Apparatus and method for processing biological sample

Abstract

Cartridge modules, devices, and methods are provided for improving contamination control during a bioanalysis or nucleic acid analysis protocol. The cartridge module (10) includes a hollow tube (36) and a container (18). The porous cover layer (23) isolates the container (18) from the surroundings while the container (18), when attached to the syringe (32), allows the tube (36) to dispense the reagent liquid (12) or biological sample (21) to the container (18) only from the container top opening (22) through the cover layer (23). The cover layer (23) substantially blocks aerosol exchange between the environment and the container (18) throughout the analysis process. And pollution to the environment due to accidental overflow of the container (18) is also prevented. The porous cover layer (23) reduces air pressure variations within the container (18) during aspiration or dispensing of the liquid.

Description

[0001] This application is a divisional application of a patent application with an application date of April 22, 2024, application number 202280070823.4, and title “Device and method for processing biological samples”.

[0002] Technical field of the invention

[0003] The present invention relates to a device and method for processing biological samples, in particular to a device and method for nucleic acid extraction, amplification reaction and detection. Background Art

[0004] Nucleic acid extraction and amplification techniques are becoming increasingly important in molecular biology, food safety, and environmental monitoring. Many biological researchers use polymerase chain reaction (PCR) in nucleic acid analysis because of its high sensitivity and specificity. Generally, PCR is performed through a thermal cycling process that denatures, anneals, and extends DNA by heating and cooling a container containing the reaction material. In real-time PCR, the instrument detects positive reactions by the accumulation of fluorescent signals. The instrument usually performs fluorescent imaging multiple times or after each thermal cycle to record progressive changes in the biological sample and detect the amount of target nucleic acid in the sample or biological sample. PCR amplification of trace amounts of DNA can be used for applications such as scientific research, forensic analysis, wildlife research, and ultrasensitive diagnosis. Isothermal amplification techniques are also used. In general, sample preparation steps such as nucleic acid extraction and purification are required before PCR, which requires multiple liquid handling steps, including aspiration, displacement, and mixing of liquids. Operations in both sample preparation steps and nucleic acid amplification steps can lead to contamination, mainly due to the generation of aerosols and / or the volatilization of liquids. The typical method to prevent nucleic acid analyzers or sample processing instruments from contaminating laboratory spaces is to seal the PCR containers during amplification and place the sample preparation instruments in a biosafety cabinet or fume hood with negative pressure, where aerosols and / or vapors generated during liquid processing are removed from the biosafety cabinet. In nucleic acid testing laboratories, in order to prevent the occurrence of such contamination, the sample preparation steps for nucleic acid extraction and the PCR steps are performed in different spaces, and the air pressure and air circulation direction of the spaces are strictly controlled. Outside of PCR laboratories, such as distributed care testing sites (POCT) such as schools, pharmacies, airports, or commercial buildings, the use of such biosafety cabinets or safety covers and controlled testing environments is technically inappropriate or not cost-effective.

[0005] Nested PCR with a preamplification step is a technique that can reduce nonspecific amplification of DNA templates. This technique usually uses two sets of primer combinations or reagents and two consecutive PCR reactions. The first set of primers is used for the initial PCR reaction. The amplicon produced by the first PCR reaction is used as the template for the second set of primers and the second amplification step. However, since the performer needs to open the container used for the first round of amplification to perform additional liquid transfer and handling of the amplicon products from the first round of amplification in preparation for the second round of amplification, the above process usually increases the possibility of contamination in the laboratory environment. In order to minimize the contamination carryover between the first and second rounds of amplification, the different steps of the process need to be physically separated from each other and preferably performed in completely independent experimental spaces. The amplicons in the nested PCR test are detected in the same way as in PCR. Therefore, when all steps are performed in a single instrument box, the nested PCR steps are difficult to automate in the same instrument due to the high possibility of contamination.

[0006] Prior to the nucleic acid amplification step, the nucleic acid extraction and amplification process often results in sample contamination from laboratory surfaces, reagents, used pipettes and pipette tips, lab coats, glove boxes, and wastebaskets. Aerosols and volatiles from sample and reagent liquids can contaminate the surrounding environment. The higher the sensitivity of the test, the more susceptible it is to contamination. Whether it is genotyping, creating NGS libraries, studying single cells, or testing sensitive clinical samples, successful aerosol contamination control is the backbone of reliable data in many experiments. In the laboratory, using unsealed well plates for extraction steps is very convenient and provides flexibility for many testing protocols. It is also low cost and easy to get in and out of liquid handling devices. However, in use, several separate rooms are required for reagent preparation, sample preparation, PCR, etc. to control aerosols and / or vapors and other contaminants.

[0007] These facilities only exist in laboratories with trained personnel, air control, and procedures. Generally, these laboratories also have procedures in place for decontamination when contamination occurs. Separate rooms, contamination prevention facilities, and trained personnel are not available in a POCT environment.

[0008] Even trace amounts of biological sample contamination can have serious negative effects, especially when these contaminants are amplified during the amplification process and subsequently lead to erroneous detection and diagnosis. Therefore, both the contamination of biological samples to the environment and the contamination of the environment to the samples need to be prevented.

[0009] Microfluidic cartridge modules are developed to provide a sealed environment, which contains internal microchannels, valves and pumps or pump interfaces, which require complex machine interfaces to operate the cartridge modules. The BioFire FilmArray system provides a new standard for molecular diagnosis of syndromic infectious diseases, which integrates sample preparation, amplification, detection and analysis functions, patent publication number: US2018 / 0214864A1. In addition, the product GeneXpert also describes the application of reaction cartridge modules. This disposable cartridge module consists of microvalves and micropumps that transfer liquids between chambers, and thermal cycling and real-time optical detection for amplification are performed in the chamber. However, due to the complex design, the production operation of this cartridge module is also complex and therefore expensive. High complexity may also lead to low performance reliability and require manufacturing with a good production system. Microfluidic cartridge modules cannot be flexibly used for different operating procedures. The number of samples that can be tested is also limited by the design of the cartridge module. In addition, the cartridge module design cannot control the cross contamination between the sample and the environment during sample preparation and PCR.

[0010] Therefore, there is a huge market demand for the development of simple and low-cost liquid handling devices for complex nucleic acid extraction and PCR sample preparation steps without aerosol and / or vapor contamination. Similarly, bioanalysis of infectious or malodorous samples such as feces is also a challenge in terms of contamination control, especially in POCT scenarios.

[0011] The present invention provides several improvements in devices, apparatuses and methods for sample preparation, amplification, detection and analysis. By using simple design and functionality, the present invention makes disposables more affordable and reliable, thereby providing a huge positive impact on bioanalysis. The invention has the potential to speed up the amplification process, provide significant contamination control, especially for POCT settings, and also solve the problems faced when performing bioanalysis on infectious or malodorous samples (such as feces). Summary of the invention

[0012] Unless otherwise specified, the terms "include" and "comprising" and their grammatical variations are intended to represent "open" or "inclusive" language such that they include the elements recited, but also allow for the inclusion of additional unlisted elements. The word "substantially" does not exclude "completely". The term biological sample has been used for biological samples containing nucleic acids, whether before or after mixing with reagent liquids. Biological samples can be derived from any biological form, such as swabs, saliva, blood, etc. "Reagent liquid" may refer to any type used in the art, such as PCR reagents, extracts, purification solutions, deamination solutions, desulfonation solutions, and types used to process biological samples for processing biological samples. In the present disclosure, the container can be in an integrated form such as a well plate, or in a decomposed form.

[0013] According to a first aspect, the present invention provides a method for improving contamination control during a biological analysis or nucleic acid analysis procedure including nucleic acid extraction, nucleic acid amplification and detection. The cartridge module comprises: at least one hollow tube; and a plurality of containers; at least one covering layer to isolate the container from the environment outside the covering layer, wherein each container allows a tube connected to a syringe or a pipette to pass through the covering layer and push and draw the reagent solution being used into and out of the container only from the opening at the top of the container, even if the tube pierces the covering layer and retracts, the covering layer can still substantially block the aerosol exchange between the environment and the container during the entire biological analysis or nucleic acid analysis process, and the covering layer can also prevent the environment from being contaminated by the overflow of the reagent solution or biological sample from the container or cartridge module, the covering layer is connected to: 1) the top opening of at least one container, or 2) the cartridge module or container, and maintains a connected air space isolated from the environment between the covering layer and the top opening of the container, the connected air space can reduce the change of air pressure in the isolated container when the tube draws or pushes the biological sample and the reagent solution. When the tube is retracted from the container, the cover layer advantageously scrapes most of the reagent solution or biological sample adhering to the bottom of the tube under the cover layer. The preferred tube is a beveled tip. The outer diameter of the tube is between 0.1 mm and 5 mm. The thickness of the cover layer is between 0.1 mm and 50 mm.

[0014] According to one embodiment, when the cover is attached to the top opening of the container, at least a portion of the cover material is porous. The porous cover helps regulate the gas pressure during liquid displacement and aspiration. The increase or decrease in the gas pressure inside the container will also affect the volume of reagent liquid or biological sample originally programmed to be pushed into or aspirated from the tube. This effect may only cause an impact on a small volume, but when the container volume is also small, the impact will become significant. Fig. 7A and 7B Further problems are described. The porous covering layer should have porosity, tortuosity, and internal pore structure to substantially block aerosols generated in nucleic acid analysis in typical laboratory practice.

[0015] According to one embodiment, at least a portion of the cover material is elastomeric. The elastomeric cover helps to substantially close the gap in the cover caused by puncture by the tube.

[0016] The material of the cover layer is any one or a combination of the following materials: a) porous elastomer, b) porous non-elastomer, c) non-porous elastomer, and d) non-porous non-elastomer. The material of the cover layer can be selected from the four types as described above to optimize between pressure regulation, aerosol blocking and spill prevention.

