A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses
Abstract
1. Introduction
2. Results
2.1. Selection of LAMP Primers
2.2. Optimization of Quantitative LAMP (qLAMP)
2.3. Evaluation of qLAMP and Visual LAMP (vLAMP) ASSAYS Limits of Detection
2.4. Optimization of the LAMP Reaction Time
2.5. Evaluation of qPCR Assay Limit of Detection
2.6. Testing on Clinical Samples
3. Discussion
4. Materials and Methods
4.1. Design of LAMP Primers
- HAdV-B, control protein E1A, region 33226–33443 (NC_011202.1, https://www.ncbi.nlm.nih.gov/nuccore/NC_011202.1/, accessed on 2 January 2022);
- HAdV-C, control protein E1B, region 3300–3489 (NC_001405.1, https://www.ncbi.nlm.nih.gov/nuccore/NC_001405/, accessed on 2 January 2022);
- HAdV-E, single-stranded DNA-binding protein E2A, region 21996–22186 (NC_003266.2, https://www.ncbi.nlm.nih.gov/nuccore/NC_003266.2/, accessed on 2 January 2022);
4.2. Design of PCR Primers
- HAdV-B—L3 gene, region 26361–26434 (NC_011202.1, https://www.ncbi.nlm.nih.gov/nuccore/NC_011202.1/, accessed on 2 January 2022);
- HAdV-C—capsid protein precursor pVI, region 25927–25996 (NC_001405.1, https://www.ncbi.nlm.nih.gov/nuccore/NC_001405/, accessed on 2 January 2022);
- HAdV-E—E3 gene, region 27467–27544 (NC_003266.2, https://www.ncbi.nlm.nih.gov/nuccore/NC_003266.2/, accessed on 2 January 2022).
4.3. Clinical and Standard Samples
4.4. Droplet Digital PCR
4.5. Quantitative LAMP (qLAMP)
4.6. Quantitative PCR (qPCR)
4.7. Visual LAMP (vLAMP)
4.8. Evaluation of the Limit of Detection, Clinical Sensitivity and Specificity of LAMP and PCR
4.9. Results Validation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Safiri, S.; Mahmoodpoor, A.; Kolahi, A.A.; Nejadghaderi, S.A.; Sullman, M.J.M.; Mansournia, M.A.; Ansarin, K.; Collins, G.S.; Kaufman, J.S.; Abdollahi, M. Global Burden of Lower Respiratory Infections during the Last Three Decades. Front. Public Health 2023, 10, 1028525. [Google Scholar] [CrossRef] [PubMed]
- Global Health Estimates: Leading Causes of DALYs. Available online: https://www.who.int/data/gho/data/themes/mortality-and-global-health-estimates/global-health-estimates-leading-causes-of-dalys (accessed on 30 October 2023).
- Morawska, L.; Allen, J.; Bahnfleth, W.; Bluyssen, P.M.; Boerstra, A.; Buonanno, G.; Cao, J.; Dancer, S.J.; Floto, A.; Franchimon, F.; et al. A Paradigm Shift to Combat Indoor Respiratory Infection. Science (1979) 2021, 372, 689–691. [Google Scholar] [CrossRef] [PubMed]
- Ljubin-Sternak, S.; Meštrović, T.; Lukšić, I.; Mijač, M.; Vraneš, J. Seasonal Coronaviruses and Other Neglected Respiratory Viruses: A Global Perspective and a Local Snapshot. Front. Public Health 2021, 9, 691163. [Google Scholar] [CrossRef]
- Matthes-Martin, S.; Feuchtinger, T.; Shaw, P.J.; Engelhard, D.; Hirsch, H.H.; Cordonnier, C.; Ljungman, P. European Guidelines for Diagnosis and Treatment of Adenovirus Infection in Leukemia and Stem Cell Transplantation: Summary of ECIL-4 (2011). Transpl. Infect. Dis. 2012, 14, 555–563. [Google Scholar] [CrossRef] [PubMed]
- Zhang, S.Y.; Luo, Y.P.; Huang, D.D.; Fan, H.; Lu, Q.B.; Wo, Y.; Chen, G.; Zhang, X.A.; Li, Y.; Tong, Y.G.; et al. Fatal Pneumonia Cases Caused by Human Adenovirus 55 in Immunocompetent Adults. Infect. Dis. 2016, 48, 40–47. [Google Scholar] [CrossRef]
