A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique.

[1]  A. Moorman,et al.  Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data , 2009, Nucleic acids research.

[2]  B. Reiser,et al.  Estimation of the Youden Index and its Associated Cutoff Point , 2005, Biometrical journal. Biometrische Zeitschrift.

[3]  Richard Cooper,et al.  Detection and Monitoring , 2018 .

[4]  Samuel Yang,et al.  PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings , 2004, The Lancet Infectious Diseases.

[5]  S. Broedersa,et al.  Guidelines for validation of qualitative real-time PCR methods , 2014 .

[6]  I. Pavlik,et al.  On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. , 2008, International journal of food microbiology.

[7]  R. Whitlock,et al.  Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. , 2011, Veterinary microbiology.

[8]  Björn Sjögreen,et al.  The real-time polymerase chain reaction. , 2006, Molecular aspects of medicine.

[9]  D. Rodríguez-Lázaro,et al.  Real-time PCR in Food Science: PCR Diagnostics. , 2013, Current issues in molecular biology.

[10]  P. Karlovsky,et al.  Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum , 2011, Analytical and bioanalytical chemistry.

[11]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[12]  D. Armbruster,et al.  Limit of blank, limit of detection and limit of quantitation. , 2008, The Clinical biochemist. Reviews.

[13]  K. Gruden,et al.  Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot , 2014, Analytical and Bioanalytical Chemistry.

[14]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[15]  Alfred Pingoud,et al.  Real‐Time Polymerase Chain Reaction , 2003, Chembiochem : a European journal of chemical biology.

[16]  Mojca Milavec,et al.  Assessment of the real-time PCR and different digital PCR platforms for DNA quantification , 2015, Analytical and Bioanalytical Chemistry.

[17]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Anne K Camper,et al.  Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. , 2006, Journal of microbiological methods.

[19]  F. Watzinger,et al.  Detection and monitoring of virus infections by real-time PCR , 2006, Molecular Aspects of Medicine.

[20]  S. Bustin.  Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. , 2000, Journal of molecular endocrinology.

[21]  M. Beer,et al.  A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health. , 2009, Veterinary microbiology.

[22]  Alison S. Devonshire,et al.  Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses , 2014, Journal of Clinical Microbiology.

[23]  M. Burns,et al.  Modelling the limit of detection in real-time quantitative PCR , 2008 .

[24]  P. Walsh,et al.  Simultaneous Amplification and Detection of Specific DNA Sequences , 1992, Bio/Technology.

[25]  Maher Chaouachi,et al.  Relative quantification in seed GMO analysis: state of art and bottlenecks , 2013, Transgenic Research.

[26]  Dieter Klein,et al.  Quantification using real-time PCR technology : applications and limitations , 2002 .

[27]  Anne K Camper,et al.  Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. , 2009, FEMS microbiology letters.

[28]  M. Koppelman,et al.  Validation of new real‐time polymerase chain reaction assays for detection of hepatitis A virus RNA and parvovirus B19 DNA , 2016, Transfusion.

[29]  M. Zwietering,et al.  Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices. , 2015, International journal of food microbiology.

[30]  R. Levin.  PCR detection of aflatoxin producing fungi and its limitations. , 2012, International journal of food microbiology.

[31]  C. E. Dawson,et al.  A comparison of nucleic acid amplification techniques for the assessment of bacterial viability , 2001, Letters in applied microbiology.

[32]  S. Bustin,et al.  Real-time quantitative PCR, pathogen detection and MIQE. , 2013, Methods in molecular biology.

[33]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[34]  Luca Bolzoni,et al.  Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia‐Romagna Region (Italy) by qPCR , 2016, MicrobiologyOpen.

[35]  J. F. Monkelbaan,et al.  Clinical consequences of polymerase chain reaction‐based diagnosis of intestinal parasitic infections , 2016, Journal of gastroenterology and hepatology.

[36]  S. Essack,et al.  Review of established and innovative detection methods for carbapenemase‐producing Gram‐negative bacteria , 2015, Journal of applied microbiology.