Cell wall-containing bacteria lysis device and cell wall-containing bacteria lysis method

Abstract

The application discloses a cell wall-containing bacteria lysis device and a cell wall-containing bacteria lysis method. The cell wall-containing bacteria lysis device comprises a fixing device for fixing a syringe with a needle tube downward, an ultrasonic generator and an ultrasonic block matched with an ultrasonic probe of the ultrasonic generator, which are arranged below the fixing device, and at least one of them can move to the other to hold the needle tube of the syringe in cooperation. A driving device is arranged above the fixing device, which is used to drive the piston rod of the syringe fixed on the fixing device to extend and retract to drive the sample solution in the sample container to flow in the needle tube for ultrasonic lysis. The lysis device can make the sample flow through the ultrasonic zone in the flow phase, which can effectively avoid the problems of excessive cavitation, local high temperature and high fragmentation of nucleic acid caused by long-time local ultrasonic action of the sample, and this method can improve the ultrasonic duty ratio and the lysis efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a device for lysing bacteria containing cell walls and a method for lysing bacteria containing cell walls. BACKGROUND

[0002] Gram-positive bacteria are common clinical infection bacteria, and their representative bacteria include Staphylococcus, Streptococcus, Enterococcus, Micrococcus, etc. The cell wall of Gram-positive bacteria is composed of a thick and dense peptidoglycan and a teichoic acid. The peptide chain of peptidoglycan is cross-linked with each other through 5 glycines, has 15-50 layers, and each layer has a thickness of 1 nm, accounting for about 50-80% of the dry weight of the cell. The teichoic acid is a polymer formed by the interconnection of ribitol or glycerol residues through a phosphodiester bond. The existence of the above special cell wall structure makes it difficult to break the wall and release nucleic acid during nucleic acid extraction, so the extraction efficiency is often low.

[0003] Common lysis methods include biological lysis, chemical lysis, and physical lysis.

[0004] Biological lysis mainly uses lysozyme digestion. The enzyme can cut the connection between the β-1, 4 glycosidic bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in peptidoglycan, destroy the peptidoglycan scaffold, and cause the cell to swell and crack under the action of internal osmotic pressure, thereby causing bacterial lysis. The action is mild, but it takes a long time and is ineffective for fungi, etc.

[0005] Chemical lysis includes strong alkali environment and denaturing agent, which makes the cell rupture, the protein denatured and precipitated, and the nucleic acid released into the aqueous phase. The strong alkali link is usually NaOH, KOH, etc., and the denaturation conditions include thermal shock, surfactants (SDS, Triton X-100, Tween 20, NP-40, CTAB, sarcosyl, Chelx-100, etc.) or strong ionic agents (guanidine isothiocyanate, guanidine hydrochloride, creatine guanidine), which are not ideal for the lysis of some cell walls, especially Gram-positive bacteria.

[0006] Physical lysis methods include freeze-thaw method, microwave method, grinding method, bead beating method, and ultrasonic method.

[0007] Among them, the working principle of the ultrasonic method is to cause vibration in the titanium probe immersed in the cell solution. A process called cavitation occurs, in which tiny bubbles form and explode, producing local shock waves and destroying the cell wall through pressure changes. This method is very suitable for plant and fungal cells. Since the ultrasonic probe is immersed in the solution, it needs to be thoroughly cleaned each time, and it is difficult to avoid cross-contamination between samples. The ultrasonic probe needs to be immersed in the solution, and the sample volume requirement is also relatively large, usually more than 5 milliliters.

[0008] Therefore, in order to solve the problems existing in the prior art, a device and method for ultrasonic lysis of biological cells are disclosed in patent No. US6686195B1.

[0009] In this device, the biological sample is loaded in a container, and the container is placed on an ultrasonic generator for ultrasonic lysis.