[0017] According to one embodiment, a layer of covering liquid is pre-displaced or provided to be displaced onto the covering layer, wherein at least the portion pierced by the tube is displaced so as to form a laminate or to soak the covering layer with the covering liquid, or to sandwich the covering liquid in the covering layer, and the covering liquid does not react with the reagent liquid and the biological sample. The covering liquid may also be on the lower side of the covering layer. The covering liquid is advantageously suitable for strengthening the isolation effect of the covering layer by blocking the gap formed on the covering layer where the tube is pierced. If the covering layer is non-porous, the isolation may be airtight. The covering liquid also improves the barrier for aerosols and overflow, especially for the improvement of the porous covering layer. With the covering liquid, especially for the non-elastomeric covering layer, the container can be kept airtightly isolated from the environment before and after the tube pierces the covering layer, for example when performing a step that does not involve high temperature. The covering liquid seals the covering layer holes of the non-elastic covering layer. When the tube passes through the covering liquid, the trace reagent liquid or biological sample remaining on the tube is covered by the covering liquid. This prevents the reagent liquid or biological sample adhering to the tube from being exposed to the environment and contaminating it. The covering liquid also covers a portion of the tube inserted into the container, so any contamination entering the container from the environment can be reduced. When the covering liquid is located below the covering layer, the covering liquid is also better protected from physical and operational damage. The preferred covering liquid is immiscible with the reagent solution and the biological sample and is capable of wetting the tube and the covering layer. The covering liquid can be a viscous liquid polymer or monomer or oil or liquid wax or any other suitable material in the art.

[0018] Those skilled in the art will appreciate that containers that are amplified by heat treatment need to be provided with a separate cover to enable heating of the containers and their transfer to an amplification module outside the box.

[0019] The present invention has several advantages as described below: 1) By isolating, once the reagent liquid and biological sample are pushed into the container, these liquids will never be exposed to the environment again, from the biological analysis or nucleic acid extraction process, amplification, imaging to disposal. Therefore, in the present invention, the contamination from the environment to the biological sample and from the biological sample to the environment is significantly reduced compared to the most advanced reagent kits. Isolation significantly prevents aerosols and / or vapors from escaping or entering the container during the analysis process. 2) The mechanism of the container with precautions and liquid communication only from the opening above the container allows the cartridge module to operate in a relatively low-complexity environment. Therefore, the cartridge module reduces cost and improves the reliability of detection and analysis without any valves, thermal cycling modules or optical detection modules. Moreover, since there is no liquid communication between the containers, the present invention reduces cross-contamination between biological samples in the containers. Compared with microchannels, this feature also allows the containers to be used on custom computer programming. 3) In addition, the low cost allows the cartridge module to be discarded after the reagent liquid is consumed, thereby further reducing the possibility of contamination. 4) The cartridge provides users with greater flexibility in selecting the baths for amplification in the device, for example, users can achieve faster thermal cycling by using several baths maintained at their respective target temperatures, rather than limiting the thermal cycling speed to the application of a single bath or heating block. 5) The simple features of the cartridge module make it easy to carry. 6) The cartridge module allows the use of disposable items commonly used in the industry, such as individual containers and well plates. 7) The cover layer isolation also solves the problem faced when performing bioanalysis of infectious or malodorous samples such as feces in a POCT environment.

[0020] According to one embodiment, the cartridge further comprises a scrubbing layer fixed to the stack, so that the covering layer, the covering fluid and the scrubbing layer form a composite layer, the scrubbing layer can be pierced by the tube, and the scrubbing layer can substantially scrub away the covering fluid adhering to the tube when the tube is retracted from the container. Without the scrubbing layer, the covering fluid may be exhausted more quickly when the number is small or the tube is long.

[0021] According to one embodiment, the cartridge further comprises at least one syringe and provides at least one feature from the following group: a) an O-ring is disposed between the plunger of the syringe and the syringe barrel, and a covering liquid is provided on the O-ring, the covering liquid being away from the pushing or suction tip of the syringe barrel, and b) at least two O-rings are disposed between the plunger of the syringe and the barrel, and a covering liquid is provided between the two O-rings, the covering liquid being immiscible and non-reactive with the reagent liquid and the biological sample. The O-ring with the covering liquid tends to make the syringe more airtight, thereby further reducing any contamination that may enter the container from the environment, and contamination that may enter the environment from the container. In addition, when the plunger is pushed down into the syringe barrel, trace amounts of reagent liquid or biological sample attached to the inner wall of the syringe plunger and the outer wall of the syringe barrel will be covered by the covering liquid. The covering helps to prevent the reagent liquid or biological sample from being exposed to the environment, thereby preventing them from contaminating the environment.

[0022] According to one embodiment, the cartridge further comprises a rigid retaining layer positioned above or below the cover layer, the rigid retaining layer having a retaining layer hole aligned above the top opening of the container to allow the tube to penetrate the cover layer. The rigid retaining layer advantageously prevents the cover layer from detaching or bending when the tube penetrates or is retracted from the container.

[0023] According to one embodiment, the cartridge further comprises at least one container holder for accommodating at least one container, the container holder being removable from the cartridge. The container holder is useful when placing containers into or out of cartridge modules, such as within a module on an apparatus for bioanalysis or nucleic acid analysis procedures including nucleic acid extraction, nucleic acid amplification and detection.

[0024] According to one embodiment, the cartridge further comprises a liquid sealant which, when pushed onto the opening at the top of at least one container, can seal the opening at the top of the container, either before or after solidification, and the liquid sealant is immiscible and non-reactive with the reagent liquid and the biological sample. The liquid sealant can pass through a tube passing through a covering layer or a laminate and be pushed out, thereby creating an air gap between the biological sample or the reagent liquid in the container. The liquid sealant can be viscous, which can provide great resistance to pressurized air leaking out of the container. When the container is heated during the amplification step, the sealing layer helps prevent aerosols or vapors from the biological sample in the container from escaping through the covering layer holes or the laminate holes. The sealing layer also controls any such contamination that may reach the container from the environment. Thanks to the air gap, fluorescence from the liquid sealant will not be captured together with fluorescence from the biological sample during imaging. The liquid sealant can be any viscous material, such as wax or glue. Wax that solidifies at room temperature makes it easier to transport the test kit. As it is drawn and pushed through the tube, it can be locally heated into liquid form.

[0025] According to one embodiment, the cover layer is provided with grooves to accommodate the cover liquid, and the grooves are interconnected. Then, the tip of the hollow tube can move between the pits while remaining immersed in the cover liquid and not exposed to the environment. If the tip of the tube is exposed to the environment after aspirating or pushing a biological sample containing nucleic acid, any dripping of the biological sample will contaminate the environment. There are several possible factors that cause unnecessary sample dripping, such as software errors or instrument errors. This embodiment avoids the possibility of such contamination because any such biological sample drops will be captured in the cover liquid.

[0026] According to one embodiment, the cover layer above at least one of the container top openings is flexible and has at least one slit or partially permeable slit to allow for easier and less effortful penetration by a blunt tube such as a pipette. Blunt tubes are preferred when the container is made of a deformable material. Sharp tubes have the potential to penetrate a deformable material.

[0027] According to one embodiment, the cover layer has a recessed top for guiding the tip of the tube before piercing the cover layer when the tip of the tube passes through the base area of ​​the recessed top, and the recessed top has inclined sides to provide a taper toward the base area, and the bottom area is smaller than the top opening of the container. When the tube is in a sharp needle form, the tapered surface is preferably a hard surface to prevent the needle from piercing the tapered surface. This provides better tolerance for misalignment between the tip of the syringe or pipette and the top opening of the small container. This feature is particularly helpful when the container is in a very narrow capillary form used to improve heat transfer efficiency for nucleic acid amplification in biological samples. The inclined sides are made of a rigid material to prevent piercing by the tip of the tube.

[0028] The cartridge module may include at least one container containing magnetic beads for binding nucleic acids from biological samples. At least one container may contain oil or water or a DNA remover as a cleaning fluid for cleaning the tube and syringe at any stage during nucleic acid analysis, including the inner and outer surfaces of the needle tube, FIG24 shows the liquid retention space, which is the area where the syringe joins the tube head. These areas retain surface shapes left by previous processing steps that the researcher does not want. When one syringe is used to process several samples, this cleaning effect also removes the biological sample from the previous processing.

[0029] According to an embodiment of a cartridge module, at least a portion of the container is made of a deformable material so that, when in use, (i) the container in its initial state or shape can expand to accommodate a reagent solution or biological sample pushed by the instrument. (ii) When the reagent solution or biological sample pushed by the instrument is drawn out of the container, the container in its initial state or shape can contract so that the buffer tube is responsive to changes in air pressure in the isolated container when the biological sample and reagent solution are drawn or pushed. This embodiment is particularly useful for non-porous covering layers. An increase or decrease in air pressure inside the container will also affect the volume of reagent solution or biological sample that is originally programmed to be pushed into or drawn from the tube. This effect may only affect a small volume, but when the container volume is also small, the effect becomes significant.

[0030] According to one embodiment, the cartridge module with air connection space further comprises: a sealing layer, which seals the single or multiple container top openings to prevent any reagent liquid and biological sample in the container from spilling out when the cartridge module is not in an upright position, the sealing layer being pierceable by a tube. During use, the device needs to create a vent in the sealing layer through a tube attached to a syringe or pipette mechanism, so that the container is connected to the air of the air connection space during subsequent aspiration or expulsion from the container into the container.

[0031] According to one embodiment, when the cover is attached to the container top opening, a plurality of isolation containers are placed vertically in a stack so that the tip of the hollow tube can pierce the cover above each container in the stack to reach the bottom container in the stack from the top container in the stack. This embodiment helps to reduce the footprint of the cartridge module, which is ideal for portable devices.

[0032] According to one embodiment, the cartridge module further comprises: a fixed pattern detectable by the device when the device automatically positions the tube over the top opening of the container.

[0033] According to one embodiment, the cartridge module also accommodates at least one sample container having a lid, at least a portion of which comprises a covering layer that can be pierced by a tube, so as to allow the tube to aspirate and push the biological sample from the sample container into the container. This improves contamination control because the biological sample is transferred into the container by the tube while the sample container is in the cartridge module. The lid and the covering layer in the lid prevent any possible spillage of the biological sample during the entire analysis process until the cartridge module is discarded.

[0034] According to a second aspect, the present invention provides an apparatus for improving contamination control during a biological analysis or nucleic acid analysis procedure including nucleic acid extraction, nucleic acid amplification and detection. The apparatus comprises: a) a receiving module for accommodating a cartridge module as described above; b) a mechanically controlled syringe or pipette mechanism for operating a syringe or pipette connected to a syringe or pipette so as to draw out or push a biological sample or reagent solution from or into a container through a top opening of the container and a covering layer or laminate or interlayer; c) an amplification module for providing isothermal heating or cyclic heating, and amplification can be performed when the container containing the biological sample is inside or outside the cartridge module; d) an optical imaging mechanism for imaging the biological sample when or after the container containing the biological sample is inside or outside the cartridge module; and e) when amplification is performed outside the cartridge module, a transfer device controlled by a robot can be configured to transfer a container held by a container holder from inside the cartridge module or inside the device to outside the cartridge module.

