BACKGROUND
since february  <dig> a novel avian influenza a  virus has emerged in china, resulting in human infections  <cit> .
as of  <dig> sept  <dig>  the chinese cdc reported  <dig> laboratory-confirmed human cases  <cit> .
according to the etiology and gene traceable results, h7n <dig> avian flu virus is a new recombinant virus .
human infection with avian influenza a  virus usually results in an influenza-like illness  with symptoms such as fever, cough with little to no sputum production, accompanied by headache, muscular soreness, and general malaise  <cit> .
most patients presented with rapidly progressing severe lower respiratory tract infections.
considering that the novel avian h7n <dig> is characteristic of mammalian adapted, scientific community widespreadly concerns that the emerging reassorted virus could cause a new influenza pandemic  <cit> .
several newly published researches indicated that the h7n <dig> virus exhibited human-type receptor-binding ability and could replicate in mammals .
h7n <dig> virus could invade epithelial cells in human lower respiratory tract and pneumonocytes  <cit> .
these biological characterizations of h7n <dig> virus increase the pandemic risk especially that the virus acquires the ability of transmitting readily among humans, and the lack of pre-existing immunity to virus of this subtype among humans  <cit> .
therefore, the risk of a pandemic caused by avian h7n <dig> virus requires rapid detection methods.
currently, the available detection methods for h7n <dig> virus include virus isolation and real-time rt-pcr assay  <cit> .
the serological detection and real-time pcr were recommended to detect avian influenza a  in  <dig> by world health organizatin   <cit> .
considering the requirement for specific equipment and trained operators of real-time rt-pcr assay, there is great demand for simple, rapid and sensitive diagnosis method for early detection of h7n <dig> to provide timely treatment and disease control.
the loop-mediated isothermal amplification  method, first described in  <dig>  has been proved to be a rapid, accurate, and cost-effective diagnostic method for infectious diseases .
previously, the lamp methods have been applied for detection of different subtypes of influenza viruses, including avian h <dig>  h <dig>  h <dig>  h <dig>  the  <dig> h1n <dig> influenza virus, seasons type a or b influenza virus, and h <dig> swine influenza virus .
all these assays showed high specificity, efficiency, and sensitivity that were similar to or even higher than real-time pcr assays.
these studies exhibited the potential of lamp as a routine diagnostic method for influenza infections.
in this study, a h7n9-specific rt-lamp assay targeting the ha or na gene of avian influenza a  virus was developed and evaluated with clinical throat swab and avian samples.
the results demonstrated that the rt-lamp assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza a  virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.
RESULTS
the primer sets of h <dig> or n <dig> specific rt-lamp comprise two outer primers , two inner primers , and one loop primer lf that recognize seven distinct regions on the target sequence of ha or na gene.
considering that the genomic stability of the emerging reassorted h7n <dig> was still unclear, the target sequences of the primers were optimized to avoid covering the hot spot in ha or na gene, such as receptor binding domain or membrane fusion loop in ha gene.
after evaluation with different dilutions of h7n <dig> viral rna, the primer set  was selected.table  <dig>
rt-lamp primer sets designed for detection of h7n <dig> virus
primer
position
a
length
sequence
h7fipb
ah7n <dig> strain a/anhui/1/ <dig> .
bfip and bip primer are long primers containing two separate recognition sequences with a tttt linker .
the detection limits of the h <dig> and n <dig> specific rt-lamp assay were both  <dig>  pfu per reaction , which was 10-fold-higher sensitive than that of real-time rt-pcr assay recommended by who .
furthermore, the cross-reactivity tests with seven respiratory viruses also revealed the high specificity of the h7/n <dig> rt-lamp assay.figure  <dig>
sensitivity and specificity of the h7/n <dig> -specific rt-lamp assay.
amplification curves of the h7/n9-special rt-lamp were performed with 10-fold serial dilutions of viral rna .
specificities of h <dig> and n <dig> rt-lamp were tested by using direct visual detection of rt-lamp with rna extracted from viral culture of h1n <dig>  h3n <dig>  h5n <dig>  piv <dig>  h9n <dig>  rsv, hadv- <dig> and h7n <dig>   detection limit of h7-specific rt-lamp was  <dig>  pfu per reaction.
the color changes under natural or uv light were only observed in the tube of h7n <dig> viral rna by using h7-specific rt-lamp assay.
detection limit of n9-specific rt-lamp was  <dig>  pfu per reaction.
the color changes were observed for n9-specific rt-lamp reaction with h7n <dig> viral rna.