- Bautista-Gogel, J.; Madsen, C.M.; Lu, X.; Sakthivel, S.K.; Froh, I.; Kamau, E.; Gerber, S.I.; Watson, J.T.; Cooper, S.S.; Schneider, E. Outbreak of Respiratory Illness Associated with Human Adenovirus Type 7 Among Persons Attending Officer Candidates School, Quantico, Virginia, 2017. J. Infect. Dis. 2020, 221, 697–700. [Google Scholar] [CrossRef]
- Kujawski, S.A.; Lu, X.; Schneider, E.; Blythe, D.; Boktor, S.; Farrehi, J.; Haupt, T.; Mcbride, D.; Stephens, E.; Sakthivel, S.K.; et al. Outbreaks of Adenovirus-Associated Respiratory Illness on 5 College Campuses in the United States, 2018–2019. Clin. Infect. Dis. 2021, 72, 1992–1999. [Google Scholar] [CrossRef] [PubMed]
- Yi, L.; Zou, L.R.; Lu, J.; Kang, M.; Song, Y.; Su, J.; Zhang, X.; Liang, L.J.; Ni, H.Z.; Ke, C.; et al. A Cluster of Adenovirus Type B55 Infection in a Neurosurgical Inpatient Department of a General Hospital in Guangdong, China. Influenza Other Respir. Viruses 2017, 11, 328–336. [Google Scholar] [CrossRef] [PubMed]
- Guo, Z.; Tong, L.; Xu, S.; Li, B.; Wang, Z.; Liu, Y. Epidemiological Analysis of an Outbreak of an Adenovirus Type 7 Infection in a Boot Camp in China. PLoS ONE 2020, 15, e0232948. [Google Scholar] [CrossRef]
- Naesens, L.; Lenaerts, L.; Andrei, G.; Snoeck, R.; Van Beers, D.; Holý, A.; Balzarini, J.; De Clercq, E. Antiadenovirus Activities of Several Classes of Nucleoside and Nucleotide Analogues. Antimicrob. Agents Chemother. 2005, 49, 1010–1016. [Google Scholar] [CrossRef]
- Morfin, F.; Dupuis-Girod, S.; Mundweiler, S.; Falcon, D.; Carrington, D.; Sedlacek, P.; Bierings, M.; Cetkovsky, P.; Kroes, A.C.M.; Van Tol, M.J.D.; et al. In Vitro Susceptibility of Adenovirus to Antiviral Drugs Is Species-Dependent. Antivir. Ther. 2005, 10, 225–229. [Google Scholar] [CrossRef]
- Kim, S.J.; Kim, K.; Park, S.B.; Hong, D.J.; Jhun, B.W. Outcomes of Early Administration of Cidofovir in Non-Immunocompromised Patients with Severe Adenovirus Pneumonia. PLoS ONE 2015, 10, e0122642. [Google Scholar] [CrossRef] [PubMed]
- Pierce, V.M.; Hodinka, R.L. Comparison of the GenMark Diagnostics ESensor Respiratory Viral Panel to Real-Time PCR for Detection of Respiratory Viruses in Children. J. Clin. Microbiol. 2012, 50, 3458–3465. [Google Scholar] [CrossRef] [PubMed]
- Wong, S.; Pabbaraju, K.; Pang, X.L.; Lee, B.E.; Fox, J.D. Detection of a Broad Range of Human Adenoviruses in Respiratory Tract Samples Using a Sensitive Multiplex Real-Time PCR Assay. J. Med. Virol. 2008, 80, 856–865. [Google Scholar] [CrossRef] [PubMed]
- Echavarria, M.; Kolavic, S.A.; Cersovsky, S.; Mitchell, F.; Sanchez, J.L.; Polyak, C.; Innis, B.L.; Binn, L.N. Detection of Adenoviruses (AdV) in Culture-Negative Environmental Samples by PCR during an AdV-Associated Respiratory Disease Outbreak. J. Clin. Microbiol. 2000, 38, 2982–2984. [Google Scholar] [CrossRef] [PubMed]
- Echavarria, M.; Sanchez, J.L.; Kolavic-Gray, S.A.; Polyak, C.S.; Mitchell-Raymundo, F.; Innis, B.L.; Vaughn, D.; Reynolds, R.; Binn, L.N. Rapid Detection of Adenovirus in Throat Swab Specimens by PCR during Respiratory Disease Outbreaks among Military Recruits. J. Clin. Microbiol. 2003, 41, 810–812. [Google Scholar] [CrossRef] [PubMed]