[0035] According to one embodiment, the device is computer programmable so as to operate the syringe or pipette device so that it presses the tube attached to the syringe against a fixed surface in the cartridge module or device and bends the tube, and then inserts the syringe and the bent tube into the cartridge module. The advantages have been described in the first aspect. Bending the tube attached to the syringe or pipette after use is advantageous so that it can be easily received in the cartridge before being discarded.

[0036] The device also includes an ultraviolet (UV) curing module for curing a UV resin deposited at the top opening of the container. This in-situ curing before amplification helps prevent vapors from the biological sample in the container from contaminating the environment through the cover layer holes formed in the cover layer due to the piercing of the tube.

[0037] According to one embodiment, the device is further computer programmable so that when a syringe or pipette is used to draw and push reagents or biological samples into and out of multiple containers, the container holder is positioned so that the section of tubing that contacts the biological sample in the container does not move out of the air connection space. This significantly reduces contamination.

[0038] According to one embodiment, the device is computer programmable to perform at least two amplification steps for a biological sample treated by a reagent solution through a covering layer with or without a covering liquid between any two consecutive amplification steps, during which the container containing the biological sample is retained inside or outside the card box module, but is retained in the device. The present invention enables the nucleic acid in the biological sample to be automatically nested and double-amplified in the same container in the presence of a covering layer or stack. There is no need to remove the PCR amplicon from the container after one amplification in order to mix it with the reagent solution before another amplification. Therefore, the method is suitable for rapid point-of-care testing (POCT) PCR processes. The product can automatically perform highly complex and time-consuming manual procedures while minimizing the possibility of contamination. In order to increase the detection sensitivity of the DNA and RNA studied by the user, more than two amplifications may be required.

[0039] According to one embodiment, the device further comprises: a detection module for detecting the relative misalignment between the tip of the tube and the top opening of the container due to piercing the covering layer; and an automatic calibration module for allowing the syringe or pipette device to compensate for the misalignment. The degree of misalignment of the tip of the tube depends on the material, thickness and other factors of the covering layer. The thinner the tube is, the easier it is to bend when it pierces the covering layer and is subjected to force during use during manufacturing and assembly to the cartridge module. Therefore, the tube may not be able to enter the container through the top opening of the container, such as for PCR or glass capillaries. During magnetic extraction, the tube may also not be able to enter a required area in a container and cannot avoid hitting the magnetic bead group. One method of finding the position of the tip of the tube is to bring the tip of the tube close to a fixed pattern on the cartridge module before or during operation of the cartridge module, and use a camera or sensor for recording images or signals of the tip of the tube and the pattern, and then calculate the coordinates of the position of the tip of the tube relative to the position of the pattern. The coordinates calculated by the system are sent to a controller in the device to generate motion instructions for moving the tip of the tube to a target position required by the test procedure. The sensor can be optical, electrical or magnetic.

[0040] According to one embodiment, the detection module on the device optically detects the misalignment of the tip relative to the fixed pattern on the cartridge module.

[0041] According to one embodiment, the apparatus further comprises: a syringe picking module for picking up at least one syringe from the cartridge module.

[0042] According to one embodiment, the apparatus further comprises: a tube picking module for mounting the tube provided in the cartridge module onto the syringe.

[0043] According to one embodiment, the device also includes: a positioning module for detecting a fixed pattern on the cartridge module and for positioning the tube above the top opening of the container when the tube is attached to a syringe or pipette device. Before and during operation of the cartridge module, the tip of the tube approaches the fixed pattern on the cartridge module, and a camera or sensor records an image or signal of the tip of the tube and the pattern, and then calculates the relative position between the tip of the tube and the pattern. The coordinates calculated by the system are sent to a controller in the device to generate motion instructions for moving the tip of the tube to a target position required by the test procedure. The sensor can be optical, electrical or magnetic.

[0044] According to one embodiment, the device also contains at least one sample container having a cover for containing a biological sample, wherein a portion of the cover comprises a covering layer, and the syringe or pipette device is programmable by a computer to suck the biological sample from the sample container by piercing the covering layer, and to push the biological sample into the container. This improves contamination control because the biological sample is transferred into the container by the tube while the sample container is in the cartridge module. The cover and the covering layer in the cover prevent any possible spillage of the biological sample during the entire analysis process until the cartridge module is discarded.

[0045] According to a third aspect, the present invention provides a method for improving contamination control during bioanalysis and nucleic acid analysis procedures, including nucleic acid extraction, nucleic acid amplification and nucleic acid detection, the method using any of the cartridge modules and devices described above; loading the cartridge module containing containers containing reagent fluids and biological samples into a receiving module; performing the bioanalysis or nucleic acid analysis within the device by piercing and retracting the cover with a tube to aspirate or push the reagent fluids and biological samples from the containers or into the containers without exposing the reagent fluids and biological samples to the environment; and then discarding the cartridge module containing the containers and the cover, reagent fluids, biological samples and tubes. The advantages have been described in the second aspect.

[0046] According to one embodiment, the method comprises: operating a syringe or pipette operating mechanism according to one of the following steps: a) after the reagent liquid or biological sample is pushed into the container, when the tip of the tube has reached the top of the reagent liquid or biological sample in the container, allowing the syringe or pipette to perform an aspiration mode for a preset time to release at least a part of the excess air pressure caused by the pushing in the airtight isolation container, b) before the tube is inserted into the container and before the aspiration, when the tip of the tube is above the reagent liquid or biological sample in the container, allowing the syringe or pipette to perform an ejection mode for a preset time to increase the air pressure in the airtight isolation container to at least partially compensate for the vacuum caused by the aspiration of the reagent or biological sample, and c) when the tube is removed from the container, when the tip of the tube reaches the covering liquid in the stack, the position of the tube is maintained for a predetermined time to release part of the reagent liquid or biological sample adhering to the tube in the covering liquid. During liquid processing, this mechanism reduces the change of air pressure when sucking and pushing liquid from the container. Due to the push of the tube, the air pressure inside the container increases. Subsequently, when the tube is retracted from the airtight insulating cover, the reagent liquid or biological sample drips at atmospheric pressure, thereby exposing the biological sample or reagent liquid to the environment. Similarly, due to the suction of the tube, the air pressure inside the container decreases. Subsequently, when the tube is retracted from the airtight insulating cover, air from the environment is forced into the tube, creating an opportunity for contamination from the environment. The increase or decrease in air pressure inside the container will also affect the volume of reagent liquid or biological sample originally programmed to be pushed into or drawn from the tube. This effect may only affect small volumes, but when the container volume is also small, the effect will become significant.

[0047] According to one embodiment, the method further comprises: when the container is in the cartridge module, pushing the covering liquid provided in at least one container to several designated areas on the covering layer. The pusher can be of any form in the art. Pushing the covering liquid to the covering layer outside or inside the device just before use helps prevent the covering liquid from shifting or being wiped off during transportation or handling.

[0048] According to one embodiment of the method, the present invention employs a tube attached to a pipette, the pipette comprising a filter and a liquid layer, the filter and the liquid layer being separated by a first gap, and the liquid layer being closer to the tip of the tube; and operating a syringe or pipette operating mechanism so as to maintain a second gap between the liquid layer and the reagent liquid or the biological sample being aspirated. The filter and the liquid layer block vapor or aerosol from the biological sample, allowing them to enter the pipette only to a limited extent, thereby reducing contamination of the equipment.

[0049] According to one embodiment, the method includes taking any steps consisting of the following group: a) injecting a liquid sealant through the cover layer to seal the top opening of the container before amplifying the biological sample in the container, and b) placing the liquid sealant on the cover layer or laminate on the top opening of the container before amplifying the biological sample in the container. The liquid sealant can be viscous enough to provide a great resistance to the pressurized aerosol and / or vapor that escapes through the cover layer hole or laminate hole when the container is heated during amplification. The liquid sealant can be cured by any means in the art. Here, the opening area at the top of the container is narrow enough to provide sufficient surface tension in retaining the liquid sealant. The liquid sealant forms an air gap above the biological sample so that during imaging, the fluorescence from the liquid sealant is not unnecessarily captured together with the fluorescence from the biological sample. The liquid sealant can be wax or glue. Wax that solidifies at room temperature makes it easier to transport the cartridge module. It can be locally heated to liquid form during the process of being sucked and pushed by the tube.

[0050] According to one embodiment, the method includes programming a syringe or pipette mechanism with a computer so that the tube pierces only a few selected locations in the covering layer on the top opening of the container so as to minimize the size of the hole pierced in the covering layer during multiple piercings.

[0051] According to one embodiment, the method further comprises at least once during the analysis: cleaning the tube and the syringe by aspirating and expelling a cleaning liquid through the tube.

[0052] According to one embodiment, the method includes: using a card box module with a sealing layer; and creating a vent in the sealing layer above the top opening of the container through a tube connected to a syringe or a pipette mechanism, so that during the subsequent process of sucking from the container or pushing into the container, the air in the container and the air connection space are interconnected.

[0053] According to one embodiment, the method includes: using the device to detect misalignment of the tube relative to the container top opening due to piercing the cover layer; and calibrating the syringe or pipette mechanism to compensate for the misalignment.

[0054] According to one embodiment, the method includes: at least once, aspirating a fill liquid in a syringe through a tube; and pushing the fill liquid so that the fill liquid: a) fills any existing liquid retention space in the tube head that may otherwise retain reagent liquid and biological sample during subsequent pushing after aspiration, or b) replaces the reagent liquid and biological sample from a previous treatment that originally occupied the liquid retention space. The fill liquid is immiscible with, heavier than, and non-reactive with the reagent liquid and biological sample. Due to the use of the fill liquid, the dilution of the reagent liquid and biological sample is maintained. Moreover, there is no loss of volume of the reagent liquid and biological sample during aspiration and pushing.

[0055] According to a fourth aspect, the present invention provides a sample container having a cover which, in use, contains a biological sample, the cover comprising at least partly a covering layer as described in the first aspect.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In the following figures, the same reference numbers refer to the same objects throughout the text.

[0058] The drawings are not drawn to scale, emphasis instead being placed on illustrating concepts.

[0059] Figure 1A are front and cross-sectional views of a cartridge module of an embodiment of the present invention with a cover layer attached to the container top opening.

[0060] Figure 1B are front and cross-sectional views of a cartridge module of an embodiment of the present invention with a cover layer attached to the cartridge module.

[0061] Figure 1C are front and cross-sectional views of a cartridge module of an embodiment of the present invention with a cover layer attached to a container.

