Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology

Authors

  • Kordo B.A. Saeed Royal Hampshire County Hospital, Winchester, UK.
  • Nusreen S. Ahmad Health Protection Agency and Southampton University Hospitals Foundation Trust, Southampton, UK.

DOI:

https://doi.org/10.5195/ijms.2013.22

Keywords:

Real-Time Polymerase Chain Reaction, Reagent Kits, Diagnostic, Molecular Diagnostic Techniques

Abstract

The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. Real-Time PCR (RT-PCR) is becoming the gold standard test for accurate, sensitive and fast diagnosis for a large range of infectious agents. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, high throughout and quantification. RT-PCR assays have advanced the diagnostic abilities of clinical laboratories particularly microbiology and infectious diseases. In this review we would like to briefly discuss RT-PCR in diagnostic microbiology laboratory, beginning with a general introduction to RT-PCR and its principles, setting up an RT-PCR, including multiplex systems and the avoidance and remediation of contamination issues. A segment of the review would be devoted to the application of RT-PCR in clinical practice concentrating on its role in the diagnosis and treatment of infectious diseases.

Metrics

Metrics Loading ...

References

1. Espy MJ, Uhl JR, Sloan LM, et al. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin Microbiol Rev 2006;19(1):165-256.
2. Swift GH, Peyton MJ, MacDonald RJ. Assessment of RNA quality by semiquantitative RT-PCR of multiple regions of a long ubiquitous mRNA. Biotechniques 2000; 28(3):524-31.
3. Mannhalter C, Koizar D, Mitterbauer G. Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA. Clin Chem Lab Med 2000; 38(2):171-7.
4. Saeed K, Ahmad N, Pallett A, Guiver M, Marsh P. Specific staphylococcal polymerase chain reaction can be a complementary tool for identifying causative organisms and guiding antibiotic management in orthopaedic infections. Curr Orthop Pract 2010; 21(6):628-31.
5. Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998; 36(5):255-69.
6. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25(2):169-93.
7. Pfaffl MW, Hageleit M. Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechn Lett 2001; 23:275-282.
8. Abdul-Careem MF, Hunter BD, Nagy E, et al. Development of a real-time PCR assay using SYBR Green chemistry for monitoring Marek’s disease virus genome load in feather tips. J Virol Methods 2006; 133(1):34-40.
9. Comprehensive list of vendors. Accessed at various time points. Available from: <http://www.appliedbiosystems.com>, <http://www.bioneer.co.kr>,
<http://discover.bio-rad.com>, <http://www.eppendorf.com/mastercycler/index.html>, <http://www.stratagene.com>, <http://www.techne.com/CatMol/quantica.htm>, <http://www.appliedbiosystems.com>, <http://www.roche-applied-science.com>, <http://www.biotrove.com>, <http://www.corbettresearch.com)>, <http://www.cepheid.com>, <http://www.biobank.co.kr/pcr/insyte.shtml>, <http://www.fluidigm.com>, <http://www.alphahelix.com/pages/superconvector.html>,<http://www.dnatechnology.com/catalog/ob_dt322_en.shtml>,<http://www.bioer.com.cn/en/shengming_66pcr.htm>,.
10. Premier Biosoft International. Accessed on 24.10.2011. Available from: <http://www.premierbiosoft.com/tech_notes/real_time_PCR.html> and Primer Bank: PCR Primers for Gene Expression Detection and Quantification. available from: <http://pga.mgh.harvard.edu/primerbank/>.
11. Protocol online. Accessed on 24.10.2011. Available from: <http://www.protocol-online.org/prot/Molecular_Biology/PCR/Real-Time_PCR/>.
12. The Gene Quantification page: Summarises all technical aspects involved in quantitative gene expression analysis using RT-PCR. It presents a lot of new and innovative PCR applications, chemistries, methods, algorithms, QC strategies, cyclers, kits, dyes, analysis methods, events, and services involved. Accessed on 24.10.2011. Available from: <http://www.genequantification.info/>.
13. Dennis Lo YM. Setting up a PCR laboratory. Methods mol med 1998; 16:11-7.
14. Corradini P, Astolfi M, Cherasco C, et al. Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografting. Blood. 1997;89:724-731.
15. van Dongen J, Macintyre EA, Gabert JA, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 concerted action investigation of minimal residual disease in acute leukemia. Leukemia 1999;13(12):1901-28.
16. Mandigers CM, Meijerink JP, Raemaekers JM, Schattenberg AV, Mensink EJ. Graft-versus-lymphoma effect of donor leucocyte infusion shown by real-time quantitative PCR analysis of t(14;18). Lancet 1998; 352(9139):1522-3.
17. Bustin S A and Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci (Lond) 2005;109(4):36579.
18. Kaltenboeck B, Wang C. Advances in real-time PCR: application to clinical laboratory diagnostics. Adv Clin Chem 2005; 40: 219-59.
19. Helb D, Jones M, Story E, et al. Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, NearPatient Technology. J Clin Microbiol 2010;48:229-37.
