scholarly journals The update and optimization of an eDNA assay to detect the invasive rusty crayfish (Faxonius rusticus)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0259084
Author(s):  
Stephanie S. Coster ◽  
Megan N. Dillon ◽  
William Moore ◽  
George T. Merovich
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Environmental Dna ◽  
Extraction Methods ◽  
Noninvasive Detection ◽  
Rusty Crayfish ◽  
Central Pennsylvania ◽  
Presence Data ◽  
Invasion Fronts ◽  
Polymerase Chain ◽  
Dna Extraction Methods

Environmental DNA (eDNA) is nuclear or mitochondrial DNA shed into the environment, and amplifying this DNA can serve as a reliable, noninvasive way to monitor aquatic systems for the presence of an invasive species. Assays based on the collection of eDNA are becoming increasingly popular, and, when optimized, can aid in effectively and efficiently tracking invasion fronts. We set out to update an eDNA assay to detect the invasive rusty crayfish, Faxonius rusticus. We tested for species specificity compared to other stream crayfish and field tested the assay at sites with known presence (N = 3) and absence (N = 4) in the Juniata River watershed in central Pennsylvania, USA. To maximize sensitivity, we field tested different storage buffers (Longmire’s buffer and ethanol), DNA extraction methods (Qiagen’s DNEasy and PowerWater kits), and quantitative polymerase chain reaction (qPCR) chemistries (TaqMan and SYBR green). Our assay confirmed the presence data and performed optimally when filter samples were stored in Longmire’s buffer, DNA was extracted with DNeasy Blood and Tissue Kit, and TaqMan qPCR chemistry was utilized. With proper sample processing, our assay allows for accurate, noninvasive detection of F. rusticus in streams.

scholarly journals Evaluation of DNA Extraction Methods for Detection of <i>Leishmania</i> by Polymerase Chain Reaction

2020 ◽  
Vol 10 (04) ◽  
pp. 265-272
Author(s):  
Letícia Surian Batalini ◽  
Silvana de Oliveira Castro ◽  
Carla Geórgia Rodrigues G. ◽  
Herintha Coeto Neitzke-Abreu ◽  
Manoel Sebastião da Costa L.
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Dna Extraction Methods

scholarly journals Validation Studies and Proficiency Testing

2002 ◽  
Vol 85 (3) ◽  
pp. 809-816 ◽  
Author(s):  
Elke Anklam ◽  
Petra Heinze ◽  
Simon Kay ◽  
Guy van den Eede ◽  
Bert Popping
Keyword(s):  
Validation Studies ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Extraction Methods ◽  
International Level ◽  
Testing Methods ◽  
Chain Reaction ◽  
Formal Validation ◽  
Food Market ◽  
Polymerase Chain

Abstract Genetically modified organisms (GMOs) entered the European food market in 1996. Current legislation demands the labeling of food products if they contain &lt;1% GMO, as assessed for each ingredient of the product. To create confidence in the testing methods and to complement enforcement requirements, there is an urgent need for internationally validated methods, which could serve as reference methods. To date, several methods have been submitted to validation trials at an international level; approaches now exist that can be used in different circumstances and for different food matrixes. Moreover, the requirement for the formal validation of methods is clearly accepted; several national and international bodies are active in organizing studies. Further validation studies, especially on the quantitative polymerase chain reaction methods, need to be performed to cover the rising demand for new extraction methods and other background matrixes, as well as for novel GMO constructs.

scholarly journals Detection and Differentiation of Phaeoisariopsis griseola Isolates with the Polymerase Chain Reaction and Group-Specific Primers

Plant Disease ◽  
1999 ◽  
Vol 83 (1) ◽  
pp. 37-42 ◽  
Author(s):  
P. Guzmán ◽  
P. Gepts ◽  
S. Temple ◽  
A. B. C. Mkandawire ◽  
R. L. Gilbertson
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Extraction Methods ◽  
Chain Reaction ◽  
Phaeoisariopsis Griseola ◽  
Geographical Regions ◽  
Polymerase Chain ◽  
Dna Extraction Methods ◽  
Group Specific Primer

Specific detection of the two major groups of Phaeoisariopsis griseola(Andean and Mesoamerican) from infected common bean (Phaseolus vulgaris) leaves was achieved by amplification of different-sized DNA fragments with polymerase chain reaction (PCR) using group-specific primer pairs. These primer pairs were designed based on DNA sequences of cloned random amplified polymorphic DNA (RAPD) fragments. Using this method, P. griseola isolates from diverse geographical regions (five countries) were differentiated into the two previously established groups. Various sources of fungal tissue and DNA extraction methods were tested in order to develop a rapid PCR-based method to detect and differentiate P. griseola isolates. A simple and rapid sonication method was developed that allowed for PCR detection of P. griseola from mycelia or synnemata and conidia collected from angular leaf spot lesions on bean leaves.

scholarly journals Comparison of Three Mycobacterial DNA Extraction Methods from Extrapulmonary Samples for PCR Assay

2013 ◽  
Vol 6 (1) ◽  
pp. 9-11
Author(s):  
Khandaker Shadia ◽  
Shaheda Anwar ◽  
Sayera Banu ◽  
Ahmed Abu Saleh ◽  
Md Ruhul Amin Miah
Keyword(s):  
Dna Extraction ◽  
Extrapulmonary Tuberculosis ◽  
Extraction Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Lysis Buffer ◽  
Dna Extraction Methods ◽  
Positive Results

Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%), 4 (20%) and 7 (35%) in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. DOI: http://dx.doi.org/10.3329/imcj.v6i1.14711 Ibrahim Med. Coll. J. 2012; 6(1): 9-11

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