WO2020211835A1 - 抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法 - Google Patents

抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法 Download PDF

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WO2020211835A1
WO2020211835A1 PCT/CN2020/085275 CN2020085275W WO2020211835A1 WO 2020211835 A1 WO2020211835 A1 WO 2020211835A1 CN 2020085275 W CN2020085275 W CN 2020085275W WO 2020211835 A1 WO2020211835 A1 WO 2020211835A1
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Prior art keywords
bacteria
hole
bacterial
pulse signal
counted
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PCT/CN2020/085275
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English (en)
French (fr)
Inventor
崔璟
唐明忠
张会翠
许朋
Original Assignee
山东鑫科生物科技股份有限公司
北京鑫骥金诺医疗器械有限公司
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Priority claimed from CN201911290900.3A external-priority patent/CN111876316A/zh
Application filed by 山东鑫科生物科技股份有限公司, 北京鑫骥金诺医疗器械有限公司 filed Critical 山东鑫科生物科技股份有限公司
Priority to JP2021562041A priority Critical patent/JP2022529281A/ja
Priority to US17/604,449 priority patent/US20220195487A1/en
Priority to EP20790711.4A priority patent/EP3957740A4/en
Publication of WO2020211835A1 publication Critical patent/WO2020211835A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the invention relates to the field of biomedicine, in particular to a method for detecting the inhibition of bacteria by antibacterial drugs, a bacteria counting device and a method thereof.
  • Rapid drug sensitivity tests can be divided into two categories: phenotypic and non-phenotypic methods.
  • Non-phenotyping methods are mainly nucleic acid-based molecular biology methods, such as real-time PCR, microarray, RNA sequencing, transcriptome and whole-genome sequencing, etc. Its advantages are: 1. Short time, such as direct positive blood culture Multiplex PCR can detect multiple drug resistance genes; 2. Digital PCR can achieve quantitative analysis; 3. Clear the corresponding drug resistance mechanism. Disadvantages are: 1. The drug resistance mechanism of bacteria is complex and huge. If it is fully used in clinical practice, the workload is too large and affects the economy and rapidity; 2.
  • Drug resistance gene detection due to the problem of genetic heterogeneity, its genetic test results The issue of consistency with the same phenotype still requires a lot of verification work; 3. New resistance mechanisms cannot be detected, but keenly discovering new resistance mechanisms is urgently needed in clinical practice; 4. Not yet applied in clinical practice, immature, Further clinical observations are needed, and the actual clinical application can only be achieved after global experts’ approval and standardization.
  • Phenotypic drug sensitivity test directly observes the reaction of bacteria to drugs in vitro, and can directly observe the sensitivity and tolerance of bacteria to antibiotics.
  • the traditional phenotypic drug sensitivity test has been fully developed, tested, verified and is in clinical practice It has been fully proved to become the reference standard of AST method.
  • this "intermediate technology” that is, those technologies developed on the basis of traditional culture methods, can be implemented earlier and has great feasibility. Arouse people's expectations.
  • cell counters refer to instruments that measure the number of platelets, white blood cells, and red blood cells.
  • the automatic cell counter is widely used.
  • the technical solution Coulter principle analysis method has always been an internationally recognized standard control method for measuring cell and particle size, and it has always occupied an important position in hematological analysis.
  • the counting device and method applied to bacteria still have the following problems: (1) At present, there is no equipment on the market that uses the resistance counting method to measure the number of bacteria. 2The diameter of the gem hole of the existing cell counter is suitable for measuring red blood cells, white blood cells, etc. with larger sizes, ensuring that the cells can pass through the gem hole one by one; the bacteria are small and cannot normally pass through the gem hole one by one, and the bacteria may pass through at the same time. The number of gem holes is greater than or equal to 2, resulting in inaccurate counting. 3When measuring red blood cells, white blood cells, etc., if the jewel hole is less than 50um, it will cause the hole blockage of the existing counting instrument, so the hole diameter of the jewel hole in the prior art is limited to 50um or more.
  • the present invention aims to provide a method for detecting the inhibition of bacteria by antibacterial drugs, a bacteria counting device and the method thereof, so as to at least solve the technical problem of long time for the detection method of bacteria by antibacterial drugs in the prior art. Two-day targeted choice of medication.
  • a method for detecting the inhibition of bacteria by antibacterial drugs includes: adding a predetermined concentration of an antibacterial drug to the bacteria to be detected and setting it as a bacterial drug mixture, and at the same time, setting the bacteria to be detected without adding the antibacterial drug as a positive control; at the time when the antibacterial drug is added When the first predetermined time interval is reached, obtain the current number of the bacteria in the bacterial drug mixture and the current number of the bacteria in the positive control; according to the current number of the bacteria in the bacterial drug mixture and the current number of the bacteria in the positive control The ratio of the quantities determines the inhibition or partial inhibition or non-inhibition of the aforementioned antibacterial drug at the aforementioned predetermined concentration on the aforementioned bacteria.
  • the ratio of the current number of the bacteria in the bacterial drug mixture to the current number of the bacteria in the positive control is equal to a first predetermined threshold, it is determined that the antibacterial drug at the predetermined concentration inhibits the bacteria.
  • the foregoing first predetermined threshold value is any value from 0 to 0.6.
  • the first predetermined threshold is any value from 0 to 0.4.
  • the ratio of the current number of the bacteria in the bacterial drug mixture to the current number of the bacteria in the positive control is equal to a second predetermined threshold, it is determined that the antibacterial drug at the predetermined concentration has a partial effect on the bacteria. Inhibited, but did not reach inhibition;
  • the second predetermined time period is separated from the time when the antibacterial drug is added, the second current number of the bacteria in the bacterial drug mixture and the second current number of the bacteria in the positive control are obtained, wherein the second predetermined time period is greater than The aforementioned first predetermined duration;
  • the ratio of the second current number of the bacteria in the bacterial drug mixture to the second current number of the bacteria in the positive control is equal to the first predetermined threshold, it is determined that the antibacterial drug at the predetermined concentration is present on the bacteria inhibition.
  • the ratio of the current number of the bacteria in the bacterial drug mixture to the current number of the bacteria in the positive control is greater than a second predetermined threshold, it is determined that the antibacterial drug at the predetermined concentration does not exist on the bacteria inhibition.
  • the first predetermined duration is any value from 0 to 1.5 hours, and the first predetermined duration is not equal to 0 hours.
  • the foregoing second predetermined threshold value is any one of 0.4 to 0.8.
  • a resistance counting method is used to obtain the current number of the bacteria in the bacterial drug mixture and the current number of the bacteria in the positive control.
  • the detection method for the inhibition of bacteria by the antibacterial drug includes the following detection steps:
  • a Prepare the above-mentioned bacterial strains: inoculate the above-mentioned bacterial strains on the culture medium, and incubate at a temperature of 20 degrees Celsius (°C) to 40 degrees Celsius (°C) for 15 hours to 24 hours, for use;
  • the current number or second current number of the bacteria in the bacterial drug mixture and the current number or second current number of the bacteria in the positive control are obtained by the resistance counting method Quantity
  • the ratio of the current number or second current number of the bacteria in the bacterial drug mixture to the current number or the second current number of the positive control is equal to any value from 0 to 0.4, it is determined
  • the antibacterial drug at the predetermined concentration inhibits the bacteria.
  • the above bacterial strains are inoculated on a blood agar medium and incubated at 37 degrees Celsius (°C) for 18 hours; and/or
  • step b prepare the above bacterial drug mixture and the above positive control, and incubate at 37°C; and/or
  • the first predetermined time length is 0.5 hour or 1 hour or 1.5 hours; the second predetermined time length is 2 hours or 2.5 hours or 3 hours.
