Rapid diagnosis of h5n1 avian influenza virus infection by newly developed influenza h5 hemagglutinin gene specific loop mediated isothermal amplification method

Journal of Virological Methods 141 (2007) 173–180 Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method Masaki Imai a , Ai Ninomiya a , Harumi Minekawa b , Tsugunori Notomi b , Toru Ishizaki c , Phan Van Tu d , Nguyen Thi Kim Tien d , Masato Tashiro a , Takato Odagiri a,∗ a Laboratory of Influenza Viruses, Department of Virology 3, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan b Eiken Chemical Company, Ltd., Tochigi 324-0036, Japan c Kyoto Prefectural Institute of Hygienic and Environmental Sciences, Kyoto 612-8369, Japan d Pasteur Institute, Ho Chi Minh City, Vietnam Received 15 June 2006; received in revised form 30 November 2006; accepted December 2006 Available online 10 January 2007 Abstract Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 at 62.5 ◦ C In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI) The sensitivity of the system was 0.1–0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006 This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR By using viral RNAs extracted from avian influenza viruses of H1–H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus © 2006 Elsevier B.V All rights reserved Keywords: H5N1 highly pathogenic avian influenza virus; RT-LAMP; RT-PCR Introduction Influenza A viruses infect humans, pigs, horses, seals and whales, as well as a variety of domestic and wild birds Each known subtype of hemagglutinin (HA, H1–H16) and neuraminidase (N1–N9) of influenza A viruses has been isolated from aquatic birds (Fouchier et al., 2005; Webster et al., 1992) Influenza A virus isolates not generally cause symptomatic infection in birds However, some influenza viruses of the H5 and H7 subtypes become highly virulent in poultry, causing outbreaks of severe systemic disease with high morbidity and mortality (Bosch et al., 1979) ∗ Corresponding author Tel.: +81 42 561 0771; fax: +81 42 561 0812 E-mail address: todagiri@nih.go.jp (T Odagiri) 0166-0934/$ – see front matter © 2006 Elsevier B.V All rights reserved doi:10.1016/j.jviromet.2006.12.004 Since the end of 2003, simultaneous outbreaks of highly pathogenic avian influenza (HPAI) caused by the influenza H5N1 virus occurred in poultry in Asian countries (Li et al., 2004; Lipatov et al., 2004), and outbreaks in both poultry and wild birds are now spreading to European, Middle Eastern and African countries (OIE, 2006) In Japan, outbreaks of HPAIH5N1 occurred in chicken farms and in privately raised pet chickens in early 2004, and the HPAI-H5N1 virus was isolated from chickens and crows living near the affected farms during the outbreaks (Mase et al., 2005) Fortunately, no human cases with influenza-like symptoms have been reported in Japan The poultry outbreaks in Japan were eradicated by the end of April 2004 (Mase et al., 2005) By contrast, in 10 countries in Southeast Asia, China, Middle East and Africa, intermittent outbreaks in poultry continue, and there have been 249 confirmed cases of human infection with 146 fatalities (WHO, 174 M Imai et al / Journal of Virological Methods 141 (2007) 173–180 2006) Some human-to-human transmissions in family clusters have also been reported in Vietnam and Thailand (Tran et al., 2004; Ungchusak et al., 2005) Consequently, improved surveillance of H5N1 virus infection and the development of a simple, rapid and highly sensitive diagnostic tool are high priorities, together with emergency countermeasures for pandemic preparedness Reverse transcriptase (RT)-PCR with subtype H5-specific primers recommended by WHO has been used in diagnostic laboratories worldwide for rapid detection of influenza H5 virus in clinical specimens (WHO, 2005) However, this assay is time-consuming and prone to cross-contamination, as the post-PCR handling steps require evaluation of the amplicon On the other hand, real-time PCR allows for the direct detection of the PCR product during the amplification process without post-PCR processing However, this method requires specialized equipment and reagents, which