Skip to main content

Advertisement

SpringerLink
Log in

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP)

  • Original Paper
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/μL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2–4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/μL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs.

Graphical abstract

The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Ricklin ME, Garcia-Nicolas O, Brechbuhl D, Python S, Zumkehr B, Posthaus H, Oevermann A, Summerfield A (2016) Japanese encephalitis virus tropism in experimentally infected pigs. Vet Res 47:34. https://doi.org/10.1186/s13567-016-0319-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Oliveira ARS, Cohnstaedt LW, Noronha LE, Mitzel D, McVey DS, Cernicchiaro N (2020) Perspectives regarding the risk of introduction of the Japanese encephalitis virus (JEV) in the United States. Front Vet Sci 7:48. https://doi.org/10.3389/fvets.2020.00048

    Article  PubMed  PubMed Central  Google Scholar 

  3. Tajima S, Shibasaki KI, Taniguchi S, Nakayama E, Maeki T, Lim CK, Saijo M (2019) E and prM proteins of genotype V Japanese encephalitis virus are required for its increased virulence in mice. Heliyon 5:e02882. https://doi.org/10.1016/j.heliyon.2019.e02882

    Article  PubMed  PubMed Central  Google Scholar 

  4. Gao X, Liu H, Li X, Fu S, Cao L, Shao N, Zhang W, Wang Q, Lu Z, Lei W, He Y, Cao Y, Wang H, Liang G (2019) Changing geographic distribution of Japanese encephalitis virus genotypes, 1935-2017. Vector Borne Zoonotic Dis 19:35–44. https://doi.org/10.1089/vbz.2018.2291

    Article  PubMed  Google Scholar 

  5. Hills SL, Walter EB, Atmar RL, Fischer M, Group AJEVW (2019) Japanese encephalitis vaccine: recommendations of the advisory committee on immunization practices. MMWR Recomm Rep 68:1–33. https://doi.org/10.15585/mmwr.rr6802a1

    Article  PubMed  PubMed Central  Google Scholar 

  6. Misra UK, Kalita J (2010) Overview: Japanese encephalitis. Prog Neurobiol 91:108–120. https://doi.org/10.1016/j.pneurobio.2010.01.008

    Article  CAS  PubMed  Google Scholar 

  7. Ricklin ME, Garcia-Nicolas O, Brechbuhl D, Python S, Zumkehr B, Nougairede A, Charrel RN, Posthaus H, Oevermann A, Summerfield A (2016) Vector-free transmission and persistence of Japanese encephalitis virus in pigs. Nat Commun 7:10832. https://doi.org/10.1038/ncomms10832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Young CL, Lyons AC, Hsu WW, Vanlandingham DL, Park SL, Bilyeu AN, Ayers VB, Hettenbach SM, Zelenka AM, Cool KR, Peterson GJ, Higgs S, Huang YS (2020) Protection of swine by potent neutralizing anti-Japanese encephalitis virus monoclonal antibodies derived from vaccination. Antivir Res 174:104675. https://doi.org/10.1016/j.antiviral.2019.104675

    Article  CAS  PubMed  Google Scholar 

  9. Kumari R, Joshi PL (2012) A review of Japanese encephalitis in Uttar Pradesh, India. WHO South-East Asia J Public Health 1:374–395

    Article  Google Scholar 

  10. Le Flohic G, Porphyre V, Barbazan P, Gonzalez JP (2013) Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology. PLoS Negl Trop Dis 7:e2208. https://doi.org/10.1371/journal.pntd.0002208

    Article  PubMed  PubMed Central  Google Scholar 

  11. Shao N, Li F, Nie K, Fu SH, Zhang WJ, He Y, Lei WW, Wang QY, Liang GD, Cao YX, Wang HY (2018) TaqMan real-time RT-PCR assay for detecting and differentiating Japanese encephalitis virus. Biomed Environ Sci 31:208–214. https://doi.org/10.3967/bes2018.026

