Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…Such assays are more sensitive to assay conditions compared with assays relying on unique signature sequences, and the occurrence of false positive signals from B. cereus strains caused by mispriming is more likely. Even though various techniques have been evaluated to enhance the specificity of SNP-based PCR assays (including TaqMan mismatch amplification mutation assay, 23 restriction site insertion-PCR, 56 tentacle or locked nucleic acids probes-based PCR 25 or high resolution melting (HRM)-PCR 53 ), they are neither as robust nor as user friendly as assays based on unique signature sequences. The chromosomal markers BA5345 (Antwerpen), PL3 (Wielinga), or BA5357 (Letant), enable unambiguous identification of B. anthracis strains, including plasmid-cured isolates.…”

“…[13][14][15] Finally, a few single nucleotide polymorphisms (SNP) have also been considered for PCR markers. Target genes include rpoB, 24,[48][49][50][51] gyrA, 25,52,53 gyrB, 54,55 plc, 20,23,53,56 purA, 57 and the 16S-23S rDNA internal spacer sequences. [58][59][60] But, so far, only the nonsense mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis, have proved to be truly unique to B. anthracis strains.…”

“…Small alterations in these conditions can result in the loss of specificity, especially with hydrolysis probes, i.e., TaqMan chemistry. 18,[23][24][25] To evaluate the wide range of PCR methods used in laboratories for B. anthracis identification, a computer-based comparative analysis of more than 300 PCR-target sequences reported in the literature was conducted. All sequences were compared against all publicly available Bacillus genomes and sorted for specificity.…”

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

“…Assays that have performed very well under highly controlled laboratory conditions sometimes fail under more routine conditions. 22 Large melting curve differentials provide robustness in the presence of variances in test conditions, such as salt, template concentrations, and temperature variations. Because each of these parameters affects the melting curves of the probe-target hybrid, they can potentially lead to false positives and negatives.…”

“…Although assays like PCR have expected concentration limits based on primer concentration, Taqman-MGB probes have failed when exposed to high concentrations of variant. 22 In general, assays relying on PCR mask potential concentration dependence problems in probe technologies because, if target is present, it is typically amplified to levels near the primer concentration. In most cases, the one to two orders of magnitude of concentrations where accuracy is achieved by existing probes is sufficient for PCR reactions.…”