are expensive when compared with the equipment necessary for RT-PCR, and thus it has not been adopted by many small, front-line laboratories Notomi et al (2000) reported a novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), which employs self-recurring strand-displacement DNA synthesis primed by a specially designed set of target-specific primers LAMP can be completed within 30–60 min, and it is simple when compared with RT-PCR More importantly, LAMP does not require any expensive or specialized equipment, as it can be undertaken in a water bath at an isothermal range from 60 to 65 ◦ C Therefore, LAMP is easy to introduce in frontline or poorly equipped laboratories Recently, this method has been used successfully to detect human pathogenic RNA viruses (Fujino et al., 2005; Hong et al., 2004; Ito et al., 2006; Okafuji et al., 2005; Poon et al., 2005, 2004; Ushio et al., 2005) Consequently, the LAMP method is now a useful diagnostic tool for those pathogens In the present study, an HPAI-H5N1 virus HA-specific RTLAMP system was developed for rapid and sensitive diagnosis of H5N1 avian influenza infection When H5N1 viruses isolated in 2004 and 2006 were used as a reference, the RT-LAMP system detected the viral HA gene with approximately 10–100-fold higher sensitivity than regular one-step RT-PCR These findings suggest that RT-LAMP is a useful diagnostic tool for H5N1 virus infection Materials and methods 2.1 Viruses and RNA isolation The reference strains of avian influenza and human influenza viruses used in this study were from the repository in the Department of Virology III of the National Institute of Infectious Diseases A/duck/Hokkaido/55/96 (H1N1), A/Hong Kong/213/2003 (H5N1), A/chicken/Yamaguchi/7/2004 (H5N1) and A/chicken/Anhui-choahu/85/2004 (H5N1) are reference viruses that have been described previously (Hulse-Post et al., 2005; Mase et al., 2005; Okazaki et al., 2000; Peiris et al., 2004) Human pathogenic respiratory viruses were provided by Dr M Noda from the National Institute of Infectious Diseases The H5 influenza viruses were titrated by plaque assays in Madin-Darby canine kidney (MDCK) cells using a standard protocol (Salomon et al., 2006) All experiments with HAPI-H5 viruses were performed in a biosafety level laboratory at the National Institute of Infectious Diseases RNA was isolated directly from 140 ␮l samples using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions RNA was stored at −70 ◦ C until used 2.2 Design of LAMP primers Influenza H5HA gene-specific RT-LAMP primers were designed using the LAMP primer design support software program (Net Laboratory, Kanagawa, Japan) Based on the sequence information obtained from the Influenza Sequence Database (http://www.flu.lanl.gov), a primer set comprising two outer, two inner and two loop primers recognizing eight distinct regions on the target sequence were chosen Because of the large sequence variation of the H5 genes, the primers were primarily targeted to HAPI-H5N1 viruses The locations, names and sequences of the primers are given in Fig 2.3 RT-LAMP RT-LAMP was carried out using a Loopamp RNA Amplification kit (Eiken Chemical, Tochigi, Japan) Five microliters of extracted RNA was added to 20 ␮l of master mix containing l× reaction mix, ␮l of enzyme mix, ␮l of fluorescence detection reagent (Eiken Chemical), 1.6 ␮M FI primer and BI primer, 0.2 ␮M outer primers (F3 primer and B3 primer) and 0.8 ␮M loop primers (LPF primer and LPB primer) The mixture was incubated using a Loopamp real-time turbidimeter (LA-320C; Eiken Chemical) for 35 at 62.5 ◦ C and then for at 80 ◦ C to terminate the reaction 2.4 Detection of RT-LAMP products To prevent cross-contamination, two detection methods were used: real-time turbidity detection and visual fluorescent detection Changes in absorbance at 650 nm were measured for real-time turbidity detection with a Loopamp real-time turbidimeter (LA-320C) A cutoff value was determined based on the mean of the negative detection control optical density (OD) Specimens with an OD of less than 0.1 were determined to be negative for H5 influenza viral RNA After RT-LAMP, fluorescence was