    Article  PubMed  Google Scholar 

  12. Chanama S, Sukprasert W, Sa-ngasang A, An A, Sangkitporn S, Kurane I, Anantapreecha S (2005) Detection of Japanese encephalitis (JE) virus-specific IgM in cerebrospinal fluid and serum samples from JE patients. Jpn J Infect Dis 58:294–296

    CAS  PubMed  Google Scholar 

  13. Cha GW, Cho JE, Ju YR, Hong YJ, Han MG, Lee WJ, Choi EY, Jeong YE (2014) Comparison of four serological tests for detecting antibodies to Japanese encephalitis virus after vaccination in children. Osong Public Health Res Perspect 5:286–291. https://doi.org/10.1016/j.phrp.2014.08.003

    Article  PubMed  PubMed Central  Google Scholar 

  14. Zanoli LM, Spoto G (2013) Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors (Basel) 3:18–43. https://doi.org/10.3390/bios3010018

    Article  CAS  Google Scholar 

  15. Yue S, Li Y, Qiao Z, Song W, Bi S (2021) Rolling circle replication for biosensing, bioimaging, and biomedicine. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2021.02.007

  16. Bi S, Yue S, Zhang S (2017) Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine. Chem Soc Rev 46:4281–4298. https://doi.org/10.1039/c7cs00055c

    Article  CAS  PubMed  Google Scholar 

  17. Lee SH, Ahn G, Kim MS, Jeong OC, Lee JH, Kwon HG, Kim YH, Ahn JY (2018) Poly-adenine-coupled LAMP barcoding to detect apple scar skin viroid. ACS Comb Sci 20:472–481. https://doi.org/10.1021/acscombsci.8b00022

    Article  CAS  PubMed  Google Scholar 

  18. Gao J, Huang X, Liu H, Zan F, Ren J (2012) Colloidal stability of gold nanoparticles modified with thiol compounds: bioconjugation and application in cancer cell imaging. Langmuir 28:4464–4471. https://doi.org/10.1021/la204289k

    Article  CAS  PubMed  Google Scholar 

  19. Lu W, Wang L, Li J, Zhao Y, Zhou Z, Shi J, Zuo X, Pan D (2015) Quantitative investigation of the poly-adenine DNA dissociation from the surface of gold nanoparticles. Sci Rep 5:10158. https://doi.org/10.1038/srep10158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pei H, Li F, Wan Y, Wei M, Liu H, Su Y, Chen N, Huang Q, Fan C (2012) Designed diblock oligonucleotide for the synthesis of spatially isolated and highly hybridizable functionalization of DNA-gold nanoparticle nanoconjugates. J Am Chem Soc 134:11876–11879. https://doi.org/10.1021/ja304118z

    Article  CAS  PubMed  Google Scholar 

  21. Liu J (2012) Adsorption of DNA onto gold nanoparticles and graphene oxide: surface science and applications. Phys Chem Chem Phys 14:10485–10496. https://doi.org/10.1039/c2cp41186e

    Article  CAS  PubMed  Google Scholar 

  22. Ahn SJ, Baek YH, Lloren KKS, Choi WS, Jeong JH, Antigua KJC, Kwon HI, Park SJ, Kim EH, Kim YI, Si YJ, Hong SB, Shin KS, Chun S, Choi YK, Song MS (2019) Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform. BMC Infect Dis 19:676. https://doi.org/10.1186/s12879-019-4277-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lopez-Jimena B, Wehner S, Harold G, Bakheit M, Frischmann S, Bekaert M, Faye O, Sall AA, Weidmann M (2018) Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome. PLoS Negl Trop Dis 12:e0006448. https://doi.org/10.1371/journal.pntd.0006448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kaneko H, Kawana T, Fukushima E, Suzutani T (2007) Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70:499–501. https://doi.org/10.1016/j.jbbm.2006.08.008

