Advanced Techniques in Diagnostic Microbiology

176 M. Loeffelholz and H. Deng
RT-PCR is often used interchangeably to describe reverse transcription PCR
and real-time PCR. To avoid confusion, “real-time” will not be abbreviated in this
chapter.
Quantitative PCR
A variety of quantitative PCR assays have been developed to accurately quantify
nucleic acid targets in clinical specimens (Clarke and McClure, 1999; Boyer and
Marcellin, 2000; Mylonakis et al., 2001). In addition to PCR, other molecular
techniques such as branched (b) DNA provide accurate quantification of nucleic
acids. Although these methods determine the amount of DNA or RNA template
in a clinical specimen, the results can be easily extrapolated to organism equivalents,
hence the use of terms bacterial load, viral load, and so forth. Quantitative
PCR results have become a valuable tool for guiding antiviral therapy, monitoring
clinical course, and predicting outcome from a variety of infectious diseases
(Hodinka, 1998; Orlando et al., 1998; Jung et al., 2000). The value of quantitative
PCR has led to commercialization of tests for such viruses as human
immunodeficiency virus (HIV), cytomegalovirus, hepatitis C virus, and hepatitis
B virus.
Nucleic acids can be quantified using an absolute standard in order to generate
concrete numbers or a relative standard to give comparative data. Absolute
standards can be used whenever definite numbers are needed. Relative standards
are useful when absolute quantities are less important than knowing how a sample
differs from a control. Fundamental RT-PCR quantification strategies are relative,
competitive, and comparative.
Relative quantitative PCR compares nucleic acid amount across a number of
serial dilutions of a sample, using a coamplified internal control for sample normalization.
Results are expressed as ratios of the sequence-specific signal to the
internal control signal. This yields a corrected relative value for the sequencespecific
product in each sample. Relative PCR uses primers for an internal control
that are multiplexed in the same PCR reaction with the target-specific primers. Internal
control and target-specific primers must be compatible—that is, they must
not produce additional bands or hybridize to each other. The signal from the internal
control is used to normalize sample data to account for variation in RT
or amplification efficiency. Common internal controls include the housekeeping
genes (or their mRNAs) β-actin and GAPDH and 18S rDNA.
Competitive RT-PCR provides absolute quantification of a nucleic acid target
in a sample. An internal control or quantification standard is added at a known
concentration to samples and coamplified with the target sequence. Addition of
the internal control to the sample prior to processing monitors for nucleic acid
recovery during this step. The internal control is often a synthetic RNA or DNA
with the same primer binding sequence (hence the term competitive PCR) but
designed to produce an amplicon slightly different in size than the target amplicon
or with a unique internal sequence allowing detection with a different probe. After