detected directly in the reaction tubes using an ultraviolet (UV) transilluminator Fluorescence detection occurs as follows: (1) calcein in the Loopamp Fluorescent Detection Reagent is initially combined with manganese ions to achieve a quenching effect; (2) amplification generates a by-product, pyrophosphate ions, which binds with and removes manganese ions from calcein, resulting in fluorescence; (3) fluorescence is intensified further as calcein combines with magnesium ions; and (4) the presence of fluorescence indicates the presence of the target gene M Imai et al / Journal of Virological Methods 141 (2007) 173–180 175 Fig (A) Positions of RT-LAMP primers on the H5 HA gene The nucleotide sequence of the A/Pigeon/Hong Kong/SF215/2001(H5N1) HA gene (GenBank Accession number AF509023) is shown The forward loop primer (LF24) and reverse loop primer (LB1) are composed of sequences complementary to the sequences between the F1cm5 and F2m3 regions and the B1cm1 and B2m1 regions, respectively Locations of primer binding sequences are underlined The box indicates the Dra III restriction site, and the numbers indicate nucleotide positions (B) Names and sequences of RT-LAMP primers 2.5 RT-PCR Samples were amplified using a one-step RNA PCR kit (Takara, Shiga, Japan) and the influenza virus H5 HA-specific primers designed previously by Imai et al (1999), with slight modifications Primer sequences were 5′ -CATACCCAACAATAAAGAGG (H5 515f) and 5′ -GTGTTCATTTTGTTAATGAT (H5 1220r) RT-PCR was performed in 25 ␮l of reaction mixture consisting of l× one-step RNA PCR buffer, 250 ␮M (each) deoxynucleoside triphosphate, mM MgCl2 , 20 U of RNAase inhibitor, 2.5 U of AMV RTase XL, 2.5 U of AMV-Optimized Taq, 0.4 ␮M forward and reverse primers and ␮l of RNA Reaction conditions were set at 50 ◦ C for 45 and 94 ◦ C for min, followed by 40 cycles of 94 ◦ C for 30 s, 50 ◦ C for 30 s and 70 ◦ C for 45 s A total of 10 ␮l of PCR products were then analyzed by 1.5% agarose gel electrophoresis in tris-buffer, and target bands were visualized by staining with ethidium bromide 2.6 Specimens A total of 53 throat swabs were collected from hospitalized patients with clinically suspected H5N1 influenza from December 2004 through January 2005, during outbreaks of H5N1 human influenza in southern Vietnam Specimens were obtained from 44 patients ranging in age from years to 76 years Samples were received by the National Institute of Infectious Diseases from the Pasteur Institute in Ho Chi Minh City, Vietnam, for diagnosis of HPAI-H5N1 During outbreaks of H5N1 influenza in the Kyoto Prefecture in Japan (Mase et al., 2005), avian throat swab specimens were collected from a total of 30 dead crows found within a 30-km radius of the affected chicken farm in Kyoto RNA was extracted in a biosafety level laboratory at Kyoto Prefectural Institute of Hygienic and Environmental Sciences and was then sent to the National Institute of Infectious Diseases for nucleic acid amplification Results 3.1 Sensitivity ofH5-RT-LAMP assay To evaluate the sensitivity of the LAMP assay with the H5 primer set, the detection limit of the assay was determined by testing serial 10-fold dilutions of H5N1 influenza A/Vietnam/JP 1203/2004 RNA of a known plaque-forming units (PFU) dose Reactions were conducted at 62.5 ◦ C for 35 min, followed by heat inactivation at 80 ◦ C for Detection of amplification products was carried out by real-time monitoring of turbidity Kinetic analysis of turbidity revealed that the H5-RT-LAMP assay was able to detect H5 virus at a level of 0.1 PFU per tube (Fig 2A) The sensitivity of the assay was also confirmed by observing the fluorescence of the solution under a UV light source, as described in Section As shown in Fig 2B, clear fluorescence signals were seen at concentrations ranging from 1000 to 0.1 PFU per tube There were no differences in sensitivity between the real-time turbidity and visual fluorescence detections for the LAMP assay When the same RNA template was subjected to one-step RT-PCR using the H5-specific primers described in Section 2, the detection limit of the system was PFU per tube (Fig 2C), confirming that