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AssigneeUniversity of Surrey
InventorsKhushboo BORAH SLATER, Johnjoe MCFADDEN, Roberto LA RAGIONE, Ravi SILVA, Muhammad AHMAD, Aurore POIRIER
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The invention relates to nucleic acid detection, and particularly, although not exclusively, to electrochemical methods for detecting target nucleic acid sequences in a sample obtained from a subject. The nucleic acid detection methods of the invention can also be used to diagnose or prognose both infectious and non-infectious diseases in a subject, or to determine whether a pathogen is drug resistant. The invention extends to apparatus for performing improved nucleic acid detection using electrochemical methods.
A method of detecting a target nucleic acid sequence in a sample obtained from a subject, the method comprising:
A method of diagnosing or prognosing a disease in a subject, the method comprising detecting a target nucleic acid sequence in a sample obtained from a subject, by:
An apparatus for detecting a target nucleic acid sequence in a sample obtained from a subject, the apparatus comprising:
The method or apparatus according to any preceding claim, wherein removing substantially all free phosphate ions from the sample comprises the use of:
The method according to any preceding claim, wherein the method further comprises measuring the concentration of free phosphate ions in the sample after substantially all the free phosphate ions have been removed.
The method according to any preceding claim, wherein amplifying the target nucleic acid sequence in the sample comprises amplification by polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or nucleic acid sequence-based amplification (NASBA).
The method according to any preceding claim, further comprising contacting the amplified products with pyrophosphatase (PPase) to generate phosphate ions (Pi).
The method according to any preceding claim, wherein using the amplified products to create an electroactive compound comprises contacting the amplified products with a phosphate-precipitating salt to produce phosphomolybdate.
The method according to any preceding claim, wherein the electrochemical signal is conducted along an electrode.
The method according to any preceding claim, wherein the electrochemical signal is detected using a two electrode system comprising a working electrode (WE) and a counter electrode (CE), or wherein the electrochemical signal is detected using a three electrode system comprising a WE, a CE and a reference electrode (RE).
The method according to any preceding claim, wherein detecting the electrochemical signal comprises conductometric sensing, potentiometric sensing or amperometric sensing.
The method according to any preceding claim, wherein detecting the electrochemical signal comprises measuring the current across a range of potentials between −1V and 1 V, or more preferably between −500 mV and 500 mV.
The apparatus according to any preceding claim, wherein the apparatus is connected to a mobile or computer device.
The method or apparatus according to any preceding claim, wherein the target nucleic acid sequence is a DNA or RNA sequence, optionally wherein the target nucleic acid sequence is selected from chromosomal DNA, mitochondrial DNA, messenger RNA, transfer RNA, ribosomal RNA, small nuclear RNA, micro RNA, small-interfering RNA, viral RNA and extrachromosomal DNA.
The method or apparatus according to any preceding claim, wherein the target nucleic acid sequence is from a virus, a bacteria, a mycoplasma, a fungus, an animal, a plant, an alga, a parasite, or a protozoan, or wherein the target nucleic acid sequence is from an antibiotic resistant pathogen.
The method according to any preceding claim, further comprising measuring the intensity of the electrochemical signal, to determine the concentration of the target nucleic acid in the sample.
The method according to any preceding claim, further comprising comparing the electrochemical signal with the electrochemical signal of a positive and/or negative control.
Dependencies are inferred from the claim text.