    Article  CAS  PubMed  Google Scholar 

  25. Wan L, Chen T, Gao J, Dong C, Wong AH, Jia Y, Mak PI, Deng CX, Martins RP (2017) A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection. Sci Rep 7:14586. https://doi.org/10.1038/s41598-017-14698-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rasti R, Nanjebe D, Karlstrom J, Muchunguzi C, Mwanga-Amumpaire J, Gantelius J, Martensson A, Rivas L, Galban F, Reutersward P, Andersson Svahn H, Alvesson HM, Boum Y 2nd, Alfven T (2017) Health care workers' perceptions of point-of-care testing in a low-income country-a qualitative study in Southwestern Uganda. PLoS One 12:e0182005. https://doi.org/10.1371/journal.pone.0182005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Niemz A, Ferguson TM, Boyle DS (2011) Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 29:240–250. https://doi.org/10.1016/j.tibtech.2011.01.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63. https://doi.org/10.1093/nar/28.12.e63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Huang W, Zhang H, Xu J, Wang S, Kong X, Ding W, Xu J, Feng J (2017) Loop-mediated isothermal amplification method for the rapid detection of Ralstonia solanacearum phylotype I mulberry strains in China. Front Plant Sci 8:76. https://doi.org/10.3389/fpls.2017.00076

    Article  PubMed  PubMed Central  Google Scholar 

  30. Li J, Hu X, Wang X, Yang J, Zhang L, Deng Q, Zhang X, Wang Z, Hou T, Li S (2021) A novel one-pot rapid diagnostic technology for COVID-19. Anal Chim Acta 1154:338310. https://doi.org/10.1016/j.aca.2021.338310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang Y, Li H, Wang Y, Zhang L, Xu J, Ye C (2017) Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of Enterococcus faecalis and Staphylococcus aureus. Front Microbiol 8:192. https://doi.org/10.3389/fmicb.2017.00192

    Article  PubMed  PubMed Central  Google Scholar 

  32. Teixeira A, Paris JL, Roumani F, Dieguez L, Prado M, Espina B, Abalde-Cela S, Garrido-Maestu A, Rodriguez-Lorenzo L (2020) Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach. Materials (Basel):13. https://doi.org/10.3390/ma13081934

  33. Kong X, Qin W, Huang X, Kong F, Schoen CD, Feng J, Wang Z, Zhang H (2016) Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola. Sci Rep 6:28935. https://doi.org/10.1038/srep28935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lee S, Kim JH, Han BK, Kim WI, Cho BK, Woo SM, Kim YH, Ahn JY (2020) Wax-printed well pads and colorimetric LAMP detection of ApxIA toxin gene. Mol Cell Toxicol 16:263–270

    Article  CAS  Google Scholar 

  35. Teoh BT, Sam SS, Tan KK, Johari J, Danlami MB, Hooi PS, Md-Esa R, AbuBakar S (2013) Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification. BMC Infect Dis 13:387. https://doi.org/10.1186/1471-2334-13-387

    Article  PubMed  PubMed Central  Google Scholar 

  36. Parida MM, Santhosh SR, Dash PK, Tripathi NK, Lakshmi V, Mamidi N, Shrivastva A, Gupta N, Saxena P, Babu JP, Rao PV, Morita K (2007) Rapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay. J Clin Microbiol 45:351–357. https://doi.org/10.1128/JCM.01734-06

    Article  CAS  PubMed  Google Scholar 

  37. Nunes MR, Vianez JL Jr, Nunes KN, da Silva SP, Lima CP, Guzman H, Martins LC, Carvalho VL, Tesh RB, Vasconcelos PF (2015) Analysis of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for yellow fever diagnostic. J Virol Methods 226:40–51. https://doi.org/10.1016/j.jviromet.2015.10.003

