A simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.
Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.
Gunawardene
Y I
YI
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Sri Lanka.
Wijesundera
W S
WS
Karunanayake
E H
EH
Chandrasekharan
N V
NV
Jayasekera
N
N
Siridewa
K
K
D000818:Animals; D019921:Biotinylation; D018509:DNA, Helminth / Q000032:analysis; D004605:Elephantiasis, Filarial / Q000175:diagnosis*; D006801:Humans; D008163:Luminescent Measurements; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity; D013188:Sri Lanka; D013997:Time Factors; D014958:Wuchereria bancrofti / Q000302:isolation & purification*
D018509:DNA, Helminth; D015345:Oligonucleotide Probes