Patent Publication Number: US-2022235407-A1

Title: Nucleic acid testing cassette

Description:
BACKGROUND 
     Technical Field 
     The invention relates to a nucleic acid testing kit, and in particular relates to a nucleic acid testing cassette. 
     Description of Related Art 
     Current nucleic acid testing kits are low in integration level and complex to use. Moreover, it is necessary for users to add some reagents manually, which often leads to mis-operations. As a result, testing results are inaccurate, and these nucleic acid testing kits are not suitable for primary users to use. 
     SUMMARY 
     To solve the problems in the prior art, the invention provides a nucleic acid testing cassette. 
     The invention provides a nucleic acid testing cassette, including a substrate, a liquid storage component, a solid-reagent storage component, and an amplification reaction region, wherein the substrate is connected to the amplification reaction region; the liquid storage component and the solid-reagent storage component are disposed on the substrate, respectively; the liquid storage component is communicated with the solid-reagent storage component through a micro flow channel; and the solid-reagent storage component is communicated with the amplification reaction region through a micro flow channel. 
     As a further improvement of the invention, the liquid storage component includes a sample storage component, an extraction-reagent storage component, and a waste-liquid immobilization cavity; the substrate is provided with a nucleic acid immobilization-extraction reaction pool; an output end of the sample storage component and an output end of the extraction-reagent storage component are communicated with an input end of the nucleic acid immobilization-extraction reaction pool through a micro flow channel, respectively; and an output end of the nucleic acid immobilization-extraction reaction pool is communicated with the solid-reagent storage component and the waste-liquid immobilization cavity through a micro flow channel, respectively. 
     As a further improvement of the invention, the extraction-reagent storage component includes a plurality of separate chambers, each of which includes a tube wall, a plunger, and a diaphragm; the plungers are disposed above the tube walls; the diaphragms are disposed below the tube walls; the tube walls, the plungers, and the diaphragms enclose a liquid-reagent storage cavity; the substrate is provided with sharp protrusions in one-to-one correspondence to the chambers; and the sharp protrusions are disposed below the diaphragms. 
     As a further improvement of the invention, the chambers at least include a first chamber storing a binding solution, a second chamber storing a rinsing solution, and a third chamber storing an eluent; the first chamber is communicated with the sample storage component through a micro flow channel; a micro flow channel between the sample storage component and the nucleic acid immobilization-extraction reaction pool is provided with a first fluid isolating valve; a micro flow channel between the second chamber and the nucleic acid immobilization-extraction reaction pool is provided with a second fluid isolating valve; a micro flow channel between the third chamber and the nucleic acid immobilization-extraction reaction pool is provided with a third fluid isolating valve; a micro flow channel between the nucleic acid immobilization-extraction reaction pool and the solid-reagent storage component is provided with a fourth fluid isolating valve; a micro flow channel between the nucleic acid immobilization-extraction reaction pool and the waste-liquid immobilization cavity is provided with a fifth fluid isolating valve; and a micro flow channel between the solid-reagent storage component and the amplification reaction region is provided with a sixth fluid isolating valve. 
     As a further improvement of the invention, the chambers further include a preamplification reagent chamber storing a preamplification reagent; and the preamplification reagent chamber is communicated with the nucleic acid immobilization-extraction reaction pool through a micro flow channel, on which a seventh fluid isolating valve is disposed. 
     As a further improvement of the invention, the substrate is provided with a plurality of grooves that are communicated with each other; a cover piece is attached to the substrate, and encloses the grooves to form micro flow channels; and the substrate is provided with a plurality valve seat structures, on which the first fluid isolating valve, the second fluid isolating valve, the third fluid isolating valve, the fourth fluid isolating valve, the fifth fluid isolating valve, and the sixth fluid isolating valve are installed, respectively. 
     As a further improvement of the invention, the waste-liquid immobilization cavity is provided with first and second air passage interfaces that may be independently controlled to be opened or closed; the sample storage component is communicated with the first air passage interface; and the third chamber or the preamplification reagent chamber is communicated with the second air passage interface. 
