EP3505252B1

EP3505252B1 - Method for extracting nucleic acid and extraction cassette thereof

Info

Publication number: EP3505252B1
Application number: EP18215506.9A
Authority: EP
Inventors: Wei-Yu Chung; Song-Bin Huang; Shing-Lun Liu; Yu-Kai Kao; Yi-Chen Li
Original assignee: Delta Electronics Inc
Current assignee: Delta Electronics Inc
Priority date: 2017-12-26
Filing date: 2018-12-21
Publication date: 2021-02-24
Anticipated expiration: 2038-12-21
Also published as: EP3505252A3; EP3505252A2

Description

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to an extraction cassette with a liquid receiving module, and a method for extracting nucleic acid from a sample.

Description of the Related Art

In a conventional liquid receiving module, the sample compartment typically has a pressure supply opening connected to a pressure source. The movement of the liquid inside the sample compartment can be controlled by the pressure supplied via the pressure supply opening. However, the sample often contains viruses, bacteria or other contaminants, and the contaminants can travel to the analyzer or the outer environment via the pressure supply opening. Such contaminants not only cause inaccurate test results, but also endanger the health of the operator.
US 2017/015993 A1 discloses an extraction cassette for extracting nucleic acid from a mixed liquid including a sample according to the preamble of claim 1. The extraction cassette comprises a liquid receiving module with several compartments for receiving the sample and liquids and a connection module for establishing certain liquid flow pathways for conducting the sample to be tested and liquids. The sample is loaded to the sample reservoir provided in a lower module. For extracting the nucleic acid, external air is induced into the lower module via air holes on a lower membrane by an air pump. Channels in the lower module communicate with corresponding channels in an upper module, and thus the liquids including the sample material are transferred via the channels provided in the upper surface of the upper module. The sample must be loaded to the sample reservoir in the lower module while the lower module and the upper module are not yet assembled together. There is not provided any sealant that seals a filling hole of the sample reservoir. Rather, the sample reservoir is open at its upper end. Communication between the liquid / sample receiving chambers in the lower module is accomplished only via the channels in the upper module. No connection holes and fluid pathways are provided in the lower module.
US 2015/024436 A1 an integrated and automated sample-to-answer system that, starting from a sample comprising biological material, generates a genetic profile in short time. The device can be used for extracting nucleic acid and comprises several integrated modules, including an analyte preparation module; a detection and analysis module and a control module. The analyte preparation module has a different configuration.
US 2006/019379 A1 and WO 2005/121963 A2 disclose similar devices for extracting nucleic acid of different configuration.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide an enhanced extraction cassette, particularly for use in a method for extracting nucleic acids, which allows particularly to restrict the area polluted by the sample to the environment inside or around the analyzer, improving the accuracy of the analysis result and/or safeguarding the health of the operator. It is a further object of the present invention to provide a corresponding enhanced method for extracting nucleic acid.
These problems are solved by an extraction cassette as claimed by claim 1, and by a method for extracting nucleic acids as claimed by claim 6. Further advantageous embodiments are the subject-matter of the dependent claims.
Utilizing the extraction cassette of the embodiment of the present invention, the sample feeding hole of the sample compartment is sealed by the sealing member. The mixed liquid inside the sample compartment is moved to the first mixing compartment by the pressure applied to the alcohol compartment. Therefore, the virus, bacteria and other contaminants of the sample in the sample compartment is prevented from leaking from the liquid receiving module. Even if the contaminants of the sample enter the alcohol compartment via the first sample compartment connection hole (above the sample compartment) and the first path, the contaminants are disinfected by the alcohol compartment and are unable to cause pollution. The extraction cassette of the embodiment of the present invention can restrict the area polluted by the sample to the environment inside or around the analyzer, improving the accuracy of the analysis result and safeguarding the health of the operator.
A detailed description is given in the following embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:

Fig. 1A is an assembled view of an extraction cassette of an embodiment of the present invention;
Fig. 1B is an exploded view of the extraction cassette of the embodiment of the present invention;
Fig. 2A is a detail of the liquid receiving module of the embodiment of the present invention;
Fig. 2B is a detail of the third path of the embodiment of the present invention;
Figs. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I show the operation of the liquid receiving module of the embodiment of the present invention; and
Figs. 4A, 4B and 4C show a method for extracting nucleic acid of the embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best-contemplated mode of carrying out the present invention. This description is made for the purpose of illustrating the general principles of the present invention and should not be taken in a limiting sense. The scope of the present invention is best determined by reference to the appended claims.
Fig. 1A is an assembled view of an extraction cassette C of an embodiment of the present invention. Fig. 1B is an exploded view of the extraction cassette C of the embodiment of the present invention. With reference to Figs. 1A and 1B, the extraction cassette C includes an extraction module 1, a liquid receiving module 2, a sampling module 3 and a connection module 4. The extraction cassette C is adapted to be disposed into an analyzer. The analyzer includes a first pressure supplying module, an analyzing module and a second pressure supplying module. The first pressure supplying module and the second pressure supplying module provide pressure toward the extraction cassette C to control the liquid movement inside the extraction cassette C. The analyzing module heats and cools the extraction cassette C, and analyzes the sample inside extraction cassette C.
With reference to Fig. 1B, the liquid receiving module 2 communicates with the extraction module 1 via the connection module 4. The compartments of the liquid receiving module 2 communicate with one another via the connection module 4. Fig. 2A is a detail of the liquid receiving module 2. With reference to Fig. 2A, the liquid receiving module 2 includes a receiving module body 29, a sample compartment 21, a sealing member 211, an alcohol compartment 22 and a first path 221. The receiving module body 29 includes a first side 291, a second side 292 and a sample feeding hole 212. The first side 291 is opposite the second side 292. The sample feeding hole 212 is formed on the first side 291. The sample compartment 21 is formed on the receiving module body 29. The sample compartment 21 communicates with the sample feeding hole 212. The sample compartment 21 includes a first sample compartment connection hole 213 and a second sample compartment connection hole 214. The first sample compartment connection hole 213 nears the first side 291 relative to the second side 292. The sealing member 211 is adapted to seal the sample feeding hole 212. The alcohol compartment 22 is formed on the receiving module body 29. The alcohol compartment 22 communicates with the first sample compartment connection hole 213 of the first sample compartment 21 via the first path 221. In one embodiment, wherein at least one portion of the first path 221 is located between the alcohol compartment 22 and the second side 292.
With reference to Fig. 2A, in one embodiment, the alcohol compartment 22 includes an alcohol compartment connection hole 222. The alcohol compartment connection hole 222 nears the second side 292 relative to the first side 291. The first path 221 connects the alcohol compartment connection hole 222 to the first sample compartment connection hole 213.
With reference to Fig. 2A, in one embodiment, the liquid receiving module 2 further includes a first mixing compartment 23 and a second path 231. The first mixing compartment 23 is formed on the receiving module body 29. The second sample compartment connection hole 214 nears the second side 292 relative to the first side 291. The second path 231 connects the second sample compartment connection hole 214 to the first mixing compartment 23.
With reference to Figs. 2A and 2B, in one embodiment, the liquid receiving module 2 further includes a second mixing compartment 24, a third path 241 and a fourth path 242. The second mixing compartment 24 is formed on the receiving module body 29. The third path 241 connects the first mixing compartment 23 to the second mixing compartment 24. The fourth path 242 connects the second mixing compartment 24 to the extraction module 1. In one embodiment, the third path 241 connects the first mixing compartment 23 to the bottom of the second mixing compartment 24.
With reference to Fig. 2A, in one embodiment, the liquid receiving module 2 further includes a first detergent compartment 25, a second detergent compartment 26, a fifth path 251 and a sixth path 261. The first detergent compartment 25 and the second detergent compartment 26 are formed on the receiving module body 29. The fifth path 251 connects the first detergent compartment 25 to the first mixing compartment 23. The sixth path 261 connects the second detergent compartment 26 to the first mixing compartment 23.
With reference to Fig. 2A, in one embodiment, the liquid receiving module further includes an eluent compartment 27 and a seventh path 271. The eluent compartment 27 is formed on the receiving module body 29, and the seventh path 271 connects the eluent compartment 27 to the extraction module 1.
Figs. 3A∼3I show the operation of the liquid receiving module 2 of the embodiment of the present invention. With reference to Fig. 3A, first, liquid receiving module 2 receives four liquids, which are the alcohol 281 (in the alcohol compartment 22), the first detergent 282 (in the first detergent compartment 25), the second detergent 283 (in the second detergent compartment 26) and the eluent 284 (in the eluent compartment 27). Next, with reference to Fig. 3B, the sample 285 is filled into the sample compartment 21. After the extraction cassette C is disposed into the analyzing module of the analyzer, the temperature of the sample 285 in the sample compartment 21 is raised to 60 degrees Celsius (60 °C). Then, with reference to Fig. 3C, after the temperature of the sample 285 in the sample compartment 21 is raised to 60 degrees Celsius (60 °C), the alcohol 281 from the alcohol compartment 22 to the sample compartment 21, wherein the sample 285 and the alcohol 281 are mixed into a mixed liquid 286 in the sample compartment 21. Next, with reference to Figs. 3D and 3E, the mixed liquid 286 with the sample 285 and the alcohol 281 is moved from the sample compartment 21 to the first mixing compartment 23 (Fig. 3D). Then, the mixed liquid 286 is moved between the first mixing compartment 23 and the second mixing compartment 24 repeatedly to mix the sample 285 and the alcohol 281 (Fig. 3E).
In one embodiment, the mixed liquid 286 with the sample 285 and the alcohol 281 is moved into the extraction module 1 and the nucleic acid is captured from the mixed liquid 286 by the extraction module 1. Then, the mixed liquid 286 is moved into a first waste-liquid compartment of the extraction module 1.
With reference to Figs. 3F and 3G, next, the first detergent 282 is progressively moved from the first detergent compartment 25 to the first mixing compartment 23 and the second mixing compartment 24 to clean the first mixing compartment 23 and the second mixing compartment 24. Then, the first detergent 282 is moved to the extraction module 1 to clean the extraction module 1. Next, the first detergent 282 is moved to the first waste-liquid compartment of the extraction module 1.
Then, with reference to Figs. 3H and 3I, the second detergent 283 is progressively moved from the second detergent compartment 26 to the first mixing compartment 23 and the second mixing compartment 24 to clean the first mixing compartment 23 and the second mixing compartment 24. Then, the second detergent 283 is moved to the extraction module 1 to clean the extraction module 1. Next, at least one portion of the second detergent 283 is moved to a second waste-liquid compartment of the extraction module 1.
Finally, the eluent 284 is from the eluent compartment 27 to the extraction module 1 to take out the nucleic acid from the extraction module 1. The eluent 284 with the nucleic acid is then moved to the sampling module 3 to be quantitatively sampled.
Utilizing the extraction cassette of the embodiment of the present invention, with reference to Figs. 2A and 3B, the sample feeding hole 212 of the sample compartment 21 is sealed by the sealing member 211. The mixed liquid 286 inside the sample compartment 21 is moved to the first mixing compartment 23 by the pressure applied to the alcohol compartment 22. Therefore, the virus, bacteria and other contaminants of the sample 285 in the sample compartment 21 is prevented from leaking from the liquid receiving module. Even if the contaminants of the sample 285 enter the alcohol compartment 22 via the first sample compartment connection hole (above the sample compartment 21) and the first path 221, the contaminants are disinfected by the alcohol compartment 22 and are unable to cause pollution. The extraction cassette of the embodiment of the present invention can restrict the area polluted by the sample 285 to the environment inside or around the analyzer, improving the accuracy of the analysis result and safeguarding the health of the operator.
With reference to Figs. 2A, 3D and 3E, utilizing the extraction cassettes of the embodiment of the present invention, the mixed liquid 286 can be moved back and forth between the first mixing chamber 23 and the second mixing chamber 24 via the third path 241. The mixed liquid 286 is thoroughly mixed, whereby the effect of sufficiently mixing the mixed liquid 286 can be achieved with a minimum space. In one embodiment, the fourth path 242 has a fourth path outlet 242A that is higher than the top of the first mixing chamber 23 and the second mixing chamber 24. The fourth path outlet 242A is higher than the liquid level of the mixed liquid 286. Thus, the mixed liquid 286 is prevented from entering the extraction module 1 unintentionally through the fourth path outlet 242A.
With reference to Fig. 2A, the liquid receiving module 2 can be integrally formed except the sealing member 211.
Figs. 4A∼4C show a method for extracting nucleic acid of an embodiment of the present invention. With reference to Fig. 4A, the method for extracting nucleic acid of the embodiment of the present invention includes the following steps. First, an extraction cassette is provided, wherein the extraction cassette includes a liquid receiving module and an extraction module, the extraction module communicates with the liquid receiving module, the liquid receiving module includes a sample compartment, an alcohol compartment, a first mixing compartment, a second mixing compartment, a first detergent compartment, a second detergent compartment and an eluent compartment (S11). Then, an alcohol is filled into the alcohol compartment, a first detergent is filled into the first detergent compartment, a second detergent is filled into the second detergent compartment, and an eluent is filled into the eluent compartment (S12). Next, a sample is filled into the sample compartment (S13). Then, the sample inside the sample compartment is heated (S14).
With reference to Fig. 4B, in one embodiment, the method further includes the following steps. The alcohol is moved from the alcohol compartment to the sample compartment, wherein the sample and the alcohol are mixed into a mixed liquid (S15). Next, the mixed liquid is moved into the first mixing compartment, and then the mixed liquid is moved between the first mixing compartment and the second mixing compartment repeatedly to mix the sample and the alcohol (S16). Next, the mixed liquid is moved into the extraction module and the nucleic acid is captured from the mixed liquid by the extraction module (S17). Then, the mixed liquid is moved into a first waste-liquid compartment of the extraction module (S18).
With reference to Fig. 4C, in one embodiment, the method further includes the following steps. The first detergent is progressively moved from the first detergent compartment to the first mixing compartment and the second mixing compartment to clean the first mixing compartment and the second mixing compartment (S21). Then, the first detergent is moved to the extraction module to clean the extraction module (S22). Next, the first detergent is moved to the first waste-liquid compartment of the extraction module (S23). Then, the second detergent is progressively moved from the second detergent compartment to the first mixing compartment and the second mixing compartment to clean the first mixing compartment and the second mixing compartment (S24). Then, the second detergent is moved to the extraction module to clean the extraction module (S25). Next, at least one portion of the second detergent is moved to a second waste-liquid compartment of the extraction module (S26). Then, the eluent is moved from the eluent compartment to the extraction module to take out the nucleic acid from the extraction module (S27).
In one experiment, the applicant used an oral cell preservation solution containing a high concentration of the nucleic acid to be tested (Mh plasmid: 106 copies/mL, CytB plasmid) as a positive sample, and an oral cell preservation solution containing no nucleic acid to be tested as a negative sample, and perform interleaved tests of positive and negative samples. It is desired to confirm that the negative sample test result after the positive sample test result has no nucleic acid detected. It has been experimentally verified that the extraction cassette of the embodiment of the present invention can effectively prevent cross-contamination.
Test 1: High concentration positive and negative samples are alternatively tested four times (test sequence: positive → negative → positive → negative)
Result: no nucleic acid detected in negative samples