  • the current number of the above-mentioned bacteria in the above-mentioned bacterial drug mixture and the above-mentioned positive control are obtained by using the flow cytometry method or the microscopic bacterial counting method or the counter measurement method or the electronic counter counting method or the viable cell counting method or the cell weight method.
  • the current number of the aforementioned bacteria are obtained by using the flow cytometry method or the microscopic bacterial counting method or the counter measurement method or the electronic counter counting method or the viable cell counting method or the cell weight method.
  • a bacteria counting device can be used to obtain the current number of bacteria of the bacterial drug mixture and the current number of bacteria of the positive control in the aforementioned method for detecting the inhibition of bacteria by antibacterial drugs.
  • the bacteria counting device includes: a sampling component for obtaining bacterial samples to be counted; a counting cell component including: a gem hole, a front pool, a back pool and an electrode, wherein the front pool and the back pool are connected through the gem hole, There is one electrode on both sides of the jewel hole, the liquid pressure between the front pool and the back pool is negative pressure, and the negative pressure is used to make the bacteria sample to be counted enter the back pool through the jewel hole from the front pool
  • the circuit control system is used to determine the number of bacteria in the bacterial sample to be counted according to the pulse signal in the case of detecting the pulse signals generated on both sides of the gem hole, wherein the pulse signal is used to indicate the bacteria to be counted
  • the aforementioned circuit control system includes: a first processor, configured to detect the aforementioned pulse signal, transmit the aforementioned pulse signal to a processing device, and obtain the number of bacteria in the aforementioned bacterial sample to be counted sent by the aforementioned processing device, wherein: The number of bacteria in the bacterial sample to be counted is determined according to the bacterial characteristic data indicated by the pulse signal; or
  • the second processor is configured to detect the pulse signal and determine the number of bacteria in the bacterial sample to be counted according to the bacterial characteristic data indicated by the pulse signal.
  • the above-mentioned circuit control system includes: a first power supply circuit for providing a constant current to the above-mentioned jewel hole through the above-mentioned electrode, wherein the above-mentioned pulse signal is generated by one or more Pulse signals generated by the above-mentioned bacteria triggered by the above-mentioned jewel hole; or
  • a second power supply circuit for supplying a constant voltage to the jewel hole through the electrode, wherein the pulse signal is generated by one or more bacteria triggered by the jewel hole when the constant voltage is supplied to the jewel hole Pulse signal.
  • the diameter of the gem hole is a diameter within a first target diameter range, wherein the first target diameter range is used to allow only one bacteria to pass through the gem hole at a time when the bacteria in the bacterial sample to be counted pass through the gem hole Gem hole; or
  • the diameter of the jewel hole is a diameter within a second target diameter range, wherein the second target diameter range is used to allow multiple bacteria to pass through the jewel hole at a time when the bacteria in the bacterial sample to be counted pass through the jewel hole.
  • the diameter of the jewel hole is 30 ⁇ m to 70 ⁇ m, and/or the length of the jewel hole is 30 ⁇ m to 100 ⁇ m .
  • the diameter of the jewel hole is 40 to 60 microns, and/or the length of the jewel hole is 40 to 70 microns .
  • the diameter of the gem hole is a diameter within the first target diameter range
  • the diameter of the gem hole is 50 microns
  • the length of the gem hole is 50 microns
  • a method for counting bacteria can be used to obtain the current number of bacteria of the bacterial drug mixture and the current number of bacteria of the positive control in the detection method for the inhibition of bacteria by antibacterial drugs.
  • the bacteria counting method includes: adding a bacterial sample to be counted to a counting cell assembly, wherein the counting cell assembly includes a gem hole, a front pool, a back pool, and an electrode, and the front pool and the back pool are connected through the gem hole, The liquid pressure between the front pool and the back pool is negative pressure, and the negative pressure is used to make the bacteria sample to be counted enter the back pool from the front pool through the jewel hole, and there is one electrode on each side of the jewel hole , When the electrode is energized, there is a predetermined resistance between the two sides of the gem hole;
  • the number of bacteria in the bacterial sample to be counted determined according to the pulse signal is obtained.
