8 - Detection of Viral Pathogens Using PCR Amplification

Abstract

This chapter focuses on the detection of viral pathogens using polymerase chain reaction (PCR) amplification. PCR is a relatively simple procedure consisting of repetitive cycles of oligonucleotide primer-directed DNA replication. A selected genetic target is copied in a three-step reaction: denaturing the target DNA, annealing, and extending the bound primers by DNA polymerase action. The use of Taq DNA polymerase resulted in increased specificity of PCR amplification reactions by permitting higher primer annealing and polymerase extension temperatures to be used, decreasing the amount of non-specific target amplification. The high analytical sensitivity of PCR-based procedures facilitates the detection of minute amounts of pathogens in biological specimens that are undetectable using more conventional technology. The use of uracil-N-gylcosylase in PCR protocols improves the sensitivity of PCR amplifications by degrading nonspecific extension products that result from low-stringency primer binding that occurs below the optimal primer annealing temperature. Direct detection of viral particles or nucleic acid is the most reliable method for diagnosis of human immunodeficiency virus (HIV) infection in newborns of HIV-infected women.