Novel fast reverse transcriptase PCR assay for molecular detection of human influenza A (H1N1) virus.
ABSTRACT A novel subtype of influenza A pandemic virus has been recently reported by the Center for Disease Control and Prevention (CDC; Atlanta,GA) and WHO in April 2009 .
JOURNAL OF CLINICAL MICROBIOLOGY, May 2010, p. 1985–1986
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 5
Novel Fast Reverse Transcriptase PCR Assay for Molecular
Detection of Human Influenza A (H1N1) Virus?
A novel subtype of influenza A pandemic virus, A(H1N1)v,
was recently reported by the Centers for Disease Control and
Prevention (CDC) (Atlanta, GA) and WHO in April 2009 (1).
Infection most commonly results in a mild respiratory tract
infection, but there are reports of severe cases with mortalities
even in patients without chronic diseases (2). Early detection
of infection is important in programs aiming to minimize the
spread of the disease (3). There are only a few validated lab-
oratory diagnostic methods for the diagnosis of the virus
A(H1N1)v; reverse transcriptase PCR (RT-PCR)-based detec-
tion methods are the fastest of these (4).
This communication describes the development of a fast
protocol for a real-time RT-PCR assay for viral detection
(“fast RT-PCR”). Assay performance was then compared with
the results obtained with the standard CDC real-time protocol.
A retrospective study of 228 previously diagnosed samples was
performed. Samples were randomly selected: 155 samples
provided negative results, and 73 provided positive results
for the new pandemic virus. RNA extraction was performed
from nasopharyngeal or oropharyngeal swabs collected with
a synthetic tip and plastic shaft (Virocult) using QIAmp
virus RNA minikit (Qiagen) and EZ1 virus minikit (Qiagen),
following the manufacturer’s instructions.
In the CDC protocol for real-time PCR amplification and
detection, the InfA primer and probe set is designed for uni-
versal detection of type A influenza viruses, the swInfA primer/
probe set is directed to all swine influenza A viruses, and the
swH1 primer/probe set specifically detects swine H1 influenza.
The fourth primer/probe set targets the human RNase P gene
and serves as an internal control for human nucleic acid (6).
The RT-PCR assays were performed using the AgPath-ID
one-step RT-PCR kit (Applied Biosystems). Briefly, 5 ?l
purified RNA was reverse transcribed and amplified in a
25-?l reaction mixture containing 12.5 ?l of 2? RT-PCR
buffer (Applied Biosystems), 1 ?l of 25? RT-PCR enzyme
mix (Applied Biosystems), 300 nM forward primer, 300 nM
reverse primer, and 75 nM probe (see the CDC protocol for
the primer/probe description ). Fast RT-PCR was per-
formed in a 7500 fast real-time PCR system (Applied Bio-
systems) and analyzed by 7500 software v2.0.1. The thermal
cycling conditions comprised a 10-min RT step at 50°C, a
10-min initial PCR activation step at 95°C, and 45 cycles of
95°C for 3 s and 55°C for 30 s each.
The results for the CDC and fast RT-PCR were in complete
agreement, with all 73 (H1N1)v-positive samples and 155 neg-
ative samples having the same findings with both assays (Table
1). Threshold cycle (CT) means were similar in all 4 sets, with
standard deviation means ranging from 1.1113 to 2.7951. The
fast RT-PCR assay took an average of 67 min to run, nearly an
hour less than the CDC assay, which took 122 min to run.
Overall, the studies herein reveal a significant benefit to the
substitution for the standard RT-PCR method by the fast RT-
PCR method, with a significant reduction in response time,
without any loss of consistency. These are major advantages
during an influenza pandemic, where accurate and rapid diag-
nosis is critical for minimizing laboratory time consumption
(5). The described protocol modification is easily introduced if
using a fast real time thermal cycler and proper enzyme such as
the 7500 fast real-time PCR system and the AgPath-ID one-
step RT-PCR kit, respectively.
TABLE 1. Results of RT-PCR analyses of 228 samples according to the standard or fast RT-PCR protocola
CTmean or SDNo. of samples
aComparison of the standard PCR (CDC protocol) and fast RT-PCR showed the fast RT-PCR to have 100% accuracy.
1. Carr, M. J., R. Gunson, A. Maclean, S. Coughlan, M. Fitzgerald, M. Scully,
B. O’Herlihy, J. Ryan, D. O’Flanagan, J. Connell, W. F. Carman, and W. W.
Hall. 2009. Development of a real-time RT-PCR for the detection of swine-
lineage influenza A (H1N1) virus infections. J. Clin. Virol. 45(3):196–199.
2. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. 2009.
Emergence of a novel swine-origin influenza A (H1N1) virus in humans.
N. Engl. J. Med. 360:2605–2615.
3. Pabbaraju, K., S. Wong, A. A. Wong, G. D. Appleyard, L. Chui, X. L. Pang,
S. K. Yanow, K. Fonseca, B. E. Lee, J. D. Fox, and J. K. Preiksaitis. 2009.
Design and validation of real-time reverse transcription-PCR assays for de-
tection of pandemic (H1N1) 2009 virus. J. Clin. Microbiol. 47:3454–3460.
4. Poon, L. L., K. H. Chan, G. J. Smith, C. S. Leung, Y. Guan, K. Y. Yuen, and
J. S. Peiris. 2009. Molecular detection of a novel human influenza (H1N1) of
pandemic potential by conventional and real-time quantitative RT-PCR as-
says. Clin. Chem. 55:1555–1558.
5. Whiley, D. M., S. Bialasiewicz, C. Bletchly, C. E. Faux, B. Harrower, A. R.
Gould, S. B. Lambert, G. R. Nimmo, M. D. Nissen, and T. P. Sloots. 2009.
Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J. Clin.
6. WHO Collaborating Centre for Influenza, Atlanta, GA. 2009. CDC protocol
of realtime RTPCR for influenza A (H1N1). Centers for Disease Control and
Prevention, Atlanta, GA.
Isa F. Dutra
Bruno F. Bettencourt
Raquel N. Fialho
Ana R. Couto
Marta S. Soares
Margarida R. Santos
Joa ˜o P. Pinheiro
Ja ´come Bruges-Armas*
Servic ¸o Especializado de Epidemiologia e Biologia Molecular
Hospital de Santo Espírito de Angra do Heroísmo
Angra do Heroísmo, Azores, Portugal
?Published ahead of print on 17 March 2010.
1986 LETTERS TO THE EDITORJ. CLIN. MICROBIOL.