the sensitivity of H5-RT-LAMP assay with a representative virus belonging to clade (WHO Global Influenza Program Surveillance 176 M Imai et al / Journal of Virological Methods 141 (2007) 173–180 The sensitivity of H5-RT-LAMP relative to that of RT-PCR was investigated by testing the same RNA preparations by onestep RT-PCR The detection limit of the RT-PCR assay for the two 2004 and one 2006 isolates was PFU per tube, which is 10–100-fold lower than that of the H5-RT-LAMP assay (Fig 3A and B) In contrast, RT-PCR detected Tern/61 and Duck/79 at PFU per tube, indicating that the detection of older H5 viruses by RT-PCR was 2–3 log units more sensitive than that by H5RT-LAMP (Fig 3j and i) 3.2 Specificity ofH5-RT-LAMP assay Fig Comparative sensitivity of RT-LAMP and RT-PCR methods H5-RTLAMP and RT-PCR were performed using A/Vietnam/JP1203/2004 (H5N1) viral RNA at concentrations ranging from 1000 to 0.001 PFU per tube (A and B) Detection limit of H5-RT-LAMP LAMP products were detected by a realtime turbidity assay using an LA-320c (A) and a fluorescence assay using a UV lamp (B) (C) Detection limit for one-step RT-PCR using the same RNA extracts used in the H5-RT-LAMP assay PCR products were observed on a 1.5% agarose gel stained with ethidium bromide Network, 2005) is approximately 10-fold higher than that of RT-PCR The detection limits of H5 influenza viruses obtained at various geographical locations and times, in particular the recent isolates belonging to clade (WHO Global Influenza Program Surveillance Network, 2005), were then determined to asses whether the H5-RT-LAMP assay is able to detect a variety of H5 influenza viruses at constant sensitivity The sensitivity determined by real-time monitoring of turbidity of clade viruses isolated from 2004 to 2006, A/chicken/Yamaguchi/7/2004 (H5N1), A/chicken/Anhuichoahu/85/2004 (H5N1) and A/Indonesia/JP283/2006 (H5N1), was within a range of 0.1–0.01 PFU per tube The sensitivity for older H5 avian viruses, A/tern/South Africa/61 (H5N3) (Tern/61) and A/duck/Hong Kong/698/79 (H5N3) (Duck/79), however, was 100 and 1000 PFU per tube, respectively (Fig 3) Thus, RT-LAMP was highly sensitive for the detection of recent H5N1 viruses belonging to both clade 1, which includes the majority of isolates from Vietnam in 2004, and clade 2, which includes isolates from China, Indonesia and Japan But, the sensitivity of RT-LAMP was low for the older H5 viruses (Tern/61 and Duck/79) Similarly, RT-LAMP was high sensitive for previous H5N1 human isolates, A/Hong Kong/156/97 (H5N1) and A/Hong Kong/213/2003 (H5N1), as well as A/Vietnam/JP 1203/2004 (H5N1) (data not shown) To determine whether the present H5 primer set was specific for H5 influenza virus amplification, the primer set was evaluated with representative avian influenza viruses and human pathogenic respiratory viruses LAMP reactions were examined by real-time turbidity detection and by visual fluorescence detection The results are summarized in Table The H5-specific primer set used for RT-LAMP amplified only the H5 influenza viruses isolated from 1961 to 2006, and nonspecific amplification products were not observed Similarly, the LAMP assay with visual fluorescence detection resulted in clear signals for the H5 influenza viruses, and false positive signals were not observed with other virus subtypes Confirmation of the specificities of the LAMP-amplified products was also conducted by agarose gel electrophoresis after ethidium bromide staining and digestion of the products with restriction enzyme Dra III Fig shows a representative positive pattern using A/Vietnam/JP 1203/2004 (H5N1) after LAMP amplification; a visibly distinct ladder-like pattern was observed on agarose gel electrophoresis (Fig 4, lane 1) and four Dra III-digested DNA fragments of 119, 90, 130 and 83 bp were generated, which corresponds to the predicted sizes (Fig 4, lane 2) 3.3 Evaluation of H5-RT-LAMP assay using clinical samples and bird samples The ability of the H5-RT-LAMP assay to detect H5 viruses in clinical samples was assessed by testing 53 throat swab samples collected from suspected patients in an HPAI-H5N1 endemic area in