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. 2004 Jan 9;72(3):484–495. doi: 10.1002/jmv.20008

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested‐PCR assays

MT Coiras 1,✉, JC Aguilar 1, ML García 1, I Casas 1, P Pérez‐Breña 1
PMCID: PMC7166637  PMID: 14748074

Abstract

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT‐nested PCR assay has been used widely for simultaneous detection of non‐related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT‐PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT‐PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT‐PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population. J. Med. Virol. 72:484–495, 2004. © 2004 Wiley‐Liss, Inc.

Keywords: multiplex RT‐PCR assay, diagnosis of respiratory infection, parainfluenza virus, human coronavirus, enterovirus, rhinovirus

REFERENCES

  1. Aguilar J, Pérez P, García ML, Cruz N, Erdman D, Echevarría JE. 2000. Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription‐PCR. J Clin Microbiol 38: 1191–1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andréoletti L, Lesay M, Deschildre A, Lambert V, Dewilde A, Wattré P. 2000. Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis. J Med Virol 61: 341–346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Apalsch AM, Green M, Ledesma‐Medina J, Nour B, Wald ER. 1995. Parainfluenza and influenza virus infections in pediatric organ transplant recipients. Clin Infect Dis 21: 394–399. [DOI] [PubMed] [Google Scholar]
  4. Arbour N, Day R, Newcombe J, Talbot PJ. 2000. Neuroinvasion by human respiratory coronaviruses. J Virol 74: 8913–8921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arola M, Ruuskanen O, Ziegler T, Salmi TT. 1995. Respiratory viral infections during anticancer treatment on children. Pediatr Infect Dis J 14: 690–694. [DOI] [PubMed] [Google Scholar]
  6. Arola A, Santti J, Ruuskanen O, Halonen P, Hyypia T. 1996. Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis. J Clin Microbiol 34: 313–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arruda E, Pitkaranta A, Witek TJ Jr, Doyle CA, Hayden FG. 1997. Frequency and natural history of rhinovirus infections in adults during autumn. J Clin Microbiol 35: 2864–2868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Atmar RL, Georghiou PR. 1993. Classification of respiratory tract Picornavirus isolates as Enteroviruses and Rhinoviruses by using reverse transcription polymerase chain reaction. J Clin Microbiol 31: 2544–2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Atmar RL, Baxter BD, Dominguez EA, Taber LH. 1996. Comparison of reverse transcription‐PCR with tissue culture and other rapid diagnostic assays for detection of type A influenza virus. J Clin Microbiol 34: 2604–2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ballabio A, Ranier JE, Chamberlain JS, Zollo M, Caskey CT. 1990. Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification. Hum Genet 84: 571–573. [DOI] [PubMed] [Google Scholar]
  11. Casas I, Powell L, Klapper PE, Cleator GM. 1995. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction. J Virol Methods 53: 25–36. [DOI] [PubMed] [Google Scholar]
  12. Casas I, Pozo F, Trallero G, Echevarria JM, Tenorio A. 1999. Viral diagnosis of neurological infection by RT multiplex PCR: A search for entero‐ and herpesviruses in a prospective study. J Med Virol 57: 145–151. [DOI] [PubMed] [Google Scholar]
  13. Coiras MT, Pérez‐Breña P, García ML, Casas I. 2003. Simultaneous detection of influenza A, B, and C viruses, respiratory syncitial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested‐PCR assay. J Med Virol 69: 132–144. [DOI] [PubMed] [Google Scholar]
  14. Dieffenbach CW, Lowe TMJ, Dveksler GS. 1993. General concepts for primer design. PCR Methods Appl 3: S30–S37. [DOI] [PubMed] [Google Scholar]
