Nucleic acid amplification device, nucleic acid amplification method, and chip for nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-11098347-B2
Application number	US-201515322000-A
Country	US
Kind code	B2
Filing date	Jul 7, 2015
Priority date	Jul 8, 2014
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

The present invention provides a reciprocal-flow-type nucleic acid amplification device comprising:heaters capable of forming a denaturation temperature zone and an extension/annealing temperature zone;a fluorescence detector capable of detecting movement of a sample solution between the two temperature zones;a pair of liquid delivery mechanisms that allow the sample solution to move between the two temperature zones and that are configured to be open to atmospheric pressure when liquid delivery stops; a substrate on which the chip for nucleic acid amplification according to claim 2 can be placed; and a control mechanism that controls driving of each liquid delivery mechanism by receiving an electrical signal from the fluorescence detector relating to movement of the sample solution from the control mechanism; the device being capable of performing real-time PCR by measuring fluorescence intensity for each thermal cycle.

First claim

The invention claimed is:

1. A reciprocal-flow-type nucleic acid amplification device comprising:
(a) heaters capable of forming a denaturation temperature zone and an extension/annealing temperature zone;
(b) at least one fluorescence detector capable of detecting movement of a sample solution between the two temperature zones;
(c) a substrate on which a chip for nucleic acid amplification can be placed, wherein the chip for nucleic acid amplification comprises at least one microchannel contacted to each temperature zone, wherein the at least one microchannel comprises:
(i) curved-channel portions each set in a denaturation temperature zone and an extension/annealing temperature zone of the nucleic acid amplification device,
(ii) a linear intermediate-channel portion connecting the curved-channel portions, and
(iii) connections at both ends of the microchannel;

(d) at least one liquid delivery mechanism selected from a microblower and a fan, which is connected to at least one of the connections of the microchannel, that allows the sample solution to move between the two temperature zones and that is configured to be open to atmospheric pressure when liquid delivery stops; and
(e) a control mechanism that controls driving of the at least one liquid delivery mechanism by receiving an electrical signal relating to movement of the sample solution from the at least one fluorescence detector,
wherein the device is configured:
to perform real-time PCR by measuring fluorescence intensity for each thermal cycle,
so that the at least one liquid delivery mechanism selected from a microblower and a fan applies or reduces pressure in the microchannel by blowing air in order to reciprocally move the sample solution between the two temperature zones, and
so that liquid delivery stops immediately after air blowing is stopped, when or immediately before the solution has reached a precise position on each temperature zone.

2. The reciprocal-flow-type nucleic acid amplification device according to claim 1 , wherein the nucleic acid amplification device comprises a pair of liquid delivery mechanisms selected from microblowers and fans, which are connected to the connections of the microchannel, that allow the sample solution to move between the two temperature zones and that are configured to be open to atmospheric pressure when liquid delivery stops.

3. A nucleic acid amplification method comprising the following steps:
step 1: placing a chip for nucleic acid amplification according to claim 1 on the substrate of the device of claim 1 in such a manner that the denaturation temperature zone includes at least one curved-microchannel portion and the extension/annealing temperature zone includes at least one curved-microchannel portion;
step 2: introducing a sample solution into the microchannel;
step 3: connecting a pair of liquid delivery mechanisms according to claim 1 to the liquid delivery mechanism connections at both ends of the microchannel; and
step 4: reciprocating the sample solution between the two curved-microchannel portions of the microchannel by the liquid delivery mechanisms to perform thermal cycling, and simultaneously measuring the fluorescence intensity of the sample solution and confirming the movement of the sample solution for each thermal cycle using the at least one fluorescence detector in the intermediate-microchannel portion to perform the nucleic acid amplification method,
wherein the nucleic acid amplification method is selected from the group consisting of polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), one-step RT-PCR, multiplex RT-PCR, real-time PCR, and real-time RT-PCR.

4. The nucleic acid amplification method according to claim 3 , wherein the measurement of fluorescence intensity is performed by simultaneously measuring two or more fluorescent wavelengths to simultaneously measure the nucleic acid amplification of multiple genes in one microchannel.

5. The nucleic acid amplification method according to claim 3 , wherein the initial concentration of a nucleic acid in the sample is determined using a calibration curve obtained by plotting Ct values as a function of concentration for a number of standards having known concentrations of the nucleic acid.

6. The nucleic acid amplification method according to claim 3 , wherein the chip comprises two or more microchannels, wherein the operation of liquid delivery through each of the microchannels can be independently controlled.

7. The nucleic acid amplification method according to claim 3 , the method further comprising:
connecting an end of a filtered pipette tip of a micropipette to one of the connections so as to introduce a sample solution into the microchannel;
removing the micropipette with the pipette tip being connected to the connection, and
then connecting the pipette tip to one of the liquid delivery mechanisms.

8. The nucleic acid amplification method according to claim 3 , wherein the volume of the sample solution introduced into the microchannel is in the range of 5 μL to 50 μL.

9. The nucleic acid amplification method according to claim 4 , wherein the initial concentration of a nucleic acid in the sample is determined using a calibration curve obtained by plotting Ct values as a function of concentration for a number of standards having known concentrations of the nucleic acid.

10. The nucleic acid amplification method according to claim 4 , wherein the chip comprises two or more microchannels, wherein the operation of liquid delivery through each of the microchannels can be independently controlled.

11. The nucleic acid amplification method according to claim 10 , the method further comprising:
connecting an end of a filtered pipette tip of a micropipette to one of the connections so as to introduce a sample solution into at least one of the microchannels;
removing the micropipette with the pipette tip being connected to the connection, and
then connecting the pipette tip to one of the liquid delivery mechanisms.

12. The nucleic acid amplification method according to claim 11 , wherein the volume of the sample solution introduced into the at least one microchannel is in the range of 5 μL to 50 μL.

Assignees

• Aist
• Kyorin Seiyaku Kk

Inventors

• Nagai Hidenori
• Furutani Shunsuke
• Hagihara Yoshihisa
• Fuchiwaki Yusuke

Classifications

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• B01L2300/1827

  using resistive heater · CPC title

• B01L2300/0883

  Serpentine channels · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• B01L2300/0654

  Lenses; Optical fibres · CPC title