Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-21
2001-04-03
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S173900, C435S259000, C514S514000
Reexamination Certificate
active
06210881
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the present invention broadly relates to nucleic acid hybridization. More specifically, the present invention relates to the reduction of substances in samples which inhibit nucleic acid hybridization events. Such events include nucleic acid probe hybridization to determine the presence and/or amount of a target nucleic acid, and nucleic acid primer hybridization for the initiation of a nucleic acid amplification process.
Nucleic acid amplification processes such as strand displacement amplification (SDA), polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA) and others are used to create multiple copies of a particular nucleic acid sequence(s) of interest (target sequence) which is present in lesser copy number in a sample. However, a number of substances commonly found in such samples cause inhibition of nucleic acid amplification processes, because of inhibition of the hybridization of primers to initiate the amplification process. Similarly, such substances inhibit direct nucleic acid probe hybridization reactions used for the detection of unamplified target nucleic acids.
An example of such nucleic acid hybridization inhibitory substances are porphyrin compounds derived from heme and hematin which are both commonly found in blood samples and inhibit PCR. (
PCR Technology
, Stockton Press, Henry A. Erlich, Ed. pp 33-34, 1989). Protocols using osmotic lysis and pelleting of nucleic and cell debris have been used to reduce the amount of these inhibitors.
Salivary samples have also been reported to contain PCR inhibitory substances. Ochert et al.,
PCR Methods and Applications
3, 365-368 (1994). Although the inhibitory substances were not identified, it was found that extended microwaving or boiling of the salivary sample totally removed PCR inhibition.
Frickhofen and Young,
J. Virol. Methods
35, 65-72 (1991), report that heating of serum samples for 45 seconds at 70° C. improves PCR amplification of viral nucleic acid sequences. This improvement is theorized to be due to heat inactivation of serum enzymes such as aprotinin, leupeptin PMSF and pepstatin which are believed to be inhibitory to PCR processes.
Another approach for removing PCR inhibitory substances from serum prior to amplification of a viral nucleic acid sequence is taught by Zeldis et al.,
J. Clin. Invest
. 84, 1503-1508 (1989). This approach involves adsorbing the virus to antibody coated microparticles, washing the microparticles, and then destroying the remaining proteins which may be inhibitory to PCR with proteinase K.
In attempting to detect
Treponema pallidum
in amniotic fluid, fetal and neonatal sera and cerebrospinal fluid by PCR, four different processes were attempted to remove PCR inhibitory compounds. Grimprel et al,
J. Clin. Microbiol
. 29, 1711-1718 (1991). Briefly, the four processes for removal of PCR inhibitory compounds were: (1) a boiling method wherein sample in a tube was placed in a boiling water bath for 10 minutes, cooled on ice, and then centrifuged; (2) a low-spin separation method wherein sample was added to sterile phosphate buffered saline and subjected to a series of centrifugations, then the pellet was resuspended and boiled for 10 minutes, after which it was cooled on ice; (3) an alkaline lysis extraction method wherein sample was boiled for 1.5 minutes in 1 M NaCl, 1 N NaOH and 0.1% SDS, then neutralized with 0.5 M Tris-HCl (pH 8.0), and then subjected to a series of extractions with phenol and chloroform-isoamyl alcohol, and precipitated with isopropyl alcohol; and (4) a spin extraction method wherein sample was subjected to low-spin separation as described in (2) above, followed by 10 minutes of boiling and one phenol-chloroform extraction before precipitation in cold absolute ethanol. The authors reported varying success of these methods dependent on the type of samples used.
With stool samples, polyethylene glycol precipitation was found to remove a significant amount of small particles and soluble substances which could be inhibitory to a reverse transcriptase-PCR process. Jiang et al,
J. Clin. Microbiol
. 30, 2529-2534 (1992). Following the precipitation, an extraction process was performed using the cationic detergent, cetyltrimethylammonium bromide (CTAB) in a high salt concentration in conjunction with phenol-chloroform extraction.
A different approach to removal of PCR inhibitory substances from stool samples is reported by Wilde et al.,
J. Clin. Microbiol
. 28, 1300-1307 (1990). Before using PCR to detect rotavirus nucleic acid from stool samples, the extraction process was modified with an added step that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps.
When performing a study to detect cytomegalovirus (CMV) in urine using PCR, it was found that urea is inhibitory to PCR. Khan et al.,
J. Clin. Pathol
. 44, 360-365 (1991). This reference reports that the PCR inhibitory effects of urea in urine are effectively removed by simple dialysis or ultracentriftigation.
Another process to remove PCR inhibitory substances from urine before detection of CMV nucleic acid is reported by Buffone et al.,
Clin. Chem
. 37, 1945-1949 (1991). This process occurs subsequent to release of the nucleic acid from the CMV organisms and uses fine glass beads to adsorb nucleic acid such that protein and other substances can be selectively eluted before recovery of the nucleic acid for amplification.
As evidenced by the references described above, most of the publication regarding nucleic acid amplification inhibition has related to PCR. However, these same substances which are inhibitory to PCR, as well as a number of other substances commonly found in clinical samples such as proteinaceous substances, EDTA, human DNA and iron have been found to be inhibitory to SDA, and other nucleic acid amplification processes as well.
Also, most of these methods to reduce or remove nucleic acid hybridization inhibiting substances involve rather time-consuming complicated steps which must be added to the sample processing methodology. Another problem with methods which utilize relatively severe processing steps or conditions, and/or require separation of target nucleic acid from other substances is the loss of some target nucleic acid sequence. Despite the ability of nucleic acid amplification processes to make multiple copies of target sequence (amplicons) from very few original targets, amplification efficiency and detection ability are improved if there are greater numbers of original targets in the sample. The greater detection ability can be very important when processing particularly difficult to detect samples such as acid fast Bacillus (AFB) smear negative
Mycobacterium tuberculosis
samples.
SUMMARY OF THE INVENTION
In order to address the problems associated with the presence of substances inhibitory to nucleic acid hybridization in samples and thus, achieve the benefits of more efficient amplification and improved detection of target nucleic acid sequences, the present invention provides a method for reducing the amount of such substances in samples by, prior to lysis of cells in the sample which contain nucleic acid to be amplified, contacting the sample with an agent which does not effectuate the release of nucleic acid from the cells, and then separating the cells from the agent.
Examples of some useful agents for use in the present invention include chaotropes such as guanidine thiocyanate, sodium perchlorate and sodium thiocyanate. Also, the separation of cells from the agent is generally accomplished by a wash and centrifugation step with a solution in which the agent is soluble.
REFERENCES:
patent: 4483920 (1984-11-01), Gillespie et al.
patent: 5010183 (1991-04-01), Macfarlane
patent: 5393672 (1995-02-01), Van Ness et al.
patent: 5482834 (1996-01-01), Gillespie
patent: 5554503 (1996-09-01), Down et al.
patent: 5571894 (1996-11-01), Wels et al.
Collis Matthew P.
Little Michael C.
Llorin Oscar J.
Becton Dickinson and Company
Highet David W.
Jones W. Gary
Tung J.
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