[0010] During lysis, the biological sample is in a static state in the container, which causes differences in the energy of the ultrasonic waves received by different regions of the biological sample. Therefore, there are problems such as excessive local temperature, excessive cavitation, and high fragmentation of nucleic acids. Although glass beads can be added to the container to ensure sufficient lysis, this requires the addition of glass beads in the container and the separation of the glass beads from the processed sample, which increases the operation process and implementation difficulty. SUMMARY

[0011] The purpose of the present application is to solve the above-mentioned problems existing in the prior art, and to provide a device and method for lysis of bacteria containing cell walls.

[0012] The purpose of the present application is achieved by the following technical solutions:

[0013] The device for lysis of bacteria containing cell walls comprises:

[0014] A fixing device is used to fix the syringe, and the needle tube of the syringe faces downward;

[0015] An ultrasonic generator and an ultrasonic stopper matched with the ultrasonic probe of the ultrasonic generator are arranged below the fixing device, and at least one of them can move towards the other to hold the needle tube of the syringe in cooperation;

[0016] A driving device is arranged above the fixing device, which is used to drive the piston rod of the syringe fixed on the fixing device to extend and retract to drive the sample solution in the sample container to flow in the needle tube for ultrasonic lysis.

[0017] Preferably, in the device for lysis of bacteria containing cell walls, the needle tube is a ferromagnetic needle tube.

[0018] Preferably, in the device for lysis of bacteria containing cell walls, a magnetic attraction mechanism is arranged between the fixing device and the ultrasonic stopper, and the magnetic attraction mechanism comprises a rotating shaft, an adsorption driving motor driving the rotating shaft to rotate, and a magnet fixed on the rotating shaft.

[0019] Preferably, in the device for lysis of bacteria containing cell walls, the fixing device and the driving device are arranged on a moving mechanism driving them to translate and lift.

[0020] Preferably, in the cell wall-containing bacterial lysis device, the ultrasonic block comprises a V-shaped positioning groove, and a hollow hole is arranged at a position of the ultrasonic block opposite to the ultrasonic probe of the ultrasonic generator.

[0021] Preferably, in the cell wall-containing bacterial lysis device, different supporting members are selectively arranged in the hollow hole.

[0022] Preferably, in the cell wall-containing bacterial lysis device, the ultrasonic generator and the ultrasonic block are arranged on an opening and closing driving assembly for driving them to move synchronously and reversely.

[0023] A cell wall-containing bacterial lysis method comprises the following steps:

[0024] S1, providing the cell wall-containing bacterial lysis device as described in any one of the above;

[0025] S2, preparing a sample solution to be lysed;

[0026] S3, lysing the sample solution by the cell wall-containing bacterial lysis device, wherein, during the lysis process, after the ultrasonic probe of the ultrasonic generator and the ultrasonic block cooperate with the needle tube of the syringe at the clamping and fixing device, the ultrasonic generator generates ultrasonic waves, and the driving device drives the piston rod of the syringe to extend and retract so that the sample solution is subjected to ultrasonic lysis in the needle tube in a flow phase.

[0027] Preferably, in the cell wall-containing bacterial lysis method, the number of pulses of the opening and closing driving motor of the opening and closing driving assembly for driving the ultrasonic generator and the ultrasonic block to clamp the needle tube is between 12000 and 13000.

[0028] Preferably, in the cell wall-containing bacterial lysis method, the driving device drives the suction and discharge flow rate of the syringe to be between 1.5 milliliters per minute and 4 milliliters per minute.

[0029] The advantages of the technical scheme of the present application mainly include:

[0030] In the lysis device of the present application, the sample can be subjected to lysis in a flow phase through the ultrasonic zone by the cooperation of the driving device and the syringe during the lysis process, which can effectively avoid problems such as excessive cavitation, local high temperature, and high fragmentation of nucleic acid caused by long-time local ultrasonic action of the sample, and this method does not need to use glass beads, can effectively simplify the operation process, and can improve the ultrasonic duty ratio of the ultrasonic generator, which is beneficial to shorten the lysis time and improve the lysis efficiency.

