title: Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus.
abstract: The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, single-tube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of (50)/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.
authors.affiliation: Division of Virology, Defence Research & Development Establishment, Gwalior, India. paridamm@rediffmail.com
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
authors.forename: Jyoti
authors.initials: J
authors.lastname: Shukla
authors.forename: Shashi
authors.initials: S
authors.lastname: Sharma
authors.forename: Sanna
authors.initials: S
authors.lastname: Ranghia Santhosh
authors.forename: Vasanthapuram
authors.initials: V
authors.lastname: Ravi
authors.forename: Reeta
authors.initials: R
authors.lastname: Mani
authors.forename: Maria
authors.initials: M
authors.lastname: Thomas
authors.forename: Shashi
authors.initials: S
authors.lastname: Khare
authors.forename: Arvind
authors.initials: A
authors.lastname: Rai
authors.forename: Radha
authors.initials: R
authors.lastname: Kant Ratho
authors.forename: Sujit
authors.initials: S
authors.lastname: Pujari
authors.forename: Bijayanti
authors.initials: B
authors.lastname: Mishra
authors.forename: Putcha Venkata
authors.initials: PV
authors.lastname: Lakshmana Rao
authors.forename: Rajagopalan
authors.initials: R
authors.lastname: Vijayaraghavan
mesh_terms: D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral