T Mokhtari-Azad et al: Comparison of Multiplex … Iranian J Publ Health, Vol. 36, No.3, 2007 , pp.1-7
Original Article
Comparison of Multiplex Nested RT-PCR with Virus Isolation for Detection of Influenza Viruses A and B *T Mokhtari-Azad 1, F Rezaie-Khollari 1, AR Nadji 1,V Salimi 1, Z Noroozbabaie 1, M Naseri 1, AR Mirzaie 1, Z Saadatmand 1, MM Gouya 2, AR Esteghamati 2, R Hamkar 1 1
Dept. of Pathobiology, School of Public Health and Institute of Health Research, Medical Sciences/University of Tehran, Iran 2 Center for Diseases Control, Ferdosi Squar, Iranshar Street, No.68, Tehran-1581615117, Iran (Received 17 Feb 2007; accepted 5 Jul 2007)
Abstract Background: Influenza A viruses bearing all known HA and NA subtypes have been isolated from avian hosts: but only the A/H1N1,A/H2N2 and A/H3N2 influenza subtypes have been associated with widespread epidemics in humans until now .While influenza B detected only in humans. Subtype H5 infection in humans occurred for the first time during 1997-1998 in Hong Kong, then China in 2003, Vietnam and Thailand in 2004 and continued to 2005-2006 in Turkey and Egypt. In this study a Multiplex Nested RT-PCR was developed for simultaneous detection of influenza virus A and B in clinical samples. Methods: Throat swab and throat wash was collected between October 2005 and September 2006 from 689 patients with a suspected influenza virus infection. Samples were transported to the National Influenza Center (Tehran University of Medical Science, School of Public Health). Each sample is divided into two parts in order to virus isolation and nested RT-PCR. Results: The RT-PCR detected in 75 cases of influenza A and B, compared to 26 cases by virus isolation and 74 cases are positive by RT-PCR. In total, 50 out of 74 RT-PCR positive specimens were negative by virus isolation. The RTPCR provides a sensitive and specific method for detecting and typing influenza viruses A and B. Conclusion: The RT-PCR provides a sensitive and specific method for detecting and typing influenza viruses A and B. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral confections to be identified with the same respiratory specimen.
Key words: Influenza viruses, RT-PCR, Isolation
Introduction Influenza viruses are enveloped, segmented negative-sense RNA viruses that belong to family orthomyxoviridae (1, 2). In human, three types of influenza, types A, B and C, have been described on the basis of antigenic differences in matrix (M) protein and nucleoprotein (NP) (3, 4). Type influenza viruses are further subtyped on the basis of antigenic differences on the external glycoprotein's, the hemagglutinin (HA) and the neuraminidase (NA) Proteins. To date 15 distinct HA (H1 to H15) and 9 NA (N1 to N9) subtypes have been identified (1, 2, 4, 5). Type A and B influenza viruses can cause a wide spectrum of illness, including lower respiratory tract disease, pneumonia, and even, in the case of type an influenza virus, encephalopathy and encephalitis (1,
3, 4, 6-8). Viruses bearing all known HA and NA subtypes have been isolated from avian hosts, but until recently only viruses of the, H1N1, H2N2 and H3N2 subtypes have been associated with widespread epidemics in humans (6-7). Subtype H5 infection in humans occurred for the first time during 1997-1998 in Hong Kong, then China in 2003, Vietnam and Thailand in 2004 and continued to 20052006 in turkey and Egypt.H5N1 prompted us to highlight the need for a highly sensitive accurate and rapid diagnostic test for the infection (9). Such a test would be important, not only in infection control but also to facilitate early antiviral therapy (1, 9). Conventional diagnostic tools, cell culture, and serologic testing require from 2 d to weeks for results. On the other hand, commercially available rapid
*Corresponding author: Tel: +98 21 88950595, Fax: + 98 21 88962343, E-mail:
[email protected]
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Iranian J Publ Health, Vol. 36, No.3, 2007, pp.1-7
antigen test are rapid and simple, but subtyping of virus is not feasible and sensitivity and specificity is low: thus, they are less useful in making therapeutic and infection control decisions (1, 3-8, 10-12). Molecular diagnosis of influenza by reverse transcription polymerase chain reaction (RT-PCR) provides a sensitive and rapid means for detection and has facilitated the typing and subtyping of virus (3-6, 10, 13). In the so-called "multiplex" format PCR assays have been designed to amplify more than one respiratory viral target in the same PCR test (13). With the strategy to detect and distinguish normally prevalent types of human influenza viruses, we have developed and evaluated an inhouse multiplex reverse transcriptase-PCR assay for influenza A types H1, H3,H5 and for influenza virus B on 689 clinical samples including throat wash and swab and compared it to isolation with cell culture.