[0031] The present application utilizes a small-volume needle tube, which not only reduces the sample consumption of a single test, but also ensures the power input per unit volume, ensuring the effect and efficiency of rapid lysis. At the same time, the needle tube adopts a ferromagnetic needle tube, which can effectively reduce the attenuation in the ultrasonic transmission process compared with a plastic container, thereby improving the lysis efficiency.

[0032] The present application can fix the position of the ferromagnetic needle tube through the ultrasonic block and magnetic attraction mechanism, which can ensure that the needle tube and the ultrasonic probe are well and stably attached during lysis, thereby maximizing the input of ultrasonic energy.

[0033] The present application can effectively ensure that the lysis effect and efficiency meet the actual needs through the research and design of many parameters such as clamping force and flow rate during lysis. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a perspective view of the lysis device of the present application, only showing part of the moving mechanism;

[0035] Figure 2 is a perspective view of part of the lysis device of the present application;

[0036] Figure 3 is a sectional view of the syringe arranged at the fixing device of the present application;

[0037] Figure 4 is a sectional view of the ultrasonic block of the present application. DETAILED DESCRIPTION

[0038] The purposes, advantages and characteristics of the present application will be illustrated and explained by the following non-limiting description of preferred embodiments. These embodiments are only typical examples of the application of the technical solutions of the present application, and any technical solutions formed by equivalent replacement or equivalent transformation fall within the scope of the present application.

[0039] In the description of the scheme, it should be noted that the orientations or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "inner", "outer" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of description and simplification of description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0040] The cell wall-containing bacterial lysis device disclosed in the present application will be described below in conjunction with the drawings, as shown in the drawings, which comprises: Figure 1 -attached Figure 3 as shown, which comprises:

[0041] a fixing device 100 for fixing a syringe 200, a needle tube 210 of which is directed downward;

[0042] an ultrasonic generator 300 and an ultrasonic block 400 matched with an ultrasonic probe of the ultrasonic generator 300, which are arranged below the fixing device 100, and at least one of which is movable to the other to make both cooperate to clamp the needle tube 210 of the syringe 200;

[0043] a driving device 500 arranged above the fixing device 100, which is used to drive the syringe 200 fixed at the fixing device 100 to extend and retract the piston rod 220 to drive the sample solution in the sample container 600 to flow in the needle tube 210 for ultrasonic lysis.

[0044] As shown in the accompanying drawings Figure 3As shown, the injector 200 can be a known feasible structure, which generally comprises a syringe barrel 230, a needle head arranged at one end of the syringe barrel 230, and a piston rod 220 movably arranged in the syringe barrel 230. The injector 200 is consumable and can be replaced after each test. The syringe barrel 230 can be a known feasible shape, and the top of the syringe barrel 230 is symmetrically provided with wing plates 231 extending to both sides, which are perpendicular to the axis of the syringe barrel 230. In order to facilitate the fixation of the injector 200 during use, the fixing device 100 comprises a groove body 110 and a side cover 120, the groove of the groove body 110 faces the side surface, and a first limiting groove 112 is formed on the bottom plate 111 of the groove body 110 to limit the needle head and enable the needle tube 210 of the needle head to extend below the groove body 110. A second limiting groove 114 opposite to the first limiting groove 112 is arranged on the top plate 113 of the groove body 110, and the second limiting groove 114 is used for the piston rod 220 to pass through. A limiting plate 115 is further arranged in the groove body 110, and of course the limiting plate 115 is not necessary, the limiting plate 115 is parallel to the top plate 113 of the groove body 110, and the spacing between the limiting plate 115 and the groove body 110 is comparable to the thickness of the wing plate 231, a third limiting groove 116 opposite to the second limiting groove 114 is arranged on the limiting plate 115, the width of the third limiting groove 116 is smaller than the diameter of the syringe barrel 230, and the depth of the groove body 110 matches the diameter of the syringe barrel 230. When it is necessary to fix the injector 200 at the fixing device 100, the needle head of the injector 200 is opposite to the first limiting groove 112, the syringe barrel 230 is opposite to the third limiting groove 116, and the wing plate 231 is opposite to the gap between the top plate 113 and the limiting plate 115, and the injector 200 is clamped into the groove body 110, at this time, the groove of the groove body 110 is closed by the side cover 120, and the syringe barrel 230 is tightly fixed in the groove body 110. The side cover 120 and the groove body 110 can be detachably connected, they can be fixed by clamping, or can be fixed by magnetic attraction, lock buckle, screwing, etc. Of course, in a more optimal way, the side cover 120 and one side of the groove body 110 are hinged, and the other side opposite to them is fixed by clamping or lock buckle, which is more convenient to operate. Or the fixing device can also adopt other known structures to realize the fixation of the injector, for example, using a clasp.

