Construction of specific DNA probes for Opisthorchis viverrini detection and an attempt to clone genes coding for diagnostic epitopes

Abstract

Conventional method of diagnosing Opisthorchis viverrini infection by microscopic examination is time-consuming. Distinguishing O. viverrini eggs from those small intestinal flukes also requires experienced personals. Immunological methods still possess some difficulties due to frequent cross reactivity of O.viverrini antigens with those of other parasites. Although from an antigen analysis, the 89 kDa protein give promising results. Unfortunately, O.viverrini cannot be cultured in vitro which making it difficult to prepare a substantial quantities of the antigenic proteins. Thus, cloning a gene encoding for some of these specific proteins may overcome this problem. This study therefore total genomic DNA from the parasite was used to construct libraries in pUC 8,9 and 12 plasmid and a bacteriophage expression vector Xgt11. Screening of these libraries with a number of different anti O.viverrini antisera were however unsuccessful. This is probably due to the interference of introns in the antigenic gene or the restriction enzymes used are not suitable to cut into the coding region. Another possibility is that the 89 kDa is a glyoprotein, thus reducing the possibility when cloned this component in E. coli. Another approach to diagnose this parasite is to develop specific DNA probes for detecting parasite eggs in stool samples. Two recombinant plasmids were selected and named pOV-P5 and pOV-A6. The pOV-P5 has a 1.4 Kb insert which cross hybridized with a 334 base pair (bp) insert fragment of pOV-A6. When using as probes to hybridize with digested genomic DNA from O.viverrini, both plasmids gave similar tandemly repeated patterns. Sequence analysis of pOV-A6 suggests it to be the smallest unit of the repeated elements. This repeated unit was found to be conserved. The pOV-P5 was mapped and compared to pOV-A6 insert. The insert fragment of pOV-P5 may probably contains one unit of the same family of repeated elements. Both DNA probes were highly specific to O.viverrini and did not cross hybridize with genomes of other parasites, common intestinal bacterial flors and hosts. The sensitivity of pOV-P5 and pOV-A6 was also evaluated. The pOV-P5 and pOV-A6 insert could detect 25 pg of total genmoic DNA and as little as 6.3 pg of pOV-P5 and 3.1 pg of pOV-A6 respectively. Various methods were used to release DNAs from O.viverrini eggs to facilitate the detection of parasite eggs in stool sample. Among the various attempts, a combination of alkaline treatment (0.1 N NaOH) and autoclaving for 15 min were found to be most efficient at releasing DNAs from the egg shell. Both probes could detect about 5 purified eggs. The pOV-A6 insert was used as a probe to evaluate its diagnostic potential. It can detect about 1,600-2,000 effs per gm of feces from human stool specimens. The level of detection of this probe is in the range of light infection. In addition, pOV-A6 cal also detect O. viverrini at other stage of life cycle e.g. metacercarias and cercaria.