     As a further improvement of the invention, the sample storage component is internally provided with a first magnetic rotor; the nucleic acid immobilization-extraction reaction pool is internally provided with a second magnetic rotor; a first magnet rotating mechanism for driving the first magnetic rotor to rotate is installed on the sample storage component; a second magnet rotating mechanism for driving the second magnetic rotor to rotate is installed on the nucleic acid immobilization-extraction reaction pool; a first heating module is installed on the nucleic acid immobilization-extraction reaction pool; and a second heating module is installed on the amplification reaction region. 
     As a further improvement of the invention, the sample storage component mainly consists of a cavity and a cover; the cavity of the sample storage component internally pre-stores nucleic-acid-capturing magnetic beads required for use in a nucleic acid extraction process. 
     As a further improvement of the invention, a porous water-absorbing material is contained in the waste-liquid immobilization cavity. 
     As a further improvement of the invention, the amplification reaction region mainly consists of a plurality of reaction wells and pipes connecting the reaction wells; the amplification reaction region is connected to the substrate through a connecting piece; and a fluorescence-imaging processing module is installed above the amplification reaction region. 
     The invention has the following beneficial effects: with the technical solutions described above, all the reagents required for nucleic acid extraction and amplification can be internally disposed on an integrated cassette, a liquid reagent is stored in the liquid storage component, and a dry-powder reagent is stored in the solid-reagent storage component, so that a user only needs to add a sample; therefore, the nucleic acid testing cassette is extremely easy to operate and truly suitable for primary users to use. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an overall schematic diagram of a nucleic acid testing cassette according to the invention. 
         FIG. 2  is a schematic diagram of a nucleic acid testing cassette according to the invention. 
         FIG. 3  is a schematic structural diagram of chambers of a nucleic acid testing cassette according to the invention. 
         FIG. 4  is a schematic diagram of a nucleic acid testing cassette with an added pre-amplification reagent chamber according to the invention. 
     
    
    
     DESCRIPTION OF THE EMBODIMENTS 
     The invention will be further described below in conjunction with the accompanying drawings and specific embodiments. 
     As shown in  FIG. 1  to  FIG. 4 , the invention provides a nucleic acid testing cassette, which is an integrated cassette, including a substrate  1 , a liquid storage component  2 , a solid-reagent storage component  3 , an amplification reaction region  4 , a cover piece  5 , rotor components  6 , fluid isolating valves  7 , and a casing. The components above are assembled and attached to form a sealed integrated cassette formed by a plurality of regions that are communicated with each other. The substrate  1  is connected to the amplification reaction region  4 ; the liquid storage component  2  and the solid-reagent storage component  3  are disposed on the substrate  1 , respectively; the liquid storage component  2  is communicated with the solid-reagent storage component  3  through a micro flow channel; and the solid-reagent storage component  3  is communicated with the amplification reaction region  4  through a micro flow channel. 
     As shown in  FIG. 1  to  FIG. 4 , the liquid storage component  2  includes a sample storage component  201 , an extraction-reagent storage component  202 , and a waste-liquid immobilization cavity  203 ; the substrate  1  is provided with a nucleic acid immobilization-extraction reaction pool  102 ; an output end of the sample storage component  201  and an output end of the extraction-reagent storage component  202  are communicated with an input end of the nucleic acid immobilization-extraction reaction pool  102  through a micro flow channel, respectively; and an output end of the nucleic acid immobilization-extraction reaction pool  102  is communicated with the solid-reagent storage component  3  and the waste-liquid immobilization cavity  203  through a micro flow channel, respectively. 
     As shown in  FIG. 1  to  FIG. 4 , the solid-reagent storage component includes a storage cartridge  301  and a connecting structure; and the immobilization-reagent storage component  3  contains a reagent for nucleic acid amplification, such as dNTP or Taq polymerase, which is prestored in the storage cartridge  301  in the form of dry powder. 
     As shown in  FIG. 1  to  FIG. 4 , the extraction-reagent storage component  202  includes a plurality of separate chambers, each of which includes a tube wall  2021 , a plunger  2022 , and a diaphragm  2023 ; the plungers  2022  are disposed above the tube walls  2021 ; the diaphragms  2023  are disposed below the tube walls  2021 ; the tube walls  2021 , the plungers  2022 , and the diaphragms  2023  enclose a liquid-reagent storage cavity; the substrate  1  is provided with sharp protrusions  103  in one-to-one correspondence to the chambers; and the sharp protrusions  103  are disposed below the diaphragms  2025  to pierce the diaphragms  2025  for releasing a liquid reagent within the liquid-reagent storage cavity. 
