A textbook in real time

Rapid-Cycle Real-Time PCR: Methods and Applications – Microbiology and Food Analysisedited by Udo Reischl et al.Springer, 2002.£30 (hbk) (ix + 258 pages)ISBN 3 540 41881 4After a brief introduction, this book begins with a superb chapter on the applications and challenges of real-time PCR for the clinical microbiology laboratory. The chapter is full of pertinent information, tables and diagrams summarizing the cutting-edge technologies being used for diagnostics in state-of-the-art laboratories. This first chapter (out of a total of 25) is itself a journey worth taking for the reader, and will be a fantastic teaching guide.The remainder of the book is divided into four sections: Bacteriology; Detection of Fungal Pathogens; Virology; and Detection of Genetically Modified Organisms. The bacteriology and virology sections make up the largest part of the book, with only one chapter each available on fungal pathogens and food analysis. Each chapter conforms to a standard format, which makes it easy for the reader to move between chapters to find complementary information. More specifically, each chapter contains the same sections: introduction; materials; procedures; results; comments; and references. The introduction section is used to describe the clinical problem and justify the use of real-time PCR technology for diagnosis. The materials section provides the reader with a detailed list of equipment and reagents, including vendor information for their own purchasing efforts. The procedural section contains sufficient experimental detail to allow readers to use the book more like a laboratory manual than a textbook. There is no shortage of informative tables (there are 127 tables in the book in total), including oligonucleotide sequence information for primers and probe synthesis, master mix formulations and assay parameters. Readers will find it easy to follow the step-by-step guide to reproduce the assay in their own laboratory. The results section of each chapter describes the analytical and diagnostic sensitivities and specificities of the assay and contains beautifully detailed, colored photographs illustrating real-time analyses simulating the computer screens seen by the instrument operator. My favorite section within each chapter was the comment section. Here, you will find especially helpful information concerning assay limitations and performance characteristics, together with inhibition and cross-reactivity issues. Even the most veteran users of real-time technologies will be able to pick up new information and applications for their efforts. Finally, the reference section at the end of each chapter provides sufficient material for additional reading and a more comprehensive look into the fields presented.On finishing this book, readers might be struck by a recurring theme of crucial importance that needs to be clearly understood. One of the potential limitations of this technology results from the use of such a small reaction volume (e.g. 20 μl), which limits the amount of target DNA that can be added to the reaction mixture. This design can reduce the sensitivity of the assay if it is not accounted for during the development and validation of the assay, and although this is not a concern when working with isolated bacterial strains, it can be problematic when testing clinical specimens directly for an infectious agent.In summary, this textbook will appeal to any clinical microbiologist interested in learning more about real-time PCR applications, especially those interested in assays designed for use on the LightCycler instrument (Roche Molecular) using hybridization probes. The book provides the reader with both strengths and weaknesses of this newest technology in an ever-growing and evolving field of molecular microbiology diagnostics. I highly recommend taking the plunge!