Rapid Detection of Subtype H10N8 Influenza Virus by One-StepReverse Transcription–Loop-Mediated Isothermal AmplificationMethods

Hongmei Bao, Xiaoxiao Feng, Yong Ma, Jianzhong Shi, Yuhui Zhao, Linlin Gu, Xiurong Wang, Hualan Chen

Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy ofAgricultural Sciences, Harbin, People’s Republic of China

We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription–loop-mediated isothermal amplifica-tion assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did notamplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and theycan serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets.

H10N8 influenza virus was initially isolated from quails in 1965in Italy (1) and subsequently was found in Australia, Sweden,

and North America (Canada and the United States). In China,H10N8 influenza virus was first detected in water samples col-lected from the Dongting Lake wetland in 2007 (2) and then in aduck at a live poultry market in Guangdong Province in 2012 (3).The first outbreak of human infection with the novel H10N8 in-fluenza virus was confirmed in December 2013: a 73-year-old fe-male with chronic diseases who had visited a local live poultrymarket succumbed to community-acquired pneumonia (4). Livepoultry markets have been shown to be key locations where ge-nome segment reassortment and interspecies transmission occurin avian influenza viruses (5–7). Human infections with influenzaA H5N1, H7N9, and H10N8 viruses are all associated with expo-sure to live poultry markets (5, 8, 9). H10N8 viruses exhibit lowpathogenicity in chickens, and the infected birds do not exhibitany symptoms (2). Therefore, a rapid and sensitive method fordiagnosing H10N8 infection is urgently required for monitoringthe prevalence of the virus and reducing human exposure to in-fected poultry (10–12). Molecular techniques, particularly loop-mediated isothermal amplification (LAMP), have exhibited highsensitivity and specificity for detecting influenza A viruses, such asH1N1, H5N1, and H7N9 (13–16). In this study, we developed andsystematically evaluated a one-step reverse transcription–LAMP(RT-LAMP) assay specific for the hemagglutinin (HA) and neur-aminidase (NA) genes of H10N8 influenza virus.

To design specific primers to amplify conserved regions of theHA and NA genes of H10N8 influenza virus, we compared andanalyzed the HA and NA gene sequences of 104 strains of H10N8influenza viruses available in GenBank and the Global Initiativeon Sharing Avian Influenza Data (GISAID). Conserved regionsexhibiting the highest levels of homology were chosen as the tem-plate (GenBank accession no. KP861987 and KP861989) for de-signing H10 and N8 LAMP primers by using the PrimerExplorerversion 4 software (https://primerexplorer.jp). These primers in-cluded outer primers (H10-F3 and H10-B3), inner primers (H10-FIP and H10-BIP), and loop primers (H10-LF and H10-LB) (Ta-ble 1). The primers were synthesized by Life Technologies(Beijing, China).

RNA was isolated from H10N8 influenza virus and otherhighly pathogenic avian influenza viruses in a biosafety level 3

laboratory at Harbin Veterinary Research Institute, Harbin,China. Viral RNA was extracted from 140 �l of virus supernatantor cloacal and tracheal swabs by using an RNeasy minikit (Qiagen,Valencia, CA, USA), according to the manufacturer’s protocol.One-step RT-LAMP (H10-RT-LAMP and N8-RT-LAMP) assayswere performed in 25-�l mixtures that contained 8 U of Bst DNApolymerase (New England BioLabs, Ipswich, MA, USA), 5 U ofavian myeloblastosis virus (AMV) reverse transcriptase (Invitro-gen, Carlsbad, CA, USA), 1.4 mM each deoxynucleoside triphos-phate (dNTP), 0.8 M betaine, 8 mM MgSO4, 1 �l of fluorescentdetection reagent (Eiken Chemical Co., Ltd., Tokyo, Japan),primers (1.6 �M inner primers [FIP and BIP], 0.2 �M outer prim-ers [F3 and B3], 0.6 �M loop primers [LF and LB]) (Table 1), and2 �l of extracted RNAs. One-step RT-LAMP reactions were per-formed at 62.5°C for 60 min in either a LA-320C Loopamp real-time turbidimeter (Teramecs, Tokyo, Japan) or a water bath andthen terminated by incubation at 90°C for 1 min; reaction mix-tures lacking templates were used as negative controls. Reactionturbidity was measured in real time, and the result was indicatedby the graph in the monitor of the real-time turbidimeter, whichverified initiation of the amplification. LAMP products were de-tected by visually inspecting the color.