     As shown in  FIG. 1  to  FIG. 4 , the chambers at least include a first chamber  202   a  storing a binding solution, a second chamber  202   b  storing a rinsing solution, and a third chamber  202   c  storing an eluent; the first chamber  202   a  is communicated with the sample storage component  201  through a micro flow channel; a micro flow channel between the sample storage component  201  and the nucleic acid immobilization-extraction reaction pool  102  is provided with a first fluid isolating valve  701 ; a micro flow channel between the second chamber  202   b  and the nucleic acid immobilization-extraction reaction pool  102  is provided with a second fluid isolating valve  702 ; a micro flow channel between the third chamber  202   c  and the nucleic acid immobilization-extraction reaction pool  102  is provided with a third fluid isolating valve  703 ; a micro flow channel between the nucleic acid immobilization-extraction reaction pool  102  and the solid-reagent storage component  3  is provided with a fourth fluid isolating valve  704 ; a micro flow channel between the nucleic acid immobilization-extraction reaction pool  102  and the waste-liquid immobilization cavity  203  is provided with a fifth fluid isolating valve  705 ; and a micro flow channel between the solid-reagent storage component  3  and the amplification reaction region  4  is provided with a sixth fluid isolating valve  706 . 
     As shown in  FIG. 1  to  FIG. 4 , the chambers further include a preamplification reagent chamber  202   d  storing a preamplification reagent; the preamplification reagent chamber  202   d  is communicated with the nucleic acid immobilization-extraction reaction pool  102  through a micro flow channel, on which a seventh fluid isolating valve  707  is disposed. According to a testing process, the preamplification reagent chamber  202   d  may further be added to prestore another necessary liquid reagent, for example, a nucleic acid amplification reagent, including a target probe, a specific primer, dNTP, Taq polymerase, a reaction buffer or the like, for use in first-step amplification of nested amplification. 
     As shown in  FIG. 1  to  FIG. 4 , the substrate  3  is provided with a plurality of grooves  101  that are communicated with each other; and the cover piece  5  is attached to the substrate  1 , and encloses the grooves  101  to form micro flow channels. After the cover piece  5  is attached to both sides of the substrate  1 , a plurality of enclosed pipes and cavities that are communicated with each other are formed. The substrate  1  is provided with a plurality of valve seat structures  105 , on which the plurality of isolating valves  7  are installed. 
     As shown in  FIG. 1  to  FIG. 4 , the waste-liquid immobilization cavity  203  is provided with first and second air passage interfaces  1071  and  1072  that may be independently controlled to be opened or closed; the sample storage component  201  is communicated with the first air passage interface  1071 ; and the third chamber  202   c  or the preamplification reagent chamber  202   d  is communicated with the second air passage interface  1072 . 
     As shown in  FIG. 1  to  FIG. 4 , the rotor components  6  include a first magnetic rotor  601  and a second magnetic rotor  602 ; the sample storage component  201  is internally provided with the first magnetic rotor  601 ; the nucleic acid immobilization-extraction reaction pool  102  is internally provided with the second magnetic rotor  602 ; a first magnet rotating mechanism for driving the first magnetic rotor  601  to rotate is installed on the sample storage component  201 ; a second magnet rotating mechanism for driving the second magnetic rotor  602  to rotate is installed on the nucleic acid immobilization-extraction reaction pool  102 ; the first magnetic rotor  601  is a mixing-pool rotor, and the second magnetic rotor  602  is an immobilization-extraction reaction pool rotor; a first heating module is installed on the nucleic acid immobilization-extraction reaction pool  102 ; and a second heating module is installed on the amplification reaction region  4 . 
     As shown in  FIG. 1  to  FIG. 4 , the sample storage component  201  mainly consists of a cavity  2011  and a cover; and the cavity  2011  of the sample storage component  201  internally pre-stores nucleic-acid-capturing magnetic beads required for use in a nucleic acid extraction process. 
     As shown in  FIG. 1  to  FIG. 4 , the waste-liquid immobilization cavity  203  contains a set of porous water-absorbing material, such as sponge and/or water absorbing paper, etc., and is provided with a waste-liquid outlet. 
