Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-07-10
2004-10-26
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S007100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06808888
ABSTRACT:
TECHNICAL FIELD
The invention lies in the field of diagnostics. More in particular, the invention lies in the field of molecular diagnostics.
BACKGROUND
The increased knowledge of the molecular basis for disease has generated an increasing demand for more and more sophisticated diagnostic methods that can help identify the exact molecular cause of disease. In particular, for infectious diseases, clinicians want to be able to rapidly identify the pathogen. Importantly, concurrent accurate typing and discrimination of different strains of a pathogen is desired. This is important, for instance, in cases where certain strains have a particular unfavorable phenotype. It is also important, for instance, in the case where the pathogen is capable of rapid mutation of its genome to counteract selective pressures induced by the patient and/or the treatment. One non-limiting example of such a pathogen is, of course, Human Immunodeficiency Virus (“HIV”). HIV is, for instance, capable of evading selective pressure induced by nucleotide analogues through mutation of the reverse transcriptase enzyme. To be able to predict which nucleotide analogue, if any, would benefit the patient, it is desired to know in advance, i.e. before treatment starts, which genotype(s) of HIV prevail in the patient.
One possibility to find the pathogen causing the disease is to harvest a sample from the patient comprising the pathogen and culturing the pathogen on suitable media in the case of a bacterial pathogen or in a suitable marker cell line for a viral and/or mycobacterium pathogen. The pathogen may be typed following and/or during culturing. This culture process can be combined with, for instance, antibiotics and/or other medicines to find the relative resistance/sensitivity of the pathogen to said medicine. This so-called culture driven testing has several advantages and is indeed routinely applied for a number of diseases.
However, generally, the considerable amount of time involved with the culture process necessitates that a treatment schedule be started prior to the identification of the causative agent. This is not desired, since the treatment started may prove to be ineffective. Moreover, for many pathogens, a culture system is as yet not available. Another problem with the culture system is the inherent variability of the procedure. Not all pathogens are equally well cultured outside the body of a patient. In addition, since viability of the pathogen is essential, differences in the handling of the sample outside the body will result in variability of the result. Moreover, the costs involved in the initiation of a screen with the culture system for a wide variety of different possible causative agents in any clinical sample are considerable.
For this reason there is a need for a rapid system for the typing of a pathogen that is versatile, reliable and at least partially able to discriminate between different variants of the pathogen. A number of different strategies have been tried. One such strategy relies on the detection of pathogen-derived nucleic acid in a sample. To be able to rapidly detect such nucleic acid, a nucleic acid amplification step is usually required.
Many nucleic acid amplification methods have been devised that are able to specifically detect a certain pathogen and possibly even a number of different strains of said pathogen. However, such methods usually require the clinician to have at least some idea of the kind of pathogen that may cause the disease in the patient. This is frequently not the case.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method for detecting, quantifying and/or simultaneous typing of a variety of different nucleic acid sequences in a sample. Preferably, said nucleic acid sequences comprise nucleic acid from a microorganism and/or derivative thereof. The microorganism can be a bacterium, a phage and/or a virus.
Preferably, said microorganism is a pathogenic microorganism. The present invention further allows discrimination between different strains of a microorganism or other sequences. The present invention is not only useful for the typing of a pathogen in a sample of a patient, but it is also applicable for the typing of a pathogen in a sample derived from an animal. Preferably, said animal has a commercial and/or emotional value to a human, such as a pet, a farm animal and/or an animal living in a natural reserve. The invention is also suitable for application in poultry and fish. The method of the invention is, of course, not only suited for the typing and/or detection of a pathogen. The method is generally suited for the typing and/or detection of nucleic acid in a sample. For instance, in the case of cellular DNA or RNA, the method can be used for creating a genetic expression profile, respectively, of the nucleic acid in the sample. Knowing the origin of the nucleic acid in the sample then allows the correlation of the profile with the origin.
In case the origin is nucleic acid of (a specific part of) an individual, the profile, or a part thereof, can be correlated to, for instance, a database of profiles, or parts thereof, of other individuals. Matching of the profile (or part thereof) to known profiles(or parts thereof) allows the correlation of the profile (or part thereof) of the individual with the phenotypes of individuals with matching profiles(or parts thereof) displayed by these other individuals. Thus, the method of the invention can be used generally for the typing and/or detection of nucleic acid in a sample.
In one aspect, the invention provides a method for amplifying nucleic acid in a sample comprising providing said sample with a set of primers comprising between 3 and 8 random bases and at least 8 essentially non-random bases, subjecting said sample to a first nucleic acid amplification reaction, providing said sample with at least one second primer comprising at least 8 bases essentially identical to said non-random bases, subjecting said sample to a second amplification reaction and detecting nucleic acid amplified in said sample. Typically only a limited amplification of nucleic acid will occur in said first nucleic acid amplification reaction. Said first and second amplification reactions are preferably performed separately, optionally including a step to remove any unused primer in said first amplification reaction. This way, the reproducibility of the method is best controlled. However, the first and the second amplification reactions may also be performed simultaneously.
Nucleic acid in said sample may be DNA and/or RNA. A double stranded nucleic acid can be denatured into essentially single stranded nucleic acid prior to the priming of synthesis of a complementary strand of nucleic acid. The complementary strand may be DNA and/or RNA. Synthesis of said complementary nucleic acid is performed under conditions and using enzymes that are known in the art, such as, for instance, conditions and enzymes commonly used for polymerase chain reaction and/or NASBA.
The number of nucleic acids amplified with the method of the invention is dependent on the amount and the complexity of the nucleic acid in the sample. When the complexity, i.e. the number of different sequences in the nucleic acid(s) is low, a small number of nucleic acids will be amplified with the method of the invention. In this case, some nucleic acids will be dominant in the amplificate, resulting in a banding pattern when the amplificate is run on a gel. On the contrary, when the complexity of the nucleic acid in the sample is high, many nucleic acids will be amplified, resulting in a smear when the amplificate is run on a gel. An example of nucleic acid with a particularly low complexity is nucleic acid derived from a small virus and/or plasmid (typically smaller than 10 kb). An example of nucleic acid with a particularly high complexity is cellular DNA (typically comprising >10
8
kb). It is clear that the mentioned examples are non-limiting. Many different complexities are possible, and additionally mixtures of low and high complexity nucl
Dekker John
Gemen Bob van
Maas Jolanda
van der Kuyl Antoinette C.
Zhang Jing
Primagen Holding B.V.
Riley Jezia
TraskBritt
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