[0045] The needle tube 210 can be processed by various feasible materials, and more preferably, the needle tube 210 is a ferromagnetic needle tube 210.

[0046] As shown in FIG. 2, the injector 200 is fixed in the fixing device 100, and the needle head of the injector 200 is opposite to the first limiting groove 112, the syringe barrel 230 is opposite to the third limiting groove 116, and the wing plate 231 is opposite to the gap between the top plate 113 and the limiting plate 115. Figure 1 , FIG. 3 shows the fixing device 100 in a state of being opened, and the injector 200 is removed from the fixing device 100. Figure 2As shown, between the fixing device 100 and the ultrasonic stopper 400, a magnetic attraction mechanism 700 is arranged, which is used to attract and fix the needle tube 210 after the syringe 200 is in place. The magnetic attraction mechanism 700 comprises a rotating shaft, an attraction driving motor 710 driving the rotating shaft to rotate, and a magnet 720 fixed on the rotating shaft. When the attraction driving motor drives the rotating shaft to rotate, the magnet rotates from a position far away from the needle tube 210 to a position in contact with the needle tube 210, thereby magnetically attracting and fixing the needle tube 210.

[0047] The driving device 500 can be various devices capable of generating linear motion, for example, it can be an electric cylinder, a gas cylinder, a hydraulic cylinder, etc. arranged in the vertical direction. Preferably, the driving device 500 comprises a bracket, on which an attraction and discharge driving motor 510 is arranged, the attraction and discharge driving motor 510 is connected to a lead screw 520, the lead screw 520 is arranged in the vertical direction, the movable nut of the lead screw 520 is connected to a clamping piece 530, the clamping piece 530 is connected to the bracket through a slide rail assembly, and the clamping piece 530 is detachably clamped with the piston rod 220. Specifically, the side surface of the clamping piece 530 is provided with a T-shaped notch matching the rod portion and the end plate of the piston rod 220, and when the syringe 200 is fixed at the fixing device 100, the upper end of the piston rod 220 is embedded in the T-shaped notch. Therefore, when the attraction and discharge driving motor 510 drives the clamping piece 530 to ascend and descend, it can drive the piston rod 220 to move up and down to realize attraction and discharge.

[0048] The sample container 600 containing the sample solution can be a known centrifuge tube or a plastic tube with the mouth sealed by a plug. In use, the needle tube 210 is inserted into the sample container to realize attraction and discharge. Of course, if necessary, a certain limiting structure can be used to limit the sample container, but this is not necessary.

[0049] As shown in the accompanying drawings, Figure 1 In order to facilitate operation, the fixing device 100, the driving device 500 and the magnetic attraction mechanism 700 are arranged on a moving mechanism 800 driving them to translate and lift. The specific structure of the moving mechanism 800 can be two mutually perpendicular linear modules, one linear module is fixed on the slider of the other linear module, and one linear module is arranged horizontally and the other linear module is arranged in the vertical direction. Of course, the moving mechanism 800 can also be other feasible structures, which are known technologies and will not be described here.

