Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1993-05-14
2004-03-23
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C514S04400A, C536S024330
Reexamination Certificate
active
06709813
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to diagnostic compositions, elements, methods and test kits for the amplification and detection of human cytomegaloviral (hCMV) DNA. In particular, it relates to improved methods of polymerase chain reaction (PCR) using test kits and buffered compositions containing “matched” primers for one or more target nucleic acids, one of which is hCMV DNA.
BACKGROUND OF THE INVENTION
Cytomegalovirus is a double stranded DNA virus, approximately 240,000 nucleotides in length, and belonging to the Herpes virus group. Human cytomegalovirus has become increasingly of interest in a wide variety of disease states. In the United States, hCMV infects up to 1% of all newborns and results in significant morbidity and mortality in congenitally infected infants. Infections from hCMV also have a major impact on immunocompromised patients, such as those afflicted with acquired immunodeficiency syndrome (AIDS) and recipients of transplanted organs. People infected with the disease often suffer impairment of some of their vital organs, including the salivary glands, eyes, brain, kidneys and liver. In children, the hCMV infection represents the leading infectious cause of mental retardation and nonhereditary sensineural deafness in children. Furthermore, hCMV is associated with a wide variety of classical syndromes including mononucleosis and interstitial pneumonia. Studies suggest that hCMV is latent in white blood cells, and that it has an oncogenic potential and a possible association with certain types of malignancy including Karposi's sarcoma.
Since hCMV is relatively common in people, a considerable effort has been made to isolate it and to diagnose disease in patients. Efforts to produce vaccines or to treat the disease continue unabated, as noted for example, in U.S. Pat. No. 5,075,213 which describes radiolabeled probes for hCMV DNA.
Diagnosis of the presence of hCMV DNA by culture requires up to three weeks and is technically difficult and expensive. Immunological assays are not presently considered reliable. Earlier diagnosis could be important for treating immunosuppressed patients and for screening blood supplies. HCMV has a large linear double stranded DNA genome composed of two unique components as well as terminal repeat sequences.
Technology to detect minute quantities of nucleic acids (including hCMV DNA) has advanced rapidly over the last two decades including the development of highly sophisticated hybridization assays using probes in amplification techniques such as PCR. Researchers have readily recognized the value of such technology to detect diseases and genetic features in human or animal test specimens. The use of probes and primers in such technology is based upon the concept of complementarity, that is the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of very small concentrations of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al) and by Mullis et al,
Methods of Enzymology
, 155, pp. 335-350 (1987), although there is a rapidly expanding volume of literature in this field. Without going into extensive detail, PCR involves hybridizing primers to the strands of a targeted nucleic acid (considered “templates”) in the presence of a polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates under the appropriate conditions. The result is the formation of primer extension products along the templates, the products having added thereto nucleotides which are complementary to the templates.
Once the primer extension products are denatured, one copy of each template has been prepared, and the cycle of priming, extending and denaturation can be carried out as many times as desired to provide an exponential increase in the amount of nucleic acid which has the same sequence as the target nucleic acid. In effect, the target nucleic acid is duplicated (or “amplified”) many times so that it is more easily detected. Despite the broad and rapid use of PCR in a variety of biological and diagnostic fields, there are still practical limitations which must be overcome to achieve the optimum success of the technology. PCR also produces considerable inefficiency in the use of expensive reagents.
It is well known that PCR is susceptible to a “carry-over” problem whereby amplified nucleic acids from one reaction may be inadvertently carried over into-subsequent reactions using “fresh” PCR reaction mixtures, and thereby causing “false” positives when testing later specimens.
One approach to this problem is to completely contain the reagents for each PCR procedure so no reagents or by-products can be carried over into later reactions. Specially designed test packs or test devices have been designed to contain PCR procedures for this reason. Such test packs are described, for example, in recently allowed U.S. Ser. No. 07/962,159 [filed Oct. 15, 1992 by Schnipelsky et al as a continuation of U.S. Ser. No. 07/673,053 (filed Mar. 21, 1991, now abandoned) which in turn is a CIP of U.S. Ser. No. 07/306,735 (filed Feb. 3, 1989, now abandoned)] now U.S. Pat. No. 5,229,297. These test devices are preferably, but not necessarily, used in PCR in combination with automatic PCR processing equipment such as that described in U.S. Pat. No. 5,089,660 (Devaney Jr.) and in U.S. Pat. No. 5,089,233 (Devaney Jr. et al). This equipment is characterized by its capability to simultaneously process several test specimens in separate test devices.
More preferably, it would be desirable to detect a multiplicity of target nucleic acids (or a multiplicity of nucleic acid sequences in the same nucleic acid) in a single test device. This is referred to herein as “multiplexing”.
In one embodiment of PCR, a specific set of primers and a capture probe (a total of three oligonucleotides) are needed for each nucleic acid sequence which is to be amplified and detected. Thus, the three oligonucleotides are complementary and specific to that targeted nucleic acid sequence. For example, in multiplexing, if three nucleic acid sequences are to be amplified and detected, typically three sets of primers and probes are needed, one set specific for each sequence. Normally, detection of the multiple sequences requires a multiplicity of test devices, and perhaps different sets of PCR conditions (that is, temperature and time conditions).
It would be desirable, however, to amplify and detect a plurality of target nucleic acids simultaneously in the same test device and using “universal” processing equipment and conditions. This cannot be done by merely selecting any set of primers and probes specific for each target nucleic acid from conventional sources. Where processing equipment is used to process several test devices simultaneously, or a single test device is designed for multiplexing, the equipment must be somehow adapted to provide optimum heating and cooling times and temperatures for each set of primers and probes, since they will all likely have individual optimum denaturaton temperatures. To adapt the equipment to randomly selected primers and probes in multiplexing would be a very expensive and cumbersome solution to the problem. Yet there is a great need for efficient, relatively inexpensive and rapid multiplexing to detect multiple nucleic acid sequences of hCMV DNA, or one or more nucleic acid sequences of hCMV DNA and one or more nucleic acid sequences of other target nucleic acids.
SUMMARY OF THE INVENTION
The problems noted above are overcome by using, in PCR, an aqueous composition buffered to a pH of from about 7 to about 9, which comprises:
(a) first and second primers which are specific to and hybridizable with, respectively, first and second nucleic acid sequences which are in opposing strands of human cytomegaloviral DNA (hCMV DNA) and which are separated from eac
Bergmeyer Lynn
Cummins Thomas J.
Findlay John Bruce
Kerschner JoAnne H.
Jones W. Gary
Ortho-Clinical Diagnostics, Inc.
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