- Pinsky, B.A.; Hayden, R.T. Cost-Effective Respiratory Virus Testing. J. Clin. Microbiol. 2019, 57. [Google Scholar] [CrossRef] [PubMed]
- Gadsby, N.J.; Hardie, A.; Claas, E.C.J.; Templeton, K.E. Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis. J. Clin. Microbiol. 2010, 48, 2213–2216. [Google Scholar] [CrossRef] [PubMed]
- Huang, H.S.; Tsai, C.L.; Chang, J.; Hsu, T.C.; Lin, S.; Lee, C.C. Multiplex PCR System for the Rapid Diagnosis of Respiratory Virus Infection: Systematic Review and Meta-Analysis. Clin. Microbiol. Infect. 2018, 24, 1055–1063. [Google Scholar] [CrossRef]
- Mahony, J.; Chong, S.; Merante, F.; Yaghoubian, S.; Sinha, T.; Lisle, C.; Janeczko, R. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay. J. Clin. Microbiol. 2007, 45, 2965–2970. [Google Scholar] [CrossRef]
- Vabret, A.; Gouarin, S.; Joannes, M.; Barranger, C.; Petitjean, J.; Corbet, S.; Brouard, J.; Lafay, F.; Duhamel, J.F.; Guillois, B.; et al. Development of a PCR-and Hybridization-Based Assay (PCR Adenovirus Consensus®) for the Detection and the Species Identification of Adenoviruses in Respiratory Specimens. J. Clin. Virol. 2004, 31, 116–122. [Google Scholar] [CrossRef] [PubMed]
- Lu, X.; Trujillo-Lopez, E.; Lott, L.; Erdman, D.D. Quantitative Real-Time PCR Assay Panel for Detection and Type-Specific Identification of Epidemic Respiratory Human Adenoviruses. J. Clin. Microbiol. 2013, 51, 1089–1093. [Google Scholar] [CrossRef]
- Oscorbin, I.P.; Shevelev, G.Y.; Pronyaeva, K.A.; Stepanov, A.A.; Shamovskaya, D.V.; Mishukova, O.V.; Pyshnyi, D.V.; Filipenko, M.L. Detection of SARS-CoV-2 Rna by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis. Int. J. Mol. Sci. 2021, 22, 5743. [Google Scholar] [CrossRef] [PubMed]
- Dolskiy, A.A.; Grishchenko, I.V.; Yudkin, D.V. Cell Cultures for Virology: Usability, Advantages, and Prospects. Int. J. Mol. Sci. 2020, 21, 7978. [Google Scholar] [CrossRef] [PubMed]
- Compton, J. Nucleic Acid Sequence-Based Amplification. Nature 1991, 350, 91–92. [Google Scholar] [CrossRef]
- Walker, G.T.; Little, M.C.; Nadeau, J.G.; Shank, D.D. Isothermal in Vitro Amplification of DNA by a Restriction Enzyme/DNA Polymerase System. Proc. Natl. Acad. Sci. USA 1992, 89, 392. [Google Scholar] [CrossRef] [PubMed]
- Lizardi, P.M.; Huang, X.; Zhu, Z.; Bray-Ward, P.; Thomas, D.C.; Ward, D.C. Mutation Detection and Single-Molecule Counting Using Isothermal Rolling-Circle Amplification. Nat. Genet. 1998, 19, 225–232. [Google Scholar] [CrossRef]
- Murakami, T.; Sumaoka, J.; Komiyama, M. Sensitive Isothermal Detection of Nucleic-Acid Sequence by Primer Generation–Rolling Circle Amplification. Nucleic Acids Res. 2009, 37, e19. [Google Scholar] [CrossRef]
- Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res. 2000, 28, e63. [Google Scholar] [CrossRef]
- Vincent, M.; Xu, Y.; Kong, H. Helicase-Dependent Isothermal DNA Amplification. EMBO Rep. 2004, 5, 795–800. [Google Scholar] [CrossRef]
- Piepenburg, O.; Williams, C.H.; Stemple, D.L.; Armes, N.A. DNA Detection Using Recombination Proteins. PLoS Biol. 2006, 4, e204. [Google Scholar] [CrossRef] [PubMed]
- Van Ness, J.; Van Ness, L.K.; Galas, D.J. Isothermal Reactions for the Amplification of Oligonucleotides. Proc. Natl. Acad. Sci. USA 2003, 100, 4504–4509. [Google Scholar] [CrossRef] [PubMed]