  • the foregoing obtaining the number of bacteria in the foregoing bacterial sample to be counted determined according to the foregoing pulse signal includes:
  • the number of bacteria in the bacterial sample to be counted is determined according to the bacterial characteristic data indicated by the pulse signal.
  • the diameter of the gem hole is a diameter within a first target diameter range, wherein the first target diameter range is used to allow only one bacteria to pass through the gem hole at a time when the bacteria in the bacterial sample to be counted pass through the gem hole Gem hole; or
  • the diameter of the jewel hole is a diameter within a second target diameter range, wherein the second target diameter range is used to allow multiple bacteria to pass through the jewel hole at a time when the bacteria in the bacterial sample to be counted pass through the jewel hole.
  • the bacteria generate voltage pulse signals when passing through the gem hole, so they can According to the above solution, the number of bacteria in the above-mentioned bacterial sample to be counted is determined according to the above-mentioned pulse signal, and the "pulse signal" can be selected as a "voltage pulse signal".
  • the bacterial characteristic data represented by the pulse signal includes: amplifying and gaining the pulse signal through a conditioning circuit, filtering out noise through low-pass filtering, filtering out over-limit values through buffering and limiting; through pulse recognition, slope recognition, Algorithms such as wave peak detection, wave valley detection, and broadband detection identify the signal with the bacterial characteristic data in the pulse signal.
  • the clogging phenomenon (plugging) of the jewel hole is divided into complete plugging and incomplete plugging, that is, the phenomenon of complete clogging of the jewel hole and incomplete clogging of the jewel hole.
  • the hole is completely blocked, the count will be abnormal and the correct result cannot be counted.
  • another solution or an algorithm will determine the number of over-limits when blocking occurs, so as to judge this The secondary data is inaccurate and it is judged as a plugging phenomenon or external interference.
  • the fixed value of the counting time has been set, and the counting time is uniform. Because the small hole voltage is basically stable within a certain range under the normal working state, if the small hole voltage rises or the count is abnormal Circumstances, it proves that the above-mentioned jewel hole has clogging or impurity interference. There are many reasons for the clogging. In most cases, it is because a variety of bacteria are not uniformly mixed, or the jewel hole is not cleaned frequently, and the accumulation of non-counted substances may occur. , Resulting in plugging holes.
  • the voltage interval that is, the voltage is divided into 3 levels, which are normal, high or abnormal.
  • the voltage becomes high it means that the detector of the above-mentioned bacteria counting device has occurred. If the hole is blocked, the higher is the micro-plugging phenomenon (that is, the incomplete plugging phenomenon), the abnormal is the complete plugging, and the normal is the non-clogging state; if the small hole voltage rises or the count is abnormal, or If the baseline is judged to be abnormal, it proves that the above-mentioned gem hole has clogging, impurities or interference.
  • the middle liquid port of the above-mentioned rear tank is under negative pressure, and the rear tank has three channels.
  • the upper and lower channels are connected to the diluent through the valve, and the liquid passing through can be called uncontaminated liquid; the middle port has a straight valve and then Lead to the pump, and then discharged as waste liquid.
  • There will also be an electrode in the middle port (the electrode is made of stainless steel for the outer electrode, and the inner electrode is platinum in the front pool).
  • the middle liquid is negative pressure to ensure the above-mentioned bacterial samples to be counted.
  • the liquid can enter the back tank from the front tank, and complete the counting in the process of passing through the jewel hole.
  • the liquid enters the upper and lower liquid inlets of the back tank and from the back tank
  • the other end of the liquid outlet is used to clean the back tank, for example, the back tank is cleaned by the liquid inlet and outlet of the back tank to enter and flow out of the liquid.
  • the diluent enters the back tank.
