A PCR-based method for the identification of the roots of 10 co-occurring grassland species in mesocosm experiments

An understanding of factors influencing the distribution of plant roots is intimately linked to our understanding of basic ecosystem functions such as nutrient flux and productivity. However, it is not usually possible to measure root distributions because it is difficult to identify the roots of different species when they are grown in mixture. This is because the roots of most species are not visually distinguishable. We designed a simple, PCR-based method for the identification of roots in mesocosm experiments, which we have applied to 10 co-occurring grassland species. Species-specific primers based on ITS sequences from GenBank were evaluated in PCR assays using either homogeneous or heterogeneous DNA templates, as well as DNA extracted from mixed-root samples from multiple combinations of species. The species-specific primers reported here produced accurate identifications, free from both false negatives and false positives, in 100% of our assays. We also evaluated the sensitivity of our system and demonstrated detection of species when they comprised as little as 0.05 ng of target DNA mixed in a total of 2.5 ng of multi-species template DNA. Our PCR-based method for root identification in mesocosms is more cost effective, and simpler to apply than previously described methods.

Download Full-text

Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

Plant Soil and Environment ◽

10.17221/2226-pse ◽

2008 ◽

Vol 53 (No. 3) ◽

pp. 97-104 ◽

Cited By ~ 12

Author(s):

M. Zouhar ◽

M. Marek ◽

O. Douda ◽

J. Mazáková ◽

P. Ryšánek

Keyword(s):

Cost Effective ◽

Healthy Plant ◽

Host Preferences ◽

Sequence Characterized Amplified Region ◽

Detection And Identification ◽

Plant Hosts ◽

Cost Effective Technique ◽

Ditylenchus Dipsaci ◽

Species Specific ◽

Template Dna

Ditylenchus dipsaci, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of D. dipsaci control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of D. dipsaci in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the D. dipsaci stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all D. dipsaci isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect D. dipsaci in artificially infested plant tissues.

Download Full-text

Improving reliability in environmental DNA detection surveys through enhanced quality control

Marine and Freshwater Research ◽

10.1071/mf15349 ◽

2017 ◽

Vol 68 (2) ◽

pp. 388 ◽

Cited By ~ 15

Author(s):

Elise M. Furlan ◽

Dianne Gleeson

Keyword(s):

Quality Control ◽

Native Species ◽

Conservation Management ◽

Environmental Dna ◽

Positive Control ◽

False Negatives ◽

Method Error ◽

Target Dna ◽

Species Specific ◽

Australian Freshwater

Species-specific environmental DNA (eDNA) surveys are increasingly being used to infer species presence in an environment. Current inadequacies in quality control increase concern for false negatives, which can have serious ramifications for both the management of invasive species and the conservation of native species. eDNA surveys involve a multi-step process to sample, capture, extract and amplify target DNA from the environment. We outline various positive control options and show that many of the commonly used controls are capable of detecting false negatives arising during the amplification stage only. We suggest a secondary, generic primer, designed to co-amplify endogenous DNA sampled during species-specific eDNA surveys, constitutes a superior positive control to monitor method success throughout all stages of eDNA analysis. We develop a species-specific European carp (Cyprinus carpio) assay and a generic fish assay for use as an endogenous control for eDNA surveys in Australian freshwater systems where fish are known to be abundant. We use these assays in a multiplex on eDNA samples that are simultaneously sampled, captured, extracted and amplified. This positive control allows us to distinguish method error from informative non-amplification results, improving reliability in eDNA surveys, which will ultimately lead to better informed conservation management decisions.

Download Full-text

Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

Journal of Clinical Microbiology ◽

10.1128/jcm.36.5.1185-1188.1998 ◽

1998 ◽

Vol 36 (5) ◽

pp. 1185-1188 ◽

Cited By ~ 34

Author(s):

Ghassan M. Matar ◽

Nada Sidani ◽

Michel Fayad ◽

Usamah Hadi

Keyword(s):

Otitis Media ◽

Otitis Media With Effusion ◽

Bacterial Species ◽

Cost Effective ◽

Universal Primers ◽

Rrna Gene ◽

Specific Primer ◽

Specific Primers ◽

Bacterial Dna ◽

Species Specific

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella)catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) aHaemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive forHaemophilus, 3 (8.6%) were positive forStreptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

