Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-01-28
2001-07-31
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091100, C435S091200, C435S325000, C435S252300, C435S243000, C536S063000
Reexamination Certificate
active
06268127
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to methods for preparing DNA from serum and plasma, particularly for use as a target in amplification reactions.
BACKGROUND OF THE INVENTION
Technology is rapidly advancing in the area of amplification and detection of nucleic acids, particularly as it relates to commercial diagnostic tests, which offer early detection of infectious diseases, cancer and genetic disorders. Highly sophisticated techniques for the amplification of minute quantities of nucleic acids, such as PCR (polymerase chain reaction), are now well known (see. U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188). The inherent sensitivity of PCR, i.e., its ability to amplify very small concentrations of a target DNA, means that low level carryover of PCR products, and contamination between specimens, can yield false positive results. Carryover and sample contamination, among other things, are a function of the number of manipulations of the sample required during processing. Therefore, a simple procedure with a minimal number of steps that expose the sample to the environment is highly desirable.
Traditionally, PCR identification of viremia due to infectious human cytomegalovirus (HCMV), and identification of other viruses and bacteria has been performed on peripheral blood leukocytes (WBCs) obtained from whole blood. Separation of WBCs from whole blood is usually required prior to extraction of the HCMV DNA from the WBCs. Procedures for this separation include erythrocyte sedimentation in a dextran solution (Rasmussen et al.,
J. Infect. Dis.
171:17714 82, 1995), differential lysis using ammonium chloride solutions (U.S. Pat. No. 5,702,884 Ekeze et al), or the use of commercially available procedures (such as, e.g., CPT-Vacutainer™ tubes from Becton-Dickinson). Typically, these procedures include a wash step which results in the removal of potential inhibitors of amplification; alternatively, the isolation of WBCs serves to remove these inhibitors, which typically reside in high concentration in plasma or serum.
Once the WBCs have been isolated, they are lysed and the DNA is extracted. This involves procedures such as, for example, boiling, sonication, or freeze-thawing of the WBCs, or the use of proteolytic enzymes and/or surfactants to lyse the cells and extract the DNA. DNA extraction may also involve an alkali lysis step (U.S. Pat. No. 5,639,599). Often, a more rigorous extraction/purification is performed to further ensure that purified DNA is obtained devoid of potential inhibitors, including, e.g., use of glass beads (Gene Clean II kits, Bio 101, Inc.), phenol-chloroform extraction procedures, polymer capture (U.S. Pat. No 5,582,988), spin-column adsorption (Qiagen QIAamp kits), and other commercially available DNA isolation kits (Puregene, Gentra Systems Inc.).
Notably, procedures used for isolating and lysing WBCs, which serve to wash or remove potential inhibitors from the WBC preparation, cannot be used with plasma and serum. As the vast majority of endogenous biochemical substances and consumed drugs, metabolites of drugs, and the like, reside and are heavily concentrated in serum and plasma, there is a need in the art for a rapid and robust procedure that could be used with serum and plasma which would remove the potential inhibitors or render them ineffective.
Extracellular HCMV nucleic acid in infected individuals is present in plasma and serum. Serum and plasma are gaining acceptance as samples of choice for detecting HCMV nucleic acid using PCR. Generally, the target DNA does not exist as free DNA but rather as a complex association of DNA, RNA, and proteins. The DNA must be extracted from the complex and denatured in order to render it available for amplification. Serum and plasma samples are typically subjected to heat, surfactants, and treated with proteases. Often additional rigorous protocols are employed to extract the target DNA, such as those described above for WBCs (Spector et al.,
J. Clin. Microbiol.
30:2359-65, 1992; Nolte et al.,
J. Clin. Microbiol.
33:1263-66, 1995; Wolf et al.,
Transplantation
56:330-4, 1993; Patel et al.,
J. Clin. Microbiol.
32:1431-4, 1994). Alkali treatment has also been used. However, this alkali treatment typically requires a high NaOH concentration followed by a neutralization step (Hansen et al.,
J. Infect. Dis.
170:1271-4, 1994).
Thus, there is a need in the art for a rapid and efficient procedure for extracting DNA from serum and plasma that is compatible with PCR amplification methods.
SUMMARY OF THE INVENTION
The present invention provides a method for extracting DNA from serum or plasma samples. The method comprises:
(i) contacting the serum or plasma with alkali to yield alkalinized serum or plasma;
(ii) heating the alkalinized serum or plasma to a temperature between about 100 and about 110° C. for a time between about 5 and about 20 minutes,
(iii) centrifuging the heated alkalinized serum or plasma; and
(iv) recovering the DNA-containing supernatant.
In a preferred aspect, prior to centrifuging the heated alkalinized serum or plasma, the heated alkalinized serum or plasma produced by step (ii) is cooled or is allowed to cool to room temperature, i.e., about 25° C., prior to centrifugation in step (iii).
In another aspect, the invention provides a method for detecting the suspected presence of a DNA-containing microorganism, for example human cytomegalovirus (HCMV), in serum or plasma. The method comprises:
(i) contacting the serum or plasma with alkali to yield alkalinized serum or plasma;
(ii) heating the alkalinized serum or plasma to a temperature between about 100 and about I 110° C. for a time between about 5 and about 20 minutes;
(iii) centrifuging the heated alkalinized serum or plasma;
(iv) recovering the DNA-containing supernatant;
(v) subjecting the DNA in the supernatant to amplification using oligonucleotide primers that recognize sequences within microorganism DNA, to form microorganism-specific amplification products; and
(vi) detecting the amplification products, wherein detection of amplification products specific to the microorganism indicates the presence of the microorganism in the serum or plasma.
In a preferred aspect, subsequent to step (ii), the heated alkalinized serum or plasma is cooled or is allowed to cool to room temperature, i.e., about 25° C., prior to centrifugation in step (iii).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a simple, rapid, and highly effective method for extracting DNA from serum and plasma samples which results in DNA preparations suitable for subsequent detection assays without further manipulation.
The method comprises the steps of:
(i) contacting the serum or plasma with alkali to yield alkalinized serum or plasma;
(ii) heating the alkalinized serum or plasma to a temperature between about 100 and about 110° C. for a time between about 5 and about 20 minutes;
(iii) centrifuging the heated alkalinized serum or plasma; and
(iv) recovering the DNA-containing supernatant.
It is preferred that subsequent to heating and prior to centrifugation, the heated alkalinized serum or plasma is cooled or is allowed to cool to room temperature, i.e., about 25° C. The cooling can be passive, i.e., by simple equilibration with room air, or active, i.e., the heated alkalinized serum or plasma can be refrigerated, placed on ice, or put in a water bath, until the temperature of the heated alkalinized serum or plasma reaches room temperature.
In practicing the present invention, the suitable alkali includes, but is not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, or a mixture of any of the foregoing. Preferably, sodium hydroxide is used. The final concentration of alkali in the mixture prepared in step (i) ranges from about 10 to about 90 mM, and most preferably from about 15 to about 50 mM. Special notice is made of an alkali concentration of about 20 mM. A preferred method involves forming a mixture of 1 part by volume of serum or plasma with about 4 parts by volume of an aqueous solution of about 25
Angie Kerry Lee
Bergmeyer Lynn
Darby & Darby
Jones W. Gary
Ortho-Clinical Diagnostics, Inc.
Taylor Janell E.
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