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qPCR to Detect RNA Viruses 197
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Ultrasensitive Quantitative PCR to Detect RNA Viruses
Susan McDonagh
1. Introduction
The use of quantitative polymerase chain reaction (qPCR) to detect RNA viruses
has become increasingly important as a prognostic marker and in patient management,
for example, in human immunodeficiency virus (HIV) and hepatitis C virus (HCV)
infection. Drug therapies can be monitored by regularly checking viral load, indicating
whether the regime is sufficient, or whether alternatives should be sought. It is therefore
crucial that the systems used are ultrasensitive and give accurate and reproducible
results.
There are several elements to consider in developing a reliable and accurate
quantitative assay. First, rapid transport and storage of samples is important because
of the unstable nature of RNA, as is the method of preparation. Samples should be
received and processed within 6 h and the relevant fractions stored at –70°C until
testing. Second, it is difficult to ascertain the efficiency of sample preparation methods;
therefore, values obtained using qPCR may be several times lower than the actual copy
number. Known standards should therefore be processed alongside samples to assess
the loss within the system (1). Third, the efficiency of the reverse transcriptase step has
been assessed to be from 5 (2) to 10% (3,4), and this must be taken into account when
calculating viral load. Finally, there is a need to amplify all viral types equally, which
can be a problem with the high mutation rate observed in RNA viruses. As a result,
primers should be chosen from highly conserved noncoding regions. such as the 5′ noncoding
region of HCV (5) and of enteroviruses (6).
Several quantitative systems that use a variety of approaches have been developed
and published. These include limiting dilution; the use of external standard curves;
co-amplification of an internal reference template; and competitive PCR using an
internal control. There are advantages and disadvantages with each of these systems,
and the system of choice will depend on several factors. These methods have been used
to detect as few as 40 copies of HCV (7), 64 and 4 copies of HIV per ml of plasma or
serum, respectively (8,9), and less than 10 copies of enterovirus (10).
1.1. Limiting Dilution
The linear relationship between the amount of template and product is the basis of
the limiting dilution method. However, this only occurs over a limited range; therefore,
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
197