Botulinum toxin field assays evaluated using cosmetic botox preparations.

Several quick tests for identifying botulinum toxins (BoNTs) are commercially available, but generally these tests have not been evaluated by independent laboratories. This study presents data on the evaluation of a number of commercial tests and demonstrates the use of cosmetic preparations of BoNT A and B as positive controls. For reference we used an in-house ELISA procedure. The cosmetic toxins, Botox(®) and Neurobloc(®), were found to be useful-that is, they had a sufficiently high toxin content to be used in test evaluation studies. Some commercial kits based on columns (ABICAP(®)) or lateral flow technology were tested for their detection limits. The ABICAP column system was found to be a useful alternative to an in-house ELISA method. In general, the lateral flow-based test systems evaluated here were not able to detect BoNT, and a large proportion of the tests showed construction failures. In conclusion, this study showed that cosmetic BoNT products have enough toxin content to be used as convenient and accessible means of testing commercially available quick tests. The lateral flow tests used in this study were not satisfactory, while the ABICAP system was found to be a good alternative to an ELISA.

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.