ABSTRACT
The use of riboprobes for the detection of RNA viral sequences is analyzed. Subgenomic fragments of cDNA from poliovirus type 2, dengue virus type 4 and human immunodeficiency virus type 1, were inserted downstream SP6 and/or T7 promoters in the transcription vectors pGEM-4z or pSP64. RNAs obtained by in vitro transcription in the presence of UTP infinity (32P) were used as probes for the detection of RNA viral sequences from infected cell lines in slot and Northern blot assays. The poliovirus riboprobe (P2-221) was able to detect specific viral sequences; thus, it could be used for the detection of the virus in serum, as well as in residual waters. The human immunodeficiency virus riboprobe (HIV-378), detected viral sequences poly A+RNA from infected cells; thus it can be used as a confirmatory test or as a tool in basic research. Finally, the dengue virus riboprobes (D4-2819 and D4-1134) detected specifically dengue 4 virus; however the sensitivity of the detection could be significantly improved amplifying viral sequences by the polymerase chain reaction (PCR) prior to probe hybridization.
Subject(s)
Dengue Virus/genetics , HIV-1/genetics , Poliovirus/genetics , RNA Probes , RNA, Viral/isolation & purification , Blotting, Northern , Cells, Cultured , Dengue/diagnosis , HIV Infections/diagnosis , Humans , Nucleic Acid Hybridization , Plasmids/genetics , Poliomyelitis/diagnosis , RNA Probes/biosynthesis , RNA, Viral/analysis , RNA, Viral/genetics , Virus CultivationABSTRACT
Sequences front a cDNA of dengue virus type 4 were cloned into transcription vectors. These sequences included the E, NS1, NS2A, NS2B, NS3 genes. RNA transcripts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. With these RNA-probes we have been able to detect molecules of serotype-specific dengue 4 viral RNA. Moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might be useful to differentiate serotypes.