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Open Access 01-12-2010 | Methodology

A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

Authors: Thanh Tran Tan, Hana Apsari Pawestri, Ngoc Nghiem My, Hien Vo Minh, Harun Syahrial, Trung Nguyen Vu, H Rogier van Doorn, Heiman FL Wertheim, Chau Nguyen Van Vinh, Ha Do Quang, Jeremy J Farrar, Hien Tran Tinh, Endang R Sedyaningsih, Menno D de Jong

Published in: Virology Journal | Issue 1/2010

Abstract

Background

The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants.

Results

We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%.

Conclusions

This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.

Available only for authorised users

WHO: Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO. 2009.

Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, Ng TK, Chan KH, Lai ST, Lim WL, Yuen KY, Guan Y: Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet 2004,363(9409):617-619. 10.1016/S0140-6736(04)15595-5PubMedCrossRef

Chen H, Smith GJ, Li KS, Wang J, Fan XH, Rayner JM, Vijaykrishna D, Zhang JX, Zhang LJ, Guo CT, Cheung CL, Xu KM, Duan L, Huang K, Qin K, Leung YH, Wu WL, Lu HR, Chen Y, Xia NS, Naipospos TS, Yuen KY, Hassan SS, Bahri S, Nguyen TD, Webster RG, Peiris JS, Guan Y: Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc Natl Acad Sci USA 2006,103(8):2845-2850. 10.1073/pnas.0511120103PubMedPubMedCentralCrossRef

Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P, Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT, Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS: Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 2004,430(6996):209-213. 10.1038/nature02746PubMedCrossRef

WHO: Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 2005,11(10):1515-1521.CrossRef

Smith GJ, Naipospos TS, Nguyen TD, de Jong MD, Vijaykrishna D, Usman TB, Hassan SS, Nguyen TV, Dao TV, Bui NA, Leung YH, Cheung CL, Rayner JM, Zhang JX, Zhang LJ, Poon LL, Li KS, Nguyen VC, Hien TT, Farrar J, Webster RG, Chen H, Peiris JS, Guan Y: Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam. Virology 2006,350(2):258-268. 10.1016/j.virol.2006.03.048PubMedCrossRef

Tiensin T, Chaitaweesub P, Songserm T, Chaisingh A, Hoonsuwan W, Buranathai C, Parakamawongsa T, Premashthira S, Amonsin A, Gilbert M, Nielen M, Stegeman A: Highly pathogenic avian influenza H5N1, Thailand, 2004. Emerg Infect Dis 2005,11(11):1664-1672.PubMedPubMedCentralCrossRef

Boltz DA, Douangngeun B, Sinthasak S, Phommachanh P, Rolston S, Chen H, Guan Y, Peiris JS, Smith JG, Webster RG: H5N1 influenza viruses in Lao People's Democratic Republic. Emerg Infect Dis 2006,12(10):1593-1595.PubMedPubMedCentralCrossRef

WHO: Online Report: Toward a Unified Nomenclature System for Highly Pathogenic Avian Influenza Virus (H5N1). Emerg Infect Dis 2008,14(7):e1.PubMedCentralCrossRef

10.

Smith GJ, Fan XH, Wang J, Li KS, Qin K, Zhang JX, Vijaykrishna D, Cheung CL, Huang K, Rayner JM, Peiris JS, Chen H, Webster RG, Guan Y: Emergence and predominance of an H5N1 influenza variant in China. Proc Natl Acad Sci USA 2006,103(45):16936-16941. 10.1073/pnas.0608157103PubMedPubMedCentralCrossRef

11.

Dung NT, Vinh Nguyen T, Vijaykrishna D, Webster RG, Guan Y, Malik Peiris JS, Smith GJ: Multiple sublineages of influenza A virus (H5N1), Vietnam, 2005-2007. Emerg Infect Dis 2008,14(4):632-636. 10.3201/eid1404.071343CrossRef

12.

Le MT, Wertheim HF, Nguyen HD, Taylor W, Hoang PV, Vuong CD, Nguyen HL, Nguyen HH, Nguyen TQ, Nguyen TV, Van TD, Ngoc BT, Bui TN, Nguyen BG, Nguyen LT, Luong ST, Phan PH, Pham HV, Nguyen T, Fox A, Nguyen CV, Do HQ, Crusat M, Farrar J, Nguyen HT, de Jong MD, Horby P: Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam. PLoS ONE 2008,3(10):e3339. 10.1371/journal.pone.0003339PubMedPubMedCentralCrossRef

13.

Payungporn S, Phakdeewirot P, Chutinimitkul S, Theamboonlers A, Keawcharoen J, Oraveerakul K, Amonsin A, Poovorawan Y: Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection. Viral Immunol 2004,17(4):588-593. 10.1089/vim.2004.17.588PubMedCrossRef

14.

Ng LF, Barr I, Nguyen T, Noor SM, Tan RS, Agathe LV, Gupta S, Khalil H, To TL, Hassan SS, Ren EC: Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay. BMC Infect Dis 2006, 6: 40. 10.1186/1471-2334-6-40PubMedPubMedCentralCrossRef

15.