[0050] As shown in the accompanying drawings, Figure 4As shown, the ultrasonic block 400 includes a main body 410, on which two convex portions 420 are formed towards the ultrasonic probe, and each of the convex portions 420 is provided with a V-shaped positioning groove 421, and the two V-shaped positioning grooves 421 are vertically opposite to each other, and the opening end of the V-shaped positioning groove 421 is towards the ultrasonic probe of the ultrasonic generator. At the same time, the two convex portions 420 are located above and below the ultrasonic probe, and the main body 410 is further provided with a hollow hole 430 between the two convex portions 420, which is opposite to the ultrasonic probe of the ultrasonic generator 300. Different supporting members can be selectively arranged in the hollow hole 430, and when clamped, the supporting members are in contact with the needle tube. The hollow hole can be a threaded hole, and the supporting member can be a bolt or a spring plunger, and the material thereof can be selected as needed, such as stainless steel, ABS, PC. And the inventor found through in-depth research that when the supporting member is an ABS spring plunger, the best lysis effect can be obtained.

[0051] When it is necessary to clamp the needle tube 210, the position of the ultrasonic block 400 can be fixed, and the ultrasonic probe of the ultrasonic generator is moved towards the needle tube 210, of course, the ultrasonic probe of the ultrasonic generator can be fixed, and the ultrasonic block 400 is moved towards the needle tube 210. More preferably, the ultrasonic generator 300 and the ultrasonic block 400 are arranged on the opening and closing driving assembly 900 which drives them to move synchronously and reversely, and the opening and closing driving assembly 900 can include an opening and closing driving motor and left and right screw rods 520 driven by the opening and closing driving motor, and the ultrasonic block 400 and the ultrasonic generator are arranged on two movable nuts of the known left and right screw rods 520, respectively. When the screw rod of the left and right screw rods 520 rotates, the two movable nuts move reversely and oppositely, so that they can be driven by the opening and closing driving motor to move synchronously and oppositely to clamp the needle tube 210 or move reversely to release the needle tube 210.

[0052] Embodiment 2

[0053] This embodiment discloses a method for lysing bacteria containing cell walls, which is illustrated by lysing Staphylococcus aureus, and includes the following steps:

[0054] S1, providing a bacteria containing cell wall lysis device as described above;

[0055] S2, preparing a sample solution to be lysed; specifically, including the following steps:

[0056] S21, in a clean bench, 10 μL of 10% glycerol Staphylococcus aureus strain is added to 5 mL of LB sterile liquid medium (Shanghai Biosynthetic Engineering Co., Ltd., brand: B540111), and stirred at 37°C and 200 RPM for 16-18 hours.

[0057] S22, centrifuge the bacterial solution obtained in S21 at a speed of 4500 rpm for 5 minutes to remove the supernatant. Then resuspend the bacterial solution with 4 ml of PBS (Shanghai Yingxuan Biotech Co., Ltd., model: E607008) and centrifuge again for 5 minutes under the same conditions as above. Then, resuspend the bacterial solution with 2 ml of known Buffer-LZ (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100) and test the 600 nm absorbance value, i.e. OD600.

[0058] S23, dilute the bacterial solution obtained in S22 to 1 OD / 0.11 ml (1 OD bacterial solution, i.e. OD600 = 1, 1 ml), and record it as a sample solution.

[0059] S3, lyse the sample solution by the cell wall-containing bacterial lysis device. During the lysis process, the ultrasonic probe of the ultrasonic generator 300 of the cell wall-containing bacterial lysis device is clamped and fixed to the needle tube 210 of the syringe 200 at the injection site of the clamping and fixing device 100, and then the ultrasonic generator 300 generates ultrasonic waves. The driving device 500 of the cell wall-containing bacterial lysis device drives the piston rod 220 of the syringe 200 to extend and retract so that the sample solution in the needle tube 210 is subjected to ultrasonic lysis in the flow phase.

