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US 2010/0136531 A1Published application
Nucleic acid detection using lateral flow methods
AssigneeTECRA International Pty Ltd.
InventorsIan Garthwaite, Philip A. Myers, Christine M. Sadek
Life of this patent
Estimating the term…
Abstract
Methods and kits for use in detecting a target nucleic acid in a sample are disclosed. In one particular application, the methods and kits allow for the detection of an undesirable micro-organism (e.g. Listeria, Salmonella or Enterobacteriaceae) in food or present on a food preparation surface.
Claims39
A method for the detection of a micro-organism present in a sample, said method comprising the steps of:
- (i) treating said sample so as to cause release of nucleic acid from any of said micro-organism present in the sample;
- (ii) amplifying a target nucleotide sequence present on said nucleic acid, said target sequence being unique or otherwise characteristic of said micro-organism, comprising the use of a pair of first and second primer sequences defining the 5′ and 3′ ends of said target sequence, said first primer sequence being labelled with a first label and said second primer sequence being labelled with a second label such that any amplification of the target sequence generates an amplicon labelled with both first and second labels;
- (iii) diluting an amount of the amplification product of step (ii) in a suitable buffer solution comprising microparticles labelled with a first agent which specifically binds to said first label and allowing said first agent to bind to said first label present;
- (iv) applying at least a portion of the buffered product or step (iii) at or adjacent to a proximal end of a lateral flow device comprising a substrate which allows constituents of said buffered product to wick or flow laterally towards a distal end of the lateral flow device, wherein at a location at or adjacent to said distal end, the lateral flow device is provided with a test region and a control region, said test region provided with a second agent which specifically binds to said second label and said control region being provided with a control agent;
- and
- (v) detecting any binding of constituents of the buffered product at said test region and at said control region.
3 more elements
A method for the detection of a micro-organism present in a sample, said method comprising the steps of:
- (i) treating said sample so as to cause release of nucleic acid from any of said micro-organism present in the sample;
- (ii) amplifying a target nucleotide sequence present on said nucleic acid, said target sequence being unique or otherwise characteristic of said micro-organism, comprising the use of a pair of first and second primer sequences defining the 5′ and 3′ ends of said target sequence, wherein the amplification of the target sequence utilises deoxyribonucleotide triphosphates (dNTPs) labelled with a first label and said second primer sequence is labelled with a second label, such that any amplification of the target sequence generates an amplicon labelled with both first and second labels;
- (iii) diluting an amount of the amplification product of step (ii) in a suitable buffer solution comprising microparticles labelled with a first agent which specifically binds to one of said first and second labels and allowing said first agent to bind to said one of said first and second labels present;
- (iv) applying at least a portion of the buffered product of step (iii) at or adjacent to a proximal end of a lateral flow device comprising a substrate which allows constituents of said buffered product to wick or flow laterally towards a distal end of the lateral flow device, wherein at a location at or adjacent to said distal end, the lateral flow device is provided with a test region and a control region, said test region being provided with a second agent which specifically binds to the other of said first and second labels which is not bound by said first agent and said control region being provided with a control agent;
- and
- (v) detecting any binding of constituents of the buffered product at said test region and at said control region.
3 more elements
(canceled)
(canceled)
10-14. (canceled)
(canceled)
29-30. (canceled)
32-35. (canceled)
A kit for the detection of a micro-organism present in a sample, said kit comprising:
- a pair of first and second primer sequences defining 5′ and 3′ ends of a target nucleotide sequence that is unique or otherwise characteristic of said micro-organism, said first primer sequence being labelled with a first label and said second primer sequence being labelled with a second label;
- a control nucleic acid and a pair of primer sequences defining the 5′ and 3′ ends of a control nucleotide sequence, wherein the control nucleic acid comprises the nucleotide sequence of SEQ ID NO:
- 3 and/or SEQ ID NO:
- 4;
- a buffer solution comprising microparticles labelled with a first agent which specifically binds to said first label;
- and
- a lateral flow device comprising a substrate with a test region and a control region, said test region being provided with a second agent which specifically binds to said second label and said control region being provided with a control agent.