     As shown in  FIG. 1  to  FIG. 4 , the amplification reaction region  4  mainly consists of a plurality of reaction wells  401  and pipes  402  connecting the reaction wells  401 ; the amplification reaction region  4  is connected to the substrate  1  through a connecting piece; and a fluorescence-imaging processing module is installed above the amplification reaction region  4 . 
     As shown in  FIG. 1  to  FIG. 4 , the substrate  1  is attached to the liquid storage component  2 , the solid-reagent storage component  3 , and the cover piece  5  together in one or more of the manners such as buckling, gluing and hot pressing. 
     As shown in  FIG. 1  to  FIG. 4 , the reaction wells  401  in the amplification reaction region  4  prestore target probes or primers for use in nucleic acid amplification in a dry form. 
     As shown in  FIG. 1  to  FIG. 4 , main bodies of the substrate  1 , the liquid storage component  2 , the solid-reagent storage component  3 , the amplification reaction region  4 , and the cover piece  5  are made of high-molecular polymers, which may be one or more of polycarbonate, polymethyl methacrylate, cycloolefin copolymer, polypropylene, and polyethylene glycol terephthalate. 
     As shown in  FIG. 1  to  FIG. 4 , the fluid isolating valves  7  are made of an elastic material with good airtightness, which may be one or more of natural rubber, silica gel, nitrile rubber, butyl rubber, fluororubber, and ethylene propylene rubber. 
     As shown in  FIG. 1  to  FIG. 4 , the casing is made of a high-molecular polymer, which may be one or more of polycarbonate, polymethyl methacrylate, cycloolefin copolymer, polypropylene, polyethylene glycol terephthalate, and acrylonitrile-butadiene-styrene copolymer. 
     Embodiment 1 for use of the nucleic acid testing cassette according to the invention is as follows. 
     Embodiment 1—Integrated Extraction and Amplification 
     1. The following describes the implementation of the integrated cassette for automated nucleic acid extraction and testing as described in the invention. 
     2. A liquid sample (such as saliva, liquefied sputum, blood, and a swab rinsing solution) was added to the cavity  2011  of the sample storage component  201 ; then the cover was closed (not shown in the drawings); and the cassette was placed into a matched external instrument to start the automated nucleic acid extraction and testing. 
     3. The external instrument included at least 2 air passage interfaces (including the first air passage interface  1071  and the second air passage interface  1072 ) that might be independently controlled to be opened or closed, 10 compression levers, 2 local heating modules, 2 rotating mechanisms (including the first magnet rotating mechanism and the second magnet rotating mechanism) including magnets, and 1 fluorescence-imaging processing module. Each of the fluid isolating valves  7  was correspondingly provided with one compression lever for controlling the opening/closing of the fluid isolating valve  7 ; and the plunger  2022  in each of the chambers was correspondingly provided with one compression lever for controlling the compression of the plunger  2022 . 
     4. When the integrated cassette already containing the sample to be tested was placed in the instrument, the plurality of compression levers closed the first fluid isolating valve  701 , the second fluid isolating vale  702 , the third fluid isolating valve  703 , and the fourth fluid isolating valve  704  respectively; the first air passage interface  1071  was closed; and here, an independent closed space was formed in the cavity  2011 . The first magnet rotating mechanism, below the cavity  2011 , on the external instrument was started, such that the sample could be fully mixed and reacted with the embedded reagents. This step was intended to release the nucleic acid to be tested from the sample. 
     5. After the mixing was completed, the air passage interface  1071  was opened such that the compression lever on the second chamber  202   a  was lowered to push the plunger  2022  to press the diaphragm  2023 , which expanded and deformed and was pierced by the sharp protrusion  103  on the substrate  1 , thereby releasing a nucleic acid binding solution within the second chamber  202   a.    
     6. While the first magnet rotating mechanism kept moving, the first magnetic rotor  601  fully mixed the released nucleic acid with the magnetic beads and the binding solution; then the first fluid isolating valve  701  was opened; and meanwhile, an air source provides a positive pressure into the cavity  2011  through the first air passage interface  1071 , thereby pushing the above mixed solution to flow in an order of “the cavity  2011 , the first fluid isolating valve  701  (opened), the nucleic acid immobilization-extraction reaction pool  102 , the fourth fluid isolating valve  704  (closed), the fifth fluid isolating valve  705  (opened), and the waste-liquid immobilization cavity  203 ”. 