[0062] Figure 2A and Figure 2B A front view, a cross-sectional front view and a sectional view, as well as a folded side view of an embodiment of the present invention are shown respectively, wherein the container is made of a deformable material. Figure 2C Shows Figure 2B What the objects in the image look like when they are in an expanded state.

[0063] Figure 3A A plan view of an embodiment of the cartridge module is shown without a cover layer. Figure 3B yes Figure 3A Perspective view of the cartridge module under the cover.

[0064] Figure 4ASeveral containers made of a deformable material and stacked are shown so that they are in a collapsed state when nothing is loaded into them. Figure 4B Shows Figure 4A The container is in an expanded state when containing the reagent solution and the biological sample.

[0065] Figure 5 is an elevational cross-sectional view of an embodiment in which a region of the cover overlaps the covering layer.

[0066] Fig. 6A and Figure 6B is a cross-sectional elevation view of an embodiment in which a cover layer isolates individual containers and multiple containers in a cartridge module, respectively.

[0067] Fig. 7A and Figure 7B A partial cross-section of an elevation is shown to illustrate the problems of using an airtight covering.

[0068] Fig. 8A An elevational cross-sectional view of an embodiment of a test kit is shown, the test kit including a cartridge module, a syringe and a pipette. Figure 8B Shows the running Fig. 8A .

[0069] Fig.9A An elevational cross-sectional view of a tube coated with a covering fluid within a container is shown in one embodiment. Fig. 9B Shows when Fig.9A The situation when the tube in is retracted from the container and coated with covering fluid on the biological sample.

[0070] Fig.10 An elevational cross-sectional view of a cartridge module with a scrubbing layer according to an embodiment of the present invention is shown.

[0071] Fig.11A and Fig. 11B An elevational cross-section of one embodiment is shown with a syringe having a cover fluid, and one and two O-rings.

[0072] Fig.12 An embodiment is shown in which the cover layer is tightly clamped by a rigid retaining layer having a rigid layer hole for the tube to be inserted.

[0073] Fig.15A and 15B An elevational cross-sectional view of an embodiment of a biological sample prior to amplification is shown, wherein the liquid sealant is pushed out above or below the stack, respectively.

[0074] Fig. 15C and Fig.15DFurther embodiments are shown in which the container creates an air gap between the liquid sealant and the biological sample.

[0075] Fig.16A A plan view of an embodiment of a cover layer is shown, wherein the grooves containing the cover fluid are interconnected. Fig. 16B Shows Fig.16A Front view and cross-sectional view of the covering layer at the cutting line AB in FIG.

[0076] Fig.17A and Fig. 17B 17C and 17D are plan views of embodiments having through holes or partial through holes in the cover layer. Fig.17D These two elevation section images show the flexible nature of the covering at the holes.

[0077] Fig.18A , 18B and Fig. 18C An elevational cross-section is shown to illustrate the operation of one embodiment where the top of the cover layer has a groove.

[0078] 19A to 19D is an elevational cross-sectional view of a device that bends the tube attached to the syringe to facilitate storage within the cartridge prior to disposal, according to one embodiment.

[0079] Fig. 20A An embodiment is shown, in which Fig. 20B As shown, the tube injects air into a sealed, isolated container prior to suction.

[0080] Fig.21A An elevational cross-sectional view of an embodiment is shown in which a tube pushes liquid into a hermetically isolated container, and Fig. 21B The tube is shown sucking air from the container after expulsion.

[0081] Fig.22A An embodiment is shown in elevational cross-section in which a tube pushes liquid into a hermetically isolated container. Fig. 22B The tube is shown retracted from the container after expulsion. Fig. 22C The tube is shown briefly held in the covering fluid.

[0082] FIG. 23A to FIG. 23C is an elevational cross-sectional view according to one embodiment for using a filter in a pipette.

[0083] Fig.24A One embodiment is shown when the tube head is attached to the tube. Fig. 24B One embodiment of a method of filling a liquid holding space with a filling liquid is shown.

[0084] Fig.25is a flow chart of an embodiment of a nested PCR method.

[0085] Fig.26 A plan view of an embodiment of a cartridge module is shown, along with a description of the extraction and sample loading sequence for PCR. DETAILED DESCRIPTION

[0086] The following description presents several preferred embodiments of the invention in sufficient detail to enable those skilled in the art to make and use the invention.

[0087] Figure 1AAn embodiment of a disposable cartridge module 10 is shown for improving contamination control during a nucleic acid analysis procedure including nucleic acid extraction and / or nucleic acid amplification. A cover layer 23 is attached to the container top opening 22. The container 18 does not have a device for liquid communication between the containers 18, and only allows the tube to push liquid to each container 18 and draw liquid from each container 18 from the container top opening 22. The tube 36 passes through the cover liquid 25 carried by the cover layer 23. The test fluid 12 in the container 18 is used for nucleic acid extraction and / or nucleic acid amplification. The cover layer 23 provides the cover liquid 25 to form a stack 55 provided by the container 18, and the cover liquid 25 is immiscible and non-reactive with the reagent solution 12 and the biological sample 21 containing nucleic acid. The cover liquid 25 is used to fill the cover layer hole 60 formed when the tube 36 pierces the cover layer 23, and the tube 36 is used to push liquid into the container 18 and / or draw liquid from the container 18. The tube 36, the covering liquid 25 and the covering layer 23 are compatible with each other to form an isolation layer to control aerosol contamination in and out of the container 18. The preferred covering liquid 23 is immiscible with the reagent solution 12 and the biological sample 21, and is capable of wetting the tube 36 and the covering layer 23, so that the reagent solution 12 or the sample 21 on the tube 36 is completely covered when the tube 36 is taken out of the container 18. The material of the covering liquid 25 can be selected from any suitable material in the art, including liquid silicone, oil and liquid wax. There are many kinds of reagent solutions 12, such as lysis buffer, magnetic bead buffer, washing buffer, elution buffer, PCR reagent, etc. Figure 21 has described the function of the magnet 49. The covering layer 23 can include a sponge material in the stack 55 and on the opening 22 at the top of the container for soaking the covering liquid 25, so as to better retain the covering liquid 25 in the covering layer 23. Some containers 18 can be loaded with reagent solutions 12 for nucleic acid extraction and amplification. The cartridge module 10 may provide at least one slot 48 to accommodate at least one or more containers 18 in use. The vertical and horizontal movement of the tube 36 needs to be provided by the device. The cover layer 23 may include an elastomeric material or a non-elastomeric material or a combination of the two. The elastomeric material may be a non-porous material, such as natural or synthetic rubber or silicone or any other suitable material in the art. The elastomeric material may be a porous material, such as a sponge. The non-elastic material may be a non-porous material, such as a plastic film or a metal film or a laminate thereof, or a porous material, such as a fiber mat. The cover layer 23 may be a laminate of more than one of the above materials. The tube 36 may be metal or plastic or any other suitable material. The cartridge module 10 may include at least one container 18 without a cover layer 23, so that the user can isolate the container 18 in use from the cover layer 23. The cover liquid 25 may be soaked in the cover layer 23 or sandwiched between the cover layer materials or form a laminate 55 with the cover layer 23. Multiple layers of cover layer 23 material are helpful, particularly when cover layer 23 comprises a non-elastic material.The cartridge module 10 may or may not include a syringe 32 or a pipette 89 or a reagent solution 12. The tube 36, syringe 32 or pipette 89 are placed in the cartridge module 10 for disposal. This embodiment provides an air connection space above the container 18 and below the cover layer 23 so that several containers 18 can communicate with each other. The empty container 18 shown in the figure helps to increase the volume of the air connection space 104. The cover layer 23 is tightly clamped by a rigid retaining layer 46, which has a hole 45 for inserting the tube 36. The tube 36 passes through the covering liquid 25 retained by the cover layer 23. Figure 1C An embodiment is shown in which a cover layer 23 is attached to a container 18, and an air connection space 104 is located above the container 18 and below the cover layer 23 so that several containers 18 can communicate with each other. Depending on the process requirements, the cartridge module 10 can accommodate more than one syringe 32 or more than one tube 36 of different types or sizes. A replacement mechanism for syringes 32 and tubes 36 is provided in the device 100. The cartridge module 10 has a mechanism for accommodating, releasing and receiving: a) a syringe 32, b) a tube 36, and c) a syringe 32 with a tube 36 installed. The cartridge module 10 contains a UV-curable resin and a cleaning liquid for cleaning the tube 36. The cartridge module 10 contains magnetic beads for nucleic acid binding, or a porous filter-like element for binding nucleic acids under vacuum, pressure and centrifugal force. The cartridge module 10 has a pattern for identifying the position of the tube tip 37.

[0088] Figure 2A An elevational cross-section view drawn from a front view of a container 18 is shown according to one embodiment, wherein the container 18 is made of a deformable material. Figure 2B When Figure 2A 1. A side view of the container 18 in the collapsed state. Figure 2C The container 18 indicated by the black arrow in the figure shows how the deformable container 18 can increase the internal capacity of the container 18 by expansion from the contracted state 6 to accommodate the reagent liquid 12 or the biological sample 21. The increase in the internal capacity can be achieved without increasing the gas pressure in the container 18. The deformable material can be appropriately selected from the prior art and needs to be non-reactive with the reagent liquid 12 and the biological sample 21.

[0089] Figure 3A A plan view of an embodiment of a cartridge module 10 is shown. Figure 3B2 is a perspective view of a cartridge module 10 having a cover layer 23 and a window 41 for receiving ultraviolet light. The cartridge module 10 can be loaded into and / or unloaded from a device (not shown) for nucleic acid extraction and a polymerase chain reaction PCR for amplifying nucleic acids. Although some containers 18 are pre-filled with reagent liquid 12 and cover liquid 25, the remaining containers are shown as empty, allowing the user to use these empty containers 18 for the specified purpose of mixing the biological sample 21 with the reagent liquid 12. The biological sample 21 defined here may or may not include one or more reagent liquids 12. Before amplification, the PCR reagent as the reagent liquid 12 is added to the amplification module in the device. The cover liquid 25 can be more than one liquid loaded in different containers 18 respectively. The device can have one or more container holding racks 33 for holding one or more tubular containers 18. The container 18 can also be in the form of a well plate having several tubular containers 18. The window 41 is shown as transparent to ultraviolet light so as to allow the ultraviolet light to cure the sealant (not shown) contained in the top opening 22 of the container 18. The ultraviolet light source may be adjustable so as to project the ultraviolet light at an upward angle, thereby protecting the biological sample 21 loaded at the bottom of the container 18 from being exposed to the ultraviolet light provided in the device. Specifically, the sealing and curing steps are performed before the amplification step. The container holder 33 may be detached from the cartridge module 10. The container 18 may be fixedly attached to the container holder 33. All parts that come into contact with the biological sample 21 and the reagent solution 12 are disposable consumables after use. The cartridge module 10 includes a liquid bath medium 75 in at least one container 18 for being heated during nucleic acid amplification in the biological sample. The device may then be pre-calibrated for heating the bath medium 75, and the user may only need to fine-tune the calibration on site. The liquid bath medium 75 also allows the liquid bath medium 75 to be easily transferred between the baths in the device and between the containers 18 in the cartridge module 18. During transport of the device, the liquid bath medium 75 may be transferred back to the container 18 in the cartridge module 10. Storing the liquid bath medium 75 in the container 18 also helps reduce the effects of evaporation when the device is not in use.

