A simplified PCR methodology for semiquantitatively analyzing dengue viruses.
The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.
Upanan
Supranee
S
Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Thailand.
Cabrera-Hernandez
Arturo
A
Ekkapongpisit
Maneerat
M
Smith
Duncan R
DR
D002213:Capsid / Q000737:chemistry; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D018716:Molecular Mimicry; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D013698:Templates, Genetic; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; C084187:prM protein, Dengue virus type 3