JP2016015922A - Nucleic acid analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid analyzer having device configuration of a liquid sending system and a nozzle cleaning method which contribute to cost reduction, downsizing, and throughput improvement while retaining analysis accuracy.SOLUTION: A nucleic acid analyzer comprises a reagent rack 106 for holding a plurality of reagent vessels; a nozzle 108 for sucking a reagent; a planar substrate 103 for holding a sample and performing a nucleic acid analysis reaction; two electromagnetic valves 109, 111 which can open/close; a syringe 110 for sucking and discharging fluid and gas; a waste fluid bottle 107; flow channels which connect the nozzle 108, the planar substrate 103, the first electromagnetic valve 109, the syringe 110, the second electromagnetic valve 111, and the waste fluid bottle 107; a container for storing dip cleaning liquid to clean the nozzle 108; drive mechanism 114 for conducting access of the nozzle 108 to the reagent rack 106; and a detector 105 for detecting a signal 104 generated in a sample in the planar substrate 103, and cleans the nozzle 108 by dipping it into the dip cleaning liquid.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a nucleic acid analyzer for analyzing a nucleic acid sequence.

  A nucleic acid analyzer is a device that detects the type of a base from a nucleic acid of a sample and determines its sequence.

  In recent years, by adding beads or nucleic acids carrying nucleic acids on a flat substrate, and reading signals such as fluorescence and pH changes generated by sequence reactions on the flat substrate using a CCD camera or various detectors. An apparatus called the next-generation sequencer that can efficiently analyze multiple nucleic acids in a massively parallel manner has appeared.

  For example, in a sequence reaction of a nucleic acid analyzer that analyzes by fluorescence detection, a complementary probe (nucleic acid fragment) labeled with fluorescence is extended onto the sample nucleic acid by DNA polymerase or DNA ligase. For each reaction, this is done by detecting fluorescence. In order to add various reaction reagents and sample nucleic acids onto a flat substrate, a liquid feeding system is used.

  In the liquid feeding system described in Patent Document 1, a reagent tray that holds a plurality of reagents and a flow path for each reagent are held. By switching valves, a target reagent or nucleic acid is sent onto a flat substrate as a reaction unit. Liquid.

  The liquid feeding system described in Patent Document 2 includes a reagent rack that holds a plurality of reagents and a sampling nozzle. The reagent and nucleic acid are sucked from the nozzle and discharged onto a flat substrate. Since one nozzle is shared among a plurality of nucleic acids and reagents, nozzle cleaning is necessary for the purpose of preventing contamination between the nucleic acids and the reagents. The nozzle moves to the cleaning tank and cleans the inside of the nozzle by discharging the cleaning water from the nozzle, and cleans the outside of the nozzle by jetting the cleaning water to the outside of the nozzle.

  Recently, the application of next-generation sequencers to diagnosis is rapidly progressing. The most important keys to the diagnostic field are cost reduction, device miniaturization, throughput improvement, and maintenance of analysis accuracy.

US 2012/0270305 JP2012-173059

  However, the system of Patent Document 1 requires a plurality of nozzles and reagent valves according to the type of reagent, and is not suitable for reducing the cost of the apparatus. Further, it is conceivable that contamination occurs in the reagent switching valve, and as a result, the analysis accuracy may be lowered.

  Moreover, in the system of patent document 2, the mechanism for a nozzle to move to a washing tank, and the mechanism in which washing water is supplied from the inside or the outside of an apparatus are required. Furthermore, a large amount of cleaning liquid after cleaning is generated as waste liquid. As a result, the apparatus becomes large and complicated, so that the apparatus is not only expensive, but also takes time for cleaning, which is not suitable for improving the throughput.

  An object of the nucleic acid analyzer of the present invention is to provide an apparatus configuration and a nozzle cleaning method of a liquid feeding system that contributes to cost reduction, apparatus miniaturization, and throughput improvement while maintaining analysis accuracy.

