The Polymerase Chain Reaction Pcr For Human Viral Diagnosis

The polymerase chain reaction (PCR) is the most sensitive method for revealing the presence of otherwise undetectable quantities of the genome of RNA or DNA of human viruses. The Polymerase Chain Reaction (PCR) for Human Viral Diagnosis addresses the urgent need to use this revolutionary technology in reference and routine diagnostic laboratories. It informs the molecular biologist of the most appropriate clinical uses for PCR and educates the clinician and medical virologist about the subtleties and benefits of gene amplification. The reader is given an understanding and appreciation of the principles of PCR and how, why, and where it should be applied. The book explains the principles behind PCR and its role in the diagnostic and public health laboratory. The application of PCR to the detection and investigation of viral latency and persistence is presented by the originators of in situ amplification. There are individual contributions from experts in their respective fields on the detection, characterization, and analysis by PCR of gastroenteritis viruses, hepatitis viruses, herpesviruses, rhinoviruses, enteroviruses, flaviviruses, polyomaviruses, human immunodeficiency virus (HIV), human T-lymphotropic virus types I and II (HTLV-I and II); and measles, mumps, rubella, influenza, rabies, and B19 viruses.

The basis for the effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent, which is based on the analysis of the clinical symptoms coupled with laboratory tests. Although rapid advance ments have been made in the laboratory diagnosis of virus diseases, the neces sary isolation of the causative virus from the clinical specimens is a relatively long procedure. Viruses which integrate into the cellular DNA (such as human immunodeficiency virus, HIV -1, or hepatitis B virus) are difficult to identify by molecular techniques, while viruses which exist in the clinical material in low concentrations are even more formidable to identify. Recently, the application of the polymerase chain reaction (peR) technique developed by K. D. Mullis and detailed in the study by Saiki et al. (1985) led to a revolution in virus diagnosis. The peR technique was rapidly applied to the diagnosis of viruses in clinical material. Volume 1 of Frontiers of Virology provides new information on the advan tages of the use of the peR for the diagnosis of many human disease-causing viruses, as well as on some problems with its use.

Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods. Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies. Provides state-of-the-art PCR methods for amplification, quantitation, in situ hybridization, and multiplex reactions Goes beyond PCR with protocols for 3SR, NASBA, LCR, SDA, and LAT Covers important virus detection methods such as in situ hybridization; Southern, dot, and slot blots; branched chain signal amplification; and chemiluminescence Includes quality control information crucial in research and clinical laboratories Most chapters are written by the inventors and principal developers of the methodologies Includes color plates, 77 figures, and 18 tables

Today the diagnosis of human and animal pathogenic viruses would be unthinkable without the polymerase chain reaction (PCR). The short-styled protocols in this volume elucidate this important technique and its application in the laboratory, including a discussion of recent advances in PCR technology. The enclosed diskette enables the user to work with interesting sequences on his own computer.

PCR, developed at Cetus Corporation/USA by Henry A. Erlich, Kary Mullis and Randall K. Saiki, is a very simple method for amplifying nucleic acids in vitro. The realization of this idea bases on the repetition of a set of three different temperatures and yields an increase of the target structure up to a factor of 106 to 1012. Therefore, this technique is predisposed for safe analysis and characterization of DNA and RNA sequences of interest, even where the starting amount of material is enormously small. Because of its sensitivity, speed and versatility this method is particularly suitable for investigations of oncogenes, tumor associated translocations, retroviral sequences, lymphokines and mainly the broad field of degenerative and inflammatory diseases of nervous system. PCR seems to be the technique which could overcome the two most important problems in that field: very small amount of material combined with the necessity of rapid diagnostic procedures in inflammatory infections. "PCR topics" will give an actual overview of basic and applied research fields on usage of polymerase chain reaction. All contributions to this book have been presented at an international congress on "Usage of Polymerase chain reaction in genetic and infectious diseases" which took place in june 1990 in Berlin. The editors wish to thank all participants for their contributions. We offer our thanks and gratitude to our coworkers and especially to our technical assistents Barbara Trampenau, Mirjana Wiirdemann and Hannelore Leonhard.

PCR in Bioanalysis offers powerful PCR-based protocols and assays in actual use or potential use in clinical medicine and commercial biology. The main focus of the book is on the commercial applications of PCR, as opposed to basic research uses. Topics covered include the measurement of hormone levels using PCR, transcription factor isolation, detection of viruses using PCR, detection of tumor contamination of stem cells, evaluation of grafts for tumor cells, and more.

With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.

One: Methodology.- I. Basic Methodology.- 1. Manipulation of DNA by PCR.- 2. Cloning PCR Products.- 3. Optimization of Multiplex PCRs.- 4. Preparation of Nucleic Acids for Archival Material.- 5. PCR Amplification of Viral DNA and Viral Host Cell mRNAs in Situ.- II. Quantitation.- 6. Quantitative PCR: An Overview.- 7. Quantification of DNAs by the Polymerase Chain Reaction Using an Internal Control.- 8. RT-PCR and mRNA Quantitation.- 9. Analysis of Human T-Cell Repertoires by PCR.- III. Nonisotopic Detection.- 10. Ultrasensitive Nonradioactive Detection of PCR Reactions: An Overview.- 11. Fluorescent Detection Methods for PCR Analysis.- 12. Enzyme-Labeled Oligonucleotides.- 13. Application of the Hybridization Protection Assay (HPA) to PCR.- IV. Instrumentation.- 14. PCR Instrumentation: Where Do We Stand?.- 15. Rapid Cycle DNA Amplification.- 16. Automating the PCR Process.- V. Sequencing.- 17. PCR and DNA Sequencing.- 18. Phage Promoter-Based Methods for Sequencing and Screening for Mutations.- 19. Capture PCR: An Efficient Method for Walking Along Chromosomal DNA and cDNA.- Two: Applications.- I. General Applications.- 20. In Vitro Evolution of Functional Nucleic Acids: High-Affinity RNA Ligands of the HIV-1 rev Protein.- 21. The Application of PCR to Forensic Science.- 22. Recreating the Past by PCR.- 23. Nonbiological Applications.- II. Genetic Analysis.- 24. RT-PCR and Gene Expression.- 25. Fingerprinting Using Arbitrarily Primed PCR: Application to Genetic Mapping, Population Biology, Epidemiology, and Detection of Differentially Expressed RNAs.- 26. Genetics, Plants, and the Polymerase Chain Reaction.- III. Assessment of Therapy Effectiveness.- 27. PCR Assessment of the Efficacy of Therapy in Philadelphia Chromosome-Positive Leukemias.- 28. The Detection of Minimal Residual Disease (MRD) in Acute Lymphoblastic Leukemia Using Clone-Specific Probes Directed against V(D)J Junctional Sequences.- 29. Assessment of Therapy Effectiveness: Infectious Disease.- 30. Gene Therapy.- IV. Diagnostics.- 31. PCR and Cancer Diagnostics: Detection and Characterization of Single Point Mutations in Oncogenes and Antioncogenes.- 32. Clinical Applications of the Polymerase Chain Reaction.- 33. Infectious Diseases.- Three: PCR and the World of Business.- 34. PCR in the Marketplace.- 35. PCR and Scientific Invention: The Trial of DuPont vs. Cetus.