Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-05-24
1998-06-23
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 4, 435 912, 935 77, 935 78, C12Q 168, C12Q 100, C12N 1500
Patent
active
057703604
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP93/02248 filed 20 Aug. 1993, published as WO94/04706 Mar. 3, 1994.
FIELD OF THE INVENTION
The present invention relates to a method for the elimination of false negative test results in assays for the detection of amplified analyte nucleic acid.
Normally, when amplified nucleic acid is to be detected in a sample, negative test results, that is results indicating that no analyte nucleic acid was originally present in the sample under investigation, can only be confirmed by carrying out a separate confirmation assay. Since the performance of separate confirmation tests is a rather time consuming event the need exists for a method for providing a control on negative test results in assays for the detection of amplified analyte nucleic acid. When each sample can be provided with a direct internal check on the assaying procedures and the amplification method no separate confirmation tests have to be performed.
BACKGROUND OF THE INVENTION
The addition of other sequences to a sample comprising analyte nucleic acid, which are capable of being coamplified with the analyte nucleic acid, has been described by Becker and Hahlbrock (Nucleic Acids Research, Vol. 17. Number 22. 1989). The method described by Becker and Hahlbrock is a method for the quantification of nucleic acid where different known amounts of internal standard, comprising a nucleic acid sequence that differs from the analyte nucleic acid by just one nucleotide (point mutation) are added to a sample containing an unknown amount of analyte nucleic acid and coamplified by PCR with the analyte nucleic acid. By introducing one base change in the internal standard sequence a specific restriction site is created and the mutant sequence is cut with the appropriate restriction enzyme before the sample containing the amplified nucleic acid is applied to an electrophoretic gel. The nucleic acid is quantified by comparing the bands in the gel representing (a part of) the mutant sequence and the analyte nucleic acid.
Becker and Hahlbrook use the internal standard as a marker in their quantification method where both analyte and marker are non-competitively amplified.
SUMMARY OF THE INVENTION
The present invention relates to a method for the elimination of false negative test results in assays for the detection of amplified analyte nucleic acid in a sample characterized in that, prior to amplification, an internal control, comprising a nucleic acid distinguishable from the analyte nucleic acid, that can be amplified with the same amplification reagents as the analyte nucleic acid, is added to the sample. The reagents used with the amplification include the amplification primers. The internal control used with the method according to the invention should resemble the analyte nucleic acid in that both are capable of annealing with the same amplification primers.
When the sample is not provided with an internal control a negative test result will give no indication of the presence of amplified nucleic acid. In this case the absence of any signal representing amplified nucleic acid can also be caused by an error in the amplification or assaying procedures and a separate confirmation test must be performed to exclude this possibility.
One advantage of the use of a method with an internal control in every sample according to the invention is that in the case of a "negative" sample (that is a sample in which no analyte was present) a signal is obtained, indicating the presence of the amplified internal control. The appearance of the signal representing the internal control confirms the negative test result and serves as a control on the followed procedures (the amplification and the detection procedure). False negative test results are thereby excluded.
The internal control used with a method according to the invention must differ from the analyte in that, upon detection, the presence of the amplified internal control is indicated by a signal that differs from the signal representing the analyte. For example, with gel electrophor
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Kievits Tim
Lens Peter Franklin
Akzo Nobel N.V.
Blackstone William M.
Gormley Mary E.
Whisenant Ethan
Zitomer Stephanie W.
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