no color change was seen in rt-lamp with other subtype of influenza viral rna and other three respiratory viruses under natural or uv light.
comparison of detection of h7n <dig> virus by using rt-lamp and rrt-pcr assay
strain
virus titer
rrt-pcr
rrt-pcr
rt-lamp
rt-lamp
and, not detectable.
discussion
the rt-lamp assay could finish within 45 minutes.
by using fluorescent detection reagent, the positive amplification could be observed by color change.
as shown in the figure 1b and d, the color of positive reaction  change from orange to green under natural light, while the positive reaction also produced bright green fluorescence under uv light.
for negative sample such as h1n <dig>  h3n <dig>  h5n <dig>  piv <dig>  h9n <dig>  rsv and hadv- <dig> virus, the color of reaction mixture did not change either under normal light or under uv light.
the h7/n9-specific rt-lamp assay was further evaluated using clinical samples and chicken samples compared with rrt-pcr assay.
the rt-lamp results showed that  <dig> samples were tested to be positive and the other  <dig> samples were negative for h7n <dig>  respectively, which could be confirmed by rrt-pcr assay.
no false positive and false negative were observed.
by using these  <dig> samples, 100% diagnostic sensitivities were achieved with h <dig> or n <dig> specific rt-lamp assay compared with rrt-pcr assays, which showed that rt-lamp results were consistent with that of rrt-pcr.
the lamp method aimed to detect  <dig> avian influenza h7n <dig> showed reliable results in the detection of avian and human clinical samples.
previously, two rt-lamp systems had been developed for detection of avian influenza subtype h <dig> virus  <cit> .
currently, some other rt-lamp assays for detection of h7n <dig> virus were also developed .
these assays showed excellent sensitivities and specificities.
our h7/n9-specific rt-lamp assays, as well as those published rt-lamp methods, were very useful for the early detection of suspected h7n <dig> infection, and could contribute to the prevention and control of h7n <dig> epidemics and potential pandemic in china and other asian countries.
CONCLUSIONS
the current study described a rapid, accurate and feasible h7/n <dig> specific rt-lamp assay with perfect sensitivities and high specificities.
it was simple and easy to perform.
this assay have the potential to provide useful tools for detection of novel h7n <dig> virus especially in resource-limited setting, which will play important roles in the prevention and control of spread of novel h7n <dig> infection.

 since february a novel avian influenza a virus has emerged in china, resulting in human infections. as of sept the chinese cdc reported laboratory-confirmed human cases. according to the etiology and gene traceable results, h7n avian flu virus is a new recombinant virus. human infection with avian influenza a virus usually results in an influenza-like illness with symptoms such as fever, cough with little to no sputum production, accompanied by headache, muscular soreness, and general malaise. most patients presented with rapidly progressing severe lower respiratory tract infections. considering that the novel avian h7n is characteristic of mammalian adapted, scientific community widespreadly concerns that the emerging reassorted virus could cause a new influenza pandemic. several newly published researches indicated that the h7n virus exhibited human-type receptor-binding ability and could replicate in mammals. h7n virus could invade epithelial cells in human lower respiratory tract and pneumonocytes. these biological characterizations of h7n virus increase the pandemic risk especially that the virus acquires the ability of transmitting readily among humans, and the lack of pre-existing immunity to virus of this subtype among humans. therefore, the risk of a pandemic caused by avian h7n virus requires rapid detection methods. currently, the available detection methods for h7n virus include virus isolation and real-time rt-pcr assay. the serological detection and real-time pcr were recommended to detect avian influenza a in by world health organizatin. considering the requirement for specific equipment and trained operators of real-time rt-pcr assay, there is great demand for simple, rapid and sensitive diagnosis method for early detection of h7n to provide timely treatment and disease control. the loop-mediated isothermal amplification method, first described in has been proved to be a rapid, accurate, and cost-effective diagnostic method for infectious diseases. previously, the lamp methods have been applied for detection of different subtypes of influenza viruses, including avian h h h h the h1n influenza virus, seasons type a or b influenza virus, and h swine influenza virus. all these assays showed high specificity, efficiency, and sensitivity that were similar to or even higher than real-time pcr assays. these studies exhibited the potential of lamp as a routine diagnostic method for influenza infections. in this study, a h7n9-specific rt-lamp assay targeting the ha or na gene of avian influenza a virus was developed and evaluated with clinical throat swab and avian samples. the results demonstrated that the rt-lamp assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza a virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.  