Hindson BJ, Ness KD, Masquelier DA et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Rose-Zerilli MJ, Barton SJ, Henderson AJ et al (2009) Copy-number variation genotyping of GSTT1 and GSTM1 gene deletions by real-time PCR.D’haene B, Vandesompele J, Hellemans J (2010) Accurate and objective copy number profiling using real-time quantitative PCR.Weaver S, Dube S, Mir A et al (2010) Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.Jiang W, Johnson C, Jayaraman J et al (2012) Copy number variation leads to considerable diversity for B but not a haplotypes of the human KIR genes encoding NK cell receptors.Buchanan JA, Scherer SW (2008) Contemplating effects of genomic structural variation.Henrichsen CN, Chaignat E, Reymond A (2009) Copy number variants, diseases and gene expression.anthracis strain CZC5 as a positive control. pseudomycoides strain BP_CZC2 lane NC, distilled water as a negative control lane BC, B.
pseudomycoides strain BP_CZC1 lane 16, B. anthracis, and field isolates in Japan (right panel) were subjected to the multiplex PCR assay developed in this study and Ba813 PCR. anthracis.įield isolates in Zambia (left panel) and clinical outbreak strains in Japan (right panel), which were genetically related to B. Multiplex PCR for Bacillus field isolates and clinical strains genetically related to B. CFU/ml in lanes 1 and 2 were not determined because of uncountable colonies. Lane 3, 2.99 × 10 3 CFU/ml lane 4, 3.83 × 10 2 CFU/ml lane 5, 2.0 × 10 CFU/ml lanes 6 and 7, 0 CFU/ml lanes 8, distilled water as a negative control.
Lane M, 100-bp DNA ladder lanes 1 to 7, serially 10-fold diluted B. All 5 bands were simultaneously detected at the template concentration of 2.99 × 10 3 CFU/ml (lane 3). anthracis strain CZC5 cultured in LB broth containing 5% (v/v) sheep blood (lanes 1 to 7). anthracis cultured in liquid media.ĭNA fragments were amplified by the multiplex PCR in the extracted DNA from B. anthracis vaccine strain Sterne 34F2 clone2 lane 9, distilled water as a negative control. anthracis vaccine strain Sterne 34F2 lane 8, B.
anthracis strain CZC5 clone1 lane 7, DNA from B. anthracis strain L158–1 lane 6, DNA from B. anthracis strain CZC5 lane 4, DNA from B. anthracis-infected hippopotamus tissue lane 3, DNA from B. anthracis-infected elephant blood lane 2, DNA from B. Lane M, 100-bp DNA ladder lane 1, DNA from B. anthracis harboring or lacking virulent plasmids. The expected DNA fragments corresponding to the characteristics of samples were amplified in all samples by the multiplex PCR assay established in this study (uppermost panel) and by three single PCRs, two duplex PCRs and PCR using a commercial kit (lower panels). PCR using primers designed in previous studies.Ĭomparison of the specificity of multiplex PCR and those of existing PCR methods. Expected amplification was simulated in silico using Amplifx 1.7.0 software.
Amplifx software#
GENETYX-MAC Network Version 15.0.5 software was used for multiple sequence alignment. The rectangles shaded with grey correspond to BA_5031 and BACI_c47770 primer sequences.
weihenstephanensis BA_5031 (A) and BACI_c47770 (B) sequences from the database and primer sequences in this study. Other Bacillus strains used in this study.īacterial DNAs used for optimization of 16S rRNA amplification in this study.īA_5031 and hBC/BT primer annealing sites on genome sequences of Bacillus cereus group strains.Īlignment of B. Bacillus anthracis strains used in this study.
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