Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-08-21
1998-10-06
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435912, 435810, 536 231, 536 2432, 536 2433, 935 8, 935 78, C07H 2104, C12Q 168, C12P 1934
Patent
active
058174605
DESCRIPTION:
BRIEF SUMMARY
This invention relates to an oligonucleotide agent which can be used to evidence at least one strain of Borrelia burgdorferi, and to the varied applications of the said oligonucleotide agent.
The spirochete Borrelia burgdorferi (Bb) is the causative agent of Lyme disease. The vector host of this spirochete is an acarid of the genus Ixodes, I. ricinus in Europe or I. dammini and I. pacificus in North America. Clinical diagnosis of the illness relies on various anatomical and pathological signs (erythema chronicum migrans (ECM), fever, search for anti-OspA antibody (glycoprotein situated at the Bb surface) and/or anti-flagelline antibody (flagellum protein) constitutes the chief for anti-Borrelia antibodies does, however, entail a number of drawbacks. It is dependent on the reactivity of the host's immune system vis-a-vis Bb, which determines the sensitivity of this type of detection system. This immuno-biochemical method also has the drawback of not being able to dissociate a possible reaction that overlaps with infectious agents closely related to Bb (for example Treponema pallidum, which is therefore be helpful to develop new diagnostic tools that overcome these difficulties. The detection of the DNA of Bb using molecular hybridization techniques that employ a nucleotide probe and a target DNA fulfils these conditions. These hybridization experiments are generally carried out on a solid support (a nitrocellulose or nylon filter) to which the target DNA is attached, or alternatively in a liquid medium, for example at the time of a genetic amplification reaction known as the "Polymerase Chain Reaction", or PCR, which requires the presence of the target DNA, two nucleotide probes or primers specific to the target DNA, and a hybridization techniques are highly sensitive (capable of detecting a single bacterial genome) and highly specific. They have the advantage that they do not depend on any physiological or pathological reaction whatsoever of the infected host organism.
Thanks to molecular hybridization studies on the entire Bb genome it is now possible to separate a large number of known strains into three major comparing the sequence of the gene coding for the OspA surface glycoprotein of different strains of Bb using a customized computer program. The results of this computer analysis are corroborated by a phylogenetic study carried out on the gene coding for 16S ribosomal RNA clinical signs observed in the course of the illness suggests a useful link between a strain's membership of a particular group and the pathology associated therewith; for example, the strains subsumed under group I show a tendency to induce arthritic signs, whilst those belonging to group II
The detection of a Bb infection is currently done by using the serum of the potentially infected patient to recognize the purified flagellar antigen fixed on an Elisa type plate. This recognition is evidenced by the addition of an anti-human antibody associated with a disclosure enzyme or
Within the area of epidemiological studies, the routine diagnosis of infections by different known groups of Bb and the follow-up of the efficacy and composition of vaccines affording protection against Bb infections, it is helpful to develop simple, specific and sensitive techniques based on the use either of synthetic DNA probes labelled non-isotopically and suited to use in molecular hybridization reactions on a solid support, or synthetic DNA primers suited to use in genetic amplification reactions (PCR). With this aim in mind, we have used computer processing to identify, within the sequence of the gene coding for the OspA surface glycoprotein of Bb, regions capable of being used as probes or primers in molecular hybridization reactions.
It is an object of the present invention to propose sequences common to all of the groups of Bb strains, which can be used either as primers in the course of genetic amplification reactions (PCR), or as a capture probe in a hybridization test on a solid support where the target DNA is "sandwiched" between the said captur
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Bollen Alex
Godfroid Edmond
La Region Wallonne
Myers Carla J.
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