SPADA Draft Documents

lab testing, they can reduce the amount of effort wasted on empirical optimization and iterative 43 re-designs and also guide validation studies. The proposed computational approaches also result 44 in higher performing assays with better sensitivity, specificity and lower limit of detection and 45 reduce the possibility of assay failure due to signature erosion. To provide clarity, an extensive 46 glossary of defined terms is provided.

47 48

Background and Rationale for Project

49

Nucleic acid-based assays, such as real time polymerase chain reaction (PCR), are the 50 mainstay of clinical diagnostics and biosurveillance. A typical PCR assay design begins with 51 computational (“ in silico ”) identification of a unique region (signature) that can support the 52 binding of primer and probe sequences for target-specific amplification as a means of detecting 53 the presence of the target organism. This is followed by wet-lab testing of the primers and probes 54 using genomic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and performance- 55 optimization of selected assays. In addition, extensive testing of the assay in the intended clinical 56 matrix is required to evaluate assay parameters, such as: limit of detection, sensitivity 57 (probability of detection) and specificity (see glossary for definitions). The sensitivity and 58 specificity of the assay are experimentally determined using a set of target (inclusivity) strains 59 and near neighbor (exclusivity) strains and matrix relevant (background) organisms. Assay 60 performance also needs to be measured in assay-specific matrices (i.e. blood, stool, water, soil, 61 etc.). Often, assays are computationally designed using a small set of available genomic/gene 62 sequences and then experimentally validated for signature presence in all available samples of 63 the target organism (the inclusivity panel) and validated for signature absence in many other 64 samples that do not contain the target (the exclusivity panel and the matrix panel). In an ideal 65

3

Made with FlippingBook flipbook maker