  15. Dowell SF, Anderson LJ, Gary HE, Erdman DD, Plouffe JF, File TM, Marston BJ, Breiman RF. 1996. Respiratory syncytial virus is an important cause of community‐acquired lower respiratory infection among hospitalised adults. J Infect Dis 174: 456–462. [DOI] [PubMed] [Google Scholar]
  16. Edwards MC, Gibbs RA. 1994. Multiplex PCR: Advantages, development, and applications. PCR Methods Appl 3: S65–S75. [DOI] [PubMed] [Google Scholar]
  17. Ellis L, Fleming DM, Zambon MC. 1997. Multiplex reverse transcription‐PCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996. J Clin Microbiol 35: 2076–2082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eugene‐Ruellan G, Freymuth F, Bahloul C, Badrane H, Vabret A, Tordo N. 1998. Detection of respiratory syncytial virus A and B and parainfluenza virus 3 sequences in respiratory tracts of infants by a single PCR with primers targeted to the L‐polymerase gene and differential hybridization. J Clin Micronbiol 36: 796–801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fan J, Henrickson KJ, Savatski LL. 1998. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription‐polymerase chain reaction‐enzyme hybridisation assay (Hexaplex). Clin Infect Dis 26(6): 1397–1402. [DOI] [PubMed] [Google Scholar]
  20. Fleming DM, Cross KW. 1993. Respiratory syncytial virus or influenza? Lancet 342: 1507–1510. [DOI] [PubMed] [Google Scholar]
  21. Freymuth F, Vabret A, Galateau‐Salle F, Ferey J, Eugene G, Petitjean J, Gennetay E, Brouard J, Jokik M, Duhamel J, Guillois B. 1997. Detection of respiratory syncytial virus, parainfluenza 3, adenovirus, and rhinovirus sequences in respiratory tract of infants by polymerase chain reaction and hybridization. Clin Diagn Virol 8: 31–40. [DOI] [PubMed] [Google Scholar]
  22. Garcia Garcia ML, Ordobas Gabin M, Calvo Reya C, Gonzalez Alvarez M, Aguilar Ruiz J, Arregui Sierra A, Perez Breña P. 2001. Viral infection of the lower respiratory tract in hospitalized infants: Etiology, clinical features, and risk factors. An Esp Pediatr 55: 101–107. [PubMed] [Google Scholar]
  23. García García ML, Aguilar Ruíz J, Echevarría Mayo JE, Calvo Rey C, Pinto Fuentes I, Ordobás Gabin M, Roman Riechmann E, Pérez Breña P. 2002. Parainfluenza virus type 4 infections. An Esp Pediatr 57: 116–120. [PubMed] [Google Scholar]
  24. Gilbert LL, Dakhama A, Bone BM, Thomas EE, Hegele RG. 1996. Diagnosis of viral respiratory tract infections in children by using a reverse transcription‐PCR panel. J Clin Microbiol 34: 140–143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Griner PF, Mayewski RJ, Mushlin AI, Greenland P. 1981. Selection and interpretation of diagnostic tests and procedures. Ann Inter Med 94: 553–600. [PubMed] [Google Scholar]
  26. Gröndahl B, Puppe W, Hoppe A, Kuhne I, Weigl JA, Schmitt HJ. 1999. Rapid identification of nine microorganisms causing acute respiratory tract infections by single‐tube multiplex reverse transcription‐PCR: Feasibility study. J Clin Microbiol 37: 1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gwaltney JM. 1980. Epidemiology of the common cold. Ann NY Acad Sci 353: 54–690. [DOI] [PubMed] [Google Scholar]
  28. Halonen P, Rocha EJ, Hierholzer J, Holloway B, Hyypia T, Hurskainen P, Pallansch M. 1995. Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid‐phase hybridisation. J Clin Microbiol 33: 648–653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. 1997. Multiplex PCR: Critical parameters and step‐by‐step protocol. BioTechniques 23: 504–511. [DOI] [PubMed] [Google Scholar]
  30. Hruskova J, Heinz F, Svandova E, Pennigerova S. 1990. Antibodies to human coronaviruses 229E and OC43 in the population of C.R. Acta Virologica 34: 346–352. [PubMed] [Google Scholar]
  31. Huttunen P, Santti J, Pulli T, Hyypia T. 1996. The major echovirus group is genetically coherent and related to coxsackie B viruses. J Gen Virol 77: 715–725. [DOI] [PubMed] [Google Scholar]
