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Literature DB >> 19633128

Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.

Ofer Peleg¹, Gad Baneth, Osnat Eyal, Jacob Inbar, Shimon Harrus.

Abstract

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.

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Year: 2009 PMID： 19633128 PMCID： PMC2753072 DOI： 10.1128/AEM.00720-09

Source DB: PubMed Journal: Appl Environ Microbiol ISSN： 0099-2240 Impact factor: 4.792

23 in total

1. Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands.

Authors: Shu-ichi Nakano; Takayuki Kanzaki; Naoki Sugimoto
Journal: J Am Chem Soc Date: 2004-02-04 Impact factor: 15.419

2. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

Authors: Q Chou; M Russell; D E Birch; J Raymond; W Bloch
Journal: Nucleic Acids Res Date: 1992-04-11 Impact factor: 16.971

3. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

Authors: Stephen A Bustin; Vladimir Benes; Jeremy A Garson; Jan Hellemans; Jim Huggett; Mikael Kubista; Reinhold Mueller; Tania Nolan; Michael W Pfaffl; Gregory L Shipley; Jo Vandesompele; Carl T Wittwer
Journal: Clin Chem Date: 2009-02-26 Impact factor: 8.327

Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.

1. Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands.

2. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

3. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

4. Maximizing sensitivity and specificity of PCR by pre-amplification heating.

5. 'Touchdown' PCR to circumvent spurious priming during gene amplification.

Review 6. PCR and the cloning of receptor subtype genes.

7. Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides.

8. Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

9. Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate syntase gene.

10. High-resolution genotyping by amplicon melting analysis using LCGreen.

Review 1. Current advances in detection and treatment of babesiosis.

2. A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.