Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1993-06-08
2001-05-01
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S091100, C435S006120
Reexamination Certificate
active
06225094
ABSTRACT:
The invention concerns a method for the genus-specific or/and species-specific detection of bacteria in a sample liquid.
After the development of hybridization techniques many methods were developed for the detection of certain nucleic acids which could be used to detect the presence or absence of particular nucleic acid segments. These methods also encompass the diagnosis of human hereditary diseases as well as of other defects at the genome level which do not necessarily lead to an actual effective disease. However, the nucleic acid tests based on the initially developed methods did not have a very high specificity and there were difficulties with respect to the accuracy of the detection in particular when testing for mutations on nucleic acids which only affected a few bases. In particular the known hybridization methods were too unspecific for the differentiation of nucleic acid variants which merely differed in one or two bases. The reason for this was firstly that very stringent conditions could not be used for the hybridization because of the use of relatively short oligonucleotides which is why the absence of hybridization of one base (a so-called “mismatch”) could usually not be detected. Another reason was the availability of often only relatively small amounts of purified nucleic acid or nucleic acids to be differentiated.
The PCR reaction was developed among others as part of the further development of the nucleic acid detection methods and can be used to amplify a nucleic acid to be detected. However, by-products are often formed in the PCR reaction which interfere with the subsequent detection by hybridization. Therefore a method was already suggested in EP 0 332 435 in which, in order to differentiate between nucleic acids which differ by at least one nucleotide, a diagnostic primer is used which is elongated with the aid of a polymerase and the nucleoside triphosphates, whereby, however, an elongation only takes place when the 3′ terminal nucleotide of the primer is complementary to the corresponding nucleotide of the nucleic acid to be detected. The presence of a possible point mutation variant is determined in this method via the presence or absence of an elongation product. This is shown in this application using particular hereditary diseases in humans as an example.
However, there are also difficulties in this technique in that some polymerases have a so-called “proof reading activity” which corrects the mismatch during the primer elongation and thus make a specific detection impossible. It was therefore still necessary to develop a method with which differences of only one base on nucleic acids can be detected reliably. In particular there was a need for a highly specific detection method for nucleic acid variants in order to detect the presence or absence of particular bacterial genera and to differentiate between them in a sample liquid. The object of the invention was therefore to provide such a highly specific method.
The invention therefore concerns a method for the genus-specific or/and species-specific detection of bacteria in a sample liquid in which bacterial RNA is hybridized with a primer which is complementary to a genus-specific or species-specific region of RNA of particular bacteria but which is not complementary at its 3′ end to the RNA of bacteria of another genus or species, the primer is elongated in the presence of a suitable polymerase and the four deoxyribonucleotides, if desired, with a concurrent or subsequent labelling of the elongation product whereby an elongation product which forms when the primer and template RNA are complementary is additionally hybridized, after denaturation, with a genus-specific or species-specific oligonucleotide and the hybridization is detected by means of the oligonucleotide label.
Within the scope of the invention the primer can also be complementary to a region of the RNA of bacteria which is in general highly conserved provided that a mismatch results at the 3′ end of the primer when it is base paired with the RNA of the other genus or species.
Within the scope of the invention the term “bacterial RNA” also includes an amplification product of the RNA which is directly formed by the bacteria. The amplification of the bacterial RNA can be carried out using known methods such as e.g. polymerase chain reaction (U.S. Pat. No. 4,683,195), nucleic acid sequence based amplification (EP-A 0 329 822) or fast enzyme linked intense chain replication effect (German Patent Application P 39 29 030.1).
Since two selection steps are used in the method according to the present invention it is possible to considerably reduce the risk of erroneous results caused by only one differentiating reaction and thus the method provides a highly specific test which can be used to differentiate between bacterial RNAs which only differ in a few bases which in turn allows a statement about the presence of a particular bacterial genus or species. Indeed it is particularly the 16S-rRNA and also the 23S-rRNA of bacterial species which often only differ in a few nucleotides. Such differences are often the only possibility of differentiating between “harmful” and “harmless” bacteria. In principle also a differentiation of individual bacterial families can be carried out according to the present invention.
In a particular preferred embodiment of the invention it is possible to determine the actual species in virtually one step after an examination for the presence of members of a genus of bacteria. A primer is used for this which is complementary to the RNA of a particular genus of bacteria but which differs from the RNA of bacteria of other genera by at least one and preferably two or three bases at its 3′ end. If bacteria of the genus being searched for are present in the sample liquid, an elongation product is formed after primer elongation and this elongation product is then hybridized with an oligonucleotide which corresponds to another region of the bacterial RNA whereby in this case the requirements for hybridization are only met for a particular species of this bacterial genus.
In the method according to the present invention it is possible to use very long primers for the hybridization of the RNA with the primer which in turn allows the use of very stringent conditions in this hybridization. As a result the primer elongation step already yields a very specific result which is substantially increased by the additional hybridization step. According to the present invention it is also possible to run additional control experiments in which primers or oligonucleotides are used which are complementary or identical to regions on the RNA which are the same for many bacterial genera and species. For control experiments there are even primers or oligonucleotides which are specific for all eubacteria. The presence and accessibility of the target RNA can be demonstrated by such control experiments and thus prevents “false negative” results. In addition blind experiments without target RNA can be carried out. By this means it is possible to avoid mistaking artefacts in the primer elongation as well as in the hybridization for the actual specific reaction. At the same time the amounts of bacteria of a particular genus or species can be estimated in-relation to the total amount of bacteria.
According to the present invention any RNA which can be shown to be different in different genera or species can be used as the RNA of bacterial origin.rRNA, mRNA or t-RNA is preferably used by selecting primers and oligonucleotides for the method according to the present invention which are complementary to them. Bacterial RNA is prepared for the method according to the present invention according to known methods.
The actual detection of the hybridization of the elongation product formed is carried out via the oligonucleotide label whereby any type of label for nucleic acids can be used in this case. According to the present invention the elongation product can in addition be labelled in order to also observe this reaction; the
Kessler Christoph
Ludwig Wolfgang
Rueger Ruediger
Schleifer Karl-Heinz
Stern Anne
Arent Fox Kintner & Plotkin & Kahn, PLLC
Roche Diagnostics GmbH
Sisson Bradley L.
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