  32. Hyypia T, Puhakka T, Ruuskanen O, Makela M, Arola A, Arstila P. 1998. Molecular diagnosis of human rhinovirus infections: Comparison with virus isolation. J Clin Microbiol 36: 2081–2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Johnson SL, Sanderson G, Pattemore PK, Smith S, Bardin PG, Bruce CB, Lambden PR, Tyrrell DAJ, Holgate ST. 1993. Use of polymerase chain reaction for diagnosis of Picornavirus infection in subjects with and without respiratory symptoms. J Clin Microbiol 31: 111–117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, Symington P, O'Toole S, Myint SH, Tyrrell DA et al. 1995. Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. BMJ 13: 1225–1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kehl SC, Henrickson KJ, Hua W, Fan J. 2001. Evaluation of the Hexaplex assay for detection of respiratory viruses in children. J Clin Microbiol 39: 1696–1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kendal AP, Pereira MS, Skehel JJ. 1982. Concepts and procedures for laboratory‐based influenza surveillance. WHO Collaborating Centers for Reference and Research on Influenza.
  37. Liolios L, Jenney A, Spelman D, Kotsimbos T, Catton M, Wesselingh S. 2001. Comparison of a multiplex reverse transcription‐PCR‐enzyme hybridisation assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens. J Clin Microbiol 39: 2779–2783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Macnaughton MR. 1982. Occurrence and frequency of coronavirus infections in humans as determined by enzyme‐linked immunosorbent assay. Infect Immun 38: 419–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McIntosh K, Kapikian AZ, Turner HC, Hartley JW, Parrott RH, Chanock RM. 1970. Seroepidemiologic studies of coronavirus infection in adults and children. Am J Epidemiol 91: 585–592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. McMillan JA, Weiner LB, Higgins AM, Macknight K. 1993. Rhinovirus infection associated with serious illness among pediatric patients. Pediatr Infect Dis J 12: 321–325. [DOI] [PubMed] [Google Scholar]
  41. Myint S. 2002. Recent advances in the rapid diagnosis of respiratory tract infection. Br Med Bull 61: 97–114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nicholson KG, Kent J, Ireland DC. 1993. Respiratory viruses and exacerbations of asthma in adults. Brit Med J 307: 982–986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Osiowy C. 1998. Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription‐PCR assay. J Clin Microbiol 36: 3149–3154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tantivanich S, Suphanaranonda K, Balachanda K, Anderson R. 1995. Detection of respiratory syncytial virus from clinical specimens: Comparison between reverse transcription polymerase chain reaction and tissue culture. Southeast Asian J Trop Med Public Health 26: 684–688. [PubMed] [Google Scholar]
  45. Trallero G, Casas I, Tenorio A, Echevarria JE, Castellanos A, Lozano A, Pérez‐Breña P. 2000. Enteroviruses in Spain: Virological and epidemiological studies over 10 years (1988–97). Epidemiol Infect 124: 497–506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Valassina M, Cuppone AM, Cusi MG, Valensin PE. 1997. Rapid detection of different RNA respiratory virus species by multiplex RT‐PCR, application to clinical specimens. Clin Diagn Virol 8: 227–232. [DOI] [PubMed] [Google Scholar]
  47. Woo PC, Chiu SS, Seto WH, Peiris M. 1997. Cost‐effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients. J Clin Microbiol 35: 1579–1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yun BY, Kim MR, Park JY, Choi EH, Lee HJ, Yun CK. 1995. Viral etiology and epidemiology of acute lower respiratory tract infections in Korean children. Pediatr Infect Dis 14: 1054–1059. [DOI] [PubMed] [Google Scholar]

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