20. Jonas D, Speck M, Daschner FD, et al. Rapid PCR-based identification of methicillin-resistant Staphylococcus aureus from screening swabs. J Clin Microbiol. 2002; 40:1821--1823.
21. Department of Health/ UK. Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infection (ARHAI). UPDATED GUIDANCE ON THE DIAGNOSIS AND REPORTING OF CLOSTRIDIUM DIFFICILE. First published March 2012 Published to DH website, in electronic PDF format only. Available at: www.dh.gov.uk/publications.
22. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 2001; 39(4):1553-8.
23. Zeaiter Z, Fournier PE, Greub G, Raoult D. Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum. J. Clin. Microbiol 2003; 41:919–925.
24. Chan EL, Antonishyn N, McDonald R, et al. The use of TaqMan PCR assay for detection of Bordetella pertussis infection from clinical specimens. Arch Pathol Lab Med 2002; 126:173–176.
25. Fenollar F, Fournier PE, Raoult D, Gérolami R, Lepidi H, Poyart C. Quantitative detection of Tropheryma whipplei DNA by realtime PCR. J Clin Microbiol 2002;40(3):1119-20.
26. Pietila J, He Q, Oksi J, Viljanen MK. Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J. Clin. Microbiol 2000; 38:2756–2759.
27. Saeed K, Pelosi E. Comparison between turnaround time and cost of herpes simplex virus testing by cell culture and polymerase chain reaction from genital swabs. Int J STD AIDS. 2010;21(4):298-9.
28. Nigrovic LE, Chiang VW. Cost analysis of enteroviral polymerase chain reaction in infants with fever and cerebrospinal fluid pleocytosis. Arch Pediatr Adolesc Med. 2000;154(8):817-21.
29. Mahony J, Chong S, Merante F, et al. 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(9):2965-70.
30. Shu B, Wu KH, Emery S, Villanueva J, et al. Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus. J Clin Microbiol. 2011;49(7):2614-9.
31. Junhui Zhai, Thomas Briese, Erhei Dai, et al. Real-Time Polymerase Chain Reaction for Detecting SARS Coronavirus, Beijing, 2003. Emerging Infectious Disease Journal. 2004;10(2):300-303.
32. Mengelle C, Pasquier C, Rostaing L. Quantitation of human cytomegaloviruse in recepiant of solid organ transplant by real-time Quantitative PCR. J Med Virol 2003;69(2):225-31.
33. Gulley ML. Molecular diagnosis of Epstein-Barr virus-related diseases. J Mol Diagn 2001;3(1):1-10.
34. Havlir DV, Bassett R, Levitan D, et al. Prevalence and predictive value of intermittent viremia with combination hiv therapy. JAMA 2001;286(2):171-9.
35. Smith CJ, Staszewski S, Sabin CA, et al. Use of viral load measured after 4 weeks of highly active antiretroviral therapy to predict virologic outcome
at 24 weeks for HIV-1-positive individuals. J Acquir Immune Defic Syndr 2004;37(1):1155-9.
36. Garson JA, Grant PR, Ayliffe U, Ferns RB, Tedder RS. Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control. J Virol Methods 2005;126(1-2):207-13.
37. Department of health/ Uk. Guidance. Hepatitis B infected healthcare workers and antiviral therapy. 16 March 2007. Available at: http://www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/DH_073164.
38. Farrugia C, Cabaret O, Botterel F, et al. Cytochrome b Gene Quantitative PCR for Diagnosing Plasmodium falciparum Infection in Travelers. J Clin Microbiol 2011;49(6):2191-5.
39. Berry A, Benoit-Vical F, Fabre R, et al. PCR-based methods to the diagnosis of imported malaria. Parasite. 2008;15:484–488.
40. Mei-Hui L, Tse-Ching C, Tseng-tong K, et al. Real-Time PCR for Quantitative Detection ofToxoplasma gondii. J. Clin. Microbiol. November 2000;38(11):4121-5.
41. Piron M, Fisa R, Casamitjana N, López-Chejade P, et al. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop. 2007;103(3):195-200.
42. Teal AE, Habura A, Ennis J, Keithly JS, Madison-Antenucci S. A new realtime PCR assay for improved detection of the parasite Babesia microti. J Clin Microbiol. 2012;50(3):903-8.
43. Wortmann G, Hochberg L, Houng HH, Sweeney C, et al. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg. 2005;73(6):999-1004.
44. Verweij JJ, Blange RA, Templeton K, et al. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 2004;42:1220–23.
45. Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R. Real-time-PCR assay for diagnosis of Entamoeba histolytica infection. J Clin Microbiol 2005;43(5):2168-72.
46. Costa C, Vidaud D, Olivi M, Bart-Delabesse E, Vidaud M, Bretagne S. Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum. J Microbiol Methods 2001; 44:263–9.
47. Veron V, Simon S, Blanchet D, Aznar C. Real-time polymerase chain reaction detection of Cryptococcus neoformans and Cryptococcus gattii in human samples. Diagn Microbiol Infect Dis 2009;65(1):69-72.
48. Botterel F, Cabaret O, Foulet F, Cordonnier C, Costa JM, Bretagne S. Clinical significance of quantifying Pneumocystis jirovecii DNA by using real-time PCR in bronchoalveolar lavage fluid from immunocompromised patients. J Clin Microbiol 2012 ;50(2):227-31.

