Nucleic acid detection or quantification method, chip and assay kit therefor, device for detecting or quantifying nucleic acid and program therefor
According to one embodiment, a method of quantifying a target nucleic acid containing a first sequence in a sample is provided. The method includes preparing a substrate on which a plurality of detection regions are arranged, forming a reaction field by placing, on the substrate, a reaction liquid containing a sample, a primer set, and an amplification enzyme, maintaining the reaction field in an isothermal amplification condition, detecting a detection signal for each of the detection regions, determining, for each of the plurality of detection regions, whether positive or negative and detecting or quantifying the target nucleic acid based on the number of positive and/or a rise time of each of the positive detection signal.
1. A method of quantifying one kind of target nucleic acid containing a first sequence in a sample, the method comprising:
preparing a substrate on which a plurality of detection regions are arranged, without attaching any nucleic acid probe on the detection regions, wherein a surface of the substrate on which the detection regions are arranged is not divided for the detection regions;
forming one reaction field by placing, on the surface of the substrate, one reaction liquid containing the sample, a primer set for isothermally amplifying the first sequence to obtain a first amplification product, and an amplification enzyme;
maintaining the reaction field in an isothermal amplification condition;
detecting a detection signal varying with an increase of the first amplification product in each of the plurality of detection regions;
determining, for each of the plurality of detection regions, whether the first amplification product exists in its vicinity (positive) or the first amplification product does not exist in its vicinity (negative) based on the result of the detecting; and
detecting or quantifying the target nucleic acid in the one reaction liquid based on the number of positive detection regions and on a rise time of the detection signal in each of the positive detection regions.
2. The method of claim 1 , wherein
the isothermal amplification condition is a LAMP reaction condition.
3. The method of claim 1 , wherein
the substrate comprises an electrode corresponding to each of the detection regions, the reaction liquid further contains a first marker substance producing the electric signal which varies with the increase of the first amplification product, and the detection signal is an electric signal obtained by oxidation or reduction reaction of the first marker substance.
4. The method of claim 3 , wherein
the first marker substance is ruthenium hexaamine.
5. The method of claim 1 , wherein
the substrate comprises an optical sensor corresponding to each of the detection regions, the reaction liquid further contains a second marker substance producing an optical signal which varies with the increase of the first amplification product, and the detection signal is an optical signal.
6. The method of claim 1 , wherein
the substrate comprises a turbidity sensor corresponding to each of the detection regions, and the detection signal is a signal correlated to a turbidity of the reaction liquid.
7. The method of claim 1 , wherein
a distance between each adjacent pair of the plurality of detection regions is 1 mm or more.
8. A chip for detecting or quantifying one kind of target nucleic acid containing a first sequence in one sample, the chip comprising:
a substrate, and a plurality detection regions disposed in one space on one surface of the substrate without attaching any nucleic acid probe on the detection regions, a distance between each adjacent pair of the plurality of detection regions being 1 mm to 10 mm,
wherein
the one surface of the substrate is not divided for the detection regions;
one reaction liquid containing the sample, a primer set for isothermally amplifying the first sequence to obtain a first amplification product, and amplification enzyme is placed on the substrate to form one reaction field which causes an amplification reaction,
the detection signal which varies with the increase of the first amplification product is detected in each of the detection regions,
each detection region is determined based on the result of the detection as to whether the first amplification product exists in its vicinity (positive) or the first amplification product does not exist in its vicinity (negative), and
the target nucleic acid in the one reaction liquid is detected or quantified from the number of positive detection regions and/or the rise time of the detection signal in each of the positive detection regions.
9. The chip of claim 8 , wherein the substrate comprises an electrode corresponding to each of the detection regions.
10. The chip of claim 8 , wherein the substrate comprises an optical sensor corresponding to each of the detection regions.
11. The chip of claim 8 , wherein the substrate comprises a turbidity sensor corresponding to each of the detection regions.
12. An assay kit comprising:
a chip of claim 8 ,
a primer set for isothermally amplifying the first sequence to acquire the first amplification product and an amplification reagent.
13. The assay kit of claim 12 , further comprising:
a first marker substance producing an electric signal which varies with the increase of the first amplification product.
14. The method of claim 1 , which quantifies only one kind of target nucleic acid.