- Dean, F.B.; Hosono, S.; Fang, L.; Wu, X.; Faruqi, A.F.; Bray-Ward, P.; Sun, Z.; Zong, Q.; Du, Y.; Du, J.; et al. Comprehensive Human Genome Amplification Using Multiple Displacement Amplification. Proc. Natl. Acad. Sci. USA 2002, 99, 5261–5266. [Google Scholar] [CrossRef] [PubMed]
- Li, Y.; Kim, H.J.; Zheng, C.; Chow, W.H.A.; Lim, J.; Keenan, B.; Pan, X.; Lemieux, B.; Kong, H. Primase-Based Whole Genome Amplification. Nucleic Acids Res. 2008, 36, e79. [Google Scholar] [CrossRef] [PubMed]
- Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Isothermal Amplification of Nucleic Acids. Chem. Rev. 2015, 115, 12491–12545. [Google Scholar] [CrossRef] [PubMed]
- Goto, M.; Honda, E.; Ogura, A.; Nomoto, A.; Hanaki, K.I. Colorimetric Detection of Loop-Mediated Isothermal Amplification Reaction by Using Hydroxy Naphthol Blue. Biotechniques 2009, 46, 167–172. [Google Scholar] [CrossRef]
- Sadeghi, Y.; Kananizadeh, P.; Moghadam, S.O.; Alizadeh, A.; Pourmand, M.R.; Mohammadi, N.; Afshar, D.; Ranjbar, R. The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Microorganisms: A Systematic Review and Meta-Analysis. Iran. J. Public. Health 2021, 50, 2172. [Google Scholar] [CrossRef]
- Meagher, R.J.; Priye, A.; Light, Y.K.; Huang, C.; Wang, E. Impact of Primer Dimers and Self-Amplifying Hairpins on Reverse Loop-Mediated Isothermal Amplification Detection of Viral. Analyst 2018, 143, 1924. [Google Scholar] [CrossRef] [PubMed]
- Jang, W.S.; Lim, D.H.; Yoon, J.; Kim, A.; Lim, M.; Nam, J.; Yanagihara, R.; Ryu, S.W.; Jung, B.K.; Ryoo, N.H.; et al. Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for on-Site Diagnosis of SARS-CoV-2. PLoS ONE 2021, 16, e0248042. [Google Scholar] [CrossRef]
- Kim, J.; Park, B.G.; Lim, D.H.; Jang, W.S.; Nam, J.; Mihn, D.C.C.; Lim, C.S. Development and Evaluation of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Differentiation of Mycobacterium Tuberculosis and Non-Tuberculosis Mycobacterium in Clinical Samples. PLoS ONE 2021, 16, e0244753. [Google Scholar] [CrossRef]
- Mahony, J.; Chong, S.; Bulir, D.; Ruyter, A.; Mwawasi, K.; Waltho, D. Multiplex Loop-Mediated Isothermal Amplification (M-LAMP) Assay for the Detection of Influenza A/H1, A/H3 and Influenza B Can Provide a Specimen-to-Result Diagnosis in 40 Min with Single Genome Copy Sensitivity. J. Clin. Virol. 2013, 58, 127–131. [Google Scholar] [CrossRef] [PubMed]
- Goikhman, Y.; Drori, Y.; Friedman, N.; Sherbany, H.; Keller, N.; Mendelson, E.; Pando, R.; Mandelboim, M. Adenovirus Load Correlates with Respiratory Disease Severity among Hospitalized Pediatric Patients. Int. J. Infect. Dis. 2020, 97, 145–150. [Google Scholar] [CrossRef] [PubMed]
- Abdullah, O.; Fall, A.; Klein, E.; Mostafa, H.H. Increased Circulation of Human Adenovirus in 2023: An Investigation of the Circulating Genotypes, Upper Respiratory Viral Loads, and Hospital Admissions in a Large Academic Medical Center. J. Clin. Microbiol. 2024, 62, e0123723. [Google Scholar] [CrossRef] [PubMed]
- Mozgovoj, M.; Graham, M.D.; Ferrufino, C.; Blanc, S.; Souto, A.F.; Pilloff, M.; Santos, M.J.D. Viral Load in Symptomatic and Asymptomatic Patients Infected with SARS-CoV-2. What Have We Learned? J. Clin. Virol. Plus 2023, 3, 100166. [Google Scholar] [CrossRef]