The nucleic acid analyzer of the present invention comprises a reagent rack that holds a plurality of reagent containers,
A nozzle for aspirating the reagent;
A flat substrate for holding a sample and performing a nucleic acid analysis reaction;
Two solenoid valves that can be opened and closed;
A syringe for aspirating and discharging liquids and gases;
A waste bottle,
A flow path connecting a nozzle, a planar substrate, a first electromagnetic valve, a syringe, a second electromagnetic valve, and a waste bottle;
A container for storing a dip cleaning liquid for cleaning the nozzle;
A drive mechanism for accessing the reagent rack of the nozzle;
A detection unit for detecting a signal generated in the sample in the planar substrate;
Clean by dipping the nozzle into the dip cleaning solution.

  The nucleic acid analyzer of the present invention has a simple apparatus configuration in which a nozzle, a flat substrate, and waste liquid are connected by a single flow path, thereby reducing the cost and size of the apparatus. In addition, by sucking an appropriate amount of air between reagents, it is not necessary to clean the inside of the nozzle, and the outside of the nozzle is cleaned by dipping into a small amount of washing liquid to eliminate the influence of contamination between reagents and maintain analysis accuracy. can do. This makes it possible to reduce the cost, size, and throughput of the device.

The block diagram of a nucleic acid analyzer. Explanatory drawing of the contamination in the flow path of a nucleic acid analyzer. Explanatory drawing of the contamination on the outer side of the nozzle of a nucleic acid analyzer. Explanatory drawing of the dip washing | cleaning method of a nucleic acid analyzer. Explanatory drawing of the hole structure which prevents the contamination of a nucleic acid analyzer.

  The nucleic acid analyzer of the present invention has a flat substrate as a reaction part, a syringe for sucking a liquid, and a waste liquid part on the downstream side of one sampling nozzle. Also, a plurality of trace amounts of cleaning liquid of about several mL to several tens of mL are retained for nozzle cleaning. By pulling the syringe and making the inside of the flow path have a negative pressure, the reagent is sucked into the nozzle. Since the nozzle, the planar substrate, and the waste liquid are connected by a single flow path, the sucked reagent is sent to the planar substrate and the waste liquid side by the operation of the syringe. When switching to a different reagent, contamination inside the nozzle and in the flow path can be prevented by sucking an appropriate amount of air between the reagents. Further, the outside of the nozzle can be cleaned by dipping into a plurality of nozzle cleaning liquids to prevent contamination. Cleaning by dipping is a simple and quick cleaning method compared to a method in which the nozzle is moved to the cleaning tank and the cleaning liquid is ejected outside the nozzle.

  A first embodiment of the present invention will be described.

[Description of nucleic acid analyzer]
FIG. 1 is a configuration diagram of a nucleic acid analyzer. The nucleic acid analyzer includes a detection unit 101 and a liquid feeding unit 102. The detection unit 101 detects a signal 104 such as fluorescence or hydrogen ion emitted from the flat substrate 103 by a sequence reaction by a detector 105 such as a CCD camera. On the flat substrate 103, the nucleic acid is immobilized on the flat substrate and various sequence reactions are performed, so that the temperature can be adjusted to an appropriate temperature. Although not shown in the figure, the base sequence information of the sample is obtained by converting the detection signal into base information by a computer.

  The liquid feeding unit 102 includes a reagent rack 106, a waste liquid tank 107, and a flow path connecting it. The flow path is formed by connecting the nozzle 108, the planar substrate 103, the substrate side electromagnetic valve 109, the syringe 110, the waste liquid side electromagnetic valve 111, and the waste liquid tank 107 with a hollow tube. Next, a reagent feeding method will be described. The plurality of reagents are held and stored in the reagent rack 106 at a temperature suitable for reagent storage. In addition to the sequence reagent, the reagent rack can hold a sample nucleic acid, a reagent for fixing the nucleic acid to the flat substrate 103, a cleaning liquid for cleaning the inside of the flow path, a cleaning liquid for cleaning the outside of the nozzle, and the like. . In addition to the sequence reagent, the reagent refers to a sample nucleic acid, a reagent for fixing the nucleic acid to the flat substrate 103, a cleaning liquid for cleaning the inside of the flow path, a cleaning liquid for cleaning the outside of the nozzle, and the like. . Each reagent is arranged on the circumference of the reagent rack 106, and when the reagent rack 106 rotates in the clockwise direction 112 and the counterclockwise direction 113, an arbitrary reagent is moved directly below the nozzle 108. By moving the nozzle 108 in the Z direction 114, the nozzle can access each reagent. This is an example, and the reagent rack 106 may not be circular but may be square, and each reagent may not be arranged on the outer periphery. Further, as long as the nozzle 108 can access the reagent, either the reagent rack 106 or the nozzle 108 may move in any direction, but most preferably, the reagent rack 106 rotates in the plane direction and the nozzle 108 moves in the vertical direction. It is a configuration.