Downloads

Published

2013-04-30

How to Cite

Saeed, K. B., & Ahmad, N. S. (2013). Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology. International Journal of Medical Students, 1(1), 28–36. https://doi.org/10.5195/ijms.2013.22

Issue

Vol. 1 No. 1 (2013)

Section

Review

Categories

License

  1. The Author retains copyright in the Work, where the term “Work” shall include all digital objects that may result in subsequent electronic publication or distribution.
  2. Upon acceptance of the Work, the author shall grant to the Publisher the right of first publication of the Work.
  3. The Author shall grant to the Publisher and its agents the nonexclusive perpetual right and license to publish, archive, and make accessible the Work in whole or in part in all forms of media now or hereafter known under a Creative Commons Attribution 4.0 International License or its equivalent, which, for the avoidance of doubt, allows others to copy, distribute, and transmit the Work under the following conditions:
    1. Attribution—other users must attribute the Work in the manner specified by the author as indicated on the journal Web site; with the understanding that the above condition can be waived with permission from the Author and that where the Work or any of its elements is in the public domain under applicable law, that status is in no way affected by the license.
    2. The Author is able to enter into separate, additional contractual arrangements for the nonexclusive distribution of the journal's published version of the Work (e.g., post it to an institutional repository or publish it in a book), as long as there is provided in the document an acknowledgment of its initial publication in this journal.
    3. Authors are permitted and encouraged to post online a prepublication manuscript (but not the Publisher’s final formatted PDF version of the Work) in institutional repositories or on their Websites prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work. Any such posting made before acceptance and publication of the Work shall be updated upon publication to include a reference to the Publisher-assigned DOI (Digital Object Identifier) and a link to the online abstract for the final published Work in the Journal.
    4. Upon Publisher’s request, the Author agrees to furnish promptly to Publisher, at the Author’s own expense, written evidence of the permissions, licenses, and consents for use of third-party material included within the Work, except as determined by Publisher to be covered by the principles of Fair Use.
    5. The Author represents and warrants that:
      1. the Work is the Author’s original work;
      2. the Author has not transferred, and will not transfer, exclusive rights in the Work to any third party;
      3. the Work is not pending review or under consideration by another publisher;
      4. the Work has not previously been published;
      5. the Work contains no misrepresentation or infringement of the Work or property of other authors or third parties; and
      6. the Work contains no libel, invasion of privacy, or other unlawful matter.
    6. The Author agrees to indemnify and hold Publisher harmless from the Author’s breach of the representations and warranties contained in Paragraph 6 above, as well as any claim or proceeding relating to Publisher’s use and publication of any content contained in the Work, including third-party content.

Enforcement of copyright

The IJMS takes the protection of copyright very seriously.

If the IJMS discovers that you have used its copyright materials in contravention of the license above, the IJMS may bring legal proceedings against you seeking reparation and an injunction to stop you using those materials. You could also be ordered to pay legal costs.

If you become aware of any use of the IJMS' copyright materials that contravenes or may contravene the license above, please report this by email to contact@ijms.org

 

Infringing material

If you become aware of any material on the website that you believe infringes your or any other person's copyright, please report this by email to contact@ijms.org