David H. -F. Teng

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise approximately 16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42-86 bp) genotyping on the LightScanner. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067 degrees C before calibration to 0.022 degrees C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086 degrees C to 0.032 degrees C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical.

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the ''FilmArray'', which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

Neurocalcins belong to a family of neuronal specific EF hand Ca2+-binding proteins defined by recoverin. Previously, we reported the cloning and initial characterization of neurocalcin in Drosophila melanogaster (Teng, D. H.-F., Chen, C.-K., and Hurley, J. B. (1994) J. Biol. Chem. 269, 31900-31907). We showed that the Drosophila neurocalcin protein (DrosNCa) is expressed in neurons and that bacterially expressed recombinant DrosNCa (rDrosNCa) can be myristoylated. Here, we present two lines of evidence that DrosNCa is fatty acylated in vivo. First, the mobility of affinity-purified native DrosNCa on two-dimensional gel electrophoresis is identical to that of myristoylated rDrosNCa and distinct from that of nonacylated rDrosNCa. Second, the membrane binding properties of native DrosNCa are similar to those of myristoylated rDrosNCa; both of these proteins bind to membranes at 0.2 mM Ca2+, whereas nonacylated rDrosNCa always remains soluble. It has been shown that recoverin inhibits the phosphorylation of rhodopsin when Ca2+ is present (Kawamura et al., 1993) and that a dependent recoverin/rhodopsin kinase interaction underlies the inhibitory effect of recoverin (Chen et al., 1995). Given the similarities between recoverin and neurocalcin, we examined the effect of DrosNCa on rhodopsin phosphorylation. We find that rDrosNCa is capable of inhibiting bovine rhodopsin phosphorylation in vitro in a Ca2+-dependent manner. The inhibitory activity of rDrosNCa is enhanced by myristoylation, and the potency of its effect is similar to that of recoverin. Two other related EF hand proteins, guanylate cyclase-activating protein-2 and calmodulin, are only poor inhibitors in these phosphorylation assays. in vitro inhibition of rhodopsin phosphorylation therefore appears to be an assayable property of a subset of recoverin-like proteins.