[0090] Figure 4A A plurality of containers 18 are shown which are made of a deformable material and are stacked so that they are in a collapsed state when not loaded with any object. Figure 4B Shows Figure 4A The container 18 is in an expanded state when containing the reagent solution 12 and the biological sample 21.

[0091] Figure 5An embodiment of the present invention is shown in which a cover 23 is embedded in a lid 35 of a container 18 and extends through the entire thickness of the lid 35, and a cover region 39 covers above and below the lid 35. A portion of the cover 23 on the lid 35 is pierceable by a tube 36, the cover 23 is embedded in the lid 23 and extends through the entire thickness of the lid 23, and has first and second cover regions 39 above and below the lid 35, the cover regions being able to resist any relative movement between the cover 23 and the lid 35 during piercing and retraction of the tube 36.

[0092] Fig. 6A The cover layer 23 is shown individually surrounding each container top opening 22 with an attachment 43 so as to individually cover each container 18 in the cartridge module 10 . Figure 6B The cover layer 23 is shown covering several containers 18 in the card box module 10 by surrounding the several containers 18 by the attachment parts 43.

[0093] Fig. 7A and Figure 7B Some practical problems of the airtight insulating cover 23 are shown. Fig. 7A As shown, the air pressure inside the container 18 increases due to the push of the tube 36. When the tube 36 is then retracted from the airtight insulating cover 23, the biological sample 21 drips from the tube tip 37 under atmospheric pressure, thereby exposing the biological sample 21 to the environment and contaminating the environment. Similarly, the air pressure inside the container 18 decreases due to the suction of the tube 36. Thereafter, when the tube 36 is retracted from the airtight insulating cover 23, air in the environment is forced into the tube 36, thereby giving the environment a chance to contaminate the inside of the tube 36.

[0094] Fig. 8A An embodiment of a kit 110 is shown that includes a cartridge module 10, a syringe 32, and a covering fluid pipette 89. A covering fluid 25 is provided in one of the containers 18, and the pipette 89 is used to push the covering fluid 25 to a selected area, such as a groove 27 on the covering layer 23, when the container 18 is in the cartridge module 10. Figure 8BAn embodiment is shown in which a pipette 89 pushes a covering fluid 25 from a container 18 onto the covering layer 23, as indicated by the dashed arrow. The solid arrow represents the process of aspirating the reagent fluid 12 from the container 18 by the syringe 32 and pushing it into the container 18 containing the biological sample 21. The pipette 89 can be of any form known in the art. Pushing the covering fluid 25 onto the covering layer 23 inside the device just before use helps protect the covering fluid 25 from being displaced or wiped off during transportation or handling. In this embodiment, the cartridge module 10 also houses a sample container 115 having a lid 116, which houses the biological sample 21 for analysis. The lid 116 includes a covering layer 23 that allows the tube 36 to pierce to aspirate the biological sample 21 and push it into the container 18. As shown Fig.25 As described in the foregoing, when the cartridge module 10 is loaded into the apparatus 100, the syringe or pipette device 84 is computer programmable so that the biological sample 21 is aspirated from the sample container 115 and pushed into the container 18 using the tube 36. This improves contamination control when the tube 36 transfers the biological sample 21 into the container 18 while the sample container 115 is in the cartridge module 10. The lid 116 and the cover 23 in the lid 116 prevent any possible spillage and aerosol leakage of the biological sample 21 during the entire analysis process until the cartridge module 10 is discarded. This allows the user to avoid the complex manual transfer operation that requires training in the use of the syringe 32 or pipette 89 to push the biological sample 21 into the container 18 without contaminating the surrounding environment. This approach is particularly important for the analysis of infectious biological samples.

[0095] Fig.9A The tube 36 of the syringe 32 is shown inserted into the container 15 through the stack 55 and pushing the biological sample 21 into the container 15. As the tube 36 passes through the covering fluid 25, a certain amount of the covering fluid 25 covers the tube 36 entering the container 18. Since the covering fluid 25 is immiscible and non-reactive with the reagent fluid 12 and the biological sample 21 in use, the covering fluid 25 does not cause contamination problems. Fig. 9B The tube 36 is shown as it is retracted from the container 18 through the stack 55. As expected, by default, the tube 36 is covered by a layer of biological sample 21, and when the tube 36 passes through the covering fluid 25, a layer of covering fluid 25 also covers the biological sample 21 layer, as expected by the present invention. Therefore, the present invention prevents trace layers of biological sample 21 from being exposed to the environment, thereby reducing contamination. The covering fluid 25 also helps to seal the cover layer hole 60 when the tube 36 pierces the cover layer. The covering fluid 25, the cover layer 23 and the tube 36 need to be compatible with each other to ensure the sealing of the cover layer hole 60. The figure only shows a portion of the cover layer 23.

[0096] Fig.10An embodiment is shown in which a scrubbing layer 28 is fixed to the stack 55 so that the covering layer 23, the covering liquid 25 and the scrubbing layer 28 form a composite layer 30 for the tube 36 to penetrate and enter the container 18. As shown, the scrubbing layer 28 helps to wipe off the excess covering liquid 25 adhering to the upper part of the tube 36, which may deplete the covering liquid 25 when the amount of the covering liquid 25 is small or the tube 36 is long.

[0097] Fig.11A An embodiment is shown in which a syringe 32 containing a biological sample 21 has two O-rings 68 between a plunger 66 and a barrel 67 of the syringe 32. The covering fluid 25 is disposed between the two O-rings 68 and away from the tip 62 that engages the barrel 67. Fig. 11B The situation is shown when the plunger 66 is further pushed into the barrel 67 to push the biological sample 21. The covering fluid 25 between the two O-rings 68 substantially covers the trace biological sample 21 adhering to the inner wall of the barrel 67 and the outer wall of the plunger 66, so that the biological sample 21 exposed to the environment is substantially maintained at a negligible level.

[0098] Fig.12 An embodiment is shown in which the cover layer 23 is tightly clamped by a rigid retaining layer 46 located below the cover layer 23, and the rigid retaining layer 46 has a rigid layer hole 45 aligned with the container top opening 22 for allowing the tube 36 to be inserted so as to penetrate the cover layer 23.

[0099] Fig.13 The cartridge module 10 is shown with an air connection space 104 and a sealing layer 120 on the container top opening 22. The device 100 is computer programmable to allow the tube 36 to pierce the laminate 55 and the sealing layer 120 so as to create a vent hole 61 in the sealing layer 120 above the container top opening 22, so that the container 18 is in air communication with the air connection space 104 during the suction and discharge of the tube 36.

[0100] FIG. 14A to FIG. 14D An embodiment of the operation of the apparatus 100 is shown. Here, the cartridge module 10 is provided with a coupling mechanism 99, which is sealingly mounted on the cartridge module wall 98 and connected to the container holder 33, so that the apparatus 100 can operate the coupling mechanism 99 from the outside of the cartridge module 10 to move the container holder 33 in the cartridge module 10 back and forth in the horizontal direction for positioning the container top opening 22 below the tube tip 37 while the container 18 is in the cartridge module 10. The syringe or pipette device 84 is computer programmable so that the tube 36 can be moved in the vertical direction so that the tube 36 does not need to be moved out of the air communication space 104 during the aspiration and expulsion of the reagent liquid 12 and the biological sample 21 from the container 18 and into the container 18. Fig.14AThe state when the tube 36 is inserted into the left outermost container 18 is shown. Fig. 14B The state when the tube 36 is lifted out of the left outermost container 18 and the tube tip 37 is not moved out of the air communication space 104 is shown. Fig. 14C The state when the tube 36 is inserted into the right outermost container 18 again is shown. Fig.14D The state is shown when the tube 36 is lifted out of the right outermost container 18 without the tube tip 37 moving out of the air communication space 104. According to an alternative embodiment (not shown), vertical movement is provided to the cartridge module 10 instead of the tube 36. The airtight coupling mechanism 99 helps to isolate the container from the environment. Alternatively, the device 100 can also operate the coupling mechanism 99 from the outside of the cartridge module to move the cartridge module in the horizontal direction, so that the coupling mechanism 99 does not need to be sealingly attached to the cartridge module wall 98.

