Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.
Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.
Deubel
V
V
Institut Pasteur, Laboratoire des Arbovirus, Paris, France.
Laille
M
M
Hugnot
J P
JP
Chungue
E
E
Guesdon
J L
JL
Drouet
M T
MT
Bassot
S
S
Chevrier
D
D
D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D015139:Blotting, Southern / Q000379:methods; D003715:Dengue / Q000097:blood; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012703:Serotyping
D015345:Oligonucleotide Probes; D012367:RNA, Viral