Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

Abstract

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved byAlu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% ho mologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes. (4,5). DNA was isolated from frozen adult female worms or from microfilariae in whole blood by the proteinase K-digestion method of Emmons et al. (6) followed by phenol extraction. For the Southern blots and cloning experiments, the DNA was further purified by ethidium bromide/CsCl centrifugation (7), which removed a band of white material, possibly from the cuticle, that was more dense than the DNA.Cloning of Repeated DNA from B. malayi. All restriction endonucleases, methylases, ligases, linkers, and 35S-labeled dideoxy sequencing reagents used in these experiments were prepared at New England Biolabs and used as described by the supplier. B. malayi DNA was cleaved with Alu I, protected with EcoRI methylase, and then ligated to 5'-phosphorylated EcoRI linkers with T4 DNA ligase. Cohesive EcoRI ends were generated by digestion with EcoRI. The linkers were separated from the Brugia DNA by electrophoresis on a 1.5% low melt agarose gel stained with ethidium bromide. The 320-base-pair (bp) and 640-bp restriction fragments were cut out and the DNA isolated (8). In two separate reactions, either the 640-bp or the 320-bp fragments were ligated to EcoRI cleaved pBR322 that had been dephosphorylated by treatment with bacterial alkaline phosphatase (9).Escherichia coli RRI cells were transformed with the recombinant plasmids, selected for growth on ampicillin, and screened for repeated sequences by hybridization with radioactively labeled B....

Detection of Brugia Parasite DNA in Human Blood by Real-Time PCR

“…The present study shows that this sequence is also a useful target for real-time PCR. Early studies estimated that there were about 30,000 copies of the HhaI repeat per haploid genome of B. malayi (ϳ10% of the total DNA) (20). Shotgun sequencing of the B. malayi genome has shown that the HhaI repeat is less abundant than this and suggests that the HhaI repeat comprises only ϳ1% of the haploid genome (7).…”

“…A control plasmid was constructed by cloning a PCR product of the B. malayi HhaI repeat (20) into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. The plasmid was propagated in Escherichia coli and purified by using a QIAprep spin miniprep kit (QIAGEN, Valencia, CA).…”

Use of restriction fragment length polymorphisms (RFLPs) to distinguish between nematodes of pathogenic significance

“…In such cases, hybridization of parasite DNA obtained from a patient or a suspected animal reservoir will definitively identify the organism for which the probe is specific, while ruling out other species. For example, species-specific probes have been generated for Brugia malayi (McReynolds, DeSimone & Williams, 1986;Sim, Mak, Cheong, Sutanto, Kurniawan, Marwoto, Franke, Campell, Wirth & Peissens, 1986;Sim, Piessens & Wirth, 1986) and Onchocerca volvulus (Perler & Karam, 1986); for the latter, a strain-specific probe, able to distinguish between forest and Savannah forms, has recently been reported (Ertmann, Unnasch, Greene, Albiez, Boateng, Denke, Ferraroni, Karam, Schulz-Key & Williams, 1987). The alternative approach is to generate probes that hybridize with the DNA of all filarial parasites, but can distinguish between the DNA of various parasites on the basis of restriction fragment length polymorphisms (RFLPs).…”

Detection of DNA of nocturnally periodic Brugia malayi in night and day blood samples by a polymerase chain reaction-ELISA-based method using an internal control DNA.

“…So far no sequence consistent differences of the Hha I repeat in different strains of B. malayi have been found and it was reported that the Hha I repeat of B. malayi is species specific and distinct from that of the closely related species B. pahangi. 20 Since the Hha I sequence is highly repeated and comprises about 13% of the whole B. malayi genome, this sequence appears to be preordained as PCR target for the development of specific and sensitive detection assays for this parasite.…”

“…In the present study we developed a PCR-ELISA method based on the Hha I repeat of B. malayi, 20 which involves the coamplification of an internal control. This method was evaluated on blood samples from persons living in an area endemic for nocturnally periodic B. malayi in central Sulawesi, Indonesia.…”