Direct PCR from serum: application to viral genome detection.

Abstract

Nucleic acids used for polymerase chain reaction (PCR) assays usually are extracted by the phenol-chloroform method or an alternative rapid purification. The acid-guanidinium 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 (,), 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 (). 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.