Samples positive negative positive negative

Mh plasmid1 Cq 25.11 N/A 26.48 N/A

CytB plasmid Cq 24.54 N/A 28.1 N/A

Test 2: Negative sample test after five high consecution positive sample tests (test sequence: positive → positive → positive → positive → negative)
Result: no nucleic acid detected in negative samples

Samples positive positive positive positive positive negative

Mh plasmid1 Cq 28.33 27.81 27.18 24.82 26.44 N/A

CytB plasmid Cq 26.89 23.51 25.85 24.68 28.58 N/A
Use of ordinal terms such as "first", "second", "third", etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having the same name (but for use of the ordinal term).
While the present invention has been described by way of example and in terms of the preferred embodiments, it should be understood that the present invention is not limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements (as would be apparent to those skilled in the art), provided that they fall within the scope of the appended claims.

Claims

An extraction cassette (C) for extracting nucleic acid from a mixed liquid (286) including a sample (285), comprising:
a liquid receiving module (2) for receiving the sample (285) and alcohol (281) and for mixing the sample (285) with the alcohol (281) to generate the mixed liquid (286); and

an extraction module (1) for capturing the nucleic acid from the mixed liquid (286); wherein

the liquid receiving module (2) communicates with the extraction module (1), and

the liquid receiving module (2) comprises a receiving module body (29) having a first side (291), a second side (292) opposite to the first side (291) and a sample feeding hole (212) formed on the first side (291); wherein

the receiving module body (29) comprises:
a sample compartment (21) formed on the receiving module body (29) and configured to receive the sample (285), wherein the sample compartment (21) communicates with the sample feeding hole (212), and

an alcohol compartment (22), formed on the receiving module body (29) and configured to receive the alcohol (281), wherein

the alcohol compartment (22) communicates with the first sample compartment (21) via a first path (221);

characterized in that

the liquid receiving module (2) further comprises a sealing member (211) adapted to seal the sample feeding hole (212), wherein

the sample compartment (21) comprises a first sample compartment connection hole (213) and a second sample compartment connection hole (214) for connecting to the extraction module (1),

the first sample compartment connection hole (213) nears the first side (291) relative to the second side (292),

