scholarly journals A new molecular diagnostic tool for surveying and monitoring Triops cancriformis populations

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3228
Author(s):  
Graham S. Sellers ◽  
Larry R. Griffin ◽  
Bernd Hänfling ◽  
Africa Gómez
Keyword(s):  
Land Development ◽  
Habitat Destruction ◽  
Molecular Diagnostic ◽  
Monitoring Methods ◽  
Specific Primers ◽  
Diapausing Eggs ◽  
Egg Banks ◽  
Triops Cancriformis ◽  
Conventional Methods ◽  
Species Specific

The tadpole shrimp, Triops cancriformis, is a freshwater crustacean listed as endangered in the UK and Europe living in ephemeral pools. Populations are threatened by habitat destruction due to land development for agriculture and increased urbanisation. Despite this, there is a lack of efficient methods for discovering and monitoring populations. Established macroinvertebrate monitoring methods, such as net sampling, are unsuitable given the organism’s life history, that include long lived diapausing eggs, benthic habits and ephemerally active populations. Conventional hatching methods, such as sediment incubation, are both time consuming and potentially confounded by bet-hedging hatching strategies of diapausing eggs. Here we develop a new molecular diagnostic method to detect viable egg banks of T. cancriformis, and compare its performance to two conventional monitoring methods involving diapausing egg hatching. We apply this method to a collection of pond sediments from the Wildfowl & Wetlands Trust Caerlaverock National Nature Reserve, which holds one of the two remaining British populations of T. cancriformis. DNA barcoding of isolated eggs, using newly designed species-specific primers for a large region of mtDNA, was used to estimate egg viability. These estimates were compared to those obtained by the conventional methods of sediment and isolation hatching. Our method outperformed the conventional methods, revealing six ponds holding viable T. cancriformis diapausing egg banks in Caerlaverock. Additionally, designed species-specific primers for a short region of mtDNA identified degraded, inviable eggs and were used to ascertain the levels of recent mortality within an egg bank. Together with efficient sugar flotation techniques to extract eggs from sediment samples, our molecular method proved to be a faster and more powerful alternative for assessing the viability and condition of T. cancriformis diapausing egg banks.

scholarly journals New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes

Insects ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 447 ◽  
Author(s):  
Linyu Zheng ◽  
Yue Zhang ◽  
Wenzhao Yang ◽  
Yiying Zeng ◽  
Fan Jiang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Dna Barcode ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Specific Primers ◽  
Oxidase Subunit ◽  
Preimaginal Stages ◽  
Specific Fragment ◽  
Species Specific

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

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.

Species‐specific primers of chitin synthase 1 gene for the differentiation of the Trichophyton mentagrophytes complex

Mycoses ◽  
1999 ◽  
Vol 42 (1‐2) ◽  
pp. 71-74 ◽  
Author(s):  
R. Kano ◽  
Y. Nakamura ◽  
T. Watari ◽  
S. Watanabe ◽  
H. Takahashi ◽  
...  
Keyword(s):  
Trichophyton Mentagrophytes ◽  
Chitin Synthase ◽  
Specific Primers ◽  
Trichophyton Mentagrophytes Complex ◽  
Species Specific

scholarly journals Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

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.

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