Payungporn S, Chutinimitkul S, Chaisingh A, Damrongwantanapokin S, Buranathai C, Amonsin A, Theamboonlers A, Poovorawan Y: Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. J Virol Methods 2006,131(2):143-147. 10.1016/j.jviromet.2005.08.004PubMedCrossRef

16.

WHO: Guidlines on Laboratory Diagnosis of Avian Influenza. 2007.

17.

Ng EK, Cheng PK, Ng AY, Hoang TL, Lim WW: Influenza A H5N1 detection. Emerg Infect Dis 2005,11(8):1303-1305.PubMedPubMedCentralCrossRef

18.

Lu YY, Yan JY, Feng Y, Xu CP, Shi W, Mao HY: Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR. Lett Appl Microbiol 2008,46(1):20-25.PubMed

19.

Chen W, He B, Li C, Zhang X, Wu W, Yin X, Fan B, Fan X, Wang J: Real-time RT-PCR for H5N1 avian influenza A virus detection. J Med Microbiol 2007,56(Pt 5):603-607. 10.1099/jmm.0.47014-0PubMedCrossRef

20.

Ellis JS, Smith JW, Braham S, Lock M, Barlow K, Zambon MC: Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans. J Clin Microbiol 2007,45(5):1535-1543. 10.1128/JCM.02007-06PubMedPubMedCentralCrossRef

21.

Braasch DA, Corey DR: Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol 2001,8(1):1-7. 10.1016/S1074-5521(00)00058-2PubMedCrossRef

22.

Ugozzoli LA, Latorra D, Puckett R, Arar K, Hamby K: Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Anal Biochem 2004,324(1):143-152. 10.1016/j.ab.2003.09.003PubMedCrossRef

23.

Reynisson E, Josefsen MH, Krause M, Hoorfar J: Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR. J Microbiol Methods 2006,66(2):206-216. 10.1016/j.mimet.2005.11.006PubMedCrossRef

24.

Higgs ES, Hayden FG, Chotpitayasunondh T, Whitworth J, Farrar J: The Southeast Asian Influenza Clinical Research Network: development and challenges for a new multilateral research endeavor. Antiviral Res 2008,78(1):64-68. 10.1016/j.antiviral.2007.10.008PubMedCrossRef

25.

CDC: New laboratory assay for diagnostic testing of avian influenza A/H5 (Asian Lineage). MMWR Morb Mortal Wkly Rep 2006,55(5):127.

26.

Tran TH, Nguyen TL, Nguyen TD, Luong TS, Pham PM, Nguyen VC, Pham TS, Vo CD, Le TQ, Ngo TT, Dao BK, Le PP, Nguyen TT, Hoang TL, Cao VT, Le TG, Nguyen DT, Le HN, Nguyen KT, Le HS, Le VT, Christiane D, Tran TT, Menno de J, Schultsz C, Cheng P, Lim W, Horby P, Farrar J: Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med 2004,350(12):1179-1188. 10.1056/NEJMoa040419PubMedCrossRef

27.

Buh Gasparic M, Cankar K, Zel J, Gruden K: Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms. BMC Biotechnol 2008, 8: 26. 10.1186/1472-6750-8-26PubMedPubMedCentralCrossRef

28.

Thomsen R, Nielsen PS, Jensen TH: Dramatically improved RNA in situ hybridization signals using LNA-modified probes. Rna 2005,11(11):1745-1748. 10.1261/rna.2139705PubMedPubMedCentralCrossRef

29.

de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, Hoang DM, Chau NV, Khanh TH, Dong VC, Qui PT, Cam BV, Ha do Q, Guan Y, Peiris JS, Chinh NT, Hien TT, Farrar J: Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 2006,12(10):1203-1207. 10.1038/nm1477PubMedPubMedCentralCrossRef

30.

Lee MS, Chang PC, Shien JH, Cheng MC, Shieh HK: Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J Virol Methods 2001,97(1-2):13-22. 10.1016/S0166-0934(01)00301-9PubMedCrossRef

31.

Bao Y, Bolotov P, Dernovoy D, Kiryutin B, Zaslavsky L, Tatusova T, Ostell J, Lipman D: The influenza virus resource at the National Center for Biotechnology Information. J Virol 2008,82(2):596-601. 10.1128/JVI.02005-07PubMedPubMedCentralCrossRef

Title: A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes
Authors: Thanh Tran Tan
Hana Apsari Pawestri
Ngoc Nghiem My
Hien Vo Minh
Harun Syahrial
Trung Nguyen Vu
H Rogier van Doorn
Heiman FL Wertheim
Chau Nguyen Van Vinh
Ha Do Quang
Jeremy J Farrar
Hien Tran Tinh
Endang R Sedyaningsih
Menno D de Jong
Publication date: 01-12-2010
Publisher: BioMed Central
Published in: Virology Journal / Issue 1/2010
Electronic ISSN: 1743-422X
DOI: https://doi.org/10.1186/1743-422X-7-46

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A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

Abstract

Background

Results

Conclusions

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