Download Full-text

Supplementary data for: Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips

Journal of Economic Entomology ◽

10.1603/ec14027sf1 ◽

2014 ◽

Keyword(s):

Western Flower Thrips ◽

Multiplex Assay ◽

Supplementary Data ◽

Specific Primers ◽

Flower Thrips ◽

Species Specific

Download Full-text

Distribution Analysis of Six PredominantBacteroidesSpecies in Normal Human Feces Using 16S rDNA-Targeted Species-Specific Primers

Microbial Ecology in Health and Disease ◽

10.3402/mehd.v14i3.8239 ◽

2002 ◽

Vol 14 (3) ◽

Cited By ~ 1

Author(s):

Yukiko Miyamoto ◽

Koichi Watanabe ◽

Ryuichiro Tanaka ◽

Kikuji Itoh

Keyword(s):

16S Rdna ◽

Distribution Analysis ◽

Human Feces ◽

Specific Primers ◽

Normal Human ◽

Species Specific

Download Full-text

Duplex DNA barcoding allows accurate species determination of morphologically similar limpets (Patella spp.) from non-destructive sampling

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315416000813 ◽

2016 ◽

Vol 97 (7) ◽

pp. 1479-1482 ◽

Cited By ~ 1

Author(s):

Thomas J. Ashton ◽

Meriem Kayoueche-Reeve ◽

Andrew J. Blight ◽

Jon Moore ◽

David M. Paterson

Keyword(s):

Dna Barcoding ◽

Species Abundance ◽

Microscopic Analysis ◽

Cost Effective ◽

Field Studies ◽

Similar Species ◽

Species Determination ◽

Destructive Sampling ◽

Species Specific ◽

Non Destructive

Accurate discrimination of two morphologically similar species of Patella limpets has been facilitated by using qPCR amplification of species-specific mitochondrial genomic regions. Cost-effective and non-destructive sampling is achieved using a mucus swab and simple sample lysis and dilution to create a PCR template. Results show 100% concurrence with dissection and microscopic analysis, and the technique has been employed successfully in field studies. The use of highly sensitive DNA barcoding techniques such as this hold great potential for improving previously challenging field assessments of species abundance.

Download Full-text

Trace Detection of Pentaerythritol Tetranitrate Using Electrochemical Gas Sensors

Journal of Sensors ◽

10.1155/2014/234607 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Praveen K. Sekhar ◽

Jie Zhou ◽

Hui Wang ◽

Eric R. Hamblin

Keyword(s):

Gas Sensors ◽

Cost Effective ◽

Pentaerythritol Tetranitrate ◽

Screening System ◽

Trace Detection ◽

False Negatives ◽

Quantitative Measurements ◽

Front End ◽

Effective Manner ◽

Single Sensor

Selective and sensitive detection of trace amounts of pentaerythritol tetranitrate (PETN) is demonstrated. The screening system is based on a sampling/concentrator front end and electrochemical potentiometric gas sensor as the detector. A single sensor is operated in the dominant hydrocarbon (HC) and nitrogen oxides (NOx) mode by varying the sensor operating condition. The potentiometric sensor with integrated heaters was used to capture the signature of PETN. Quantitative measurements based on hydrocarbon and nitrogen oxide sensor responses indicated that the detector sensitivity scaled proportionally with the mass of the explosives (10 μg down to 200 ng). The ratio of the HC integrated peak area to the NOxintegrated peak area is identified as an indicator of selectivity. The HC/NOxratio is unique for PETN and has a range from 1.7 to 2.7. This detection technique has the potential to become an orthogonal technique to the existing explosive screening technologies for reducing the number of false positives/false negatives in a cost-effective manner.

Download Full-text

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽

10.1163/156854109x428016 ◽

2009 ◽

Vol 11 (6) ◽

pp. 847-857 ◽

Cited By ~ 21

Author(s):

Lieven Waeyenberge ◽

Nicole Viaene ◽

Maurice Moens

Keyword(s):

Internal Control ◽

Dna Sequences ◽

Rrna Gene ◽

Duplex Pcr ◽

Specific Primers ◽

5.8S Rrna Gene ◽

5.8S Rrna ◽

Wide Range ◽

Pcr Products ◽

Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Download Full-text