     7. During this process, when magnetic-bead particles flowed to the nucleic acid immobilization-extraction reaction pool  102  along with the liquid, the second magnet rotating mechanism below the nucleic acid immobilization-extraction reaction pool  102  kept still; and under the action of a magnetic force, the magnetic-bead particles were immobilized in the nucleic acid immobilization-extraction reaction pool  102 , and the liquid finally entered the waste-liquid immobilization cavity  203 . 
     8. Then, the compression levers were adjusted in position, the first fluid isolating valve  701  was closed, and the second fluid isolating valve  702  was opened. Meanwhile, the compression lever on the second chamber  202   b  was lowered to push the plunger  2022  to press the diaphragm  2023 , which expanded and deformed and was pierced by the sharp protrusion  103  on the substrate  1 , thereby releasing a nucleic acid rinsing solution from the second chamber  202   b . It should be specially noted that, during this process, the compression lever could release the liquid in the chamber by one compression, or release the liquid in batches at a fixed amount by controlling the lowering height, thereby performing rinsing more than once. 
     9. The nucleic acid rinsing solution released from the second chamber  202   b  entered the nucleic acid immobilization-extraction reaction pool  102  through the second fluid isolating valve  702 , where the nucleic acid immobilization-extraction reaction pool  102  was a cavity with a low depth-to-width ratio and had a projection shape that might be round, rhombic, olivary, gourd-shaped or the like, and the foregoing various shapes guaranteed that the liquid might fully fill the cavity. After the nucleic acid immobilization-extraction reaction pool  102  was fully filled with the rinsing solution, the second magnet rotating mechanism below the nucleic acid immobilization-extraction reaction pool  102  started moving to drive the second magnetic rotor  602  to fully mix the magnetic beads immobilized in the cavity in the preceding step with the rinsing solution. 
     10. Then, the second magnet rotating mechanism stopped moving, and under the action of the magnetic force, the magnetic-bead particles suspending in the cavity after standing for a period of time were re-immobilized in the nucleic acid immobilization-extraction reaction pool  102 . 
     11. After the immobilization of the magnetic beads was completed, the second air passage interface  1072  and the third fluid isolating valve  703  were opened; and under the action of an external air source, the rinsing solution within the nucleic acid immobilization-extraction reaction pool  102  was pushed into the waste-liquid immobilization cavity  203 . 
     12. After the rinsing step was completed, the third fluid isolating valve  703  was kept opened, and the compression lever on the third chamber  203  was lowered to push the plunger  2032  to press the diaphragm  2033 , which expanded and deformed and was pierced by the sharp protrusion  103  on the substrate  1 , thereby releasing a nucleic acid eluent within the third chamber  202   c.    
     13. The nucleic acid eluent released from the third chamber  202   c  entered the nucleic acid immobilization-extraction reaction pool  102  through the third fluid isolating valve  703 . After the nucleic acid immobilization-extraction reaction pool  102  was fully filled with the eluent, the second magnet rotating mechanism below the nucleic acid immobilization-extraction reaction pool  102  started moving to drive the second magnetic rotor  602  to fully mix the magnetic beads immobilized in the cavity in the preceding step with the eluent; and meanwhile, the first heating module disposed below the nucleic acid immobilization-extraction reaction pool  102  was started. For “mixing plus heating”, a better effect was achieved by controlling the temperature to be 50-80° C. and the time to be 180-600 s. 
     14. After the above operations were completed, the fourth fluid isolating valve  704  was opened, and the fifth fluid isolating valve  705  was closed; and under the action of the external air source, the eluent entered the storage cartridge  301  of the solid-reagent storage component  3  from the nucleic acid immobilization-extraction reaction pool  102 , and was mixed with the reagent therein. 
     15. The compression lever above the storage cartridge  301  was lowered to push the mixed reagent to enter the reaction wells  401  of the amplification reaction region  4 . 
     16. Then, by lowering the compression lever to close the sixth fluid isolating valve  706 , each of the reaction wells  401  was isolated from the outside; the second heating module below the amplification reaction region  4  was opened; and the fluorescence-imaging processing module above the amplification reaction region  4  was opened. While the reaction temperature was controlled, a nucleic acid amplification reaction started occurred in each of the independent reaction wells; and the fluorescence-imaging processing module tested a target gene based on a light signal of each of the reaction wells. 
