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Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification

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Abstract

To detect several RNA viruses simultaneously, a method based on multiplex ligation reaction combined with multiplex qPCR or multiplex PCR+capillary electrophoresis was established to detect four RNA viruses: human immunodeficiency virus (HIV), hepatitis C (HCV), influenza A virus (IAV) H1N1 and H5N1. The experimental conditions including ligation probe concentration, annealing procedure, ligation temperature and ligase dosage were optimized intensively. We found that the specificity of the ligation reaction was affected by the probe concentration predominantly, high-probe concentration (100 nM) resulted in splint-independent ligation with efficiency comparable to that with RNA splint. The sensitivity of the ligation reaction was affected by the annealing mode apparently as the sensitivity of the step-down annealing mode was 100 times higher than that of the isothermal annealing at 37 °C. Under the optimized condition, this assay could detect virus RNA as low as 16 viral copies per reaction in doubleplex and triplex real-time quantitative PCR detection with satisfactory specificity and precision. By multiplex PCR+capillary electrophoresis, four RNA viruses could be detected in one tube with the sensitivity of 10 copies per reaction.

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References

  1. Rojas M, Monsalve DM, PachecoY A-A, Ramirez-Santana C, Ansari AA, Gershwin ME, Anaya JM (2020) Ebola virus disease: an emerging and re-emerging viral threat. J Autoimmun. https://doi.org/10.1016/j.jaut.2019.102375

    Article  PubMed  PubMed Central  Google Scholar 

  2. Povoa TF, Alves AM, Oliveira CA, Nuovo GJ, Chagas VL, Paes MV (2014) The pathology of severe dengue in multiple organs of human fatal cases: histopathology, ultrastructure and virus replication. PLoS ONE 9:e83386. https://doi.org/10.1371/journal.pone.0083386

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  3. Cevik M, Kuppalli K, Kindrachuk J, Peiris M (2020) Virology, transmission, and pathogenesis of SARS-CoV-2. BMJ 371:m3862. https://doi.org/10.1136/bmj.m3862

    Article  PubMed  Google Scholar 

  4. Altawalah H, Essa S, Ezzikouri S, Al-Nakib W (2019) Hepatitis B virus, hepatitis C virus and human immunodeficiency virus infections among people who inject drugs in Kuwait: A cross-sectional study. Sci Rep 9:6292. https://doi.org/10.1038/s41598-019-42810-w

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  5. Yamazaki S, Kondo M, Sudo K, Ueda T, Fujiwara H, Hasegawa N, Kato S (2016) Qualitative real-time PCR assay for HIV-1 and HIV-2 RNA. Jpn J Infect Dis 69:367–372. https://doi.org/10.7883/yoken.JJID.2015.309

    Article  PubMed  CAS  Google Scholar 

  6. Yilmaz G (2001) Diagnosis of HIV infection and laboratory monitoring of its therapy. J Clin Virol 21:187–196. https://doi.org/10.1016/s1386-6532(01)00165-2

    Article  PubMed  CAS  Google Scholar 

  7. Gu Y, Hsu AC, Pang Z, Pan H, Zuo X, Wang G, Zheng J, Wang F (2019) Role of the innate cytokine storm induced by the Influenza A virus. Viral Immunol 32:244–251. https://doi.org/10.1089/vim.2019.0032

    Article  PubMed  CAS  Google Scholar 

  8. Ibrahim N, Jamaluddin ND, Tan LL, Mohd Yusof NY (2021) A review on the development of gold and silver nanoparticles-based biosensor as a detection strategy of emerging and pathogenic RNA virus. Sensors (Basel) 21:5114. https://doi.org/10.3390/s21155114

    Article  PubMed  ADS  CAS  Google Scholar 

  9. Chin CD, Linder V, Sia SK (2007) Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip 7:41–57. https://doi.org/10.1039/b611455e

    Article  PubMed  CAS  Google Scholar 

  10. Fernandez-Carballo BL, McBeth C, McGuiness I, Kalashnikov M, Baum C, Borros S, Sharon A, Sauer-Budge AF (2018) Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection. Anal Bioanal Chem 410:33–43. https://doi.org/10.1007/s00216-017-0689-8

    Article  PubMed  CAS  Google Scholar 

  11. Reta DH, Tessema TS, Ashenef AS, Desta AF, Labisso WL, Gizaw ST, Abay SM, Melka DS, Reta FA (2020) Molecular and immunological diagnostic techniques of medical viruses. Int J Microbiol 2020:8832728. https://doi.org/10.1155/2020/8832728

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Tan AS, Nerurkar SN, Tan WCC, Goh D, Lai CPT, Poh Sheng Yeong J (2020) The virological, immunological, and imaging approaches for COVID-19 diagnosis and research. SLAS Technol 25:522–544. https://doi.org/10.1177/2472630320950248

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Cui X, Shi Y, Zhao L, Gu S, Wei C, Yang Y, Wen S, Chen H, Ge J (2018) Application of real-time quantitative PCR to detect mink circovirus in naturally and experimentally infected minks. Front Microbiol 9:937. https://doi.org/10.3389/fmicb.2018.00937

    Article  PubMed  PubMed Central  Google Scholar 

  14. St John A, Price CP (2014) Existing and emerging technologies for point of-care testing. Clin Biochem Rev 35:155–167

    PubMed  PubMed Central  Google Scholar 

  15. Stuppia L, Antonucci I, Palka G, Gatta V (2012) Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci 13:3245–3276. https://doi.org/10.3390/ijms13033245

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Woo CH, Jang S, Shin G, Jung GY, Lee JW (2020) Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription. Nat Biomed Eng 4:1168–1179. https://doi.org/10.1038/s41551-020-00617-5

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Conn V, Conn SJ (2019) SplintQuant: a method for accurately quantifying circular RNA transcript abundance without reverse transcription bias. RNA 25:1202–1210. https://doi.org/10.1261/rna.070953.119

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Zhou C, Huang R, Zhou X, Xing D (2020) Sensitive and specific microRNA detection by RNA dependent DNA ligation and rolling circle optical signal amplification. Talanta 216:120954. https://doi.org/10.1016/j.talanta.2020.120954

    Article  PubMed  CAS  Google Scholar 

  19. Xiao Y, Wang Y, Tang Q, Wei L, Zhang X, Jia G (2018) An elongation- and ligation-based qPCR amplification method for the radio labeling-free detection of locus-specific N(6)-methyladenosine modification. Angew Chem Int Ed Engl 57:15995–16000. https://doi.org/10.1002/anie.201807942

    Article  PubMed  CAS  Google Scholar 

  20. Wang P, Ma C, Zhang X, Chen LZ, Yi LY, Liu X, Lu QW, Cao Y, Gao S (2021) A ligation/recombinase polymerase amplification assay for rapid detection of SARS-CoV-2. Front Cell Infect Microbiol 11:680728. https://doi.org/10.3389/fcimb.2021.680728

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Park Y, Roh J, Kim S (2022) Performance evaluation of the aptima assays in comparison with the cobas 6800 assays for the detection of HIV-1, HBV, and HCV in clinical samples. Ann Lab Med 42(4):447–456. https://doi.org/10.3343/alm.2022.42.4.447

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Li ZG, Wu XS, Wang L, Liu Y, Zhu L (2020) Study on the temperature control technology of microfluidic PCR for the blood nucleic acid test. Electron Meas Technol 43:152–156. https://doi.org/10.19651/j.cnki.emt.2004204

    Article  Google Scholar 

  23. Kim SM, Kim J, Noh S, Sohn H, Lee T (2020) Recent development of aptasensor for Influenza virus detection. Biochip J 14(4):327–339. https://doi.org/10.1007/s13206-020-4401-2

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Jia Y, Han J, Wang H, Hong W, Wang H, Zhang M, Li Z (2021) Ultrasensitive quantification of multiplexed mRNA variants via splice-junction anchored DNA probes and SplintR ligase-initiated PCR. Chem Commun (Camb) 57:10011–10014. https://doi.org/10.1039/d1cc03033g

    Article  PubMed  CAS  Google Scholar 

  25. Kuhn H, Frank-Kamenetskii MD (2005) Template-independent ligation of single-stranded DNA by T4 DNA ligase. FEBS J 272:5991–6000. https://doi.org/10.1111/j.1742-4658.2005.04954.x

    Article  PubMed  CAS  Google Scholar 

  26. Krzywkowski T, Nilsson M (2017) Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy. Nucleic Acids Res 45:e161. https://doi.org/10.1093/nar/gkx708

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Guo Y, Yang H, Ren W, Gu H, Xu G, Xu H (2020) A noise-free, ultrasensitive and accurate miRNAs detection using streptavidin coated magnetic microsphere based stem-loop ligation PCR. Talanta 213:120845. https://doi.org/10.1016/j.talanta.2020.120845

    Article  PubMed  CAS  Google Scholar 

  28. Huang HS, Tsai CL, Chang J, Hsu TC, Lin S, Lee CC (2018) Multiplex PCR system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis. Clin Microbiol Infect 24:1055–1063. https://doi.org/10.1016/j.cmi.2017.11.018

    Article  PubMed  CAS  Google Scholar 

  29. Minkus CL, Bispo PJM, Papaliodis GN, Sobrin L (2019) Real-Time multiplex PCR analysis in infectious uveitis. Semin Ophthalmol 34:252–255. https://doi.org/10.1080/08820538.2019.1620803

    Article  PubMed  Google Scholar 

  30. Hawkins SFC, Guest PC (2017) Multiplex analyses using real-time quantitative PCR. Methods Mol Biol 1546:125–133. https://doi.org/10.1007/978-1-4939-6730-8_8

    Article  PubMed  CAS  Google Scholar 

  31. Huang Q, Chen D, Du C, Liu Q, Lin S, Liang L, Xu Y, Liao Y, Li Q (2022) Highly multiplex PCR assays by coupling the 5’-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters. Proc Natl Acad Sci U S A 119(9):e2110672119. https://doi.org/10.1073/pnas.2110672119

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Funding

This work was supported by the National Key R&D Program of China (2018YFA0801101).

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Authors and Affiliations

  1. College of Biological Science and Medical Engineering, Donghua University, #2999 North Renmin Road, Songjiang District, Shanghai, 201620, China

    Lijun Qian, Junhua Xiao, Kai Li & Yuxun Zhou

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  1. Lijun Qian
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  2. Junhua Xiao
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  3. Kai Li
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  4. Yuxun Zhou
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Contributions

JHX and YXZ: designed and supervised the research, LJQ and KL: performed the experiments and analyzed the data, LJQ and YXZ: wrote the manuscript.

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Correspondence to Yuxun Zhou.

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Qian, L., Xiao, J., Li, K. et al. Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification. Curr Microbiol 81, 75 (2024). https://doi.org/10.1007/s00284-023-03582-9

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