[0060] Specifically, a certain amount of sample solution can be first added to the sample container, and then the needle tube 210 of the syringe 200 on the clamping and fixing device 100 is inserted into the sample container containing the sample solution. Of course, the syringe 200 can also be inserted into the sample container containing the sample solution first, and then the syringe 200 is fixed at the clamping and fixing device 100 and connected with the driving device 500. Alternatively, a certain amount of sample solution can be first sucked by the syringe fixed on the clamping and fixing device, and then the sample container is inserted on the needle tube.

[0061] Then, the lysis device is started, and the control device of the lysis device controls the moving mechanism 800 to move the clamping and fixing device 100 and the driving device 500 as a whole so that the needle tube 210 of the syringe 200 on the clamping and fixing device 100 is located between the ultrasonic block 400 and the ultrasonic probe of the ultrasonic generator. Then, the control device controls the magnetic attraction device to act to attract and fix the needle tube 210. Then, the control device controls the opening and closing driving device 500 to drive the ultrasonic block 400 and the ultrasonic generator 300 to move towards each other to clamp the needle tube 210. Subsequently, the control device controls the driving device 500 to drive the telescopic rod to move up and down to realize suction and discharge, and at the same time, the ultrasonic generator 300 generates ultrasonic waves to lyse the biological sample flowing in the needle tube 210.

[0062] Example 3

[0063] The inventor further found that the clamping degree of the ultrasonic probe and the ultrasonic block 400 clamping the needle tube 210 has a significant impact on the transmission efficiency of ultrasonic waves during the lysis process, and too loose or too tight clamping will cause a decrease in transmission efficiency. The clamping force during clamping can be controlled by the number of pulses of the opening and closing drive motor during clamping, so the inventor further researched this;

[0064] During lysis, 250 μL of sample solution was taken from each experimental group for processing using the lysis device of the application. During lysis, the suction and discharge flow rate was controlled at 3 mL / min. The number of pulses of the opening and closing drive motor during clamping was controlled at 11000, 12000, 12500, 13000, and 13500 for different experimental groups. During lysis, the ultrasonic generator worked in a mode of 10 s power-on and 2 s power-off. The ultrasonic generator was turned on during suction and discharge. One cycle of suction and discharge was one cycle, and the process was repeated for 40 cycles. The gram-negative bacteria control group was not subjected to ultrasonic treatment, i.e., it was directly subjected to accounting and purification without lysis. Each group had two parallel repeats.

[0065] During nucleic acid purification, 110 μL of the lysis solution was taken and subjected to known magnetic bead method general genomic DNA extraction kit DP705 according to the instruction manual steps to complete the purification. The specific purification process is known technology, which is not described here.

[0066] During detection, the known qPCR method was used for detection. Specifically, 5 μL of the purified product was taken, 1 Unit Taq DNA Polymerase (Beijing Zhenxingjin Bioengineering, brand: AP401), 2 μL 10x Taq Buffer (buffer), 0.8 μL 2.5 mM dNTPs, 0.1 μM probe, and RNase-free water was added to 20 μL. The amplification program was enzyme activation at 95°C for 2 min; temperature cycle at 95°C for 5 s, 60°C for 15 s, 45 cycles. The detailed operation is known technology, which is not described here.

[0067] The final test results are as follows. The Ct mean value of the gram-negative bacteria control group was 20.8,

[0068] When the number of pulses was 11000, 12000, 12500, 13000, and 13500, the final Ct mean value was 17.0, 15.9, 16.6, 16.12, and 16.7, respectively.

[0069] Through the above research, it can be found that when the number of pulses when the opening and closing driving motor drives the ultrasonic generator 300 and the ultrasonic block 400 to clamp the needle tube 210 is between 12000-13000, a better lysis effect can be obtained, especially when the number of pulses is 12000, the optimal lysis effect can be obtained.

[0070] Example 4

[0071] The inventors found that the effects obtained when using different resuspension matrices for lysis also differ. In the specific experiment, the bacterial liquid was cultured according to the process of S21.

