Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-02-25
2004-12-14
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06830887
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a method and kit for quantifying and at least partially sequencing a nucleic acid analyte that is present in a sample. The nucleic acid analyte may be from an infectious organism that is present in a patient sample.
Academic and commercial interest in nucleic acid diagnostics has, to date, focused on qualitative assays. This type of assay determines the presence or absence in a patient sample of a specific gene mutation or infectious pathogen. Molecular assays which achieve these goals are well known. Many rely on amplification techniques, known to those skilled in the art such as the polymerase chain reaction (PCR), NASBA or 3SR, with or without hybridization probing. Others such as Digene Hybrid Capture Assays (DiGene Diagnostics Inc.) do not require amplification prior to detection and are generally less sensitive. Assays have been developed for many infectious pathogens such as
Chlamydia trachomatis
, Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2), and human papilloma virus (HPV). Some of these tests have been launched commercially by Roche Diagnostic Systems, Abbott Laboratories and others.
Quantitative assays of nucleic acid analytes also prove useful in diagnosis of a variety of medical disorders. For example, viral load in HIV infection may be correlated with increased risk of clinical progression of HIV disease (Mellors. J. W. et al. (1995). Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion. Ann. Intern. Med. 122: 573-579). While this example is best known, other quantitative applications also have clinical and commercial interest, such as quantitation of human papilloma virus in PAP smears. (Cuzick, J. et al. (1994) Type-specific human papillomavirus DNA in abnormal smears as a predictor of high-grade cervical intraepithelial neoplasia. Br. J. Cancer 69:167-171; Bavin P. J. et al. (1993) Use of semi-quantitative PCR for human papillomavirus DNA type 16 to identify women with high grade cervical disease in a population presenting with a mildly dyskaryotic smear report. Br. J. Cancer 67:602-605.)).
Notwithstanding their usefulness, quantitative assays of nucleic acid analytes have lagged behind in development. The delay may in part be attributed to technology barriers. Most instruments and methods provide inadequate dynamic range for measuring quantities, thus requiring labor intensive techniques such as multiple serial dilutions and repeat reactions. Further, until recently, PCR methods have been perceived as unreliable for quantitation due to the possibility of contamination and non-linear enzyme kinetics.
The AMPLICOR HIV-1 MONITOR (Roche Molecular Systems) test is a quantitative molecular assay for HIV RNA levels in blood. The assay is performed on HIV-1 and a subset of HIV-2 RNA found in 200 &mgr;L of blood plasma. The plasma sample is lysed and RNA is reverse transcribed then amplified by PCR. The reaction products are quantified by a probe based photometric assay and compared to the levels of a control RNA of known quantity that is added to the plasma sample. The control RNA is reverse transcribed along with the sample RNA and co-amplified using the same amplification primers. Six serial dilutions are necessary to detect across the full range of detectable viral load: 400 copies to 750,000 copies per ml. The test requires that for samples over 750,000 copies, (over 2.2 million copies per ml have been detected) the original patient sample must be diluted. The AMPLICOR assay therefore quantifies across the full range of possible values by a series of multiple dilutions. The AMPLICOR assay does not determine which sub-type or sub-types of HIV-1 are present, and it does not establish if HIV-2 was amplified.
Other quantitative HIV assays have been reported. Some of these papers, incorporated herein by reference, include:
Mulder, J et al. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: Application to acute retroviral infection. J. Clin. Micro. 32:292-300
Dewar, R. L. et al, 1994 Application of branched DNA signal amplification to monitor human immunodeficiency virus type 1 burden in human plasma. J. Infect. Dis. 170:1172-1179
van Gemen, B. et al. 1993 Quantification of HIV-1-1 RNA in plasma using NASBA during HIV-1-1 primary infection. J. Vir. Meth. 43:177-188.
The possibility of integrating a quantitative nucleic acid assay with a qualitative assay, such as sequencing of the nucleic acid, has not been achieved or proposed by previous workers. The advantage of an integrated test would be enormous, however, particularly in the case of evaluation of HIV infections. For example, not only could pathogen load be determined but also the exact serovar (or variety) of the pathogen could be determined. This would allow doctors and patients to know if treatments were effecting not only the quantity but also the variety of pathogen. In addition, a simplified assay would provide substantial economies of scale.
It is an object of the present invention to provide a method and kit for quantifying and determining the nucleic acid sequence of a nucleic acid analyte that is present in a patient sample.
It is a further object of the present invention to provide a method and kit for the quantifying and genotyping of HIV virus present in a patient sample.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved by a method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte, comprising the steps of:
(a) preparing a reaction mixture containing the sample and a known amount of an internal quantitation standard;
(b) combining at least a first aliquot of the reaction mixture with a set of amplification reagents effective to amplify nucleic acids in the reaction mixture, said reagents including at least one primer pair which is effective to amplify a first region of the nucleic acid analyte if present in the sample to produce a first amplified sample fragment and to amplify at least a portion of the internal quantitation standard to produce a control fragment;
(c) amplifying nucleic acid analyte from the sample and the internal quantitation standard in the reaction mixture using the first pair amplification product mixture containing first amplified sample fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragments when the nucleic acid analyte is not present in the sample;
(d) analyzing the relative amounts of first amplified sample fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample; and
(e) determining the sequence of the first amplified sample fragments in the amplification mixture to determine the qualitative characteristics of any nucleic acid analyte in the sample using at least a first sequencing primer.
In accordance with an embodiment of the method of the invention, the internal quantification fragment is derived from the analyte nucleic acid by the incorporation of a plurality of sequence variations. These sequence variations include at least a first sequence variation effective to render the internal quantitation standard distinguishable from the first amplified sample fragment, and a second sequence variation effective to substantially eliminate the production of sequencing products from interaction of the internal quantitation standard and the first sequencing primer.
For evaluation of HIV and other genes which contain multiple regions of potential genetic variations and interest, this method is preferably utilized as part of a broader method in which the genome is evaluated in several parts. For example, in the case of HIV-1, the genome may be evaluated in one part for evaluation of the protease gene and three parts for evaluation of the beginning, middle and end portions of the reverse transcriptase gene. In accordance with this embodiment of the invention, separate aliquots of the reaction mixture containing
Bayer HealthCare LLC
Holland & Hart LLP
Horlick Kenneth R.
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