Journal for Veterinary Medicine, Biotechnology and Biosafety
Volume
5, Issue 2, June 2019, Pages 15–21
ISSN 2411-3174 (print version) ISSN 2411-0388
(online version)
VALIDATION OF ANTHRAX
SPECIFIC pagA QUANTITATIVE PCR FOR
DETECTION OF BACILLUS ANTHRACIS pXO1 PLASMID
Biloivan O. V. 1,
Stegniy B. T. 1, Gerilovych A. P. 1,
Solodiankin O. S. 1, Popp C. 2,
Schwarz J. 2
1 National
Scientific Center ‘Institute of Experimental and Clinical Veterinary
Medicine’, Kharkiv, Ukraine, e-mail: silverscreen91@gmail.com
2 Bundeswehr
Institute of Microbiology, Munich, Germany
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PDF (print version)
Citation for print version: Biloivan, O. V., Stegniy, B. T.,
Gerilovych, A. P., Solodiankin, O. S., Popp, C. and
Schwarz, J. (2019) ‘Validation of Anthrax specific pagA
quantitative PCR for detection of Bacillus anthracis pXO1 plasmid’,
Journal for Veterinary Medicine,
Biotechnology and Biosafety, 5(2), pp. 15–21.
Download
PDF (online version)
Citation for online version: Biloivan, O. V., Stegniy, B. T.,
Gerilovych, A. P., Solodiankin, O. S., Popp, C. and
Schwarz, J. (2019) ‘Validation of Anthrax specific pagA
quantitative PCR for detection of Bacillus anthracis pXO1 plasmid’,
Journal for Veterinary Medicine,
Biotechnology and Biosafety. [Online] 5(2), pp. 15–21. DOI: 10.36016/JVMBBS-2019-5-2-3.
Summary. 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).
Keywords: anthrax, plasmid, validation, quantitative
PCR
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