  When the tip of the nozzle 108 is immersed in the reagent, the waste-side solenoid valve 111 is closed, the substrate-side solenoid valve 109 is opened, and the syringe 110 is pulled in the suction direction 115. The reagent is sucked into the flow path. The reagent sucked from the flow path is sent to the reaction substrate 103 on the downstream side and further to the syringe 110. By adjusting the amount by which the syringe is pulled in the suction direction 115, it is possible to adjust the amount of the reagent to be sucked, that is, how far the fluid is sent in the flow path. The reagent and air sucked into the syringe 110 can be discharged to the waste liquid tank 107 by pushing the syringe 110 in the discharge direction 116 with the substrate side electromagnetic valve 109 closed and the waste liquid side electromagnetic valve 111 opened. Similarly, when the nozzle 108 is not immersed in the reagent, the waste liquid side solenoid valve 111 is closed, the substrate side solenoid valve 109 is opened, and the syringe 110 is pulled in the suction direction 115, air is sucked into the flow path.

  Next, a contamination prevention method will be described. Since the apparatus feeds a plurality of reagents through one nozzle 108 and one flow path from there to the waste liquid tank 107, contamination occurs in which different reagents are mixed with each other. The locations where contamination occurs are roughly divided into two locations inside the flow path and outside the nozzle. Inter-reagent contamination causes the reaction solution to dilute, the target reaction is inhibited by the contaminated reagent, or an unintended reaction occurs due to the contaminated reagent. There is a fear.

  Contamination in the flow path occurs by aspirating different reagents in succession. FIG. 2 shows an explanatory diagram thereof. The figure is an enlarged schematic view of a part of the flow path 203. The upper figure 201 shows a state in which contamination occurs, and the lower figure 202 shows a state in which contamination is prevented. As shown in FIG. 201 above, when different reagents of the reagent A 204 and the reagent B 205 are continuously sucked, different reagents are adjacent to each other in the flow path 203, so that the reagents are mixed from the interface 206. Therefore, in the present invention, as shown in FIG. 202 below, an appropriate amount of air 207 is sucked between the reagents A204 and B205. Accordingly, the air layer 207 serves as a wall so that different reagents 204 and 205 do not mix with each other, and contamination can be prevented. This eliminates the need for cleaning the flow path when aspirating different reagents.

  Next, contamination outside the nozzle will be described. FIG. 3 shows an explanatory diagram thereof. During reagent aspiration, the nozzle 301 accesses the reagent 302 in the reagent rack 106 as shown in FIG. As shown in FIG. 3B, after the reagent is aspirated, when the nozzle 301 is separated from the reagent 302, the reagent 303 may adhere to the outside of the nozzle. As illustrated in FIG. 3C, when the next different reagent 304 is accessed while the reagent 303 is attached to the outside of the nozzle, the components of the previous reagent 303 are brought into the next reagent 304. The brought-in reagent 303 is sent together with the reagent 304 to the flat substrate 103 which is a reaction field. In the present invention, contamination outside the nozzle can be prevented by the nozzle cleaning method as shown in FIG. When the different reagents are sucked in the order of the reagent 401 and the reagent 402, the reagent 401 is sucked as shown in FIG. 4A, the nozzle 403 is moved away from the reagent 401, and an appropriate amount of air is sucked. The previous reagent component 405 is removed from the outside of the nozzle 403 by dipping into the cleaning liquid 404 as in -2. At this time, it is desirable that the depth at which the nozzle 403 is dipped into the cleaning liquid 404 is deeper than the depth at which the reagent 401 and the reagent 402 are accessed. A cleaning method for dipping in a cleaning solution is called dip cleaning. It is desirable that there are two or more cleaning liquids 404, and the effect of removing the pre-reagent component 405 attached to the nozzle 403 is increased by repeating the dip cleaning for the cleaning liquids 404 in two or more containers a plurality of times. For example, when two cleaning liquids 404 are used, after the pre-reagent 401 is aspirated, the first cleaning liquid is dip-cleaned, and then the second cleaning liquid is similarly dip-cleaned, and the post-reagent 402 is accessed. As shown in FIG. 4C, contamination from the outside of the nozzle 403 is prevented by accessing the next reagent 402 with the nozzle 403 after dip cleaning from which the previous reagent component 405 has been removed. The dip cleaning liquid 404 may be held in the reagent rack 106 together with the reagents. Accordingly, the customer can install the cleaning liquid 404 simultaneously with the supply of the reagent into the apparatus, and the distance that the nozzle accesses the reagent and the cleaning liquid can be shortened, and the apparatus throughput can be improved.

  On the other hand, a nucleic acid analyzer that cleans the outside of the nozzle by ejecting water to the outside of the nozzle is known. However, a pump mechanism and a washing area for ejecting water, a large amount of washing water and waste liquid are stored in the apparatus. And a supply and discharge system is needed. In addition, in the case of the jet cleaning method, not only does the customer need to supply a large amount of water or remove the waste liquid, but it also takes time to move the nozzle to the cleaning area and spray the cleaning water. Therefore, throughput does not increase. The dip cleaning of the present invention has a cleaning effect even with a small amount of cleaning liquid as compared with the jet cleaning method, and solves the disadvantages of the jet method described above.

  A second embodiment of the present invention will be described.

  In the dip cleaning described in the first embodiment, the amount of the pre-reagent brought in and the amount of the dip cleaning liquid brought in, that is, the amount attached to the nozzle are key. The amount of adhesion to the nozzle varies depending on the distance between the nozzle and the liquid. Since the liquid is more likely to adhere to the nozzle as the nozzle axis deviates from the liquid surface, the nozzle axis and the liquid surface must be vertical. Moreover, it became clear from our experiment that the amount of liquid adhering to the nozzle varies greatly depending on the speed at which the nozzle moves away from the liquid. We experimentally verified the effect of nozzle speed. When the speed at which the nozzle moves away from the liquid is fast (125 mm / sec), the pre-reagent to post-reagent is about 3.5 times faster than when the nozzle speed is slow (25 mm / sec). The result was that there was a lot of carry-in. Therefore, the nucleic acid analyzer of the present invention is an apparatus that is perpendicular to the nozzle and the liquid surface, and the speed at which the nozzle is separated can be set to a speed suitable for analysis accuracy.

  Furthermore, according to our experimental data, it is known that the amount of each cleaning solution container for dip cleaning, the number of dip times for each cleaning solution container, and the dip time for the dip cleaning solution have little effect on the nozzle adhesion amount. A device that can be set to any value within the range of achieving analysis accuracy.

  A third embodiment of the present invention will be described.

  As described in Example 2, in dip cleaning, keeping the amount of reagent and dip cleaning liquid attached to the nozzle small is the key to analysis accuracy. The amount of liquid adhesion can be reduced by the material and shape outside the nozzle. For example, the liquid breakage is improved by keeping the nozzle tip at an acute angle. Further, the surface of the nozzle can be processed with Teflon (registered trademark) or the like, or the surface of the nozzle can be polished to eliminate surface irregularities, thereby making it water repellent and reducing the amount of liquid attached to the nozzle. As a result, the amount of contamination is reduced.

