Skip to main content

Advertisement

SpringerLink
Log in

Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl2 concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10−5 to 6 × 10−7 ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.

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

Similar content being viewed by others

References

  1. Markoulatos, P., Georgopoulou, A., Siafakas, N., Plakokefalos, E., Tzanakaki, G., & Kourea-Kremastinou, J. (2001). Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay. Journal of Clinical Microbiology, 39, 4426–4432.

    Article  CAS  Google Scholar 

  2. Kubota, N., Wada, K., Ito, Y., Shimoyama, Y., Nakamura, S., Nishiyama, Y., et al. (2008). One-step multiplex real-time PCR assay to analyse the latency patterns of Epstein-Barr virus infection. Journal of Virological Methods, 147, 26–36.

    Article  CAS  Google Scholar 

  3. Caballero, O. L., Menezes, C. L. P., Costa, M. C. S. L., Fernandes, S. C., Anacleto, T. M., de Manoel Oliveira, R., et al. (1997). Highly sensitive single step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients. Journal of Clinical Microbiology, 35, 3192–3197.

    CAS  Google Scholar 

  4. Ogawa, H., Suzutani, T., Baba, Y., Koyano, S., Nozawa, N., Ishibashi, K., et al. (2007). Etiology of severe sensorineural hearing loss in children: Independent impact of congenital cytomegalovirus infection and GJB2 mutations. Journal of Infectious Diseases, 195, 782–788.

    Article  CAS  Google Scholar 

  5. Lima, H. E., Oliveira, M. B., Valente, B. G., Afonso, D. A. F., Darocha, W. D., Souza, M. C. M., et al. (2007). Genotyping of Chlamydia trachomatis from endocervical specimens in Brazil. Sexually Transmitted Diseases, 34, 709–717.

    Article  CAS  Google Scholar 

  6. Joseph, P., Calderon, M. M., Gilman, R. H., Quispe, M. L., Cok, J., Ticona, E., et al. (2002). Optimization and evaluation of a PCR assay for detecting toxoplasmic encephalitis in patients with AIDS. Journal of Clinical Microbiology, 40, 4499–4503.

    Article  CAS  Google Scholar 

  7. Speers, D. J. (2006). Clinical applications of molecular biology for infectious diseases. Clinical Biochemical Review, 27, 39–51.

    Google Scholar 

  8. Switaj, K., Master, A., Skrzypczak, M., & Zaborowski, P. (2005). Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clinical Microbiology & Infection, 11, 170–176.

    Article  CAS  Google Scholar 

  9. Barken, K. B., Haagensen, J. A. J., & Tolker-Nielsen, T. (2007). Advances in nucleic acid-based diagnostics of bacterial infections. Clinica Chimica Acta, 384, 1–11.

    Article  CAS  Google Scholar 

  10. Ratcliff, R. M., Chang, G., Kok, T., & Sloots, T. P. (2007). Molecular diagnosis of medical viruses. Current Issues in Molecular Biology, 9, 87–102.

    CAS  Google Scholar 

  11. Elnifro, E. M., Cooper, R. J., Klapper, P. E., Yeo, A. C., & Tullo, A. B. (2000). Multiplex polymerase chain reaction for diagnosis of viral and chlamydial keratoconjunctivitis. Investigative Ophthalmology and Visual Science, 41, 1818–1822.

    CAS  Google Scholar 

  12. Elnifro, E. M., Ashshi, A. M., Cooper, R. J., & Kapper, P. E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clinical Microbiology Reviews, 13, 559–570.

    Article  CAS  Google Scholar 

  13. Sambrook, J., Fritisch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (p. 626). New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.

    Google Scholar 

  14. Nishiwaki, M., Yamamoto, T., Tone, S., Murai, T., Ohkawara, T., Matsunami, T., et al. (2008). Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications. Journal of Clinical Microbiology, 46, 1161–1168.

    Article  CAS  Google Scholar 

  15. Sanguinetti, C. J., Dias Neto, E., & Simpson, A. J. (1994). Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques, 17(5), 914–921.

    CAS  Google Scholar 

  16. Sanger, F., & Coulson, A. R. (1975). A rapid method for de termining sequences in DNA by primed synthesis with DNA polymerase. Journal of Molecular Biology, 94, 441–448.

    Article  CAS  Google Scholar 

  17. McIver, C. J., Jacques, C. F. H., Chow, S. S. W., Munro, S. C., Scott, G. M., Roberts, J. A., et al. (2005). Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections. Journal of Clinical Microbiology, 43, 5102–5110.

    Article  CAS  Google Scholar 

  18. Tanaka, T., Kogawa, K., Sasa, H., Nonoyama, S., Furuya, K., & Sato, K. (2009). Rapid and simultaneous detection of 6 types of human herpes virus (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus human herpes virus 6A/B, and human herpes virus 7) by multiplex PCR assay. Biomedical Research, 30, 279–285.

    Article  CAS  Google Scholar 

  19. Schaade, L., Kockelkorn, P., Ritter, K., & Kleines, M. (2000). Detection of cytomegalovirus DNA in human specimens by LightCycler PCR. Journal of Clinical Microbiology, 38, 4006–4009.

    Google Scholar 

Download references

Acknowledgments

This study was supported by Grants from the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).

Author information

Authors and Affiliations

  1. Departamento de Pesquisa e Desenvolvimento, Biocod Biotecnologia Ltda, Belo Horizonte, MG, Brazil

    Danielle A. G. Zauli, Carla Lisandre Paula de Menezes & Cristiane Lommez de Oliveira

Authors
  1. Danielle A. G. Zauli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carla Lisandre Paula de Menezes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cristiane Lommez de Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Danielle A. G. Zauli.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zauli, D.A.G., de Menezes, C.L.P. & de Oliveira, C.L. Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus. Mol Biotechnol 54, 1004–1009 (2013). https://doi.org/10.1007/s12033-013-9652-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-013-9652-x

Keywords

Search

Navigation