  • the liquid that flows out is the waste liquid, which can also contain the sample and the diluent.
  • the upper and lower channels are connected to each other, which is 1 minute 2. 1 is the main channel to the diluent, and 2 is connected to the upper and lower channels of the back tank, and the middle channel, that is, there are electrodes. Channel.
  • the middle liquid port of the back tank is closed with negative pressure.
  • One alternative way is to start applying positive pressure through a pressurizing pump to generate pressure in the back tank and back flush the jewel hole to eliminate the jewel hole The phenomenon of complete blockage or incomplete blockage of the above-mentioned jewel hole occurs.
  • Another alternative is to feed liquid through the upper and lower liquid ports of the back tank to generate pressure in the back tank and backwash the jewel hole to eliminate the complete blockage of the jewel hole or the incomplete jewel hole. Blockage phenomenon.
  • counting pool component may also optionally include:
  • the recoil assembly is used for recoil to eliminate the blockage of the jewel hole when the jewel hole is blocked. Further, the negative pressure of the liquid in the back tank is closed, and the liquid is fed in from the two liquid ports of the back tank respectively to generate pressure in the back tank, backwash the gem hole, and eliminate the blockage of the gem hole. Alternatively, a positive pressure is applied by a pressurizing pump to generate pressure in the back pool to backwash the jewel hole to eliminate the blockage of the jewel hole.
  • counting pool component may also optionally include:
  • the burning component is used to burn and eliminate the blockage of the gem hole when the gem hole is blocked.
  • the above-mentioned burning assembly is used to provide a voltage higher than a predetermined voltage value to the above-mentioned jewel hole through the above-mentioned electrode when the above-mentioned jewel hole is blocked, so as to melt the blocking material in the above-mentioned jewel hole.
  • the computer connected to the above-mentioned bacteria counting device detects the blocked hole, that is, the computer's alarm or prompt message, it can be artificially executed high-pressure burning to eliminate the blocked hole, that is, manually click the operation button on the computer (PC side),
  • Start the high-voltage burning circuit that is, the normal count is DC voltage (relative low-voltage part), when burning, it is DC high-voltage, and the burning method is high and low-voltage fast switching.
  • High-frequency is formed during the high-voltage burning process, which is on and off. At the moment of electricity, arcing discharges will occur on both sides of the gem hole, and the generated electric spark just burns off the plugging material in the gem hole.
  • Another optional method of burning to eliminate blocking holes is to use DC high voltage for burning when the normal count is to provide a stable low pressure component through the switch circuit. Because it is high pressure during burning, the test solution is heated and boiled, and the protein The ingredients are melted and eliminated to achieve the effect of burning and eliminating blocked holes.
  • the voltage of the aforementioned predetermined voltage value is a voltage of 90 volts to 110 volts.
  • the voltage of the aforementioned predetermined voltage value is a voltage of 110 volts.
  • the front pool is made of plastic material.
  • the aforementioned front pool is made of polyoxymethylene material.
  • the above-mentioned back tank is made of plastic material.
  • the above-mentioned back tank is made of polyoxymethylene material.
  • Plastic materials especially polyoxymethylene materials, have good machining performance, which is easy to ensure the size of the front pool and the back pool, and the structure is more stable.
  • the embodiment of the present invention realizes the automatic application of the device for measuring the number of bacteria by using the resistance counting method, solves the problems of slow time and low efficiency of the current bacteria counting, and realizes the effect of fast and accurate bacteria counting.
  • the improved gem hole in the embodiment of the present invention ensures that bacteria can pass through the micro-holes one by one, prevents the overlap phenomenon from affecting the measurement of the number of bacteria, and realizes that the resistance counting method is used to measure the number of bacteria, which is accurate and efficient; the addition of high-pressure recoil and Burning function prevents hole plugging. If there is hole plugging, a high-pressure recoil design is added to the rear pool to eliminate the complete blockage of the above-mentioned jewel hole or the incomplete blockage of the above-mentioned jewel hole.
