title: A sample-in-answer-out instrument for the detection of multiple respiratory pathogens in unprepared nasopharyngeal swab samples.
abstract: Multiplex RT-PCR suspension array assays provide a powerful tool for identifying the causative agent(s) of respiratory infections. These assays are time consuming and laborious on a time-per-sample basis if only a few samples require processing. To address this shortcoming and provide an automated solution for fast detection and identification of viral pathogens, we developed the first automated multiplex RT-PCR suspension array instrument capable of handling unprepared clinical samples. The instrument requires less than 3 minutes of hands-on time for a result generated in approximately 2.5 hours. In analytical studies, the instrument performed as well as manually performed assays. The performance of the instrument and loaded multiplex viral detection assay was then tested using unprepared nasopharyngeal samples. The instrument-performed assay detected 61 of 71 RSV positive samples, for a sensitivity of 85.9%. Adenovirus (n = 5) and influenza B (n = 3) were less prevalent in the sample set, but detected to similar levels, 80% and 75%, respectively. The same sample set was also tested using FDA approved immuno-assay rapid tests, and the instrument was found to be more sensitive than the rapid tests with the sole exception being influenza A (n = 16), which was poorly detected due to significant sequence mismatches between the influenza A primer/probe set included in the multiplex mixture and the circulating influenza A strains. Overall, these data demonstrate the developed prototype platform performs multiplex array assays as well as hand-performed assays, and that the instrument's sensitivity and specificity are dictated by the quality of the loaded multiplex assay.
authors.affiliation: Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA. jack.regan@lifetech.com
authors.forename: John
authors.initials: J
authors.lastname: Regan
authors.forename: Sonia
authors.initials: S
authors.lastname: Létant
authors.forename: Kristl
authors.initials: K
authors.lastname: Adams
authors.forename: Nga
authors.initials: N
authors.lastname: Nguyen
authors.forename: Robert
authors.initials: R
authors.lastname: Derlet
authors.forename: Stuart
authors.initials: S
authors.lastname: Cohen
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Vitalis
authors.forename: Lance
authors.initials: L
authors.lastname: Tammero
authors.forename: Josue
authors.initials: J
authors.lastname: Ortiz
authors.forename: Mary
authors.initials: M
authors.lastname: McBride
authors.forename: James
authors.initials: J
authors.lastname: Birch
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D009305:Nasopharynx / Q000821:virology*; D020411:Oligonucleotide Array Sequence Analysis; D012367:RNA, Viral / Q000032:analysis; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014780:Viruses / Q000302:isolation & purification*
chemical_list: D012367:RNA, Viral