title: Automated, simple, and efficient influenza RNA extraction from clinical respiratory swabs using TruTip and epMotion.
abstract: BACKGROUND
  Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance.
  
  
  OBJECTIVE
  To automate TruTip influenza RNA extraction using an Eppendorf epMotion robotic liquid handler, and to compare its performance to the bioMerieux easyMAG and Qiagen QIAcube instruments.
  
  
  METHODS
  Extraction efficacy and reproducibility of the automated TruTip/epMotion protocol was assessed from influenza-negative respiratory samples spiked with influenza A and B viruses. Clinical extraction performance from 170 influenza A and B-positive respiratory swabs was also evaluated and compared using influenza A and B real-time RT-PCR assays.
  
  
  RESULTS
  TruTip/epMotion extraction efficacy was 100% in influenza virus-spiked samples with at least 745 influenza A and 370 influenza B input gene copies per extraction, and exhibited high reproducibility over four log10 concentrations of virus (<1% CV). RNA yields between the three automated methods differed by less than 0.5 log10 gene copies. 99% of clinical specimens that were PCR-positive after easyMAG or QIAcube extraction were also positive following TruTip extraction. Overall Ct value differences obtained between TruTip/epMotion and easyMAG/QIAcube clinical extracts ranged from 1.24 to 1.91. Pairwise comparisons of Ct values showed a high correlation of the TruTip/epMotion protocol to the other methods (R2>0.90).
  
  
  CONCLUSIONS
  The automated TruTip/epMotion protocol is a simple and rapid extraction method that reproducibly purifies influenza RNA from respiratory swabs, with comparable efficacy and efficiency to both the easyMAG and QIAcube instruments.
authors.affiliation: Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, United States. sbg03@health.state.ny.us
authors.forename: Sara B
authors.initials: SB
authors.lastname: Griesemer
authors.forename: Rebecca
authors.initials: R
authors.lastname: Holmberg
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Cooney
authors.forename: Nitu
authors.initials: N
authors.lastname: Thakore
authors.forename: Alissa
authors.initials: A
authors.lastname: Gindlesperger
authors.forename: Christopher
authors.initials: C
authors.lastname: Knickerbocker
authors.forename: Darrell P
authors.initials: DP
authors.lastname: Chandler
authors.forename: Kirsten
authors.initials: K
authors.lastname: St George
mesh_terms: D057205:Automation, Laboratory / Q000379:methods*; D006801:Humans; D007251:Influenza, Human / Q000175:diagnosis*; D009975:Orthomyxoviridae / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012137:Respiratory System / Q000821:virology*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
chemical_list: D012367:RNA, Viral
keywords: ABI; Applied Biosystems; Automated extraction; C(t); CDC; CV; Centers for Disease Control and Prevention; EPM; Influenza; NPS; RNA; RT-PCR; Respiratory swabs; SD; SPT/LPT; TruTip; WC-LVD; Wadsworth Center Laboratory of Viral Diseases; coefficients of variation; crossing threshold; epMotion; nasopharyngeal swab; pRhMK; primary rheusus monkey kidney; reverse transcriptase-polymerase chain reaction; small/large pore tips; standard deviation