southern Vietnam All samples were tested by both RTLAMP and one-step RT-PCR Of the 53 samples evaluated, H5-RT-LAMP gave 19 positive cases, whereas RT-PCR gave 17 positive cases PCR products were not detected in samples from which no H5 viral RNA could be detected by H5-RT-LAMP (Table 2) The data indicate that two H5-RT-LAMP-positive specimens were missed by RT-PCR H5N1 viruses were isolated from of the 17 positive samples by both methods For the other 11 samples, we performed blind passage in MDCK cells, but H5N1 viruses could not be recovered This result suggests that these samples contained fewer viruses or that they lacked viral infectivity LAMP products were examined by agarose gel electrophoresis and digested with Dra III to confirm whether LAMP-positive reactions in the clinical samples were the consequence of H5 virus detection All positive cases exhibited a positive electrophoresis pattern as shown in Fig (data M Imai et al / Journal of Virological Methods 141 (2007) 173–180 177 Fig Sensitivity of RT-LAMP and RT-PCR for the detection of different isolates of the H5 influenza virus Serial 10-fold dilutions of: (a and f) A/chicken/Yamaguchi/7/2004 (H5N1); (b and g) A/chicken/Anhui-choahu/85/2004 (H5N1); (c and h) A/Indonesia/JP283/2006 (H5N1); (d and i) A/duck/Hong Kong/698/79 (H5N3); and (e and j) A/tern/South Africa/61 (H5N3) RNAs were amplified by H5-RT-LAMP (A) or one-step RT-PCR (B) Viral RNA concentrations ranging from 1000 to 0.001 PFU per tube were tested LAMP products were detected by a real-time turbidity assay using an LA-320c not shown), suggesting that the specimen surely contained H5 virus During the H5N1 outbreaks in chicken farms in the Kyoto prefecture in 2004, a number of dead crows were found near the affected farms (Mase et al., 2005) The throat swabs were examined by H5-RT-LAMP to investigate whether H5N1 viruses could be detected in these crows (Table 3) The samples were also subjected to RT-PCR Of the 30 samples tested, 26 samples were negative by both methods Two samples were found to be H5-positive by both methods and H5N1 virus was iso- lated from these samples Another two samples were found to be positive by H5-RT-LAMP, but not by RT-PCR, although H5N1 virus was not recovered from these samples These H5positive reactants were verified to be positive by digestion with Dra III Discussion In the present study, a rapid and highly sensitive diagnostic system based on RT-LAMP technology was developed to 178 M Imai et al / Journal of Virological Methods 141 (2007) 173–180 Table Virus strains used for validation and specificity of H5-RT-LAMP Source and Accession numbersa Isolate A/duck/Hokkaido/55/96 (H1N1) A/New Caledonia/20/99 (H1N1) A/duck/Hong Kong/278/78 (H2N9) A/duck/Ukraine/1/63 (H3N8) A/Panama/2007/99 (H3N2) A/duck/Hong Kong/365/78 (H4N6) A/tern/South Africa/61 (H5N3) A/duck/Hong Kong/698/79 (H5N3) A/Hong Kong/156/97 (H5N1) A/Hong Kong/213/2003 (H5N1) A/Vietnam/JP1203/2004 (H5N1)b A/chicken/Yamaguchi/7/2004 (H5N1) A/chicken/Anhui-choahu/85/2004 (H5N1) A/Indonesia/JP283/2006 (H5N1)b A/shearwater/Australia/1/72 (H6N5) A/duck/Hong Kong/293/78 (H7N2) A/turkey/Ontario/6118/68 (H8N4) A/duck/Hong Kong/702/79 (H9N5) A/chick/Germany/N/49 (H10N7) A/duck/England/56 (H11N6) A/duck/Alberta/60/76 (H12N6) A/gull/Maryland/704/77 (H13N6) A/mallard/Gurjev/263/82 (H14N5) A/duck/Australia/341/83 (H15N8) B/Shanghai/361/2002 B/Shandong/7/97 Respiratory Syncytial virus Rhinovirus Adenovirus Parainfluenza virus Human metapneumovirus RT-LAMP A AY289929 CY005546 CY006038 DQ487340 CY006027 U20460 AF082039 AF046088 B (AB212054) ISDN38687 C (AB166862) D ISDN133316 D90303 CY006029 D90304 AY206672 M21647 D90306 D90307 D90308 M35997 L43916 ISDN38226 AF299384 E E E E E Turbidity Fluorescence − − − − − − + + + + + + + + − − − − − − − − − − − − − − − − − − − − − − − + + + + + + + + − − − − − − − − − − − − − − − − − a A, viruses provided by Dr H Kida B, viruses provided by Dr W Lim C, viruses provided by Dr S Yamaguchi D, viruses provided by Dr M Peiris E, viruses provided by Dr M Noda b A/Vietnam/JP1203/2004(H5Nl) and A/Indonesia/JP283/2006 (H5N1) are identical to A/Vietnam/1203/2004 (H5N1) and A/Indonesia/283H/2006 (H5N1), respectively detect the HPAI-H5N1 virus Current Asian HPAI-H5N1 viruses are classified into two clades based on phylogenetic analyses of the HA genes Vietnamese and Thailand H5N1 isolates in 2004 belong to clade 1, and Indonesian, Chinese and Japanese H5N1 isolates belong to clade (WHO Global Influenza Program Surveillance Network, 2005) The H5-RT-LAMP assay detected both clades with a detection limit of 0.1–0.01 PFU (Figs and 3) When the sensitivity of H5-RT-LAMP was compared to that of standard one-step RT-PCR in an HAPI-H5N1 virus dilution series, H5-RT-LAMP was approximately 10–100- fold more sensitive than RT-PCR The greater sensitivity of H5-RT-LAMP was also reflected in H5 virus detection in the throat swabs collected from humans in Vietnam and from dead crows found near an affected chicken farm in Japan; some positive samples detected by H5-RT-LAMP were missed by RT-PCR (Tables and 3) Furthermore, validation of the specificity of H5-RT-LAMP using 15 HA subtype RNAs and clinical samples revealed that there was no cross-reactivity with other HA subtype RNAs or host-derived RNA These findings suggest that the system possesses reliable specificity in addition to high sensitivity Table Comparison of RT-LAMP and RT-PCR for detection of H5 influenza virus in throat swabs collected from patients Table Comparison of RT-LAMP and RT-PCR for detection of H5 influenza virus in swabs collected from crows RT-LAMP result (no of samples) RT-LAMP result (no of samples) No of samples with the indicated result by RT-PCR Positive Negative Positive (19) Negative (34) 17 34 Total (53) 17 36 No of samples with the indicated result by RT-PCR Positive Negative Positive (4) Negative (26) 2 26 Total (30) 28 M Imai et al / Journal of Virological Methods 141 (2007) 173–180 Fig Restriction enzyme analysis of RT-LAMP products LAMP products derived from A/Vietnam/JP 1203/2004 (H5N1) were used for Dra III restriction analysis followed by agarose gel electrophoresis and ethidium bromide staining Lane M, DNA size markers; lane 1, RT-LAMP products from A/Vietnam/JP1203/2004 (H5N1) virus; lane 2, RT-LAMP products from A/Vietnam/JP 1203/2004 (H5N1) virus digested with Dra III In the development of the H5-RT-LAMP assay, several RTLAMP primer sets were evaluated Each primer set was designed after analysis of the sequence alignment of HPAI-H5N1 viruses isolated in China and Hong Kong from 1996 to 2003 All primer sets were based on these conserved regions of the HPAI-H5N1 virus HA gene However, the other primer sets evaluated were able to detect only some of the HPAI-H5N1 virus isolates (data not shown) The H5-RT-LAMP primer set presented here demonstrated highly effective detection of the recent HPAI-H5N1 viruses belonging to both clade and clade (Figs and 3) Sequence mismatches between primers and their target genes influence the sensitivity of the LAMP method One or zero nucleotide mismatches were identified within primer binding sites for the H5-RT-LAMP assay with the recent HPAI-H5N1 virus isolates examined In contrast, and 15 mismatches were observed with Duck/79 and Tern/61 viruses, respectively This genetic diversity at primer binding sites was reflected in the findings that the H5-RT-LAMP assay detected recent HPAI-H5N1 isolates with high sensitivity but it could not efficiently detect older H5 isolates (Duck/79 and Tern/61) Low sensitivity to the older H5 viruses (Duck/79 and Tern/61) can be attributable to the following reasons: (1) H5-RT-LAMP requires six primers, which recognize eight distinct regions on the target H5 HA gene, so that the range of selected regions for the primer sequence is limited; and (2) the sensitivity of RT-LAMP is influenced greatly 179 by sequence diversity among H5 influenza viruses Therefore, it is impossible theoretically to select all six primers from the conserved regions of the HA gene This is one of the disadvantages of the H5-RT-LAMP system Therefore, the primer sequences for RT-LAMP need to be updated at appropriate intervals for new outbreaks in order to maintain the uniform sensitivity on HPAI-H5N1 diagnosis The LAMP assay offers practical