title: Clinical evaluation of NucliSENS magnetic extraction and NucliSENS analyte-specific reagents for real-time detection of human metapneumovirus in pediatric respiratory specimens.
abstract: In this study, we evaluated the NucliSENS miniMAG (MM) and easyMAG (EM) nucleic acid extraction platforms (bioMérieux, Durham, NC) in combination with the NucliSENS EasyQ basic kit and analyte-specific reagents (ASRs) (bioMérieux) for the detection of human metapneumovirus (hMPV) in respiratory samples. Total nucleic acids from pediatric clinical samples (n = 653) and an hMPV-specific inhibition control (h-IC) were coextracted using the MM and/or the EM. Nucleic acid sequence-based amplification and real-time molecular beacon detection of hMPV were performed using a NucliSENS EasyQ analyzer (bioMérieux). Positive results were confirmed using an in-house-validated reverse transcriptase PCR ASR-based assay. The inclusion of the h-IC monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. The percentages of samples with inhibited amplification of the h-IC after initial NA extraction by EM and MM were 1.88% and 3.17%, respectively. After reprocessing of a new aliquot, the final h-IC inhibition rates were 0% (EM) and 1.06% (MM). The limit of detection of the assay was between 2 (EM extraction) and 10 (MM extraction) RNA copies/reaction, and specificity was 100% when testing viral respiratory isolates and clinical samples. hMPV was detected in 5.6% of pediatric samples tested and was also detected in three coinfections with respiratory syncytial virus (RSV). hMPV was the second most frequently detected respiratory virus in children of 0 to 2 years of age, after RSV. In summary, NucliSENS extraction and ASRs provided a sensitive and specific method for the detection of hMPV in respiratory samples.
authors.affiliation: North Shore-Long Island Jewish Health System Laboratories, Molecular Diagnostics, Department of Laboratory Medicine, 10 Nevada Drive, Lake Success, NY 11042, USA. cginocch@nshs.edu
authors.forename: Christine C
authors.initials: CC
authors.lastname: Ginocchio
authors.forename: Ryhana
authors.initials: R
authors.lastname: Manji
authors.forename: Madhavi
authors.initials: M
authors.lastname: Lotlikar
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
mesh_terms: D002675:Child, Preschool; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D029121:Metapneumovirus / Q000235:genetics / Q000302:isolation & purification*; D015335:Molecular Probes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018184:Paramyxoviridae Infections / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D012137:Respiratory System / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D015335:Molecular Probes; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic