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A Multiplex PCR Assay Mediated by Universal Primer for the Diagnosis of Human Meningitis Caused by Six Common Bacteria

  • Genetics of Microorganisms
  • Published:
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Abstract

The present study aimed to develop a universal primer-multiplex PCR (UP-M-PCR) assay for the detection of six common bacteria associated with human meningitis. One optimal universal primer (UP) was selected from three UPs by comparing their sensitivities and specificities. All specific primers were tagged with the UP sequence at 5' end, and applied to the multiplex PCR system. The multiplex system was further optimized and assessed. This UP-M-PCR can successfully detect the six meningitis-associated pathogens with high specificity, and the sensitivity could reach up to 10 copies. In the identification of clinical specimens, six positive cases infected with Streptococcus agalactiae, Staphylococcus aureus, and Streptococcus pneumoniae were confirmed. The newly developed multiplex PCR system can be used to detect the six pathogens associated with human bacterial meningitis with high specificity and sensitivity.

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References

  1. Khan, S.P, Ghani, R., and Yaqub, Z., Single step PCR for the identification of Low Density Lipoprotein Receptor (LDL-R) gene mutations, Pak. J. Med. Sci., 2014, vol. 30, p. 830.

    PubMed  PubMed Central  Google Scholar 

  2. Pallisgaard, N., Hokland, P., Riishøj, D.C., et al., Multiplex reverse transcription–polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia, Blood, 1998, vol. 92, pp. 574–588.

    CAS  PubMed  Google Scholar 

  3. Sauné, L., Auger, P., Migeon, A., et al., Isolation, characterization and PCR multiplexing of microsatellite loci for a mite crop pest, Tetranychus urticae (Acari: Tetranychidae), BMC Res. Notes, 2015, vol. 8, p. 247.

    PubMed  PubMed Central  Google Scholar 

  4. Datukishvili, N., Kutateladze, T., Gabriadze, I., et al., New multiplex PCR methods for rapid screening of genetically modified organisms in foods, Front Microbiol., 2015, vol. 6, p. 757.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Hu, Y., Duan, Q., Chen, Y., et al., A novel multiplex rt-PCR assay for the detection of four chromosomal translocations of leukemia, Genet. Test Mol. Bioma, 2014, vol. 18, pp. 810–819.

    Article  CAS  Google Scholar 

  6. Chang, S.S., Hsieh, W.H., Liu, T.S., et al., Multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis–a systemic review and metaanalysis, PLoS One, 2013, vol. 8. e62323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang, M., Xie, Z., Xie, L., et al., Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay, J. Virol. Methods, 2015, vol. 224, pp. 9–15.

    Article  CAS  PubMed  Google Scholar 

  8. Oetting, W.S., Lee, H.K., Flanders, D.J., et al., Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers, Genomics, 1995, vol. 30, pp. 450–458.

    Article  CAS  PubMed  Google Scholar 

  9. Shuber, A.P., Grondin, V.J., and Klinger, K.W., A simplified procedure for developing multiplex PCRs, Genome Res., 1995, vol. 5, pp. 488–493.

    Article  CAS  PubMed  Google Scholar 

  10. Blacket, M.J., Robin, C., Good, R.T., et al., Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence, Mol. Ecol. Resour., 2012, vol. 12, pp. 456–463.

    Article  CAS  PubMed  Google Scholar 

  11. Ge, C., Cui, Y.N., Jing, P.Y., et al., An alternative suite of universal primers for genotyping in multiplex PCR, PLoS One, vol. 9, p. e92826

  12. Missiaggia, A. and Grattapaglia, D., Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers, Genet. Mol. Res., 2006, vol. 5, pp. 72–78.

    CAS  PubMed  Google Scholar 

  13. Heath, K.E., Day, I.N., and Humphries, S.E., Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene, J. Med. Genet., 2000, vol. 37, pp. 272–280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen, Z.Y., Liu, Y.L., Duan, W.B., et al., Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing, J. Nanosci. Nanotechnol., 2014, vol. 14, pp. 4363–4370.

    Article  CAS  PubMed  Google Scholar 

  15. Xu, W.T., Yuan, Y.F., Luo, Y.B., et al., Event-specific detection of stacked genetically modified maize Bt11 × GA21 by UP-M-PCR and real-time PCR, J. Agric. Food Chem., 2008, vol. 57, pp. 395–402.

    Article  Google Scholar 

  16. Xu, W.T., Zhai, Z.F., Huang, K.L., et al., A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis, PLoS One, 2012, vol. 7, p. 1.

    Article  CAS  Google Scholar 

  17. Zhang, C.J., Xu, W.T., Zhai, Z.F., et al., Universal primer-multiplex-polymerase chain reaction (UP-MPCR) and capillary electrophoresis-laser-induced fluorescence analysis for the simultaneous detection of six genetically modified maize lines, J. Agric. Food Chem., 2011, vol. 59, pp. 5188–5194.

    Article  CAS  PubMed  Google Scholar 

  18. Yuan, Y.F., Xu, W.T., Zhai, Z.F., et al., Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples, J. Food Sci., 2009, vol. 74, pp. 446–452.

    Google Scholar 

  19. Hoogman, M., Van de Beek, D., Weisfelt, M., et al., Cognitive outcome in adults after bacterial meningitis, J. Neurol. Neurosurg. Psychiatry, 2007, vol. 78, pp. 1092–1096.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Kim, K.S., Acute bacterial meningitis in infants and children, Lancet Infect. Dis., 2010, vol. 10, pp. 32–42.

    Article  CAS  PubMed  Google Scholar 

  21. Ghotaslou, R., Farajnia, S., Yeganeh, E., et al., Detection of acute childhood meningitis by PCR, culture and agglutination tests in Tabriz, Iran, Acta Med. Iran, 2012, vol. 50, pp. 192–196.

    Google Scholar 

  22. Brouwer, M.C., Tunkel, A.R., and van de Beek, D., Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis, Clin. Microbiol. Rev., 2010, vol. 23, pp. 467–492.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kleines, M., Scheithauer, S., Schiefer, J., et al., Application of viral cerebrospinal fluid PCR testing for diagnosis of central nervous system disorders: a retrospective 11-year experience, Diag. Micr. Infect. Dis., 2014, vol. 80, pp. 207–215.

    Article  CAS  Google Scholar 

  24. Bøving, M.K., Pedersen, L.N., and Møller J.K., Eightplex PCR and liquid-array detection of bacterial and viral pathogens in cerebrospinal fluid from patients with suspected meningitis, J. Clin. Microbiol., 2009, vol. 47, pp. 908–913.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Chiba, N., Murayama, S.Y., Morozumi, M., et al., Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR, J. Infect. Chemother., 2009, vol. 15, pp. 92–98.

    Article  CAS  PubMed  Google Scholar 

  26. Thigpen, M.C., Whitney, C.G., Messonnier, N.E., et al., Bacterial meningitis in the United States, 1998–2007, New Engl. J. Med., 2011, vol. 364, pp. 2016–2025.

    Article  CAS  PubMed  Google Scholar 

  27. Shin, S.Y., Kwon, K.C., Park, J.W., et al., Evaluation of the Seeplex® Meningitis ACE Detection kit for the detection of 12 common bacterial and viral pathogens of acute meningitis, Ann. Lab. Med., 2012, vol. 32, pp. 44–49.

    Article  CAS  PubMed  Google Scholar 

  28. Liu, B., Zhou, L., Wang, Q., et al., A mutation-sensitive switch assay to detect five clinically significant epidermal growth factor receptor mutations, Genet. Test Mol. Bioma, 2015, vol. 19, pp. 316–323.

    Article  CAS  Google Scholar 

  29. Elnifro, E.M., Ashshi, A.M., Cooper, R.J., et al., Multiplex PCR: optimization and application in diagnostic virology, Clin. Microbiol. Rev., 2000, vol. 13, pp. 559–570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Onodera, K., d’Offay, J., and Melcher, U., Nylon membrane-immobilized PCR for detection of bovine viruses, Biotechniques, 2002, vol. 32, pp. 74–76.

    CAS  PubMed  Google Scholar 

  31. Henegariu, O., Heerema, N.A., Dlouhy, S.R., et al., Multiplex PCR: critical parameters and step-by-step protocol, Biotechniques, 1997, vol. 23, pp. 504–511.

    CAS  PubMed  Google Scholar 

  32. Wei, T., Lu, G., and Clover, G., Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses, J. Virol. Methods, 2009, vol. 151, pp. 132–139.

    Article  Google Scholar 

  33. Lee, C., Wu, J.S., Shiue, Y.L., et al., MultiPrimer, Appl. Bioinf., 2006, vol. 5, pp. 99–109.

    Article  CAS  Google Scholar 

  34. Piriyapongsa, J., Ngamphiw, C., Assawamakin, A., et al., RExPrimer: an integrated primer designing tool increases PCR effectiveness by avoiding 3'SNP-inprimer and mis-priming from structural variation, BMC Genomics, 2009, vol. 10, p. S4.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Shen, Z.Y., Qu, W.B., Wang, W., et al., MPprimer: a program for reliable multiplex PCR primer design, BMC Bioinf., 2010, vol. 11, p. 143.

    Article  Google Scholar 

  36. Brownie, J., Shawcross, S., Theaker, J., et al., The elimination of primer-dimer accumulation in PCR, Nucleic Acids Res., 1997, vol. 25, pp. 3235–3241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hecker, K.H. and Roux, K.H., High and low annealing temperatures increase both specificity and yield in touchdown and step down PCR, Biotechniques, 1996, vol. 20, pp. 478–485.

    CAS  PubMed  Google Scholar 

  38. Favaro, M., Savini, V., Favalli, C., et al., A multi-target real-time PCR assay for rapid identification of meningitis-associated microorganisms, Mol. Biotechnol., 2013, vol. 53, pp. 74–79.

    Article  CAS  PubMed  Google Scholar 

  39. Zhu, H.F., Wang, Q., Wen, L.Q., et al., Development of a multiplex PCR assay for detection and genogrouping of Neisseria meningitides, J. Clin. Microbiol., 2012, vol. 50, pp. 46–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author notes

  1. Q.Q. Duan and S.Q. Lu contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Diagnostics, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China

    Q. Q. Duan, S. N. Shen, B. S. Xi, X. N. Wang & W. P. Sun

  2. Emergency Department, The First Affiliated Hospital of Soochow University, Suzhou, China

    S. Q. Lu

  3. Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China

    Y. X. Hu

Authors
  1. Q. Q. Duan
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  2. S. Q. Lu
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  3. Y. X. Hu
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  4. S. N. Shen
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Correspondence to W. P. Sun.

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Duan, Q.Q., Lu, S.Q., Hu, Y.X. et al. A Multiplex PCR Assay Mediated by Universal Primer for the Diagnosis of Human Meningitis Caused by Six Common Bacteria. Russ J Genet 54, 423–430 (2018). https://doi.org/10.1134/S1022795418040075

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  • DOI: https://doi.org/10.1134/S1022795418040075

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