Materials and Methods Clinical specimens Throat swab (virocult) and throat wash was collected between October 2005 and September 2006 from 689 patients with a suspected influenza virus infection. Samples were transported to the National influenza center. (Tehran University of Medical Science, school of public health). Virus isolation Virus was isolated using MDCK cells, maintained in Dulbecco's Modified Eagle medium (supplemented with 10% fetal calf serum), 100 Iu/ml penicillin 100 mg/ml streptomycin and 2 m μ l-glutamine. The cells were seeded onto tubes either 24 or 48 h prior to sample inoculation. The samples which were processed as fresh as possible, were inoculated (100 μ L) induplicate on to mono layers of MDCK cells. Trypsin (0.25 ng/ml) was added to the medium for isolation in MDCK cells only. The cultures were maintained in serumfree medium at 34° C for 3-7 d (7). RNA extraction and cDNA synthesis RNA extracted from 150 μ L of samples using the Neucleospin viral RNA (Neucleospin RNA extraction kit) according to the manufactures' in-
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struction. cDNA synthesis was carried out in 30 μ L reaction mixture containing 6μ L of 5X RT Buffer, 2.5 μ L of mixed dntps (2.5mμ each), 1 μ L of m-mulv enzyme (fermentase), 2.5 μ L Random hexamer (Fermentase), 0.5 μ L Ransine (Fermentase) and 17.5 μ L RNA template, and Incubate 22° C for 10 min, 37° C for 30 min and 94° C for 5 min. Primer design The oligonucleotide primers designed to amplify A/H5 placed in HA Region of influenza virus. The G+C contents, melting temperature and lengths of primers were chosen and analyzed by suing oligo 5 primer design software (National Biosciences Inc.) to ensure that they not only met the essential criteria for optimal PCR primer but also could be used together in a multiplex PCR under similar conditions already determined to be effective for detection and subtyping of influenza A and B virus (Table: 1) (14). PCR Each primer pair was used at 5 pmol in primary amplification and 25 pmol in the secondary amplification. For the primary PCR 10 μ L of cDNA was added to 40 μ L of a reaction mixture containing 5 μ L of 10X PCR buffer (fermentase), 1.5 μ L of Mgcl2 (50mm), 2 μ L of dntps Mixed (10mM) and 1.5u of tag polymerase. Amplification with a DNA Engine thermocycler (eppendorf) consisted of 1cycle at 94° C for 2 min, followed by 35 cycles of 94° C for 1 min, 50° C for 1 min, 72° C for 1. min 5 microliters of primary product was then transferred to 45 μ L of the secondary amplification mixture as above, The samples were then incubated for 1 cycle at 94° C for 2 min and then 35 cycles of 94° C 1 min, 60° C min, and 72 °C 1 min. Ampilicons were visualized by ethidium bromide staining following electrophoresis on 1.5% agarose gels (14).
T Mokhtari-Azad et al: Comparison of Multiplex …
Table 1: Properties of primers used for influenza virus multiplex PCR G+C Content (%)
Optimal annealing Temp (°C)
Maximum annealing Temp (°C)
Amplicon size (bp)
Sensitivity (PFU)
52
63
1015
NA²
52
62
883
NA
53
64
900
NA
55
70
351
NA
Melting temp (°C)
40
Sequence(5´→3´)
Gene position
Amplification steps and primer¹
AH ΙA(f)
CAGATGCAGACACA ATATGT
HA
55
AH Ι FΙΙ(r)
AAACCGGCAATGGC TCCAAA
HA
72
50
AH3A(f)
CAGATTGAAGTGAC TAATGC
HA
55
40
AH3DΙΙ(r)
GTTTCTCTGGTACAT TCCGC
HA
62
50
BHA A(f)
GTGACTGGTGTGAT ACCACT
HA
56
50
BHA DΙΙ(r)
TGTTTTCACCCATAT TGGGC
HA
65
45
H5F(f)
ACTCCAATGGGGGCGA TAAAC
HA
50
H52R(r)
TCTGCATTGTAACGACC CATTG
HA
45
ATAGGCTACCATGCG AACAA CTTAGTCCTGTAACC ATCCT
HA
63
45
HA
55
45
Primary
Secondary AH ΙB(f) AH Ι EΙΙ(r) AH3B(f)
AGCAAAGCTTTCAG CAACTG
HA
63
45
AH3CΙΙ(r)
GCTTCCATTTGGAGT GATGC
HA
65
50
BHA B(f)
CATTTTGCAAATCTC AAAGC
HA
61
35
BHA CΙΙ(r)
TGGAGGCAATCTGC TTCACC
HA
68
55
H5R(f)
CAACGGCCTCAAACTG AGTGT
HA
50
H5 inhouse(r)
CAT(A/C)CACCC(C/T)CT CACCATCGGGGAA
HA
55
52
62
944