[0101] Fig.15A The liquid sealant 73 is shown being disposed on the stack 55 before the biological sample 21 in the container 18 is amplified, and then being cured by a heat or light source or chemical in the device 100 while the container is in the cartridge module 10. Fig. 15BAn alternative embodiment is represented in which there is a well layer 74 below the cover layer 23, the well layer 74 having a sealing hole 75, which can be sealed by injecting a liquid sealant 73 into the sealing hole 75 before heat treatment for amplification of the biological sample 21 in the container 18. The tube 36 can inject the liquid sealant 73 into the sealing hole 75 through the cover layer hole 60. When the container 18 is located in the card box module 10, the heat or light or chemical treatment cures the liquid sealant 73. This helps to prevent any aerosols and / or vapors generated during the heat treatment from escaping from the cover layer hole 60. Any such contaminants that enter the container 18 from the environment are also controlled. In the case of nested PCR, the liquid sealant 73 is used only before the second amplification. If the sealant is used and cured before the first amplification, it is impossible for the device to add reagent liquid 12 through the layer of hardened sealant liquid 73 before the second amplification. In the case of nested PCR, after the device completes the first amplification, the sealant liquid 73 used in the first amplification may be in a liquid state or may be converted back to a liquid state (e.g., by heating the liquid sealant 73 in the form of wax) to enable the tube 36 to draw or push liquid from or to the container 18 in preparation for PCR. If the liquid sealant 73 is solidified before the first amplification, it is not possible to add the reagent liquid 12 through the hardened liquid sealant 73 before the second amplification. Alternatively, without using the sealant liquid 73, the cover layer 23 may be directly pierced by the tube 36 after the first amplification is completed so that the tube 36 can draw and / or push liquid from or to the container 18 before the first amplification. In an alternative embodiment, the sealant liquid 73 may have a high viscosity and cannot be hardened or solidified before the first amplification. In another embodiment, the sealant liquid 73 may be a wax-like material that can be hardened or solidified before the first amplification and can be softened by heating after the first amplification to enable the tube 36 to draw or push liquid from or to the container 18 before the second amplification. The liquid sealant may be cured by ultraviolet light or heat or chemicals and may be any suitable material known in the art, such as oils, polymers, monomers, and waxes. Fig.15DA container 18 is shown in which an air gap 16 is created and maintained between a liquid sealant 73 and a biological sample 21. Here, this embodiment selects a combination of a liquid sealant 73 and a container 18 so that the container can hold the liquid sealant 73 at the top opening 22 of the container when the liquid sealant 73 is pushed in. A syringe or pipette device 84 is used to aspirate the liquid sealant 73 and push it near the top opening 22 of the container. The biological sample 21 passes through the liquid sealant 73 and is then pushed into the container 18. Here, the covering liquid 25 is located below the covering layer 23. Before amplification, the liquid sealant 73 can be cured so that it becomes hard. The liquid sealant 73 can be in the form of wax or glue. Curing is completed by exposing the liquid sealant 73 to ultraviolet light. The ultraviolet light can be irradiated to the liquid sealant 73 at an upward angle so that the biological sample 21 is not exposed. In-line curing helps to further integrate and automate the process before heat treatment to avoid the problem of evaporation of the biological sample 21 that causes contamination inside the device. Any other method known in the art for curing may also be employed, such as by chemical or thermal treatment. In the case of nested PCR, the liquid sealant 73 is used only before the second amplification. If the sealant is used and cured before the first amplification, it is impossible for the device to add the reagent liquid 12 through the layer of already hardened sealant liquid 73 before the second amplification. The liquid sealant 73 can be non-reactive and immiscible with the reagent liquid 12 and the biological sample 21. The air gap 16 is used to further isolate the biological sample 21 or the reagent liquid 12 from the environment.

[0102] Fig.16A A plan view of an embodiment of a covering layer 23 is shown, in which two recesses 27 containing a covering liquid 25 are connected to each other. Fig. 16B Shows Fig.14A AB cutting line in the vertical cross-sectional view of the cover layer 23, wherein the tip 37 of the hollow tube 36 moves between the interconnected grooves 27 while being kept immersed in the cover liquid 25 and not exposed to the environment. The tube 36 sucks out the biological sample 21 from the container 18. As shown by the box arrow, after the biological sample 21 is sucked by the tube 36, it is retracted from the container 18 and moves above the container 18. After this, the tube 36 enters the container 18 by piercing the cover layer 23. Then the tube 36 pushes the biological sample 21 onto the reagent solution 12 in the container 18. The tube 36 can be repeatedly aspirated and pushed to mix the two liquids. After pushing, the tube 36 will be retracted from the container 18, and this step is not shown here. Therefore, during this operation, the tip 37 of the tube is kept in the container 18 or in the cover liquid 25. When the tip 37 leaves a container 18 and moves to another container, it is not exposed to the environment. This prevents the biological sample 21 from dripping from the tip 37 and being exposed to the environment.

[0103] Fig.17A The plan view of FIG. 2 shows a single-directional through hole 96 on the cover layer 23 . Fig. 17B The plan view of FIG. 2 shows a cross-shaped through hole 96 in the cover layer 23, particularly for a blunt tube 36 such as used with a pipette 89. Any other shape may also be used. Fig. 17C The elevational cross-sectional view of shows that when the tube pierces the hole 96, the cover bends downward. Fig.17D The elevational cross-sectional view of shows that when the tube 36 is retracted from the container 18 through the hole 96, the cover layer 23 is substantially bent back to its original planar shape. Because the hole 96 is substantially bent back to its original shape, cross contamination between the interior of the container 18 and the surrounding environment is controlled. The cover liquid 25 can also be pushed over the hole 96 to enhance isolation. The hole 96 needs to be narrow enough to prevent the cover liquid 25 from leaking into the container 18.

[0104] Fig.18A , Fig.18B and Fig. 18C An embodiment of a container 15 for use in a nucleic acid analysis procedure including nucleic acid extraction or nucleic acid amplification is shown. As shown, the container 15 has a cover layer 23 that covers the container top opening 14 and isolates the container 15. The cover layer 23 has a recessed top 76 for guiding the tip 37 of a tube 36 in use before the tip 37 pierces the cover layer 23 through the base 72 of the recessed top 76. The recessed top 76 has an inclined side 71 that serves as a guide surface to provide a taper to the base 72, allowing the tube 36 to reach the base 72, which is smaller than or equal to the size of the container top opening 14. The rigidity of the inclined side 71 is higher than the rigidity of the cover layer 23, so that the tip 37 slides along the inclined side 71 and reaches the base area 72 without piercing the surface 71, as shown in FIG. Fig.18B As shown, in order to pierce the cover layer 23, as Fig. 18C This provides better tolerance for misalignment between the tip 37 of the syringe 32 and the top opening 14 of the small container. The inclined surface 71 can be made of materials such as metal or hard plastic.

[0105] Fig.19A An embodiment is shown in which the tube 36 attached to the syringe 32 is bent to allow the cartridge module 10 to contain the tube 36 after use and before disposal. Fig.19B , Fig.19C and Fig.19D The block arrows show how the syringe or pipette device 84 in the apparatus 100 is operated step by step to press the tube 36 sideways and downward against a fixed surface 42 in the cartridge module 10 or in the apparatus 100 in order to bend the tube 36. Thereafter, the syringe 32 with the bent tube 36 is mounted on the cartridge module 10, as shown in FIG. Fig.19AHere, the hollow tube 36 is flexible and can be made of any suitable material such as metal.

[0106] Fig. 20A It is shown that when the device inserts the tube 36 into the container 18 and before aspiration, the syringe operating device 84 can be programmed by a computer so that when the tip 37 of the tube 36 reaches the test fluid 12 or biological sample 21 in the container 18, the syringe 32 performs the spray mode for a predetermined period of time to create an excess gas pressure in the airtightly isolated container 18, and the excess gas pressure in the container 18 helps to release the gas in the container 18. Fig. 20B The tube 36 is shown as the partial vacuum created after the test fluid 12 or biological sample 21 is drawn in.

[0107] Fig.21A When the tube 36 is removed from the container 18 after the ejection, the syringe operating device (not shown) can be computer-programmed to operate according to the following steps: Fig. 21B As shown, when the tip 37 of the tube 36 has reached the top of the test fluid 12 or biological sample 21 in the container 18, the syringe 32 performs the aspiration mode for a predetermined period of time to release at least a portion of the excess air pressure in the airtight container 18. The curved arrow shows the aspiration of the excess air pressure. The excess air pressure is generated by the test fluid 12 or biological sample 21 being pushed into the airtight container 18. When the tip 37 of the tube leaves the cover 23, reducing the excess air pressure in the container 18 helps to discharge less of the test fluid 12 or biological sample 21 from the container 18.

[0108] Fig.22A An embodiment of the device is shown. When the tube 36 is removed from the container 18, the syringe operating mechanism can be programmed by a computer to operate according to the following steps: Fig. 22B As shown, when the tip 37 of the tube 36 has reached between the covering layer 23 and the covering fluid 25, the tube 36 remains in this position for a predetermined period of time in order to release the test fluid 12 or biological sample 21 attached to the tube 36. Fig. 22C yes Fig. 22B Magnified view of the dotted circle area.

[0109] Fig.23A A method is shown in which a pipette device 84 is used to aspirate or push a biological sample 21 through a filter 17 . Fig. 23B Another embodiment is shown in which the pipette device 84 aspirates and pushes the biological sample through a liquid layer 94 and the filter 17, and a first gap 93 is maintained between the liquid layer 94 and the filter 17. Fig.23C show Fig. 23BThe biological sample 21 in the container 18 is aspirated so that the biological sample 21, the liquid layer 94 and the filter 17 remain separated from each other. A second gap 95 is maintained between the biological sample 21 and the liquid layer 94. This significantly reduces nucleic acid aerosol and / or vapor contamination during suction and discharge from the container 18 to the container 18.

[0110] Fig.24A An embodiment is described in which the tube 36 is attached to the tube head 62 with glue 109. The tube top end 112 of the tube 36 protrudes above the bottom 110 of the tube head 62 to prevent the glue 109 from blocking the tube top end 112. This forms a liquid retention space 108. Fig. 24B The device is shown to draw the filling liquid 107 into the syringe 32 with the tube 36, and then push out the filling liquid 107, and then the liquid retention space 108 filled with the filling liquid 107 is shown, so that the filling liquid 107 fills any liquid retention space 108 present in the tube head 62, which would otherwise retain the reagent liquid 12 and the biological sample 21 during the pushing period after the aspiration. The filling liquid also replaces any reagent liquid 12 or biological sample 21 from the previous processing step retained in the liquid retention space 108. This two-step method of aspiration and pushing can be performed several times before one or more of the following steps: 1) aspirating the purified biological sample 21 from the elution buffer containing the purified nucleic acid. In direct PCR or other direct amplification procedures that do not require a nucleic acid purification step, 2) aspirating the PCR reagent liquid 12 to prepare the final reaction mixture containing purified nucleic acids, 3) aspirating the final reaction mixture to load into the PCR container 18, and 4) any step in the nested PCR procedure. The nucleic acid extraction step or PCR step is optional. Other sample processing can also be performed on the cartridge module 10 with the tubes 36, including direct PCR with release of nucleic acid from the sample and no purification step, isothermal PCR with or without a nucleic acid purification step.