To evaluate the sensitivities of the H10-RT-LAMP and N8-RT-LAMP assays, in vitro RNA transcripts of the HA and NA genesfrom the H10N8 virus were prepared with T7 Cap Scribe (Roche,Penzberg, Germany), according to the manufacturer’s instruc-

Received 10 August 2015 Returned for modification 8 September 2015Accepted 14 September 2015

Accepted manuscript posted online 16 September 2015

Citation Bao H, Feng X, Ma Y, Shi J, Zhao Y, Gu L, Wang X, Chen H. 2015. Rapiddetection of subtype H10N8 influenza virus by one-step reverse transcription–loop-mediated isothermal amplification methods. J Clin Microbiol 53:3884 –3887.doi:10.1128/JCM.02165-15.

Editor: A. M. Caliendo

Address correspondence to Xiurong Wang, wxr@hvri.ac.cn, or Hualan Chen,chenhualan@caas.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02165-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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tions, using H10N8 virus (A/chicken/JiangXi/S3581/13) RNA as atemplate. RNA was quantified and then 10-fold serially dilutedfrom 1 � 106 copies/�l to 1 � 10�1 copies/�l and used as thetemplate for the RT-LAMP and RT-PCR assays (see the supple-mental material). All reactions were performed in triplicate. Akinetic analysis of turbidity revealed that the lower detection limitof the H10-RT-LAMP and N8-RT-LAMP assays was 1.0 � 101

copies per reaction mixture (Fig. 1A and D). Assay sensitivity wasalso confirmed through visual inspection (Fig. 1B and E); a cleargreen color was observed at concentrations ranging from 1.0 �106 to 1.0 � 101 copies/�l. The sensitivity levels measured usingreal-time turbidity analysis and visual inspection did not differmarkedly. When the same RNA template was used in one-stepRT-PCR with H10- and N8-specific primers, the detection limit ofthe system was 1.0 � 102 copies (Fig. 1C and F). This confirmedthat the RT-LAMP assay was approximately 10-fold more sensi-tive than RT-PCR.

The specificities of the one-step RT-LAMP assays were evalu-ated by using influenza virus reference strains of the H1 to H15and N1 to N9 subtypes and other avian respiratory pathogens:Newcastle disease virus, avian infectious bronchitis virus, and in-fectious laryngotracheitis virus (see Table S1 in the supplementalmaterial). All tested samples were negative, except for the H10N8virus (A/chicken/JiangXi/S3581/13). These results indicated thatthe one-step RT-LAMP method can be used to specifically amplifyH10N8 influenza virus in the absence of cross-reactivity with ei-ther other avian influenza subtype viruses or other avian patho-genic viruses.

To examine clinical sensitivity and specificity, a total of 192samples (44 samples from chickens, 82 samples from ducks, and66 samples from the environment), including tracheal swabs andcloacal swabs that had been collected from live poultry markets inJiangxi, Hunan, and Zhejiang Provinces, were tested using theRT-LAMP assays, RT-PCR, and viral isolation. From these 192samples, 6 positives were obtained using the RT-LAMP assays andvirus isolation, whereas 5 positives were obtained using RT-PCR.Thus, the positive rates of the RT-LAMP assays, viral isolation,

and RT-PCRs were 3.1% (6/192), 3.1% (6/192), and 2.6% (5/192),respectively. The results of the RT-LAMP assays were consistentwith those of the virus isolation. Although 60 min was used for theRT-LAMP assay reactions, most of the amplification reactions forclinical samples were finished within 24 min (data not shown).These results suggested that the RT-LAMP assays were more effi-cient, practical, and rapid diagnostic methods for the detection ofthe H10N8 virus from clinical samples.

To further evaluate the ability of the RT-LAMP assays to detectthe H10N8 virus, 10 6-week-old specific-pathogen-free (SPF)White Leghorn chickens (group 1) were inoculated intranasallywith 106.0 50% embryo infective dose (EID50) of H10N8 virus(A/chicken/JiangXi/S3581/13) in a volume of 0.1 ml. Controlchickens (group 2, n � 10) were inoculated with 0.1 ml of sterileallantoic fluid collected from normal SPF embryonated chickeneggs. Tracheal and cloacal swabs collected on days 3, 5, 7, 9, 11, and13 postinfection from all chickens were detected using the RT-LAMP assays, RT-PCR, and viral isolation. In group 1, 10 out of 60tracheal samples and 39 out of 60 cloacal samples tested positivewhen viral isolation was used, 11 tracheal and 39 cloacal samplestested positive when RT-LAMP assays were used, and 9 trachealand 37 cloacal samples tested positive when RT-PCR was used(Table 2). Thus, the positive rates of tracheal and cloacal samplesin the infection group were 16.7% (10/60) and 65.0% (39/60) forviral isolation, 18.3% (11/60) and 65.0% (39/60) for the RT-LAMP assay, and 15.0% (9/60) and 61.7% (37/60) for the RT-PCRassay, respectively. All samples collected from group 2 chickenswere negative using all three methods. Four positive samplesamong the experimentally infected samples that were detectedusing the RT-LAMP assays were missed when the one-step RT-PCR assays were used. These results further indicated that theH10N8 virus-specific RT-LAMP assay is more sensitive than theRT-PCR assay for detecting H10N8 virus in specimens whose viraltiters are extremely low. This agrees with results obtained previ-ously using RT-LAMP assays specific for the H5, H7, and H9subtypes of influenza virus (14–17).