    Article  CAS  PubMed  Google Scholar 

  38. Lamb LE, Bartolone SN, Tree MO, Conway MJ, Rossignol J, Smith CP, Chancellor MB (2018) Rapid detection of Zika virus in urine samples and infected mosquitos by reverse transcription-loop-mediated isothermal amplification. Sci Rep 8:3803. https://doi.org/10.1038/s41598-018-22102-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ganguli A, Mostafa A, Berger J, Aydin MY, Sun F, Ramirez SAS, Valera E, Cunningham BT, King WP, Bashir R (2020) Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc Natl Acad Sci U S A 117:22727–22735. https://doi.org/10.1073/pnas.2014739117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ma YD, Li KH, Chen YH, Lee YM, Chou ST, Lai YY, Huang PC, Ma HP, Lee GB (2019) A sample-to-answer, portable platform for rapid detection of pathogens with a smartphone interface. Lab Chip 19:3804–3814. https://doi.org/10.1039/c9lc00797k

    Article  CAS  PubMed  Google Scholar 

  41. Huang SH, Yang TC, Tsai MH, Tsai IS, Lu HC, Chuang PH, Wan L, Lin YJ, Lai CH, Lin CW (2008) Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus. Nanotechnology 19:405101. https://doi.org/10.1088/0957-4484/19/40/405101

    Article  CAS  PubMed  Google Scholar 

  42. Pantawane PB, Dhanze H, Ravi Kumar G, M RG, Dudhe NC, Bhilegaonkar KN (2019) TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes. Anim Biotechnol 30:267–272. https://doi.org/10.1080/10495398.2018.1481417

    Article  CAS  PubMed  Google Scholar 

  43. Wu X, Lin H, Chen S, Xiao L, Yang M, An W, Wang Y, Yao X, Yang Z (2017) Development and application of a reverse transcriptase droplet digital PCR (RT-ddPCR) for sensitive and rapid detection of Japanese encephalitis virus. J Virol Methods 248:166–171. https://doi.org/10.1016/j.jviromet.2017.06.015

    Article  CAS  PubMed  Google Scholar 

  44. Liu H, Liu ZJ, Jing J, Ren JQ, Liu YY, Guo HH, Fan M, Lu HJ, Jin NY (2012) Reverse transcription loop-mediated isothermal amplification for rapid detection of Japanese encephalitis virus in swine and mosquitoes. Vector Borne Zoonotic Dis 12:1042–1052. https://doi.org/10.1089/vbz.2012.0991

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Professor Yun Hee Baek for providing the DENV types 2–4 and Zika nucleic acid. We thank all the participants who worked in BSL-3 for their support in cultivating all the viruses. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2019R1A2C1010860) and a Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Crop Viruses and Pests Response Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (321108-04).

Author information

Author notes

  1. Gna Ahn and Se Hee Lee contributed equally to this work.

Authors and Affiliations

  1. Center for Ecology and Environmental Toxicology, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea

    Gna Ahn,  Yang-Hoon Kim & Ji-Young Ahn

  2. Department of Biological Sciences and Biotechnology, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea

    Se Hee Lee,  Yang-Hoon Kim & Ji-Young Ahn

  3. College of Medicine and Medical Research Institute, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea

    Min-Suk Song

  4. Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, South Korea

    Beom-Ku Han

Authors
  1. Gna Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Se Hee Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Min-Suk Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beom-Ku Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yang-Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ji-Young Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Gna Ahn and Se Hee Lee performed the experiments and analyzed the data; Min-Suk Song and Beom-Ku Han contributed clinical materials/analysis tools; Yang-Hoon Kim and Ji-Young Ahn wrote the paper.

Corresponding authors

Correspondence to Yang-Hoon Kim or Ji-Young Ahn.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supporting information

ESM 1

(DOCX 1631 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ahn, G., Lee, S.H., Song, MS. et al. JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP). Microchim Acta 188, 333 (2021). https://doi.org/10.1007/s00604-021-04986-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-021-04986-9

Keywords

Search

Navigation