advantages when compared with conventional PCR H5-RT-LAMP requires an isothermal reaction performed at 62.5 ◦ C, which eliminates the need for a thermal cycler Moreover, the reaction time is only 35 min, and the diagnostic results can be obtained within h of obtaining the specimen Consequently, H5-RT-LAMP is beneficial for the routine diagnosis of HPAI-H5N1 infection Another advantage of the LAMP assay is that real-time monitoring of the reaction is possible (Mori et al., 2004) The real-time technique increases the speed of the assay and reduces the risk of carry-over contamination in the post-PCR process However, many small, front-line laboratories may not have access to expensive real-time LAMP detection equipment Alternatively, the LAMP reaction can be visualized by simply placing a reaction tube directly on a UV transilluminator, when the fluorescence dye reagent is added into the reaction mixture The data confirmed that the visual florescence detection assay was compatible with the real-time turbidity detection assay (Fig 2), indicating that this detection system is useful for poorly equipped laboratories in developing countries Viral isolation from tissue culture is highly sensitive when clinical specimens are transported under appropriate conditions and stored at suitable temperatures In an analysis of patient samples from Vietnam, tissue culture showed high false-negative rates The reduced percentage of positive specimens via tissue culture may be due to viral inactivation caused by improper specimen collection, storage and/or transport Indeed, we were not able to isolate H5 influenza viruses from some clinical samples that contained high levels of H5 viral RNA, as judged by band signal intensity after PCR amplification To improve the quality of clinical specimens, health care providers must be instructed on the appropriate collection, storage and transportation of patient specimens In summary, H5-RT-LAMP was significantly more rapid and more sensitive than RT-PCR Obtaining results within h is important for rapid diagnostic testing, and thus the present method provides a screening tool for differentiating HAPIH5N1 infection from the other respiratory diseases that occur throughout Asia Since 2006, human HPAI-H5N1 cases have been recognized in Azerbaijan, Djibouti, Egypt, Iraq and Turkey (WHO, 2006) and the ability of H5-RT-LAMP to detect HPAIH5N1 viruses from these countries is under study Acknowledgements We thank Drs H Kida (Hokkaido University, Japan), W Lim (University of Hong Kong, Hong Kong), S Yamaguchi (National Institute of Animal Health, Japan), M Peiris (University of Hong Kong, Hong Kong) and M Noda (National Institute of Infectious Diseases, Japan) for providing viruses 180 M Imai et al / Journal of Virological Methods 141 (2007) 173–180 and Dr N.T Long (Pasteur Institute, Vietnam) for collection of clinical samples These studies were supported by a Grant from the Regulatory Science Project of the Ministry of Health, Labor and Welfare References Bosch, F.X., Orlich, M., Klenk, H.D., Rott, R., 1979 The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses Virology 95, 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Virus in Specimens from Humans, available at www.who.int/csr/disease/avian influenza/country/cases table 2006 06 06/ en/i ndex.html World Health Organization (WHO), 2006 Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO, available at www.who.int/csr/disease/avian influenza/country/cases table 2006 09 25/ en/index.html  ... pathogens In the present study, an HPAI -H5N1 virus HA-specific RTLAMP system was developed for rapid and sensitive diagnosis of H5N1 avian influenza infection When H5N1 viruses isolated in 2004 and 2006... A/Vietnam/JP1203/2004(H5Nl) and A/Indonesia/JP283/2006 (H5N1) are identical to A/Vietnam/1203/2004 (H5N1) and A/Indonesia/283H/2006 (H5N1) , respectively detect the HPAI -H5N1 virus Current Asian HPAI -H5N1 viruses... Africa/61 (H5N3) A/duck/Hong Kong/698/79 (H5N3) A/Hong Kong/156/97 (H5N1) A/Hong Kong/213/2003 (H5N1) A/Vietnam/JP1203/2004 (H5N1) b A/chicken/Yamaguchi/7/2004 (H5N1) A/chicken/Anhui-choahu/85/2004 (H5N1)