- Platten, M.; Hoffmann, D.; Grosser, R.; Wisplinghoff, F.; Wisplinghoff, H.; Wiesmüller, G.; Schildgen, O.; Schildgen, V. SARS-CoV-2, CT-Values, and Infectivity—Conclusions to Be Drawn from Side Observations. Viruses 2021, 13, 1459. [Google Scholar] [CrossRef] [PubMed]
- Oscorbin, I.P.; Belousova, E.A.; Boyarskikh, U.A.; Zakabunin, A.I.; Khrapov, E.A.; Filipenko, M.L. Derivatives of Bst-like Gss-Polymerase with Improved Processivity and Inhibitor Tolerance. Nucleic Acids Res. 2017, 45, 9595. [Google Scholar] [CrossRef]
- Okonechnikov, K.; Golosova, O.; Fursov, M.; Varlamov, A.; Vaskin, Y.; Efremov, I.; German Grehov, O.G.; Kandrov, D.; Rasputin, K.; Syabro, M.; et al. Unipro UGENE: A Unified Bioinformatics Toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef]
Target | LAMP Primer | Sequence 3′–5′ |
---|---|---|
HAdV-B | F3 | AGAGACGGAAGAATCATGTT |
B3 | AGAAAATGCTCCTTCTTTTGG | |
LF | GCGATAGATGCCATCTGC | |
LB | GCTTCCAACAAAGCCTCC | |
FIP | CTTTTTCACCAACACAGTGGGGGCAGTACTTCAAATTGTAGATCG | |
BIP | TCAAAAGAAATGCGATTTTCAAGGTCTTTTGTTTTTGGATGTGCG | |
HAdV-C | F3 | GGGGTGTTTGACATGACCAT |
B3 | CATCGCTAGAGCCAAACTCA | |
LF | TCTGCACCTGGTGCGGGTC | |
LB | GGATGTGACCGAGGAGCTGAGG | |
FIP | GTTTACCGCCACACTCGCAGGGATCTGGAAGGTGCTGAGGT | |
BIP | TAGGAACCAGCCTGTGATGCTGCCAGCACCAAGTGATCGG | |
HAdV-E | F3 | GCGAGAGTTGCGGTACAC |
B3 | GCGATTGCAACCACAAGC | |
LF | TGGCGAGCGTGAAGCATC | |
LB | GCGTTGGCCATCCCAAAGG | |
FIP | ATCACCGACGCGACGGTGCGGGGTTGCAGCACTGG | |
BIP | CCGTCCACGTCGAGGTCTTCGCTGCGTGCCCACCATG |
Target | Primer | Sequence 3′–5′ |
---|---|---|
HAdV-B | F | GTATCACAAGAGCGAAGACCAA |
R | GCGCGCAGTACTTGTTGAAGAG | |
P | R6G-CAGCGCACTCTCGAGGACGCCG-BHQ1 | |
HAdV-C | F | CTCCGGGGTTGAAACTCACT |
R | TAGTCCTCAGGTACAAATTTGCG | |
P | ROX-AGGTAAGCCGACGTCCACAGCCCC-BHQ2 | |
HAdV-E | F | CGCCGTTGTTAACCACTGCTAC |
R | CTTCTGCGGGTGGAAAAAGTAAG | |
P | FAM-CGGAGTCCTGCTAAACGGTCCCG-BHQ1 | |
ALB | F | GACTTGCCAAGACATATGAAACC |
R | TCCAACAATAAACCTACCACTTTG | |
P | Cy5.5-TGCTGTGCCGCTGCAGATCC-BHQ3 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Koryukov, M.A.; Oscorbin, I.P.; Novikova, L.M.; Gordukova, M.A.; Turina, I.E.; Galeeva, E.V.; Kudlay, D.A.; Filipenko, M.L. A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses. Int. J. Mol. Sci. 2024, 25, 7215. https://doi.org/10.3390/ijms25137215
Koryukov MA, Oscorbin IP, Novikova LM, Gordukova MA, Turina IE, Galeeva EV, Kudlay DA, Filipenko ML. A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses. International Journal of Molecular Sciences. 2024; 25(13):7215. https://doi.org/10.3390/ijms25137215
Chicago/Turabian StyleKoryukov, Maksim A., Igor P. Oscorbin, Lidiya M. Novikova, Maria A. Gordukova, Irina E. Turina, Elena V. Galeeva, Dmitry A. Kudlay, and Maxim L. Filipenko. 2024. "A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses" International Journal of Molecular Sciences 25, no. 13: 7215. https://doi.org/10.3390/ijms25137215
APA StyleKoryukov, M. A., Oscorbin, I. P., Novikova, L. M., Gordukova, M. A., Turina, I. E., Galeeva, E. V., Kudlay, D. A., & Filipenko, M. L. (2024). A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses. International Journal of Molecular Sciences, 25(13), 7215. https://doi.org/10.3390/ijms25137215