Embodiment 4 of the present invention will be described.
As described in Example 2, in dip cleaning, keeping the amount of reagent and dip cleaning liquid attached to the nozzle small is the key to analysis accuracy. As shown in FIG. 5, you may provide the structure which wipes the liquid adhering to a nozzle. As shown in FIG. 5A, a hole structure 502 close to the diameter of the nozzle 503 is provided on the upper side of the reagent container 501. As shown in FIG. 5B, the liquid 504 is attached to the outside of the nozzle and is separated from the liquid surface. Further, the attached liquid is wiped off when passing through the minute hole structure 502, and as shown in FIG. Thus, the liquid 504 is not attached to the outside of the nozzle 503. The minute hole structure 502 is preferably a variable structure such as rubber or sponge. For example, when the outer diameter 505 at the tip of the nozzle is different from the outer diameter 506 at the upper part thereof, the hole structure 502 can be expanded and contracted to move the inside of the hole structure 502 regardless of the outer diameter of the nozzle 503 and wipe the liquid 504. be able to.

  The hole structure may be formed in the lid portion of the reagent container, or the hole structure 502 may be formed by piercing without the hole structure 502 initially. For example, the lid structure of the reagent container is sealed with a film, and the hole structure 502 can be formed by piercing the film with a nozzle 503 or an object of such shape inside or outside the nucleic acid analyzer. it can.

  By adopting such a structure, the amount of contamination that the reagent and the dip cleaning liquid bring into the reagent to be accessed next can be reduced.

  A fifth embodiment of the present invention will be described.

  The dip cleaning solution 404 described in Example 1 may be a component that does not adversely affect the result of nucleic acid analysis even if it is not water. For example, a detergent component such as a surfactant may be included according to the liquidity of the front and rear reagents so that the components of the pre-reagent 405 attached to the nozzle 403 can be easily dissolved in the cleaning liquid 404. In addition, for example, a component common to the buffer component of the front and rear reagents may be included. Thereby, the dip cleaning may be performed more efficiently than when the dip cleaning liquid is water.

  Embodiment 6 of the present invention will be described.

  In the dip cleaning described in the first embodiment, different cleaning liquid containers may be used depending on the type of reagent accessed before and after. Further, as described in Example 5, the components of the cleaning liquid may be changed according to the corresponding reagents before and after. For example, it is assumed that there are a reagent A and a reagent B for which contamination between reagents is particularly avoided, and the solution is fed in the order of reagent A → reagent C → reagent B. In the flow path, since there is a reagent C and air between the reagent A and the reagent B, the reagent A and the reagent B do not contaminate each other. However, when the reagent A and the reagent C are dipped with a common cleaning solution, the reagent A may be slightly brought into the cleaning solution. If the next reagent C is aspirated and washed with the same washing liquid as that of the reagent A, there is a possibility that the reagent A already brought in may contaminate the reagent B accessed next through the washing liquid. In this case, the contamination from the reagent A to the reagent B can be completely avoided by separating the cleaning liquid of the reagent A and the reagent C into separate containers. Thus, by separating the cleaning liquid container and the type of the cleaning liquid according to the characteristics of the preceding and subsequent reagents, the influence of contamination is further reduced, so that data accuracy is maintained.

  A seventh embodiment of the present invention will be described.

  As shown in FIG. 1 shown in Example 1, this nucleic acid analyzer includes a substrate-side electromagnetic valve 109 and a waste liquid-side electromagnetic valve 111 in a flow path. Both solenoid valves 109 and 111 can be opened and closed. As described above, when the substrate side solenoid valve 109 is opened, the waste side solenoid valve 111 is closed, and the syringe 110 is pulled in the suction direction 115, suction is performed from the nozzle 108. Yes. Conversely, when the syringe 110 is pushed in the discharge direction 116, the liquid or air in the flow path from the syringe 110 to the nozzle 108 flows in the opposite direction to the suction. Therefore, the reagent in the flow path can freely reciprocate in the flow path by the combination of opening and closing of the electromagnetic valves 109 and 111 and the operation directions 115 and 116 of the syringe 110. In general, it is known that the reaction is accelerated by stirring. The reagent is reciprocated on the flat substrate 103 by reciprocating the syringe 110 by an appropriate amount. As a result, the sequence reaction and the fixation of the nucleic acid sample are accelerated as in the case of stirring, and the throughput of the nucleic acid analyzer can be improved.

