. duodenalis in free-living lizards, although further studies are needed to distinguish between actual infection and mechanical dissemination of cysts. The detection of zoonotic and livestock-specific assemblages of G. duodenalis demonstrates the wide environmental contamination by this parasite, possibly due to human activities.

scholarly journals First Report of Downy Mildew Caused by a Peronospora Species on Sweet Basil (Ocimum basilicum) in Massachusetts

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 318-318 ◽  
Author(s):  
R. L. Wick ◽  
N. J. Brazee
Keyword(s):  
South Africa ◽  
Downy Mildew ◽  
Pcr Amplification ◽  
Infected Leaf ◽  
Its2 Region ◽  
Academic Press ◽  
First Report ◽  
Sweet Basil ◽  
Foliar Blight ◽  
Leaf Tissues

In September of 2008, downy mildew was discovered to be causing a serious foliar blight of sweet basil at several farms and greenhouses in Massachusetts. Infected leaves had chlorotic vein-bounded patches and diffuse chlorosis, and a characteristic gray, fuzzy growth was on the abaxial surface. Microscopic observations revealed branched sporangiophores that measured 187.5 to 325 μm (average 285 μm) long. Sporangia measured 22.5 to 30 × 20 to 22.5 μm (average 26.7 × 20.9 μm). No oospores were found. Sporangium measurements are comparable to unnamed Peronospora species reported previously on basil from Italy, Switzerland, and South Africa (1,2). Sequence analyses were conducted on five isolates of ‘Nufar’ basil by extracting DNA from a sporangial suspension washed from leaves and infected leaf tissues using the Qiagen DNeasy plant tissue kit (Qiagen, Valencia, CA). PCR amplification of the ITS1, 5.8S, and ITS2 region was performed using primers ITS6 and ITS4 (3). The sequences of the five isolates were identical. BLAST analyses of the sequences revealed a 99% similarity to the unnamed Peronospora species on sweet basil in Europe and South Africa (1,2). To our knowledge, this is the first report of a Peronospora species on sweet basil in Massachusetts. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) A. McLeod et al. Plant Dis. 90:1115, 2006. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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