4 more elements
A kit for the detection of a micro-organism present in a sample, said kit comprising:
- deoxyribonucleotide triphosphates (dNTPs) labelled with a first label;
- a pair of first and second primer sequences defining 5′ and 3′ ends of a target nucleotide sequence that is unique or otherwise characteristic of said micro-organism, said second primer sequence being labelled with a second label;
- a control nucleic acid and a pair of primer sequences defining the 5′ and 3′ ends of a control nucleotide sequence, wherein the control nucleic acid comprises the nucleotide sequence of SEQ ID NO:
- 3 and/or SEQ ID NO:
- 4;
- a buffer solution comprising microparticles labelled with a first agent which specifically binds to said first label;
- and
- a lateral flow device comprising a substrate with a test region and a control region, said test region being provided with a second agent which specifically binds to said second label and said control region being provided with a control agent.
5 more elements
38-39. (canceled)
A method for the detection of a nucleic acid in a sample, said method comprising the steps of:
- (i) heating said sample at a temperature in the range of 85 to 100° C. so as to cause release of nucleic acid from any cell or other nucleic acid-containing structure present in the sample;
- (ii) amplifying a target nucleotide sequence present on said nucleic acid, comprising the use of a pair of first and second primer sequences defining the 5′ and 3′ ends of said target sequence, said first primer sequence being labelled with a first label and said second primer sequence being labelled with a second label such that any amplification of the target sequence generates an amplicon labelled with both first and second labels;
- (iii) diluting an amount of the amplification product of step (ii) in a suitable buffer solution comprising microparticles labelled with a first agent which specifically binds to said first label and allowing said first agent to bind to said first label present;
- (iv) applying at least a portion of the buffered product of step (iii) at or adjacent to a proximal end of a lateral flow device comprising a substrate which—allows constituents of said buffered product to wick or flow laterally towards a distal end of the lateral flow device, wherein at a location at or adjacent to said distal end, the lateral flow device is provided with a test region and a control region, said test region being provided with a second agent which specifically binds to said second label and said control region being provided with a control agent;
- and
- (v) detecting any binding of constituents of the buffered product at said test region and at said control region.
3 more elements
A method for the detection of a nucleic acid in a sample, said method comprising the steps of:
- (i) heating said sample at a temperature in the range of 85 to 100° C. so as to cause release of nucleic acid from any cell or other nucleic acid-containing structure present in the sample;
- (ii) amplifying a target nucleotide sequence present on said nucleic acid, comprising the use of a pair of first and second primer sequences defining the 5′ and 3′ ends of said target sequence, wherein the amplification of the target sequence utilises deoxyribonucleotide triphosphates (dNTPs) labelled with a first label and said second primer is labelled with a second label, such that any amplification of the target sequence generates an amplicon labelled with both first and second labels;
- (iii) diluting an amount of the amplification product of step (ii) in a suitable buffer solution comprising microparticles labelled with a first agent which specifically binds to one of said first and second labels and allowing said first agent to bind to said one of said first and second labels present;
- (iv) applying the buffered product of step (iii) at or adjacent to a proximal end of a lateral flow device comprising a substrate which allows constituents of said buffered product to wick or flow laterally towards a distal end of the lateral flow device, wherein at a location at or adjacent to said distal end, the lateral flow device is provided with a test region and a control region, said test region being provided with a second agent which specifically binds to the other of said first and second labels which is not bound by said first agent and said control region being provided with a control agent;
- and
- (v) detecting any binding of constituents of the buffered product at said test region and at said control region.
3 more elements
42-44. (canceled)
46-53. (canceled)
55-57. (canceled)
59-62. (canceled)
Dependencies are inferred from the claim text.