Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-11-24
2001-02-06
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S024300
Reexamination Certificate
active
06183999
ABSTRACT:
The invention relates to a procedure for the detection of high virus concentrations in blood plasma and/or blood serum by means of the polymerase chain reaction, the detection of parvoviruses, in particular of parvovirus B19, being of particular importance.
It is known that viral contaminants which are contained in plasma protein solutions also can pass into the plasma products prepared therefrom and then can lead to infections in patients to whom blood plasma products of this type are administered. In order to prevent this danger, various safety measures have been developed. Among them, the examination of the donor for certain virus infections and the inactivation and elimination of viruses before, during and after the production of the plasma products from the donor blood have been given very particular importance.
Frequently, donor blood is contaminated with parvovirus B19. Parvoviruses lead to serious disorders only in exceptional cases, but for safety reasons donor blood which is contaminated with high titers of parvoviruses should not be used for the production of plasma products. On the other hand, low titers of parvoviruses are tolerable in donor blood, because physical and chemical methods are available with which small residual amounts of viruses in the plasma to be processed can be removed during the preparation of plasma proteins.
It is therefore desirable to identify donor blood having exceptionally high concentrations of parvoviruses in time and to exclude it from processing into plasma products.
It was therefore the object to develop a detection procedure for high-titer plasma protein solutions which only indicates the presence of exceptionally large amounts of pathogenic viruses, in particular of parvoviruses, but in the presence of small amounts of parvoviruses shows no reaction.
As is known, the polymerase chain reaction (PCR) is a very effective method for the detection of small amounts of a known nucleic acid sequence in a sample (Erlich H. A., Gelfand D., Sninsky J. J. (1991), Science 252, pp. 1643-1651; PCR Protocols. Current methods and applications (1993) edited by B. A. White, Humana Press, Totowa, N.J., ISBN 0-89603-244-2). If the sequence of the virus DNA is already known, a primer pair can be synthesized which is complementary to regions on single strands lying opposite to one another and flanks the desired DNA sequence. Under the conditions known per se of a PCR, large amounts of a specific DNA can then be amplified in vitro by succession of, as a rule, more than 30 reaction cycles. By means of the PCR cycles, a DNA fragment of a specific size, which is composed of the lengths of the two primers plus the length of the virus DNA between them, is only amplified if the desired virus DNA is present in the sample.
The PCR technique is so sensitive that using it extremely small amounts of the DNA can be detected with great reliability. The great sensitivity of the PCR also leads to positive results if the virus DNA to be detected is present in a plasma sample in such small amounts that as a result the practical usefulness of the donation, from which the sample derives, for the preparation of blood plasma products is not adversely affected. For example, the customary qualitative PCR technique is not suitable for the detection of the parvovirus B19 DNA, since no differentiation can be made between high and low parvovirus B19 concentrations and also plasma samples having a low parvovirus B19 titer appear to be strongly virally contaminated.
It has now been found that the procedure for the detection of high concentrations of viruses in blood plasma and/or blood serum by means of the polymerase chain reaction can be efficiently modified for practical use if the sensitivity of the polymerase chain reaction is restricted by the use of suboptimal nucleic acid extraction, amplification or detection conditions.
This procedure is suitable for the detection of high concentrations of nucleic acids of pathogenic viruses. The use of this procedure is particularly advantageous for the detection of high nucleic acid concentrations of parvoviruses. According to the invention, the sensitivity of the PCR for the detection of the nucleic acids of parvoviruses is so greatly restricted that the nucleic acids of the parvoviruses can only be detected in samples whose DNA content is greater than 10
6
to 10
7
genome equivalents. Samples having a lower parvovirus DNA content are no longer recognized as positive and can be subjected to further processing to give plasma products if small amounts of parvovirus possibly still present are removed during the production procedure.
Several procedures are available for restricting the sensitivity of the PCR. Thus it is possible, for example, to carry out the nucleic acid extraction under conditions in which only small amounts of nucleic acids are isolated or it is possible to work with highly dilute solutions in which only small amounts of nucleic acids are contained. Suboptimal amplification conditions can be established, for example, by carrying out the addition of the primers to the DNA single strand (annealing) and the amplification at unusually low temperatures and restricting the number of the PCR cycles or “slowing down” the amplification by reproduction of the primer concentration below the optimal range. Finally, it is also possible to select the detection conditions such that only particularly high concentrations of parvovirus DNA are recognized.
As is known, the customary PCR begins with a denaturation of the nucleic acids extracted from the investigation sample at 90° C. or higher, for, for example, 20 seconds to 1 minute.
In the course of this, these are split up into single strands, to which the primers are normally added at a temperature of 45 to 650° C. The amplification is then carried out at 72° C.
In contrast, according to the invention the annealing and/or the amplification is carried out at a temperature of about 52° C. Additionally, the number of PCR cycles is restricted to 30. By this means, the reaction rate of the PCR is considerably restricted and the amount of the parvovirus DNA formed is considerably reduced by the reduction of the number of cycles.
The primers employed are, for example, the following oligonucleotides.
SEQ ID NO. 1:
5′
ATG GGC CGC CAA GTA CAG G A
3′
SEQ ID No. 2:
5′
CAG GCA CAG CTA CAC TTC CAG GC
3′
It has now proven to be particularly advantageous in the procedure according to the invention to carry out the detection of the amplified parvovirus DNA section by means of a probe which carries two fluorescent dyes. This labeled probe, which is added to the DNA between the two primers characterized by the abovementioned sequences, then has, for example, the following sequence:
SEQ ID No. 3:
5′
FAM-TGG TGG TCT GGG ATG AAG GTA TTA TT-TAMRA
3′
This sequence can be obtained commercially under the name TaqMan® and is intended for use in the 5′-nuclease assay, the TaqMan Assay. This method is described in detail by Livak K. J., Flood S. J. A., Marmaro J., Giusti W., Deetz K., Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Method and Appl. 1995; 4:357-362 and which is incorporated by reference.
The particular characteristic of this probe is that the fluorescence of the (FAM) attached to the 5′ end of the probe, the reporter, is reduced by the vicinity of the second fluorescent dye (TAMRA), the quencher, arranged at the 3′ end of the primer.
In the course of the amplification, the new DNA strand is formed under the action of a thermostable DNA polymerase, preferably of Taq DNA polymerase. In the course of this, the DNA polymerase displaces the probe not only from the single strand, but splits it up by means of its endonucleolytic activity and at the same time liberates the two fluorescent dyes. The fluorescence of the reporter dye is now no longer suppressed by the quencher dye and increases. If the fluorescence at the reporter wavelength an
Groener Albrecht
Weimer Thomas
Aventis Behring GmbH
Brusca John S.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Kim Young
LandOfFree
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