Advances in Real-time PCR: Application to Clinical Laboratory Diagnostics

Overview

Journal Adv Clin Chem

Specialty Biochemistry

Date 2005 Dec 17

PMID 16355924

Citations 42

Authors

Bernhard Kaltenboeck

Chengming Wang

Affiliations

Soon will be listed here.

Abstract

The polymerase chain reaction (PCR) has become one of the most important tools in molecular diagnostics, providing exquisite sensitivity and specificity for detection of nucleic acid targets. Real-time monitoring of PCR has simplified and accelerated PCR laboratory procedures and has increased information obtained from specimens including routine quantification and differentiation of amplification products. Clinical diagnostic applications and uses of real-time PCR are growing exponentially, real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses likely will emerge. This review analyzes the scope of present and potential future clinical diagnostic applications of this powerful technique. Critical discussions focus on basic concepts, variations, data analysis, instrument platforms, signal detection formats, sample collection, assay design, and execution of real-time PCR.

Citing Articles

Development and Assessment of a Multiple-Analysis System for Diagnosing Malaria and Other Blood Parasite Infections in Humans and Non-Human Primates.

Ceballos-Caro A, Anton-Berenguer V, Lanza M, Renelies-Hamilton J, Barciela A, Koster P Diagnostics (Basel). 2025; 15(5).

PMID: 40075867 PMC: 11899344. DOI: 10.3390/diagnostics15050620.

TRP36-ELISA for detection: Concordance with TaqMan real-time PCR and point-of-care testing.

Khumtub K, Sumpavong P, Satchasataporn K, Fa-Ngoen C, Kaewmongkol S, Kaewmongkol G Heliyon. 2024; 10(21):e39652.

PMID: 39524768 PMC: 11550044. DOI: 10.1016/j.heliyon.2024.e39652.

Dual-Emission Fluorescence Resonance Energy Transfer (FRET) PCR Discriminates Pullorum and Gallinarum.

Gong J, Iduu N, Zhang D, Chenoweth K, Wei L, Yang Y Microorganisms. 2024; 12(9).

PMID: 39338489 PMC: 11433795. DOI: 10.3390/microorganisms12091815.

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention.

Omar R, Boissinot M, Huletsky A, Bergeron M Infect Dis Rep. 2024; 16(2):216-227.

PMID: 38525764 PMC: 10961803. DOI: 10.3390/idr16020017.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Huang K, Zhang J, Li J, Qiu H, Wei L, Yang Y Genes (Basel). 2024; 15(2).

PMID: 38397205 PMC: 10888005. DOI: 10.3390/genes15020215.

References

Wittwer C, Ririe K, Andrew R, David D, Gundry R, Balis U . The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques. 1997; 22(1):176-81. DOI: 10.2144/97221pf02. View

Al-Soud W, Jonsson L, Radstrom P . Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol. 2000; 38(1):345-50. PMC: 88721. DOI: 10.1128/JCM.38.1.345-350.2000. View

Fuchs B, Zhang K, Rock M, Bolander M, Sarkar G . High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR. Mol Biotechnol. 2000; 12(3):237-40. DOI: 10.1385/MB:12:3:237. View

Kim K, Seo J, Wheeler K, Park C, Kim D, Park S . Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis. FEMS Immunol Med Microbiol. 2005; 43(2):301-10. DOI: 10.1016/j.femsim.2004.10.005. View

Hoebeeck J, van der Luijt R, Poppe B, Smet E, Yigit N, Claes K . Rapid detection of VHL exon deletions using real-time quantitative PCR. Lab Invest. 2004; 85(1):24-33. DOI: 10.1038/labinvest.3700209. View

Bustin S . Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000; 25(2):169-93. DOI: 10.1677/jme.0.0250169. View

Espy M, Uhl J, Sloan L, Rosenblatt J, Cockerill 3rd F, Smith T . Detection of vaccinia virus, herpes simplex virus, varicella-zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents. Mayo Clin Proc. 2002; 77(7):624-8. DOI: 10.4065/77.7.624. View

Spanakis E . Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls. Nucleic Acids Res. 1993; 21(16):3809-19. PMC: 309896. DOI: 10.1093/nar/21.16.3809. View

Yu A, Lau L, Fung Y . Boosting the sensitivity of real-time polymerase-chain-reaction testing for SARS. N Engl J Med. 2004; 350(15):1577-9. DOI: 10.1056/NEJM200404083501523. View

10.

Wittwer C, Reed G, Gundry C, Vandersteen J, Pryor R . High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003; 49(6 Pt 1):853-60. DOI: 10.1373/49.6.853. View

11.

Ho S, Su L, Chan W, Chua S, Lim R, Biswas A . Ultrarapid prenatal detection of Down syndrome using real-time multiplex polymerase chain reaction (PCR) in amniotic fluid. Ann Acad Med Singap. 2005; 33(5 Suppl):S91-2. View

12.

Zerr D, Huang M, Corey L, Erickson M, Parker H, Frenkel L . Sensitive method for detection of human herpesviruses 6 and 7 in saliva collected in field studies. J Clin Microbiol. 2000; 38(5):1981-3. PMC: 86642. DOI: 10.1128/JCM.38.5.1981-1983.2000. View

13.

Osumi K, Fukui T, Kiyoi H, Kasai M, Kodera Y, Kudo K . Rapid screening of leukemia fusion transcripts in acute leukemia by real-time PCR. Leuk Lymphoma. 2003; 43(12):2291-9. DOI: 10.1080/1042819021000040206. View

14.

Panicker G, Vickery M, Bej A . Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish. Can J Microbiol. 2005; 50(11):911-22. DOI: 10.1139/w04-085. View

15.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

16.

Svanvik N, Westman G, Wang D, Kubista M . Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal Biochem. 2000; 281(1):26-35. DOI: 10.1006/abio.2000.4534. View

17.

Zhong X, Holzgreve W, Hahn S . Risk free simultaneous prenatal identification of fetal Rhesus D status and sex by multiplex real-time PCR using cell free fetal DNA in maternal plasma. Swiss Med Wkly. 2001; 131(5-6):70-4. DOI: 10.4414/smw.2001.09660. View

18.

Tyagi S, Kramer F . Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996; 14(3):303-8. DOI: 10.1038/nbt0396-303. View

19.

Aldous E, Collins M, McGoldrick A, Alexander D . Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay. Vet Microbiol. 2001; 80(3):201-12. DOI: 10.1016/s0378-1135(00)00371-0. View

20.

Sabersheikh S, Saunders N . Quantification of virulence-associated gene transcripts in epidemic methicillin resistant Staphylococcus aureus by real-time PCR. Mol Cell Probes. 2004; 18(1):23-31. DOI: 10.1016/j.mcp.2003.07.009. View