Loop-mediated isothermal amplification for influenza A (H5N1) virus

Abstract

We describe a 1-step reverse-transcription loop-mediated isothermal amplification assay for detection of highly pathogenic avian influenza A (H5N1) viruses. The assay was tested by using a panel of highly pathogenic H5N1 subtypes isolated over the past 10 years and clinical specimens. The assay produced negative results for all non-H5N1 subtypes.

Figure

Detection of influenza (H5) virus by loop-mediated isothermal amplification (LAMP). A) Serially diluted RNA from A/Vietnam/1203/2004 was tested by the reverse transcription (RT)–LAMP (upper panel) and RT-PCR (lower panel) assays. The viral titers used in these reactions are indicated. Viral RNA was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. For a typical 25-μL reaction, 2 μL of sample was mixed with 2× in-house reaction buffer (40 mmol/L Tris-HCl, pH 8.8; 20 mmol/L KCl; 16 mmol/L MgSO₄; 20 mmol/L [NH₄]₂SO₄; 0.2% Tween 20 [v/v]; 1.6 mol/L betaine; 2.8 mmol/L each dNTP), 50 U Bst DNA polymerase (New England Biolabs, Ipswitch, MA, USA), 8 U avian myeloblastosis virus reverse transcriptase (Invitrogen, Gaithersburg, MD, USA), 40 pmol/L primers FIP and BIP, 20 pmol/L primers LPF and LPR, and 5 pmol/L primers F3 and B3. Reaction mixtures were incubated at 60ºC for 120 min, and the turbidity of these reactions was examined by use of a turbidity meter (LA-200, Treamecs; Kyoto, Japan) in real time. The turbidities of these reactions 5–20 min after incubation were taken as the baseline. The threshold value for a positive reaction was set to be 10× above the standard deviation of the baseline. For the H5-specific RT-PCR assay, primers H5-1 (5′-GCCATTCCACAACATACACCC-3′) and H5-3 (5′-CTCCCCTGCTCATTGCTATG-3′) were used according to the protocol optimized by the World Health Organization H5 Reference Laboratory Network (7). Positive (219 bp) and nonspecific products from the PCR reaction are highlighted by the arrow and arrowhead, respectively. B) Detection of H5 virus in postmortem lung tissues from a patient with influenza (H5). Signals from the tested samples, positive control, and water control are indicated. C) Direct detection of influenza (H5) viruses from culture supernatants. Heat-treated supernatant from cells infected with A/Vietnam/1203/2004 were serially diluted and directly used as input in the LAMP assay. The plaque-forming units (pfu) of influenza (H5) virus in these reactions are shown.