scholarly journals Development of species-specific Cichla species eDNA primers for alien invasive species (AIS) monitoring in Malaysia

2021 ◽  
Vol 4 ◽  
Author(s):  
O. Nurul Fizatul Nabilah ◽  
A. R. Ramizah ◽  
A. B. Adibah ◽  
S. Syazwan ◽  
A.G. Intan Faraha ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Environmental Dna ◽  
Coi Gene ◽  
Alien Invasive Species ◽  
Survey Method ◽  
Specific Primers ◽  
Invasive Fishes ◽  
Specific Amplification ◽  
Species Specific ◽  
High Level

Peacock bass or the cichlids are known locally as top predator fishes which are invasive in Malaysia freshwater system. Detection probabilities for these fishes are typically low, especially using conventional capture-survey method due to the fish’s behaviour of hiding beneath the water’s surface. Hence, the environmental DNA (eDNA) monitoring is a relatively new approach that can be used to assess the distribution of these invasive fishes. Here, we report the strategy to develop small fragment (280- 400 bp) specific-specific primers for three selected invasive Cichla species namely, C. ocellaris, C. monoculus, and C. kelberi based on mitochondrial DNA (mtDNA) sequences. Current research showed that the developed species-specific primers from cytochrome oxidase I (COI) gene has high resolution at species level. Species-specific amplification tests also proved the specificity of the developed primers, securing the high- level species identification potential which may help in controlling the spread of alien invasive fish species.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
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.

scholarly journals First record of the semi-slug Omalonyx unguis (d’Orbigny, 1837) (Gastropoda, Succineidae) in the Misiones Province, Argentina

Check List ◽  
2018 ◽  
Vol 14 (4) ◽  
pp. 705-712
Author(s):  
Leila B. Guzmán ◽  
Enzo N. Serniotti ◽  
Roberto E. Vogler ◽  
Ariel A. Beltramino ◽  
Alejandra Rumi ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Morphological Characteristics ◽  
First Record ◽  
Coi Gene ◽  
Cytochrome Oxidase Subunit ◽  
Specific Identification ◽  
Molluscan Fauna ◽  
Misiones Province ◽  
Species Specific ◽  
Northeastern Argentina

Omalonyx unguis (d’Orbigny, 1837) is a semi-slug inhabiting the Paraná river basin. This species belongs to Succineidae, a family comprising a few representatives in South America. In this work, we provide the first record for the species from Misiones Province, Argentina. Previous records available for Omalonyx in Misiones were identified to the genus level. We examined morphological characteristics of the reproductive system and used DNA sequences from cytochrome oxidase subunit I (COI) gene for species-specific identification. These new distributional data contribute to consolidate the knowledge of the molluscan fauna in northeastern Argentina.

scholarly journals LATEST INFORMATION ON THE DISTRIBUTION OF FUSARIUM LANGSETHIAE, THE PRODUCER OF T-2 AND HT-2 TOXINS, IN RUSSIA

2020 ◽  
Vol 103 (3) ◽  
pp. 201-206
Author(s):  
O. P. Gavrilova ◽  
T. Yu. Gagkaeva
Keyword(s):  
High Performance ◽  
Geographical Area ◽  
Intraspecific Diversity ◽  
Specific Primers ◽  
Toxic Metabolites ◽  
Central Regions ◽  
Species Specific ◽  
High Level ◽  
Grain Contamination ◽  
Fusarium Fungi

The annual monitoring of grain contamination with Fusarium fungi and the identification of their species composition showed the widespread distribution of F. langsethiae producing dangerous T-2 and HT-2 toxins in the Northwestern and Central regions of Russia. Mycological analysis of grain samples harvested in 2018–2019 allowed revealing the new places of F. langsethiae distribution, including Urals. The top infection rate of the oats grain by F. langsethiae in 2019 reached 14 %. The identification of F. langsethiae strains was supported by PCR with species-specific primers. The analysis of toxic metabolites in F. langsethiae by the combination of high-performance liquid chromatography and tandem mass spectrometry revealed the high level of T-2 and HT-2 toxins. The considerable total amounts of T-2 and HT-2 toxins (165–1230 μg/kg) were found in the grain samples infected with this species. Further clarification of the geographical area of F. langsethiae and the study of its intraspecific diversity are needed to understand the distribution of this toxin-producing fungus.