  Further, when the syringe 110 is reciprocated with the nozzle 108 accessing the reagent, the reagent is repeatedly sucked and discharged, and has a function of mixing a plurality of reagents in the reagent rack 107 and stirring the reagents. it can.

101 Detection Unit 102 Liquid Supply Unit 103 Flat Substrate 104 Signal 105 Detector 106 Reagent Rack 107 Waste Liquid Tank 108 Nozzle 109 Substrate Side Solenoid Valve 110 Syringe 111 Waste Liquid Side Solenoid Valve 112 Clockwise Direction 113 Counterclockwise Direction 114 Nozzle Z Direction 115 Syringe Suction direction 116 Syringe discharge direction 201 Upper figure 202 Lower figure 203 Flow path 204 Reagent A
205 Reagent B
206 Interface 207 Air 301 Nozzle 302 Reagent 303 Reagent 304 Different reagent 401 Reagent 402 Different reagent 403 Nozzle 404 Cleaning liquid 405 Reagent component 501 Reagent container 502 Hole structure 503 Nozzle 504 Liquid 505 Nozzle tip outer diameter 506 Nozzle top outer diameter 506

Claims (18)

A reagent rack for holding a plurality of reagent containers;
A nozzle for aspirating the reagent;
A flat substrate for holding a sample and performing a nucleic acid analysis reaction;
Two solenoid valves that can be opened and closed;
A syringe for aspirating and discharging liquids and gases;
A waste bottle,
A flow path connecting a nozzle, a planar substrate, a first electromagnetic valve, a syringe, a second electromagnetic valve, and a waste bottle;
A container for storing a dip cleaning liquid for cleaning the nozzle;
A drive mechanism for accessing the reagent rack of the nozzle;
A detection unit for detecting a signal generated in the sample in the planar substrate;
A nucleic acid analyzer characterized by cleaning by dipping a nozzle into a dip cleaning solution. In claim 1,
A nucleic acid analyzer comprising two or more containers for storing dip cleaning liquids. In claim 1,
A nucleic acid analyzer, wherein air is sucked between different reagents inside a flow path. In claim 1,
The drive mechanism drives the nozzle in the vertical direction,
A nucleic acid analyzer characterized in that the rising speed of the nozzle can be varied within an arbitrary range. In claim 1,
The drive mechanism drives the nozzle perpendicular to the liquid level of the reagent container.
In claim 1,
A nucleic acid analyzer characterized in that the nozzle is processed to be water repellent. In claim 1,
A nucleic acid analyzer characterized in that the nozzle tip has an acute angle. In claim 1,
A nucleic acid analyzer characterized by having a hole structure for wiping the nozzle in the upper part of the reagent container. In claim 1,
The dip cleaning solution contains a surfactant, and the nucleic acid analyzer is characterized in that In claim 1,
The nucleic acid analyzer, wherein the dip cleaning liquid contains a salt. In claim 1,
A nucleic acid analyzer characterized in that a plurality of dip washing liquids are selectively used depending on the type of reagent before and after washing. In claim 1,
A nucleic acid analyzer characterized by using a plurality of dip cleaning liquid compositions depending on the types of reagents before and after cleaning the dip cleaning liquid. In claim 1,
A nucleic acid analyzer characterized in that a reaction on a flat substrate is promoted by reciprocating a liquid on the flat substrate by the syringe. In claim 1,
A nucleic acid analyzer which mixes or stirs a reagent by performing a suction / discharge operation from a nozzle. In claim 1,
A nucleic acid analyzer characterized by washing one nozzle with two or more dip washing liquids when sucking different reagents. In claim 8,
A nucleic acid analyzer characterized in that the hole structure is stretchable. In claim 8,
A nucleic acid analyzer, wherein the hole structure is formed in a reagent container lid. In claim 8,
The nucleic acid analyzer, wherein the hole structure is formed by breaking through a film.