Effect of enzyme system on detection of pathogen targets in primary clinical specimens.

<p>Data shown are difference in Ct value between reactions using Quanta One-step RT-PCR ToughMix and AgPath-ID One-step RT-PCR kit when testing TNA extracted from NP/OP swabs (A) or blood (B). Each data point represents the difference in Ct value between the two reactions for an individual clinical specimen. Median difference is indicated (<b>―</b>) for assays with ≥2 positive results. *Targets that were only detected using AgPath always occurred when Ct values were >33.</p

Cumulative improvement of pathogen detection with optimization of experimental parameters.

<p>Potential improvement in Ct value (number of cycles) achieved by optimizing each experimental parameter. *Number of cycles gained varies based on organism, target, and specimen type. ^#Theoretical improvement calculated based on assumption of 3.3 cycle difference with 10-fold change in nucleic acid concentration.</p

Effect of lytic enzyme treatment on extraction of nucleic acid from blood specimens.

<p>Average Ct value of individual real-time PCR reactions (<i>n</i> = 4) containing TNA extracted from healthy donor blood spiked with serial dilutions of <i>S. pyogenes</i> (A), <i>S. aureus</i> (B), or <i>K. pneumoniae</i> (C) without treatment or after incubation with TE buffer alone or TE buffer with lytic enzymes (lysozyme, lysostaphin, and mutanolysin) at 37°C for 30 min. (D) Ct values of serial dilutions of <i>K. pneumoniae</i> spiked into saline (to mimic NP/OP swab) or blood. Error bars display standard deviation. *<i>p</i><0.0001 compared to no treatment. ^#<i>p</i><0.05 compared to same concentration of organisms in saline.</p

ANISA-specific TAC configurations.

<p>Original ANISA NP/OP TAC, Original ANISA Blood TAC, Optimization TAC, Revised ANISA NP/OP TAC, Revised ANISA Blood TAC. Target designations include: MYPN, <i>Mycoplasma pneumoniae</i>; CHTR, <i>Chlamydia trachomatis</i>; CHPN, <i>Chlamydophila pneumoniae</i>; URUP, <i>Ureaplasma</i> spp.; BOP1, <i>Bordetella pertussis</i>; ADEV, Adenovirus; FLUA, Influenza A; FLUB, Influenza B; PIV1, Parainfluenza virus 1; PIV2, Parainfluenza virus 2; PIV3, Parainfluenza virus 3; RESV, Respiratory Syncytial Virus; HPEV, Human Parechovirus; IPCO, Internal Positive Control; GADH, Glyceraldehyde phosphate dehydrogenase (Manufacturing control); ENTV, Enterovirus; HMPV, Human Metapneumovirus; RUBV, Rubella virus; STPN, <i>Streptococcus pneumoniae</i>; KLPN, <i>Klebsiella pneumoniae</i>; ECSH, <i>Escherichia coli</i>/<i>Shigella</i> spp.; RHIV, Rhinovirus; GBST, Group B <i>Streptococcus</i>; HSV1, Herpes Simplex Virus 1; HSV2, Herpes Simplex Virus 2; RNP3, Human RNaseP; STAU, <i>Staphylococcus aureus</i>; GAST, Group A <i>Streptococcus</i>; PSAE, <i>Pseudomonas aeruginosa</i>; HIAT, <i>Haemophilus influenzae</i>; HITB, <i>Haemophilus influenzae</i> type B; SALS, <i>Salmonella</i> spp.; ABAU, <i>Acinetobacter baumannii</i>; CYMV, Cytomegalovirus; TOXG, <i>Toxoplasma gondii</i>; NMEN, <i>Neisseria meningitidis</i>. All oligonucleotides were spotted at 1× final concentration except where noted by * where concentration is 2×. ^#Duplex assay consisting of RNP3 assay with FAM-labeled probe and IPCO assay with VIC-labeled probe.</p