[0111] Fig.251 is a flow chart of an apparatus 100 according to an embodiment of the present invention for nested PCR. The apparatus 100 is used to improve contamination control during nucleic acid analysis procedures including nucleic acid extraction, nucleic acid amplification and detection. In this embodiment, the cartridge module 10 and the biological sample 21 are loaded into the device 100 through the receiving module 102. The syringe or pipette device 84 transfers the bath medium 75 from the container 18 in the cartridge module 10 to the bath (not shown) in the amplification module 101. The syringe or pipette device 84 is used to operate the hollow tube 36 along the syringe 36 or pipette 89 to aspirate and push from the top opening 22 of the container through the covering layer 23 at a predetermined position. The syringe or pipette device 84 mixes the biological sample 21 with the reagent solution 12 in the container 18 with the covering layer 23 for extraction and PCR. The transfer device 85 then transfers the container 18 from the cartridge module 10 to the amplification module 101. In the amplification module 101, the transfer device 85 performs a first thermal cycle for the biological sample 21 in the container 18 having the covering fluid 23 and held by the container holding rack 33. Alternatively, the amplification module 101 may provide isothermal heating instead of cyclic heating as shown in the figure for amplification of the nucleic acid in the container 18. The transfer device 85 then transfers the container 18 held by the container holding rack 33 back to the cartridge module 10. With the container 18 in the cartridge module 10, the syringe or pipette device 84 further mixes the reagent solution 12 with the biological sample 21 through the covering layer 23. The transfer device 85 then performs a second thermal cycle for the biological sample 21 in the container having the covering layer 23 in the amplification module 101. During and after amplification, the imaging mechanism 19 performs fluorescent imaging. Since in this embodiment, the amplification is performed outside the cartridge module 10, the transfer device 85 controlled by the robot is configurable so as to position the container 18 held by the container holding rack 33 outside the cartridge module 10. Alternatively, the container 18 can be retained in the cartridge module 10 to undergo amplification of the device 100. Thereafter, the transfer device 85 transfers the container 18 held by the container holding frame 33 back to the cartridge module 10. Then, the syringe or pipette device 84 transfers the bath medium 75 from the bath to the container 18 in the cartridge module 10. Thereafter, the cartridge module 10 is unloaded from the device 100. As soon as the reagent solution 12 is consumed, the cartridge module 10 is discarded. The device 100 is computer programmable to process the biological sample 21 with the reagent solution 12 while the container 18 is in the cartridge module 10, and to perform at least one of nucleic acid extraction, nucleic acid amplification and detection for the biological sample 21. This embodiment of nested PCR has a wide range of applications in rapid point-of-care testing (POCT). The imaging module 19 can perform optical imaging when the container 18 is inside or outside the cartridge module 10.The concept of using the tube 36 and the cover layer 23, and the cover layer and the cover fluid 25 described above, allows extraction and nested PCR with dual amplification to be performed in the device 100 without removing the cartridge module 10 during the process. In a nested PCR, the container 18 used for the first amplification, when mixed with the reagent 12 for the second PCR, and during the second amplification, all should be covered with one or more cover layers 23 on a single container 18 or on all containers 18.

[0112] Fig.26 is a plan view of a cartridge module, which partially depicts a flow chart of an extraction sequence and PCR, and FIG. 1 may also be referred to.

[0113] P26 Extraction Order

[0114] 1. Mix the biological sample in the container 18 using the tube 36

[0115] 2. Pipette the biological sample 21 into the lysis container 18 and mix using the tube 36

[0116] 3. Pipette the binding buffer (magnetic beads) into the lysis container 18 and mix using tube 36

[0117] 4. Move the magnet 49 upward from position B to position A and keep it at position A for a predetermined period of time.

[0118] 5. While the magnet 49 is raised, mix the solution in the lysis vessel 18

[0119] 6. Discard the supernatant into a waste container 18

[0120] 7. Move magnet 49 downward from position A to position B

[0121] 8. Aspirate the Wash 1 Buffer and push it into the Lysis Container 18 and mix using tube 36

[0122] 9. Move the magnet 49 upward from position B to position A and keep it at position A for a predetermined time.

[0123] 10. While the magnet 49 is raised, mix the solution in the lysis vessel 18

[0124] 11. Discard the supernatant into a waste container 18

[0125] 12. Move magnet 49 downward from position A to position B

[0126] 13. Aspirate the Wash 2 Buffer and push it into the Lysis Container 18 and mix using tube 36

[0127] 14. Move the magnet 49 upward from position B to position A and keep it at position A for a predetermined time.

[0128] 15. While the magnet 49 is raised, mix the solution in the lysis vessel 18

[0129] 16. Discard the supernatant into a waste container 18

[0130] 17. Move magnet 49 downward from position A to position B

[0131] 18. Start heating the cracking vessel to the predetermined temperature

[0132] 19. Clean pipe 36 at clean 1 container 18, clean 2 container 18 and clean 3 container 18

[0133] 20. Stop heating the cracking vessel 18

[0134] 21. Pipette 100ul of elution buffer into lysis container 18 and mix using tube 36

[0135] 22. Move the magnet 49 upward from position B to position A and keep it at position A for a predetermined time.

[0136] 23. While the magnet 49 is raised, mix the solution in the lysis vessel 18

[0137] 24. Move magnet 49 downward from position A to position B

[0138] 25. The eluate is extracted for PCR

[0139] Here, all mixing steps are performed by several rounds of aspiration and displacement through tube 36. Here, in step 1, the biological sample 21 is mixed in the container 18. In step 2, a certain volume of sample 21 is aspirated and displaced into the extraction container 18 containing the lysate and mixed. In step 3, another volume of binding buffer with magnetic beads is added to the lysis container 18 through tube 36. Then, in step 4, the magnet 49 is then moved upward from position B to position A near the lysis container 18 and maintained for a predetermined period of time. In step 5, while the magnet 49 is in position A, the solution in the lysis container 18 is mixed when the magnetic beads attached with DNA and RNA are located on the wall of the lysis container 18 and facing the magnet 49. In step 6, the tube 36 transfers the supernatant / waste liquid from the lysis container to the binding buffer container 18. Then, in step 7, the magnet 49 is moved downward from position A to position B. In step 8, a certain volume of wash 1 buffer is sucked from the wash 1 buffer container 18 and pushed into the lysis container 18, and then mixed. In step 9, the magnet 49 is moved upward from position B to position A again and is maintained at position A for a predetermined time. In step 10, the solution in the lysis container 18 is mixed while the magnet 49 is raised. Then, in step 11, the tube 36 transfers the supernatant / waste liquid from the lysis container 18 to the wash 1 buffer container 18. Then, in step 12, the magnet 49 is moved downward from position A to position B. In step 13, a certain volume of wash 2 buffer is sucked from the wash 1 buffer container 18 and pushed into the lysis container 18, and then mixed. In step 14, the magnet 49 is moved upward from position B to position A again and is maintained at position A for a predetermined time. In step 15, the solution in the lysis container 18 is mixed while the magnet 49 is raised. Then, in step 16, tube 36 transfers the supernatant / waste liquid from lysis container 18 to wash 2 buffer container 18. Then, in step 17, magnet 49 is moved downward from position A to position B. Subsequently, in step 18, lysis container 18 is heated to a predetermined temperature for a predetermined period of time until the washing in wash 3 container is completed. In step 19, in wash 1 container 18, the device cleans tube 36 by several rounds of aspiration and displacement, and then cleans tube 36 in wash 2 container 18 in the same manner, and then cleans tube 36 in wash 3 container 18 in the same manner. Then, in step 20, the heating of lysis container 18 is stopped. In step 21, tube 36 aspirates a certain volume of elution buffer from elution buffer container 18, and then is displaced into lysis container 18 and mixed. In step 22, magnet 49 is moved upward again from position B to position A and is maintained in position A for a predetermined period of time. In step 23, while the magnet 49 is raised, the solutions in the lysis vessel 18 are mixed.Then, in step 24, magnet 49 is moved down from position A to position B. Then, in step 25, tube 36 aspirates the eluent for PCR. Figure 24 also illustrates a sample loading sequence for PCR after the previous extraction sequence. Here, in step 26, PCR buffer is mixed in PCR buffer container 18. In step 40, a predetermined volume of PCR buffer is aspirated from PCR buffer container 18 and pushed into PCR enzyme container 18 containing PCR buffer enzyme, and then mixed to form PCR premix. In step 28, a predetermined volume of PCR premix is ​​aspirated from PCR enzyme container and pushed into negative control container 18 and then mixed. In step 29, a predetermined volume of negative control PCR premix that has been mixed is aspirated and pushed into glass capillary container 1 (not shown). In step 30, iron 49 is moved up from position B to position A again and is kept in position A for a predetermined time. In step 31, a predetermined volume of liquid is aspirated from the extraction tube and pushed into the PCR enzyme container and then mixed. In step 32, a predetermined volume of biological sample 21 from the PCR enzyme container 18 is aspirated and pushed into the glass capillary 2 (not shown). In step 33, a predetermined volume of liquid is aspirated from the PCR buffer container 18 and pushed into the PCR enzyme container 18 and then mixed. In step 33, a predetermined volume of solution is aspirated from the PCR enzyme container and pushed into the glass capillary container 3 as a positive control-PC. In step 34, a predetermined volume of liquid is aspirated from the UV buffer container 18 and pushed into the glass capillary container 18. Subsequently, in step 35, the glass capillary container 18 is sealed with UV light. Then PCR is performed.

[0140] A peeling layer (not shown) may be arranged above the stack 55 to prevent displacement of the covering liquid 25 when the container 18 is not in an upright position, and the peeling layer 31 may be peeled off before piercing. Alternatively, the peeling layer may be pierced by a suitable tube 36 for pushing liquid into the container 18 and / or sucking liquid from the container 18 through the stack 32.

[0141] The disclosed bath configurations and constructions are not limiting in scope for achieving any type of thermal profile. Any user specified thermal profile may be achieved by properly placing the containers 18 in a specified sequence into baths maintained at predetermined temperatures for a specified period of time. The containers 18 may be rapidly moved across additional baths in the direction of baths that do not contribute to the thermal profile (if any).

[0142] The tube 37 may be wedge-shaped to more easily pierce the cover 23. Typically, the outer diameter of the tube 36 or the tube 36 with a beveled or wedge-shaped tip is between 0.1 mm and 5 mm, and preferably less than 1 mm. The thicker the cover 23, the greater the chance that the cover hole 60 can be completely closed when the tube 36 is removed. The preferred cover 23 thickness is greater than 1 mm. When the cover liquid 25 is stored at 10 degrees Celsius to 50 degrees Celsius, it remains in liquid form for at least one month.

[0143] A vibration device (not shown) can be used to vibrate the container 18 at a predetermined frequency, amplitude and time to disperse the magnetic beads in the biological sample 21 during use. This is another method of suction and push mixing. As an alternative, the user can also use a press-release device (not shown) to alternately press and release the container 18 in the cartridge module 10 for mixing the biological sample 21 in the container 18, and the container 18 is elastic or deformable when pressed. The device 100 can also include a motorized platform (not shown) to position the container top opening 22 below the tip 37 while the container 18 remains in the cartridge module 10.