scholarly journals Identification protocol for sixArmillariaspecies from northeastern North America

2010 ◽  
Vol 40 (3) ◽  
pp. 536-548 ◽  
Author(s):  
J. A. McLaughlin ◽  
T. Hsiang
Keyword(s):  
Dna Sequences ◽  
Fragment Length Polymorphism ◽  
Intergenic Spacer ◽  
Polymorphism Analysis ◽  
Specific Primers ◽  
Northeastern North America ◽  
Armillaria Ostoyae ◽  
Armillaria Gallica ◽  
Species Specific ◽  
Restriction Fragment Length

DNA sequences (~3 kb long) extending from the intergenic spacer 1 (IGS1) region to the 18S gene were obtained for isolates of Armillaria ostoyae , Armillaria calvescens , Armillaria gallica , and Armillaria sinapina . Additional investigation of 16 A. ostoyae, 11 Armillaria gemina , 21 A. calvescens, 18 A. gallica, and 15 A. sinapina isolates produced 117 sequences spanning the 3′ end of the IGS1 through the 5S gene and into the 5′ end of the IGS2 region. Additional sequences spanning the 3′ IGS2 to 5′ 18S gene region were obtained for two A. ostoyae, three A. gemina, two A. calvescens, two A. gallica, and three A. sinapina isolates. This is the first report of complete IGS2 sequences from Armillaria spp. A species identification protocol involving species-specific primers and restriction fragment length polymorphism analysis was devised based on species-specific polymorphisms. The protocol successfully identified all 16 A. ostoyae, 11 A. gemina, three of three Armillaria mellea , 18 A. gallica, 14 of 15 A. sinapina (11/12 diploid and 3/3 haploid), and 14 of 21 A. calvescens (13/15 diploid and 1/6 haploid) isolates included in this study. To the best of our knowledge, this success rate has not been matched by other methods.

scholarly journals Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

1999 ◽  
Vol 37 (12) ◽  
pp. 3906-3911 ◽  
Author(s):  
Eugene J. Leys ◽  
James H. Smith ◽  
Sharon R. Lyons ◽  
Ann L. Griffen
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Sequences ◽  
Allelic Variation ◽  
Intergenic Spacer ◽  
Heteroduplex Analysis ◽  
Clinical Samples ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Species Specific ◽  
Multiple Strains

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types ofP. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivaliswas detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains ofP. gingivalis in large numbers of samples.

scholarly journals Using eDNA to biomonitor the fish community in a tropical oligotrophic lake

2018 ◽  
Author(s):  
Martha Valdez-Moreno ◽  
Natalia V. Ivanova ◽  
Manuel Elías-Gutiérrez ◽  
Stephanie L. Pedersen ◽  
Kyrylo Bessonov ◽  
...  
Keyword(s):  
Aquatic Ecosystems ◽  
Fish Community ◽  
Fish Fauna ◽  
Environmental Dna ◽  
Reference Sequence ◽  
Coi Gene ◽  
Ion Torrent ◽  
Sequence Database ◽  
Ion Torrent Pgm ◽  
Invasive Fishes

AbstractEnvironmental DNA (eDNA) is an effective approach for detecting vertebrates and plants, especially in aquatic ecosystems, but prior studies have largely examined eDNA in cool temperate settings. By contrast, this study employs eDNA to survey the fish fauna in tropical Lake Bacalar (Mexico) with the additional goal of assessing the possible presence of invasive fishes, such as Amazon sailfin catfish. Sediment and water samples were collected from eight stations in Lake Bacalar on three occasions over a 4-month interval. Each sample was stored in the presence or absence of lysis buffer to compare eDNA recovery. Short fragments (184-187 bp) of the cytochrome c oxidase I (COI) gene were amplified using fusion primers and then sequenced on Ion Torrent PGM and S5 before their source species were determined using a custom reference sequence database constructed on BOLD. In total, eDNA sequences were recovered from 75 species of vertebrates including 47 fishes, 15 birds, 7 mammals, 5 reptiles, and 1 amphibian. Although all species are known from this region, 6 fish species represent new records for the study area, while 2 require verification. Sequences for five species (2 birds, 2 mammals, 1 reptile) were only detected from sediments, while sequences from 52 species were only recovered from water. Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment to confirm our methods were appropriate for its detection. We developed protocols that enabled the recovery of eDNA from tropical oligotrophic aquatic ecosystems, and confirmed their effectiveness in detecting diverse species of vertebrates including an invasive species of Amazon catfish.

scholarly journals Clonal Stability of RAPD Markers in Three Rhododendron Species

1995 ◽  
Vol 13 (1) ◽  
pp. 43-46 ◽  
Author(s):  
M. Javed Iqbal ◽  
D. W. Paden ◽  
A. Lane Rayburn
Keyword(s):  
Dna Sequences ◽  
Rapd Markers ◽  
Cultivar Identification ◽  
Rapd Analysis ◽  
Randomly Amplified Polymorphic Dna ◽  
Plant Identification ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
The Individual ◽  
Species Specific

Abstract Amplification profiles produced by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA sequences (RAPD) have the potential for species and cultivar identification. Since most rhododendron plants are vegetatively propagated, it is imperative that RAPD profiles be stable during this propagation. Three species of rhododendron, Rhododendron arborescens, R. atlanticum and R. yedoense var. poukhanense were used to produce species specific amplification profiles. Stability of amplification profiles among individually cloned plants of each species were studied. Ten plants of R. atlanticum, 9 of R. arborescens, and 10 of R. yedoense var. poukhanense were studied with 10 random primers. No polymorphism was observed among individual plants of R. atlanticum and R. arborescens with all the primers. The amplification product of one plant of R. yedoense var. poukhanense showed a difference of one band with one primer. The rest of the profiles with 9 primers were identical in all plants of this species. In order to ascertain that RAPD markers can indeed reveal real genetic differences among plants, F2 plants of two hybrids were analyzed. In contrast to the clonally propagated plants, extensive polymorphisms were observed among the individual F2 plants. Thus, RAPD analysis can be used to detect genetic variability. This stability of RAPD profiles in clonally propagated rhododendron indicates the usefulness of these markers in plant identification.

scholarly journals A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1083
Author(s):  
Zhendong Cai ◽  
Song Zhou ◽  
Qianqian Liu ◽  
Hui Ma ◽  
Xinyi Yuan ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Animal Species ◽  
Meat Product ◽  
Meat Products ◽  
Pcr Assay ◽  
Specific Primers ◽  
Meat Species ◽  
Pcr Method ◽  
Species Specific ◽  
Simultaneous Identification

Multiplex PCR methods have been frequently used for authentication of meat product adulteration. Through screening of new species-specific primers designed based on the mitochondrial DNA sequences, a septuple PCR method is ultimately developed and optimized to simultaneously detect seven species including turkey (110 bp), goose (194 bp), pig (254 bp), sheep (329 bp), beef (473 bp), chicken (612 bp) and duck (718 bp) in one reaction. The proposed method has been validated to be specific, sensitive, robust and inexpensive. Taken together, the developed septuple PCR assay is reliable and efficient, not only to authenticate animal species in commercial meat products, but also easily feasible in a general laboratory without special infrastructures.

scholarly journals Environmental DNA and Specific Primers for Detecting the Invasive Species Ectopleura crocea (Hydrozoa: Anthoathecata) in Seawater Samples

2020 ◽  
Vol 12 (6) ◽  
pp. 2360 ◽  
Author(s):  
Philjae Kim ◽  
Tae Joong Yoon ◽  
Sook Shin
Keyword(s):  
Invasive Species ◽  
Survey Methods ◽  
Environmental Dna ◽  
Visual Assessment ◽  
Molecular Techniques ◽  
Mitochondrial Cytochrome ◽  
Specific Primer ◽  
Specific Primers ◽  
Seawater Samples ◽  
Species Specific

In marine environments, environmental DNA (eDNA) can be effectively detected and possibly quantified when combined with molecular techniques, as demonstrated by several recent studies. In this study, we developed a species-specific primer set and a probe to detect the distribution and biomass of an invasive hydrozoan in South Korea, Ectopleura crocea. These molecular markers were designed to amplify a 187 bp region based on mitochondrial cytochrome c oxidase subunit I (COI) of E. crocea and were tested on seawater samples from 35 Korean harbors in 2017. Of the 35 sites we investigated, only nine harbors returned positive detections when using traditional survey methods, while surveys based on the use of eDNA techniques detected E. crocea DNA in all seawater samples. These results suggest that eDNA surveys based on molecular techniques are more effective at identifying species distribution and estimating biomass than traditional surveys based on visual assessment of morphology.

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