Development of positive control assays for the detection of Bacillus anthracis plasmids PXO1 AND PXO2 via PCR

Biloivan, O. V.; Stegniy, B. T.; Solodiankin, Oleksii; Gerilovych, Anton

doi:10.31073/vet_biotech32(1)-3

Cited by 2 publications

(3 citation statements)

References 5 publications

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“…Testing of the diagnostic tool was conducted using previously-developed recombinant positive controls, namely p-pagA-TZ57R/T and p-capC-TZ57R/T in various dilutions (Fig. 1) (Biloivan et al, 2018). The positive control sample p-dhp61-CR2.1-TOPO for detecting of anthrax dhp61 specific chromosomal marker, developed by Antwerpen et al (2008) and kindly provided by colleagues from the Bundeswehr Institute of Microbiology (Munich, Germany), was also included as the component of this test kit.…”

Section: Introductionmentioning

confidence: 99%

“…For convenience in a diagnostic laboratory setting, primer and probe mixtures were prepared in equal volumes, with three mixtures in total for each of the genetic markers (dhp61, pagA, and capC). Additionally, the test kit includes positive control samples for detecting the plasmid markers of B. anthracis (pagA and capC), which were prepared previously (Biloivan et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Biloivan,

Popp,

Schwarz

2023

J. Vet. Med. Biotechnol. Biosafety

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This paper represents preliminary trials of the ‘Anthrax-DNA-test’, diagnostical tool for the detection of anthrax DNA. It includes recombinant positive controls p-pagA-TZ57R/T and p-capC-TZ57R/T for the detection of anthrax plasmid markers, as well as p-dhp61-CR2.1-TOPO, positive control for the detection of Bacillus anthracis chromosomal marker. Besides, three mixtures of primers and probes for the detection of each genetic marker (dhp61, pagA, and capC) and ready-to-use ‘RT-PCR МаsterМіx’ PCR diluent were also included. Concentrations of MgCl2 and Taq-polymerase obtained during qPCR validation procedure were considered when preparing the diluent. To determine specificity, qPCR was conducted with heterological panel of DNA of pathogenic bacteria and viruses causing diseases with similar to anthrax clinical signs. To determine repeatability of the results when using ‘Anthrax-DNA-test’ PCR test kit, samples were studied twice. The sensibility of the kit was analyzed by serial dilutions of p-dhp61-CR2.1-TOPO, p-pagA-TZ57R/T and p-capC-TZ57R/T plasmid DNAs containing fragments of anthrax chromosome and plasmids. To compare the tool’s ability to identify anthrax DNA, classical PCR was carried out using ANT-PA_F/R and ANT-CAP_F/R primers recommended by OIE for the detection of pXO1 and pXO2 plasmid DNA. Sensitivity testing has shown that the test kit is able to identify all positive samples. It has been found that the diagnostics tool detects anthrax DNA in recombinant positive control samples containing B. anthracis chromosomal and plasmid DNA fragments in serial dilutions from 1:100 to 1:1,000 with Ct values of 25.29–34.70. The specificity of this diagnostic tool is proved by the absence of Ct in heterological samples. Besides, repeatability of trial results has been found, which is proved by complete congruence in duplicates with each of the tested sample

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Biloivan,

Popp,

Schwarz

2023

J. Vet. Med. Biotechnol. Biosafety

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“…Materials and methods. To carry out these studies, we used pagA TZ57 R/T recombinant positive control, which had been prepared before using TA-cloning method (Biloivan et al, 2018). To reduce the risk of contamination in the laboratory when working with plasmid DNA, we obtained pagA insert from it using classical PCR with M13 forward and M13 reverse primers (Chandra and Wikel, 2005).…”

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confidence: 99%

Validation of Anthrax specific pagA quantitative PCR for detection of Bacillus anthracis pXO1 plasmid

Biloivan

Stegniy

Gerilovych

et al. 2019

J. Vet. Med. Biotechnol. Biosafety

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This paper represents qPCR validation results for the detection of Bacillus anthracis pagA pXO1 plasmid marker. The aim of the work was to transfer, implement and validate anthrax specific pagA qPCR assay for the detection of pagA, the genetic marker of the pXO1 plasmid of Bacillus anthracis. qPCR was conducted using the Applied Biosystems Fast 7500 Real-time PCR system including Applied Biosystem specific reagents (AmpliTaq Gold). Anthrax pXO1 pagA primers (pagA_forward, pagA_reverse) and TaqMan pagA probe. Data analysis and statistical calculations were performed using Microsoft Excel. The limit of detection (probit analysis) was calculated using the Statgraphics software. Robustness of qPCR was adjusted by optimization of amplification parameters (annealing temperature) and concentration of reaction components (MgCl2, primers, probe and Taq polymerase). In order to test the repeatability and precision of the qPCR assay after optimization, the variation within the experiment (Intra-assay variability) and between several independent experiments (Inter-assay variability) was evaluated. Probit analysis with serial dilutions of positive control with five replicates per dilution was carried out to define the 95% limit of detection (LOD). To determine if the CT value correlates with the amount of template DNA, the linearity of qPCR was analyzed. The standard curve was generated and the linear regression line and the coefficient of correlation (R2) were calculated. To define the ability to detect sequence of interest (sensitivity), we tested mixed panel of Bacillus anthracis DNAs. As the result, pagA marker could be detected in all tested strains . To find out the specificity of our assay, we also tested DNA of various strains of B. cereus, B. thuringiensis, B. mycoides, and B. globigii (potential cross-reacting organisms) as well as DNA samples of various pathogenic bacteria and viruses which cause similar clinical symptoms as anthrax (differential diagnosis relevant organisms).

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Development of positive control assays for the detection of Bacillus anthracis plasmids PXO1 AND PXO2 via PCR

Cited by 2 publications

References 5 publications

Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Development of in-house diagnostic tool for the detection of Anthrax genetic material in real-time PCR

Validation of Anthrax specific pagA quantitative PCR for detection of Bacillus anthracis pXO1 plasmid

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