Bioanalytical PCR: ready for Prime time?

Humana Press 1997. US$69.50 (xi + 281 pages)ISBN 0-896-03497-6The ability to amplify specific nucleic acid sequences from biological sources that have been previously beyond the reach of molecular analysis has dramatically expanded our investigative and analytical capabilities. During 15 years of tinkering with PCR, the time is well overdue for defining exactly how the technology can be used as an analytical tool. The first step in that process is to identify key areas in which good ground work has already been done and where innovation can still be applied. To this end, numerous authors and editors have tried to compile examples of the best ways to use PCR in recent years. But with the plethora of PCR methodology books available today, it can be time consuming to distinguish the good works from the excellent ones. Because PCR in Bioanalysis is excellent, the serious PCR-based investigator can be saved valuable time by turning straight to this volume. It incorporates a wide range of applications and approaches in an accessible and contemporary volume. Despite the fact that the collection of 24 chapters covers a wide range of interests, the diversity is not a distraction because all the presentations have a bioanalytical focus. The first chapter, which presents an overview of the theory, practice and performance of PCR, is one of the best that I have read, and it has become required reading by my laboratory personnel, regardless of their experience. I was grateful that this single chapter distills the essential aspects of performing analytical PCR, and other related amplification technologies, in the minimum of space and with the utmost clarity.The remainder of the volume is conceptually divided into general topics that cover: (1) PCR applications in animal and agricultural science, including transgenic mice and transgenic plant molecular biology; and (2) PCR detection and diagnosis of human genetic and infectious diseases, including a small collection of papers that describe innovative approaches to analyzing genetic heterogeneity in cancerous tissue and to finding termination mutations in large, complex genes.In the former section, several papers focus on the use of PCR for detecting and differentiating between strains of viruses that infect bovine and porcine species. This section addresses issues of disease transmission and specimen processing; a focus that is equally useful to clinical virologists who study disease transmission and diagnosis in humans. Bovine immunodeficiency-like virus (BIV) has been known for 25 years; the hard lessons that have been learned from inconclusive serological data and the difficulty of virus isolation in that field can be very instructive today for those working on HIV and who are turning to PCR for significantly improved diagnostic power, sensitivity and resolution.The most highly refined reports in the volume are those that are directed toward the diagnosis of infection in human disease, and which are readily implementable for fieldwork. The editor did an excellent job of choosing among many possible areas of infectious disease to arrive at an instructive sampling of papers that focus on those pathogens (1) that are difficult, or impossible, to cultivate; (2) that exist as low level but transmissible asymptomatic infections; and (3) that require rapid diagnosis for the making of critical therapeutic decisions. In the paper that addresses the sexually transmitted diseases, attention is paid to the fact that improved sensitivity allows the testing of alternative, less invasive specimen types, thus increasing patient compliance, an issue that is not normally considered in methodology textbooks. The chapter on tuberculosis diagnostics contains an in-depth discussion of the potential savings in health-care costs, not only from one molecular testing scheme but also from three alternative approaches, extending the discussion of the impact of PCR bioanalysis into the realm of patient management and the improvement of health care. Other strong points in this category are two reports that use innovative non-isotopic approaches to focus on the devastating worldwide problem of enterotoxigenic Escherichia coli, and the difficulty that is encountered when attempting to type closely related bacterial strains. For Legionella infection, where typing is not an issue, a PCR strategy is presented that covers all 16 species, thus ensuring that a subtle, but important, diagnosis is not missed. A creative methodology is also presented that allows for more than just the detection of HIV RNA – it also allows the discrimination of the RNA subspecies, an approach that is valuable for investigating the progression of the viral life cycle and the progressive stages on the infection.One strength of this volume is that it addresses the traditional weaknesses of PCR. One of the shortcomings of PCR is that it is primarily, by nature, a sequence-dependent technique and is thus not particularly quantitative in its generic form. Although the power of PCR in mutational analysis has been well established for a few, well-defined, human genetic lesions, the challenge now is to extend its analytical capabilities of PCR to the detection of mutations in large genes or in genes that have multiple mutations that cover a wide range of sequence information. In this context, several laboratories have combined PCR with other sequence-independent molecular methods to extend the analytical power of the assay. This could lead to the analysis of additional, medically important, genes that have evaded a comprehensive analysis to date, such as the adenomatous polyposis coli (APC) gene. With regard to the quantitative use of PCR, several approaches are presented in detail that demonstrate how a quantitative component can be incorporated into a PCR analysis, whether by using an endogenous or exogenous template or an RNA or DNA source. Enough detail is presented for even the mildly intrepid bench worker to customize internal control templates for their specific targets. This ability to achieve quantitative PCR will be a key issue as the assay gains acceptance as a bioanalytical tool.Will PCR be an incisive bioanalytical tool? This book does not necessarily make it so or guarantee its success. The success of PCR as a bioanalytical tool will be up to those in the field who will have to use the tool correctly and with regard to its highly sensitive nature. This book only presents some useful, contemporary examples of what can be done if it is done correctly; it points the way for investigators to apply the technology analytically, if attention is paid to the parameters, the limits and to the controls, internal or otherwise.