the alcohol compartment (22) comprises an alcohol compartment connection hole (222) that nears the second side (292) relative to the first side (291),

the first path (221) connects the alcohol compartment connection hole (222) to the first sample compartment connection hole (213) for transferring the alcohol (281) from the alcohol compartment (22) to the sample compartment (21), and

at least one portion of the first path (221) is located between the alcohol compartment (22) and the second side (292).
The extraction cassette as claimed in claim 1, wherein the liquid receiving module (29) further comprises a first mixing compartment (23) and a second path (231), the first mixing compartment (23) is formed on the receiving module body (29), the second sample compartment connection hole (214) nears the second side (292) relative to the first side (291), and the second path (231) connects the second sample compartment connection hole (214) to the first mixing compartment (23).
The extraction cassette as claimed in claim 2, wherein the liquid receiving module (29) further comprises a second mixing compartment (24), a third path (241) and a fourth path (242), the second mixing compartment (24) is formed on the receiving module body (29), the third path (241) connects the first mixing compartment (23) to the second mixing compartment (24), the fourth path (242) connects the second mixing compartment (24) to the extraction module (1), the fourth path (242) comprises a fourth path outlet (242A) that nears the second side (292) relative to the first mixing compartment (23) and the second mixing compartment (24).
The extraction cassette as claimed in claim 3 or 4, wherein the liquid receiving module (2) further comprises a first detergent compartment (25), a second detergent compartment (26), a fifth path (251) and a sixth path (261), the first detergent compartment (25) and the second detergent compartment (26) are formed on the receiving module body (29), the fifth path (251) connects the first detergent compartment (25) to the first mixing compartment (23), and the sixth path (261) connects the second detergent compartment (26) to the first mixing compartment (23).
The extraction cassette as claimed in any of the preceding claims, wherein the liquid receiving module (2) further comprises an eluent compartment (27) and a seventh path (271), the eluent compartment (27) is formed on the receiving module body (29), and the seventh path (271) connects the eluent compartment (27) to the extraction module (1).
A method for extracting nucleic acid from a sample (285) using an extraction cassette (C) as claimed in any of claims 1 to 5, comprising steps of:
filling an alcohol (281) into the alcohol compartment (22) of the receiving module body (29);

(29); filling a sample (285) into the sample compartment (21) of the receiving module body

heating the sample (285) inside the sample compartment (21) and sealing the sample feeding hole (212) of the sample compartment (21) of the receiving module body (29) by means of a sealing member (211);

moving the alcohol (281) from the alcohol compartment (22) to the sample compartment (21), wherein the sample and the alcohol are mixed into a mixed liquid (286); and

moving the mixed liquid (286) into the extraction module (1) and capturing the nucleic acid from the mixed liquid by the extraction module (1).
The method as claimed in claim 6, further comprising:
filling a first detergent (282) into a first detergent compartment (25) of the receiving module body (29),

filling a second detergent (283) into a second detergent compartment (26) of the receiving module body (29), and

filling an eluent (284) into an eluent compartment (27) of the receiving module body (29).
The method as claimed in claim 7, further comprising steps of:
moving the mixed liquid (286) into a first mixing compartment (23) of the receiving module body (29); and

moving the mixed liquid (286) between the first mixing compartment (23) and a second mixing compartment (24) of the receiving module body (29) repeatedly to mix the sample (285) and the alcohol (281).
The method as claimed in claim 8, further comprising steps of:
moving the mixed liquid (286) into a first waste-liquid compartment of the extraction module (1).
The method as claimed in claim 8 or 9, further comprising steps of:
progressively moving the first detergent (282) from the first detergent compartment (25) to the first mixing compartment (23) and the second mixing compartment (24) to clean the first mixing compartment and the second mixing compartment;

moving the first detergent (282) to the extraction module (1) to clean the extraction module; and

moving the first detergent (282) to the first waste-liquid compartment of the extraction module (1).
The method as claimed in any of claims 8 to 10, further comprising steps of:
progressively moving the second detergent (283) from the second detergent compartment (26) to the first mixing compartment (23) and the second mixing compartment (24) to clean the first mixing compartment and the second mixing compartment;

moving the second detergent (283) to the extraction module (1) to clean the extraction module; and

moving at least one portion of the second detergent (283) to a second waste-liquid compartment of the extraction module (1).
The method as claimed in claim 11, further comprising a step of:
moving the eluent (284) from the eluent compartment (27) to the extraction module (1) to take out the nucleic acid from the extraction module (1).