Dataset Information

Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

ABSTRACT: The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.

SUBMITTER: Gryta A

PROVIDER: S-EPMC7309186 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

Gryta Agata A Frąc Magdalena M

Sensors (Basel, Switzerland) 20200609 11

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial div ...[more]

PMID: 32527006

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Project description:Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Analysis for both mutations is increasingly being performed on patients exhibiting hypercoagulability. The goal of the current study was to evaluate the performance of primer-engineered multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of factor V Leiden and prothrombin G20210A. Primer-engineered multiplex PCR-RFLP methods for the detection of factor V Leiden and prothrombin G20210A from the medical literature were reviewed. A modified method was optimized in which both mutations generate HindIII RFLPs and the prothrombin amplicon contains an invariant HindIII recognition site to assess the completeness of endonuclease digestion. Digested amplification products were analyzed by agarose gel electrophoresis in a single gel lane and visualized by ethidium bromide. Primer-engineered multiplex PCR-RFLP was used to analyze 205 human genomic DNA samples whose factor V Leiden genotypes had been previously determined by MnlI PCR-RFLP. Complete concordance for factor V Leiden genotypes was observed between the two methods in the 205-sample cohort comprising 139 wild-type, 62 heterozygous mutant, and four homozygous mutant individuals. For prothrombin G20210A, primer-engineered multiplex PCR-RFLP identified 196 wild-type and nine heterozygous mutant individuals in the 205-sample cohort. To independently verify prothrombin genotypes, the nine heterozygous mutants and an additional 11 wild-type patient samples (representing 10% of patient samples) were subjected to DNA sequencing. Complete concordance was observed between DNA sequencing and primer-engineered multiplex PCR-RFLP results. In further validation, 123 of the DNA samples consisting of four heterozygous mutant and 119 wild type individuals were genotyped with the Invader Assay for Factor II (prothrombin G20210A). Results showed 100% concordance between the Invader Assay and primer-engineered multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on single restriction endonuclease digestion has been evaluated and shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations. The method is robust and readily adaptable to the clinical molecular diagnostic laboratory.

Dataset Information

Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

Publications

Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

Similar Datasets

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