the primer sets of h or n specific rt-lamp comprise two outer primers, two inner primers, and one loop primer lf that recognize seven distinct regions on the target sequence of ha or na gene. considering that the genomic stability of the emerging reassorted h7n was still unclear, the target sequences of the primers were optimized to avoid covering the hot spot in ha or na gene, such as receptor binding domain or membrane fusion loop in ha gene. after evaluation with different dilutions of h7n viral rna, the primer set was selected.table rt-lamp primer sets designed for detection of h7n virus primer position a length sequence h7fipb ah7n strain a/anhui/1/. bfip and bip primer are long primers containing two separate recognition sequences with a tttt linker. the detection limits of the h and n specific rt-lamp assay were both pfu per reaction, which was 10-fold-higher sensitive than that of real-time rt-pcr assay recommended by who. furthermore, the cross-reactivity tests with seven respiratory viruses also revealed the high specificity of the h7/n rt-lamp assay.figure sensitivity and specificity of the h7/n -specific rt-lamp assay. amplification curves of the h7/n9-special rt-lamp were performed with 10-fold serial dilutions of viral rna. specificities of h and n rt-lamp were tested by using direct visual detection of rt-lamp with rna extracted from viral culture of h1n h3n h5n piv h9n rsv, hadv- and h7n detection limit of h7-specific rt-lamp was pfu per reaction. the color changes under natural or uv light were only observed in the tube of h7n viral rna by using h7-specific rt-lamp assay. detection limit of n9-specific rt-lamp was pfu per reaction. the color changes were observed for n9-specific rt-lamp reaction with h7n viral rna. no color change was seen in rt-lamp with other subtype of influenza viral rna and other three respiratory viruses under natural or uv light. comparison of detection of h7n virus by using rt-lamp and rrt-pcr assay strain virus titer rrt-pcr rrt-pcr rt-lamp rt-lamp and, not detectable. discussion the rt-lamp assay could finish within 45 minutes. by using fluorescent detection reagent, the positive amplification could be observed by color change. as shown in the figure 1b and d, the color of positive reaction change from orange to green under natural light, while the positive reaction also produced bright green fluorescence under uv light. for negative sample such as h1n h3n h5n piv h9n rsv and hadv- virus, the color of reaction mixture did not change either under normal light or under uv light. the h7/n9-specific rt-lamp assay was further evaluated using clinical samples and chicken samples compared with rrt-pcr assay. the rt-lamp results showed that samples were tested to be positive and the other samples were negative for h7n respectively, which could be confirmed by rrt-pcr assay. no false positive and false negative were observed. by using these samples, 100% diagnostic sensitivities were achieved with h or n specific rt-lamp assay compared with rrt-pcr assays, which showed that rt-lamp results were consistent with that of rrt-pcr. the lamp method aimed to detect avian influenza h7n showed reliable results in the detection of avian and human clinical samples. previously, two rt-lamp systems had been developed for detection of avian influenza subtype h virus. currently, some other rt-lamp assays for detection of h7n virus were also developed. these assays showed excellent sensitivities and specificities. our h7/n9-specific rt-lamp assays, as well as those published rt-lamp methods, were very useful for the early detection of suspected h7n infection, and could contribute to the prevention and control of h7n epidemics and potential pandemic in china and other asian countries.  the current study described a rapid, accurate and feasible h7/n specific rt-lamp assay with perfect sensitivities and high specificities. it was simple and easy to perform. this assay have the potential to provide useful tools for detection of novel h7n virus especially in resource-limited setting, which will play important roles in the prevention and control of spread of novel h7n infection. 
there is great demand for simple and rapid diagnostic method for early detection of h7n to provide timely treatment and disease control. the detection limits of the h7- and n9-specific rt-lamp assay were both approximately pfu per reaction. no cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. the aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification assay for detection of h7n virus. the assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity. the h7/n specific rt-lamp assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza a virus, especially in the hospitals and laboratories without sophisticated diagnostic systems. the emerged human infection with avian influenza a virus in china since has aroused global concerns.
 the emerged human infection with avian influenza a virus in china since has aroused global concerns. there is great demand for simple and rapid diagnostic method for early detection of h7n to provide timely treatment and disease control. the aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification assay for detection of h7n virus.  the detection limits of the h7- and n9-specific rt-lamp assay were both approximately pfu per reaction. no cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. the assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity.  the h7/n specific rt-lamp assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza a virus, especially in the hospitals and laboratories without sophisticated diagnostic systems. keywords influenza virush7n subtypereverse transcription-loop-mediated isothermal amplificationmolecular diagnosisissue-copyright-statement© the author 2014 