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Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.

ABSTRACT: Bacterial-derived DNA fragments (BDNAs) have been shown to be present in a dialysis fluid, to pass through dialyzer membranes, and to induce interleukin 6 (IL-6) in mononuclear cells. DNA fragments are thought to be derived from microorganisms inhabiting hemodialysis water and fluid. The primary aim of the present study was to develop two degenerated TaqMan real-time quantitative-PCR (Q-PCR) for detection of a broad range of bacterial DNA that specifically detect 16S ribosomal DNA (rDNA) (862 and 241 bp) and evaluate the efficiency of the Bellco Selecta resin to capture the BDNAs in the dialysis fluid. For this purpose, we decided to compare measurements of unfragmented samples (9.8 × 105 Escherichia coli genome) with artificially fragmented DNA samples. We assessed two broad-range real-time PCR targeting bacterial 16S rRNA genes for detection of fragmented and unfragmented bacterial DNA in the dialytic fluid and demonstrated that Bellco Selecta resin is capable of retaining these types of bacterial DNA.

SUBMITTER: Bergallo M

PROVIDER: S-EPMC7161787 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.

Bergallo Massimiliano M Loiacono Elisa E Rizzo Giovanna G Caiazzo Marialuisa M Palladino Giuseppe G Amore Alessandro A

Biotechnology and applied biochemistry 20161215 3

Bacterial-derived DNA fragments (BDNAs) have been shown to be present in a dialysis fluid, to pass through dialyzer membranes, and to induce interleukin 6 (IL-6) in mononuclear cells. DNA fragments are thought to be derived from microorganisms inhabiting hemodialysis water and fluid. The primary aim of the present study was to develop two degenerated TaqMan real-time quantitative-PCR (Q-PCR) for detection of a broad range of bacterial DNA that specifically detect 16S ribosomal DNA (rDNA) (862 an ...[more]

PMID: 27067406

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Project description:BACKGROUND:DNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. METHODS:Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. RESULTS:COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. CONCLUSIONS:The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Project description:BACKGROUND:The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples. METHODS:A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples. RESULTS:Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/?L and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/?L of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA. CONCLUSIONS:This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

Dataset Information

Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.

Publications

Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.

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