     Embodiment 2—Integrated Nucleic Acid Extraction and Nested Amplification 
     1. The following describes another implementation of the integrated cassette for automated nucleic acid extraction and testing as described in the invention. 
     2. In this embodiment, the operations in a first stage were substantially the same as Steps 1-13 in Embodiment 1. That is, the nucleic acid eluent in the third chamber  202   c  was injected into the nucleic acid immobilization-extraction reaction pool  102 , but by controlling a lowering distance of a corresponding plunger  2032 , the eluent was not allowed to fully fill the nucleic acid immobilization and extraction reaction pool  102 . 
     3. After the nucleic acid elution was completed, the first fluid isolating valve  701 , the second fluid isolating valve  702 , the third fluid isolating valve  703 , the fourth fluid isolating valve  704 , the fifth fluid isolating valve  705 , and the seventh fluid isolating valve  707  were kept opened; the plunger  2032  above the preamplification reagent chamber  202   d  was pressed down to release a certain amount of preamplification reagent into the nucleic acid immobilization-extraction reaction pool  102 ; the second magnet rotating mechanism below the nucleic acid immobilization-extraction reaction pool  102  moved to drive the second magnetic rotor  602  to fully mix the nucleic acid eluted in the preceding step with the preamplification reagent, where an optimal effect might be achieved when the mixing time was 5-60 s. 
     4. After the mixing was completed, the first fluid isolating valve  701 , the second fluid isolating valve  702 , the third fluid isolating valve  703 , the fourth fluid isolating valve  704 , the fifth fluid isolating valve  705 , and the seventh fluid isolating valve  707  were closed; and the first heating module below the nucleic acid immobilization-extraction reaction pool  102  was started to perform the preamplification reaction of the nucleic acid. The amplification reaction might be isothermal nucleic acid amplification, or variable-temperature nucleic acid amplification. 
     5. After the preamplification was completed, the fifth fluid isolating valve  705  and the seventh fluid isolating valve  707  were opened; a flow rate of the external air source was controlled to allow part of amplification products to enter the waste-liquid immobilization cavity  203  through the fourth fluid isolating valve  704  and the fifth fluid isolating valve  705 . The rest of the amplification products might account for 1/10- 1/100 of a total amount in the nucleic acid immobilization-extraction reaction pool  102 , or might be another appropriate ratio required based on the actual amplification reaction. 
     6. The third fluid isolating valve  703  was opened; the fourth fluid isolating valve  704  was closed; the plunger  2032  above the third chamber  202   c  was pressed down to release the nucleic acid eluent to enter and fully fill the nucleic acid immobilization-extraction reaction pool  102 . Here, the nucleic acid eluent plays a role of diluting the preamplification products. The second magnet rotating mechanism below the nucleic acid immobilization-extraction reaction pool  102  moved to drive the second magnetic rotor  602  to fully mix the preamplification products from the preceding step with the eluent, where an optimal effect might be achieved when the mixing time was 5-60 s. 
     7. After the operations described above were completed, the fourth fluid isolating valve  704  was opened; the fifth fluid isolating valve  705  was closed; and under the action of the external air source, the diluted preamplification products entered the storage cartridge  301  of the solid-reagent storage component  3  from the nucleic acid immobilization-extraction reaction pool  102 , and were mixed with the reagent therein. 
     8. Subsequent operations were substantially the same as Steps 15-16 in Embodiment 1. 
     The nucleic acid testing cassette according to the invention has the following advantages: 
     1. all the reagents, including a liquid reagent and a dry-powder reagent, required for nucleic acid extraction and amplification can be internally disposed on an integrated cassette (i.e., a chip), and a user only needs to add a sample, such that the nucleic aid testing cassette is extremely easy to operate and truly suitable for primary users to use; 
     2. the nucleic acid testing cassette is applicable to nested amplification, and the sensitivity is greatly improved; and 
     3. all the components are made of common materials by using common processes in the medical industry, such that the cost is greatly reduced. 
     The description above provides further detailed explanation of the invention in conjunction with the specific preferred embodiments, and it should not be deemed that the specific implementation of the invention is limited to these explanations. For those of ordinary skill in the art to which the invention belongs, several simple deductions or substitutions can also be made without departing from the conception of the invention, and should be construed as falling within the protection scope of the invention.