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.

Polymerase chain reaction-based technique for the detection of Wuchereria bancrofti in human blood samples, hydrocele fluid, and mosquito vectors.

Oligonucleotide primers were designed to amplify a 490-basepair DNA fragment in the 5' end of the pWb 12 repeated DNA sequence in Wuchereria bancrofti for specific amplification of W. bancrofti DNA by the polymerase chain reaction (PCR). A single microfilaria in 100 microliter of blood or added to 1 ml of blood, a single third-stage larva in a pool of 20 uninfected mosquitoes, or 0.4 pg of W. bancrofti genomic DNA added to 100 microliter of human blood or serum can be detected by this PCR method. The parasite DNA in human blood and hydrocele samples and in mosquitoes was isolated free of any PCR inhibitors using simple purification techniques. Detection of PCR products was carried out by agarose gel electrophoresis, followed by staining with ethidium bromide and visualization under ultraviolet illumination. The results indicate that the PCR method is species-specific, rapid, and more sensitive than that of DNA probes and routine microscopy.

Cloning and characterization of a repetitive DNA sequence specific for Wuchereria bancrofti.

A genomic library constructed in a bacteriophage lambda replacement vector (EMBL3) with Wuchereria bancrofti DNA partially digested with Sau 3A I was screened with 32P-labeled W. bancrofti total DNA, and a strongly reactive recombinant, EMBL3Wb34, was isolated. This clone contained an approximately 16-kb insert that showed some cross-hybridization with Brugia malayi and B. pahangi DNA. However, a 969-bp subclone from EMBL3Wb34, designated pWb12, hybridized only with W. bancrofti DNA and was able to detect as little as 300 pg. Furthermore, pWb12 could detect DNA from a single infective larva or one microfilaria. It has a moderate copy number (450-700) and appears to be interspersed within the parasite genome. The nucleotide sequence contains 66% A+T and 34% G+C and shows no notable internal repeats.

Purification of simian immunodeficiency virus, SIVMAC251, and of its external envelope glycoprotein, gp148.

Two-phase extraction in a system composed of dextran and polyethylene glycol was used to purify simian immunodeficiency virus, SIVMAC251 (32H isolate) from 25 l of culture supernatant. The virus partitioned to the interphase with 80% recovery of gag peptide p27 and reverse transcriptase and an about 25% recovery of the external env glycoprotein, gp148. The virus was treated with octylglycoside and its subcomponents separated. Two gag-p27 containing fractions were obtained; gag-1, which also contained reverse transcriptase and nucleopeptides, and gag-2, which contained the major portion of the p27. The env gp148 was purified by chromatography through a series of lectin columns. The prepared materials are characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immuno- and lectin blotting.

Characterization of glycoproteins from Chlamydia trachomatis using lectins.

Glycoproteins in Chlamydia trachomatis, serotype L1, elementary bodies were studied by lectin blotting. A panel of 23 lectins representing a variety of sugar specificities was used. The pattern of lectin-binding specificities at a peptide band was studied in order to determine the type and structure of its glycoconjugate. To establish chlamydial origin of the glycopeptide bands in the blot, control samples from non-infected host cell membranes were run in parallel. Terminal mannosidic structures were demonstrated in a 72 kDa glycopeptide (gp72) by its selective binding of Galanthus nivalis lectin (GNA). Sialic acids were found in two chlamydial glycopeptides, gp40 and gp64, which appear to carry O-linked glycoconjugates as they bound the peanut agglutinin (PNA, both gp40 and gp64) and jackfruit lectin (Jac, only gp40). Such structures were also present in other chlamydial glycopeptides. Lectins with specificities for fucose in different links, galactose and N-acetyl glucosamine bound to several chlamydial peptides. On the basis of our results we suggest an alternative mechanism for uptake of chlamydial elementary bodies into host cells, namely phagocytosis mediated by eukaryotic cell surface lectins.