[0144] The container 18 can be in any shape and made of any material known in the art. The container 18 can be made in the form of a small diameter glass capillary, such as a glass tube with an outer diameter between 0.1 mm and 4 mm and an inner diameter between 0.02 mm and 3 mm. If the temperature of each bath is appropriate, the user can advantageously perform simultaneous PCR for different biological samples 21.

[0145] From the foregoing description, it will be understood by those skilled in the art that numerous changes or modifications may be made in the details of design, construction and operation without departing from the present invention as set forth in the claims.

Claims

1. A cartridge module (10) for improving contamination control during a bioanalysis or nucleic acid analysis procedure including nucleic acid extraction, nucleic acid amplification and detection, the cartridge module (10) comprising: at least one hollow tube (36); several containers (18); as well as at least one covering layer (23) to isolate the container (18) from the environment outside the covering layer (23), wherein each container (18) allows a tube (36) attached to a syringe (32) or a pipette (89) in use to push a reagent solution (12) in use or a biological sample (21) in use to be pushed into or aspirated from the container (18) only through the container top opening (22), During the entire biological analysis or nucleic acid analysis process, even after the tube (36) pierces the covering layer (23) and retracts, the covering layer (23) still substantially blocks the aerosol exchange between the environment and the container (18). The cover layer (23) also prevents any environmental contamination caused by the overflow of the reagent solution (12) or the biological sample (21) from the container (18) or the cartridge module (10). The cover layer (23) is attached to: The cartridge module (10) or container (18) maintains an air connection space (104) between a covering layer (23) and a plurality of container top openings (104) placed together, wherein the covering layer (23) isolates the air connection space (104) from the environment of the cartridge module (10) so as to reduce changes in air pressure within the isolated container (18) during suction or expulsion of a biological sample (21) and a reagent solution (12) by a tube (36).

2. The cartridge module (10) according to claim 1, wherein at least a portion of the material of the cover layer (23) is an elastomer.

3. The cartridge module (10) according to claim 1 further comprises: A covering liquid (25) is previously pushed or provided and is ready to be pushed at least on the covering layer (23) where the tube (36) pierces, so as to produce a stack (55) in the covering layer (23) or allow the covering layer (23) to enclose the covering liquid (25), which is non-reactive and immiscible with the reagent solution (12) and the biological sample (21), and the covering liquid (25) wets the tube (36) and the covering layer (23).

4. The card box module (10) according to claim 1 further comprises: A scrubbing layer (28) is fixed to the stack (55) so that the covering layer (23), the covering liquid (25) and the scrubbing layer (28) form a composite layer (30), the scrubbing layer (28) is pierceable by the tube (36), and when the tube (36) is retracted from the container (18), the scrubbing layer (28) substantially wipes off excess covering liquid (25) adhering to the tube (36).

5. The cartridge module (10) according to claim 1 further comprises at least one injector (32) and provides at least one feature from the following group: An O-ring (68) is provided between the plunger (66) and the barrel (67) of the syringe (32), a covering fluid (25) is provided above the O-ring (68), the covering fluid (25) being remote from a tube head (62) attached to the barrel (67), and At least two O-rings (68) are provided between the plunger (66) and the barrel (67) of the syringe (32), and a covering liquid (25) is provided between the two O-rings (68). The covering liquid (25) is immiscible and non-reactive with the reagent liquid (12) and the biological sample (21).

6. The card box module (10) according to claim 1 further comprises: A rigid retaining layer (46) is located above or below the covering layer (23), and the rigid retaining layer (46) has a plurality of retaining layer holes (45) aligned with the container top opening (22) for allowing the tube (36) to pierce the covering layer (23).

7. The card box module (10) according to claim 1 further comprises: At least one container holding frame (33) for holding at least one container (18), wherein the container holding frame (33) is detachable from the cartridge module (10).

8. The cartridge module (10) according to claim 1 further comprises: A liquid sealant (73) which, when pushed above or below the covering layer (23) on at least one of the container top openings (22), whether solidified or not, is capable of sealing a covering layer hole (60) made in the covering layer (23) by piercing the covering layer (23) with a tube (36), the liquid sealant (73) being immiscible and non-reactive with a reagent solution (12) and a biological sample (21).

9. The cartridge module (10) according to claim 4, wherein the covering layer (23) has a plurality of grooves (27) for containing the covering liquid (25), and the grooves (27) are interconnected.

10. A card box module (10) according to claim 1, wherein above the top opening (22) of the container, the covering layer (23) has a recessed top (76) with an inclined side (71) so as to provide a tapered area to a base area (72) smaller than the top opening (22) of the container, and the inclined side (71) is used to guide the tip (37) of a hollow tube (36) in use into the base area (72) before the tip (37) pierces the covering layer (23) through the base area (72).

11. The cartridge module (10) according to claim 1, wherein at least a portion of the container (18) is made of a deformable material so that when in use, (i) the container (18) in its initial state or shape can expand to accommodate the reagent solution (12) or biological sample (21) pushed out by the device, and (ii) when the reagent solution (12) or biological sample (21) pushed out by the device is sucked out of the container (18), the container (18) in the initial state or shape can shrink, This helps reduce changes in air pressure in the isolated container (18) during the period when the tube (36) draws or pushes the biological sample (21) and the reagent solution (12).

12. The card box module (10) according to claim 1, wherein the card box module (10) having the air communication space (104) further comprises: A sealing layer (120) seals the top opening (22) of a single or multiple containers to prevent any reagent liquid (12) and biological sample (21) from overflowing from the container (18) when the card box module (10) is not in an upright position, and the sealing layer (120) can be pierced by the tube (36).

13. An apparatus (100) for improving contamination control during a biological analysis or a nucleic acid analysis procedure including nucleic acid extraction, nucleic acid amplification and detection, the apparatus (100) comprising: A receiving module (102) for receiving a cartridge module (10) according to any preceding claim; A syringe or pipette device (84) controlled by a robot for operating a hollow tube (36) attached to a syringe (36) or a pipette (89) to draw a biological sample (21) or a reagent solution (12) from a container (18) or to push it into a container (18) through a container top opening (22) and through a covering layer (23) or a laminate (55) or an interlayer; An amplification module (101) for providing isothermal heating or cyclic heating for amplification of nucleic acids in a biological sample (21) in a container (18), wherein the amplification is performed when the container (18) is inside or outside the cartridge module (10); an optical imaging module (19) for imaging the biological sample (21) during or after amplification when the container (18) containing the biological sample (21) is inside or outside the cartridge module (10); When amplification is performed outside the cartridge module (10), a robot-controlled transfer device (85) can be configured to position the container (18) held by the holding rack (33) provided in the cartridge module (10) or the device (100) outside the cartridge module (10), and return the container holding rack (33) with the container (18) to the cartridge module (10); and The device (100) is computer programmable to provide a container holding frame (33) or a cartridge module (10) with a displacement in a substantially horizontal direction in the cartridge module (10) so that the portion of the tube (36) in contact with the biological sample (21) in the container (18) will not move out of the air connection space (104) during the period in which the syringe or pipette operating device (84) draws the reagent liquid (12) and the biological sample (21) from and pushes them into the plurality of containers (18).

14. The apparatus (100) of claim 22 being computer programmable to operate a syringe or pipette device (84) to: The tube (36) attached to the syringe (32) is pressed against a fixed surface in the cartridge module (10) or the device (100) and the tube (36) is bent, and then the syringe (32) together with the tube (36) is installed into the cartridge module (10).

15. A method for improving contamination control during a bioanalysis or nucleic acid analysis procedure including nucleic acid extraction, nucleic acid amplification and detection, the method comprising: Use of a cartridge module (10) and a device (100) according to any one of the preceding claims; Loading a cartridge module (10) with a container (18) containing a reagent solution (12) and a biological sample (21) into a receiving module (102); Performing biological analysis or nucleic acid analysis in the device (100) by piercing the cover layer (23) and retracting the tube (36) from the cover layer (23) and by aspirating and expelling the reagent liquid (12) and the biological sample (21) from the container (18) without exposing the reagent liquid (12) and the biological sample (21) to the environment; cleaning the tube (36) at least once during the analysis by aspirating and pushing a cleaning fluid through the tube (36); At least once, aspirating the filling fluid (107) in the syringe (32) using the tube (36); and Push out the filling liquid (107) so that the filling liquid (107): Fill any existing liquid retention space (108) within the tube tip (62) that may have otherwise retained the reagent fluid (12) and biological sample (21) during the subsequent expulsion after aspiration, or The liquid holding space (108) originally occupied by the reagent liquid (12) and the biological sample (21) from the previous treatment is replaced. The filling liquid (107) is immiscible and non-reactive with the reagent liquid (12) and the biological sample (21), and the filling liquid (107) is heavier than the reagent liquid (12) and the biological sample (21); and After analysis, the cartridge module (10), which comprises the container (18) with the cover layer (23), the reagent fluid (12), the biological sample (21) and the tube (36), is discarded.

16. The method according to claim 33 comprises: Attaching the tube (36) to a pipette (89) containing a filter (17) and a liquid layer (94) separated by a first gap (93), the liquid layer (94) being closer to the tip (37); and The syringe or pipette operating device (84) is operated to maintain a second gap (95) between the liquid layer (94) and the aspirated reagent liquid (12) or biological sample (21).

17. The method according to claim 33 comprises: Take any of the following steps: Prior to amplifying the biological sample (21) in the container (18), a liquid sealant (73) is injected through the cover layer (23) to seal the container top opening (22), and Prior to amplification of the biological sample (21) in the container (18), a liquid sealant (73) is pushed onto the cover layer (23) or laminate (55) over the top opening (22) of the container.

18. The method according to claim 33 comprises: Using the card box module (10) with the sealing layer (120) according to claim 18; as well as A vent hole (61) is made in the sealing layer (120) above the top opening (22) of the container using a tube (36) attached to a syringe or pipette device (84) so ​​that air can flow between the container (18) and the air communication space (104) during the subsequent suction from the container (18) or push to the container (18).

19. The method according to claim 33 further comprises the steps of: While the container (18) containing the biological sample (21) is maintained outside or inside the card box module (10) but maintained inside the device (100), at least two amplification steps are performed in the amplification module (101) of the device (100), and the biological sample (21) is processed by the reagent liquid (12) between any two consecutive amplification steps and through the covering layer (23) or the stack (55).

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