Rotatable discs and systems and methods for sample analysis
The present invention describes a method and device enabling sample processing, fast polymerase chain reaction amplification, and real-time reading of a large number of targets simultaneously while using a volume of reaction compatible with molecular assays. The present invention can be part of a cartridge allowing sample to answer nucleic acid analytic system (NAAT) or can be used as a stand-alone device.
1 . A method of detecting the presence or absence of a target nucleic acid in a sample, the method comprising:
(a) providing a rotatable PCR disc comprising a closed loading chamber, a plurality of reaction chambers, a channel connecting the closed loading chamber to the plurality of reaction chambers, and an exit valve connecting the closed loading chamber to the channel, wherein the closed loading chamber contains the sample;
(b) heating the sample in the closed loading chamber in a hyperbaric condition to a temperature above 100° C. to produce a heat-treated sample;
(c) opening the exit valve, thereby opening the closed loading chamber, and rotating the rotatable PCR disc such that the heat-treated sample flows to the plurality of reaction chambers via the channel; and
(d) amplifying the target nucleic acid, if present, in the heat-treated sample in the plurality of reaction chambers.
2 . The method of claim 1 , wherein, in (d), each of the plurality of reaction chambers comprises a PCR reaction mixture comprising a primer, a polymerase, and a plurality of nucleotides, and wherein (d) further comprises rotating the rotatable PCR disc to bring the plurality of reaction chambers adjacent to a first heating element, wherein the first heating element heats the plurality of reaction chambers to a denaturing temperature of 90° C. to 99° C.
3 . The method of claim 2 , wherein (d) further comprises rotating the rotatable PCR disc to bring the plurality of reaction chambers adjacent to a second heating element, wherein the second heating element heats the plurality of reaction chambers to an annealing temperature of 50° C. to 74° C.
4 . The method of claim 3 , wherein the heat-treated sample comprises the target nucleic acid, and wherein the method further comprises (e) detecting the target nucleic acid or an amplification product thereof.
5 . The method of claim 4 , wherein the target nucleic acid or the amplification product thereof is detectable after amplifying the target nucleic acid from 10 to 50 molecular amplification cycles.
6 . The method of claim 4 , wherein the target nucleic acid or the amplification product thereof is detectable after amplifying the target nucleic acid from 28 to 35 molecular amplification cycles.
7 . The method of claim 4 , further comprising sequencing the target nucleic acid or the amplification product thereof.
8 . The method of claim 4 , further comprising, prior to flowing the heat-treated sample into the plurality of reaction chambers, mixing the heat-treated sample with a PCR reagent selected from: a polymerase, a plurality of nucleotides, buffer, reverse transcriptase, primers, and fluorescent probes.
9 . The method of claim 4 , wherein the method further comprises hybridizing a fluorescent probe to the target nucleic acid or the amplification product thereof, exposing the plurality of reaction chambers to an excitation light, and detecting an emission wavelength corresponding to a presence of the target nucleic acid or the amplification product thereof.
10 . The method of claim 1 , further comprising, after (c) and prior to (d), heating and compressing the channel, thereby sealing the channel and preventing fluid communication between the plurality of reaction chambers.
11 . The method of claim 1 , wherein the sample is a bodily sample comprising the target nucleic acid, wherein:
(i) the bodily sample is selected from a blood sample, a lacrimal fluid sample, a saliva sample, a mucus sample, a sputum sample, a feces sample, a cerebrospinal fluid sample, and a urine sample; and
(ii) the target nucleic acid is not extracted, isolated, or otherwise purified from the bodily sample prior to the heating in (b).
12 . The method of claim 1 , wherein, in (a), the sample comprises a plurality of molecular amplification inhibitors.
13 . The method of claim 12 , wherein the plurality of molecular amplification inhibitors comprises: (i) a plurality of agents that are capable of binding to the target nucleic acid if present, (ii) a plurality of DNases, (iii) a plurality of RNases, or (iv) a plurality of proteases.
14 . The method of claim 13 , wherein the heating of the sample inactivates at least 60% of the plurality of molecular amplification inhibitors.
15 . The method of claim 1 , wherein the sample comprises one or more reagents selected from: a chelating agent, a single stranded nucleic acid binding protein, a reducing agent, and a stabilizer.
16 . The method of claim 15 , wherein the sample comprises a chelating agent, a single stranded nucleic acid binding protein, and a reducing agent.
17 . The method of claim 16 , wherein the sample further comprises a stabilizer.
18 . The method of claim 17 , wherein the stabilizer is selected from bovine serum albumin and gelatin.
19 . The method of claim 15 , wherein the sample further comprises a protease or a nuclease inhibitor.
20 . The method of claim 1 , wherein the heating of the sample in the hyperbaric condition occurs over a ramp time from a first temperature to a second temperature, wherein the second temperature is the temperature above 100° C., wherein the ramp time is from about 3 seconds to about 50 seconds.
21 . The method of claim 20 , further comprising maintaining the sample at the second temperature for a maintenance time from about 5 seconds to about 120 seconds to produce the heat-treated sample, and subsequently cooling the heat-treated sample.
22 . The method of claim 1 , wherein the heating of the sample in the hyperbaric condition occurs at a temperature ramp rate of about 5° C. per second to about 50° C. per second.
23 . The method of claim 1 , wherein the heating of the sample in the hyperbaric condition comprises heating the sample to a temperature of about 105° C. to about 160° C.
24 . The method of claim 1 , wherein the heating in (b) comprises induction heating.
25 . The method of claim 1 , wherein (c) comprises performing the rotation of the rotatable PCR disc at a speed from about 500 RPM to about 15,000 RPM.
26 . The method of claim 1 , wherein each of the plurality of reaction chambers has a volume of 5 μL to 100 μL.
27 . The method of claim 1 , wherein the rotatable PCR disc is composed at least in part of a thermoplastic material.
28 . The method of claim 27 , wherein the channel is composed at least in part of a thermoplastic material.
29 . The method of claim 1 , wherein the closed loading chamber is composed at least in part of a metal.
30 . The method of claim 1 , wherein the heat-treated sample comprises the target nucleic acid, wherein the target nucleic acid comprises RNA.