Abstract:
Filariasis is a major public health problem confined mainly to tropical and sub
tropical countries, around the world (Walls and Schantz, 1986). Estimates show
that a sizable proportion of the world’s population is affected with disease
(UNDP/WHO, 1986). Filariasis is transmitted to human by the infective bites of
mosquito vectors.
The identification of individual species, a requirement of epidemiologic studies and
control programs, has traditionally relied upon techniques such as mating
incompatibility, isozyme analysis, cytological methods, hydrocarbon analysis, etc..
Owing to the limitations of these techniques, the last few years have seen many
developments in DNA based technologies for identification. These methods
ultimately rely upon either the use of DNA probes in hybridization assays or the
polymerase chain reaction (PCR).
This study reports the cloning and isolation of recombinant clones containing
repetitive sequences of C. quinquefasciatus for the development of specific DNA
probes which could be used for the identification of mosquito adult, larvae and
pupae.
Culex quinquefasciatus genomic DNA was partially cleaved with the restriction
enzyme Sau 3A-1 to obtain the maximum number of fragments in the size range 0
- 9 kb. These fragments were cloned into the bacteriophage vector ƛZap and
packed in vitro. The library was then amplified and percentage of recombinants
were determined. To isolate recombinant clones containing repetitive sequences of
C. quinquefasciatus for the development of DNA probes, an aliquot of the library
was plated out and plaque lifts obtained were screened with P-labeled C.
quinquefasciatus genomic DNA.
Plaques giving strong hybridization signals on filters were marked and picked into
to SM buffer. These repetitive recombinant clones were stored at 4°C for further
analysis.
