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Direct PCR from Serum 161
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Direct PCR from Serum
Application to Viral Genome Detection
Kenji Abe
1. Introduction
Nucleic acids used for polymerase chain reaction (PCR) assays usually are extracted
by the phenol-chloroform method or an alternative rapid purification. The acidguanidinium
thiocyanate-phenol-chloroform method for RNA extraction and proteinase
K digestion-phenol-chloroform method for DNA extraction from serum samples are
used widely for PCR assays (1,2), but usually at least 3 h are needed for this step.
Detection of RNA is more difficult and complex than that of DNA, mainly because of
the contaminating RNase and the need to conduct an additional reverse transcription
(RT) step. Additionally, another problem is the high cost of the reagents for RNA
extraction. Recently, we reported that viral RNA and DNA are readily amplified
directly from serum without purification of nucleic acids by direct (RT) PCR (3). The
method is sensitive because as little as 1 µL of the initial serum produces a clearly
visible amplified fragment, and there is no difference in the sensitivity and stability
between the direct PCR and conventional PCR assay. Interestingly, the results of the
direct PCR are much better when 1 to 2 µL is used rather than 3 to 5 µL of serum.
This inability to amplify RNA/DNA from serum may be to the result of the masking
of the target RNA/DNA by coagulated proteins during the initial heat denaturation or
the presence in the serum of inhibitors (probably such as lipoprotein) of the enzyme
reaction. The sensitivity of the direct PCR assay allows detection of as few as one copy
of viral genome. This technique not only eliminates the risk of cross contamination
during nucleic acid extraction but also is cost and time saving. In this chapter, we
describe the method of the direct (RT) PCR assay and apply it for detection of hepatitis
B virus (HBV) DNA and hepatitis C virus (HCV) RNA in serum specimens.
2. Materials
1. Thermal cycler.
2. AmpliTaq Gold DNA polymerase 5 U/µL (Perkin–Elmer).
3. 10× reaction buffer containing 15 mM MgCl 2 (supplied with AmpliTaq Gold DNA
polymerase kit).
4. RNase inhibitor 40 U/µL (Promega).
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
161