[0072] In S22, the bacterial liquid obtained by culturing in S21 was also centrifuged at a speed of 4500 rpm for 5 min, and the supernatant was removed. Then, 4 mL of PBS Buffer (Shanghai Yingyee Biotech Co., Ltd., model: E607008) was used for resuspension, and the same condition was used for centrifugation for 5 min again.

[0073] Group one was resuspended using 2 mL Buffer-LZ (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100), and the 600 nm absorbance value was tested.

[0074] Group two was resuspended using 2 mL PBS Buffer, and the 600 nm absorbance value was tested.

[0075] The bacterial liquid of group one and group two was diluted to obtain a sample solution according to the process of S23.

[0076] During lysis, 250 μL of the bacterial liquid to be tested was taken from each of the two experimental groups and treated using the device, the liquid suction and discharge rate was 1.5 mL / min, the number of pulses when the opening and closing driving motor clamped was 11000, the ultrasonic generator worked in the mode of power on for 10 s and power off for 2 s, and the ultrasonic generator was turned on when discharging, one cycle of liquid suction and discharge was one cycle, and the lysis time was 10 min. The gram-negative bacteria control group was not treated by ultrasonic; the gram-positive bacteria control group was treated with lysozyme for 90 min, and each group had two parallel repeats.

[0077] The nucleic acid purification and detection process is the same as the above experimental process, which is not repeated here.

[0078] The test results are as follows:

[0079] The Ct average of the gram-negative bacteria control group was 21.5, the Ct average of the gram-positive bacteria control group was 15.0, the Ct average of the experimental group one was 17.5, and the Ct average of the experimental group two was 15.7. The test results of group two were obviously better than those of group one, therefore, PBS buffer (PBS buffer) was preferably used for resuspension.

[0080] Example 5

[0081] The inventors further found that the suction and discharge flow rate is also an important factor affecting the lysis effect and efficiency during lysis, and thus the inventors further studied this factor.

[0082] During the experiment, the sample solution was prepared according to the above processes S21-S23, which will not be repeated here.

[0083] During lysis, 250 μL of sample solution was taken from each experimental group and treated using the lysis device of the present application. The suction and discharge flow rates of different experimental groups were 0.6 mL / min, 0.75 mL / min, 1.5 mL / min, 3 mL / min and 4.5 mL / min, respectively. The pulse number of the clamping driving motor was 12000. The ultrasonic generator worked in a manner of being powered on for 10 s and being powered off for 2 s. The ultrasonic generator was turned on during liquid discharge. One cycle of liquid suction and discharge was one cycle, and the process was repeated for 40 cycles. The gram-negative bacteria control group was not treated by ultrasonic, and the gram-positive bacteria control group was treated by lysozyme for 90 min. Each group had two parallel repeats.

[0084] The nucleic acid purification and detection process was the same as in the above examples, which will not be repeated here.

[0085] The test results are as follows:

[0086]

[0087]

[0088] As can be seen from the above, the time consumption of each experimental group is significantly less than that of the control group. When the suction and discharge flow rate is 1.5 mL / min-4.5 mL / min, the effect is equivalent to that of the gram-positive bacteria control group, and even better. Considering the time consumption and lysis effect, the condition of 3 mL / min is the most optimal.

[0089] Example 6

[0090] The inventors found that the number of injection cycles during lysis is also an important factor affecting the lysis effect and efficiency, and thus the inventors further studied this factor.

[0091] The preparation process of the sample solution was the same as in the above examples, which will not be repeated here.

[0092] At the time of lysis, 250 μL of sample solution was taken in each experimental group and treated using the lysis device of the application, the suction and discharge flow rate was 3 mL / min, the number of pulses when the opening and closing drive motor clamped was 12000, the ultrasonic generator worked in the mode of power-on for 10 s and power-off for 2 s, and the ultrasonic generator was started when discharging, one cycle of suction and discharge was one cycle, and different experimental groups were repeated for 5, 10, 20, 30, 40 and 50 cycles. The gram-negative bacteria control group was not treated by ultrasonic, and the gram-positive bacteria control group was treated by lysozyme for 90 min, and each group had two parallel repeats.

[0093] The nucleic acid purification and detection process is the same as in the above examples, which will not be repeated here.

[0094] The test results are as follows:

[0095]

[0096]

[0097] As can be seen from the above, the time consumption of each experimental group is significantly less than that of the gram-positive bacteria control group, and when the cycle number is greater than 30, the effect is equivalent to that of the gram-positive bacteria control group. Considering the time consumption and effect, 40 cycles are the best.

[0098] Example 7

[0099] The inventors found that whether the magnetic attraction mechanism attracts the needle tube, whether the ultrasonic stopper is provided with a support and different supports have a significant impact on the lysis effect, so the inventors further studied this:

[0100] The preparation of sample solution is the same as in the above examples, which will not be repeated here.

[0101] At the time of lysis, 250 μL of sample solution was taken in each experimental group and treated using the lysis device of the application, the suction and discharge flow rate was 3 mL / min, the number of pulses when the opening and closing drive motor clamped was 11500, the ultrasonic generator worked in the mode of power-on for 10 s and power-off for 2 s, and the ultrasonic generator was started when discharging, one cycle of suction and discharge was one cycle, and 40 cycles were repeated.

[0102] In experimental group 1, no support was provided at the ultrasonic stopper and the magnetic attraction mechanism did not attract the needle tube.

[0103] In experimental group 2, an ABS material screw was provided at the ultrasonic stopper and the magnetic attraction mechanism attracted the needle tube.

[0104] In experimental group 3, an ABS material screw was provided at the ultrasonic stopper and the magnetic attraction mechanism did not attract the needle tube.

[0105] In experimental group 4, an ABS spring plunger was provided at the ultrasonic stopper and the magnetic attraction mechanism attracted the needle tube.

[0106] In the experimental group 5, a stainless steel screw is arranged at the ultrasonic block, and the magnetic attraction mechanism is used to adsorb the needle tube.

[0107] In the experimental group 6, a PC screw is arranged at the ultrasonic block, and the magnetic attraction mechanism is used to adsorb the needle tube.

[0108] The gram-negative bacteria control group is not subjected to ultrasonic treatment, and the gram-positive bacteria control group is subjected to lysozyme treatment for 90 min, and each group has two parallel repeats.

[0109] The nucleic acid purification and detection process is the same as the above examples, which will not be repeated here.

[0110] The test results are as follows:

[0111] Mean Ct Experiment 1 19.4 Experiment 2 16.6 Experiment 3 17.3 Experiment 4 16.3 Experiment 5 19.7 Experiment 6 19.9 Gram-negative control 21.6 Gram-positive control 16

[0112] As can be seen from the above, setting the support and the magnetic attraction mechanism to adsorb the needle tube can improve the lysis effect, and the effect of ABS material is obviously better than that of stainless steel and PC material, and the effect of the spring plunger of ABS material is the best.

[0113] The above experimental data is only listed for convenience of explanation, and it does not mean that only these experiments are actually performed to obtain the above research results.

[0114] The present application has various embodiments, and all technical solutions formed by equivalent transformation or equivalent transformation fall within the protection scope of the present application.

Claims

1. A cell wall containing bacterial lysis device, characterized in that, The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device.

2. The cell wall-containing bacterial lysis device of claim 1, wherein: The application relates to a cell wall bacteria lysis device.

3. The cell wall-containing bacterial lysis device of claim 1, wherein: The application relates to a cell wall bacteria lysis device.

4. The cell wall-containing bacterial lysis device of claim 3, wherein: The application relates to a cell wall bacteria lysis device.

5. The cell wall-containing bacterial lysing apparatus of any one of claims 1-4, wherein: The application relates to a cell wall bacteria lysis device.

6. A method for lysis of a cell wall-containing bacterium, characterized in that, The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device.

7. The cell wall-containing bacterial lysis method according to claim 6, characterized in that: The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria lysis device. The application relates to a cell wall bacteria

Images