In summary, HA- and NA-specific RT-LAMP assays were de-

TABLE 1 RT-LAMP and RT-PCR primers designed for detecting HA and NA gene sequences of H10N8 influenza viruses

Primera Length (k-mer) Sequence (5= to 3=)b

H10-F3 18 CTGGTATGGTTTCAGACAH10-B3 21 GACGTTACCGATTTGGTGTTCH10-FIP 42 GATCAATAGCTGCCTGAGTACTTCAAAATGCTCAGGGCACAGH10-BIP 47 AATCACTGGGAAACTGAATAGACTGATCTCACTGAACTCAGATTCTAH10-LB 22 AACCAATACTGAGTTCGAGTCAH10-LF 18 TGTAATCAGCGGCCTGGCN8-F3 18 GACAATTGGACCGGAACCN8-B3 20 CTAATGGTCCTTCCCATCCN8-FIP 45 CTCCTCTTGGGGTGTCACTGTGTTGGTGATTTCTCCAGATN8-BIP 40 GGATCATGCACTAGCCCAATGATACATCATTGCCCTGCCN8-LF 24 GAGACCTGCACACAAATATCCGACTN8-LB 21 GGGATACGGAGTTAAGGGATTTGGH10-F 22 CTGCTGATTACAAGAGTACTCAH10-R 21 CTCTGTATTGTGAATGGTCATN8-F 19 CTGCATGTCGTGAGCATCAN8-R 19 ACCACGCCACAGCTTCAAAa The primers of H10-F3, H10-B3, H10-FIP, H10-BIP, H10-LB, and H10-LF were used in the H10-RT-LAMP assay. The primers of N8-F3, N8-B3, N8-FIP, N8-BIP, N8-LB, andN8-LF were used in the N8-RT-LAMP assay. The primers of H10-F and H10-R were used in H10-RT-PCR. The primers of N8-F and N8-R were used in N8-RT-PCR.b The underlined regions indicate the F1c region within the FIP primer. The italicized regions indicate the F2 region within the FIP primer. The bolded regions indicate B1c regionwithin the BIP primer. The underlined and italicized regions indicate the B2 region within the BIP primer.

RT-LAMP for Rapid Detection of H10N8 Influenza Virus

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veloped and systematically evaluated for use in the detection ofH10N8 influenza virus in experimentally infected and clinicalspecimens. The one-step RT-LAMP assays were more sensitivethan routine RT-PCRs and more rapid than virus isolation for thedetection of H10N8 viruses. More importantly, the assays can beused to detect, with high sensitivity and specificity, H10N8 virusfrom chicken samples; thus, the assays can serve as an extremelyeffective tool for detecting and monitoring H10N8 virus in livepoultry markets or poultry farms and for contributing to the con-trol of H10N8 virus infection.

ACKNOWLEDGMENTS

This study was supported by the 12th 5-Year Plan of National Science andTechnology of Rural Areas (grant 2012AA101303), the National NaturalScience Foundation of China (grant 31470127), and the InternationalS&T Cooperation Program of China (grant 2014DFR31260).

We declare no conflicts of interest.

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FIG 1 Relative sensitivities of RT-LAMP and RT-PCR methods. H10-RT-LAMP and N8-RT-LAMP assays and RT-PCR were performed using A/chicken/JiangXi/S3581/13 (H10N8) viral RNA at concentrations ranging from 1 � 106 copies/�l to 1 � 10�1 copies/�l. (A and B) Detection limit of H10-RT-LAMP assay.LAMP products were detected through a real-time turbidity measurement in an LA-320C turbidimeter (A) or using a fluorescence assay (B). (C) Detection limitof one-step RT-PCR measured using the same RNA extracts as those used for the H10-RT-LAMP assay. (D and E) Detection limit of N8-RT-LAMP assay. LAMPproducts were detected through a real-time turbidity measurement in an LA-320C turbidimeter (D) or using a fluorescence assay (E). (F) Detection limit ofone-step RT-PCR measured using the same RNA extracts as those used for the N8-RT-LAMP assay. PCR products were visualized on a 1.5% agarose gel stainedwith ethidium bromide.

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