Amplification and detection process

Details Amplification and detection process Amplification and detection process

1995-01-24

1998-12-15

Campbell, Eggerton A.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 6, C12P 1934, C12Q 168

Patent

active

058495444

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

Hybridisation methods are widely utilised in testing for the presence of particular nucleic acid sequences, identifying and/or quantifying such sequences.
Various improvements and modifications have been introduced, to improve the specificity and sensitivity of the reaction. For example, a number of recently developed in vitro nucleic acid amplification methods have greatly increased the sensitivity of detection. These methods include: ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), Q.sub..beta. replicase based methods, strand displacement amplification (SDA) and notably polymerase chain reaction (PCR).
In a recent development, two separate PCR amplifications have been used to both amplify and label the target nucleic acid sequence. The labelled sequence is then immobilised on a solid phase carrier, and testing is carried out using a reagent specific to the label. (See D J Kemp et al, "Colorimetric detection of specific DNA segments amplified by polymerase chain reactions", Proc Natl Acad Sci USA 86, pp 2423-2427, 1989.)
However, in prior art methods, including that of Kemp et al, solution amplified nucleic acids have been detected in a second vessel by capture of hybridisation to solid phase capture reagents (two vessel assays).
Solid phase techniques have been used in relation to synthesis procedures (e.g. chemical synthesis of amino acid and nucleic acid sequences) and assay procedures (as in the final step of the procedure of Kemp et al, see above).
However, it has not hitherto been possible to conduct an in solution amplification process for nucleic acid sequences, and identification/quantification procedures, in the same reaction vessel.


SUMMARY OF THE INVENTION

This method for detecting a target nucleic acid sequence involves amplification and detection in the same vessel and comprises: provided with a solid phase capture probe comprising a nucleic acid sequence capable of hybridising to at least a portion of said amplified target nucleic acid sequence, said capture probe being incapable of participating or not participating in standard nucleic acid sequence amplification processes, sequence into contact with said capture probe under conditions which allow said amplified target nucleic acid sequence to be bound by said capture probe, and
In a further aspect, the present invention provides an assay system or kit, for detecting a target nucleic acid sequence in a sample suspected of comprising said target nucleic acid sequence, comprising: hybridising to at least a portion of said amplified target nucleic acid sequence, said capture probe being immobilised on a solid phase support which forms a part of or is insertable into a container for the sample, and said capture probe being incapable of participating in standard nucleic acid sequence amplification processes, said capture probe.
A sample suspected of comprising a particular nucleic acid sequence is placed within a reaction vessel. If said particular nucleic acid sequence is present within the sample, then it is amplified freely in solution, by any means, to give a nucleic acid product (or derivative or analogue) carrying a detector tag and captured by a complementary solid phase capture probe present in the reaction vessel. The complementary solid phase capture probe is a nucleic acid sequence (or derivative or analogue) which is incapable of participating in amplification of said particular nucleic acid sequence (e.g. will not act as a primer). Preferably, the capture probe hybridises to a central section of the amplified nucleic acid sequence--away from any primer or primer complementary nucleic acid sequences. Thus, false positives arising from amplified primer-multimers (e.g. primer-dimers) are avoided. The captured nucleic acid sequence (or derivative or analogue) is then identified via the detector tag and/or quantified by conventional means (e.g. by fluorescence, for products carrying a fluorescent tag).
Preferably, said particular nucleic acid sequence is amplified prior to c

REFERENCES:
patent: 5232829 (1993-08-01), Longiarv et al.
patent: 5635347 (1997-06-01), Link et al.
Heller et al, J. Clin, Microbiol. 29:638-641 (1991).
Sanger et al, PNAS 74: 5463-5467 (1977).

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