  • the burning function eliminates hole blocking, that is, when the hole diameter becomes smaller, the phenomenon of complete blockage of the above-mentioned jewel hole or incomplete blockage of the above-mentioned jewel hole is not easy to occur.
  • Targeted design of bacteria counting signal conditioning circuit adding signal conditioning circuit to filter out non-bacterial signals, accurately identify bacteria characteristic signals, and reduce misjudgment.
  • Fig. 1 schematically shows a schematic diagram of a complete machine of a bacteria counting device according to an embodiment of the present invention
  • Figure 1-1 schematically shows a schematic diagram of a movement sample suction process of a whole bacteria counting device according to an embodiment of the present invention
  • Figure 1-2 schematically shows a schematic diagram of adding a sample to be tested into a counting cell assembly of a complete bacteria counting device according to an embodiment of the present invention
  • Fig. 2 schematically shows a structural diagram of a counter cell assembly according to an embodiment of the present invention
  • Figure 2-1 schematically shows a partially symmetrical cross-sectional structure diagram of Figure 2 according to an embodiment of the present invention
  • Figure 2-2 schematically shows a cross-sectional structure diagram of a gem hole according to an embodiment of the present invention
  • FIG. 3 schematically shows a structural diagram of a sampling assembly according to an embodiment of the present invention
  • Figure 3-1 schematically shows a structural schematic cross-sectional view of a matching relationship between a sampling needle and a swab according to an embodiment of the present invention
  • Figure 3-2 schematically shows a schematic structural diagram of a reagent plate according to an embodiment of the present invention
  • Fig. 4 schematically shows a schematic diagram of the working principle of resistance counting according to an embodiment of the present invention
  • Figure 4-1 schematically shows a schematic diagram of the working principle of a liquid path diagram of a bacteria counting device according to an embodiment of the present invention
  • Fig. 5 schematically shows a flow chart of a signal conditioning circuit according to an embodiment of the present invention
  • Fig. 6 is a schematic diagram of an optional Escherichia coli broth culture in an embodiment of the present invention for changes in bacterial counts at different times;
  • Fig. 7 is a schematic diagram of turbidity changes in an optional Escherichia coli broth culture at different times according to an embodiment of the present invention
  • FIG. 8 is a schematic diagram of an optional observation result of broth bacterial culture turbidity and bacterial count changes according to an embodiment of the present invention.
  • Example 9 is a schematic diagram of the results of the growth experiment in Example 3 of the present invention.
  • Figure 10 is a schematic diagram of the results of comparison between 2h and 24h in Example 3 of the present invention.
  • Example 11 is a schematic diagram of the results of the sensitivity coincidence rate in Example 3 of the present invention.
  • the essence of the drug susceptibility test of the present invention is to observe the effect of antibiotics on bacterial growth, metabolism and reproduction. According to the situation of the effect of drugs on bacterial growth, metabolism and reproduction observed in vitro tests (ie Inhibition of bacteria), combined with clinical and pharmacokinetic conditions to infer the effectiveness of future medication.
  • the traditional method monitors the killing effect of antibiotics on the bacteria through the change of the number of bacteria in the liquid or solid medium. It observes the bacterial population. If the accurate number of each individual bacteria can be monitored, rather than the sum of the bacterial populations Change trend detection can quickly detect the effect of its drugs on bacteria early and quickly. Therefore, there will be a major breakthrough in the time of drug susceptibility testing.
  • the technical solution of the present invention creatively invented the method of counting bacteria for rapid detection of bacterial drug sensitivity from another angle.
  • one of the technical solutions of the present invention is to creatively apply the most mature, reliable, fastest, and most economical resistance counting method (Coulter principle) of human blood cell count to the detection method for the inhibition of bacteria by antibacterial drugs.
  • the detection of rapid antibiotic sensitivity test is realized.
  • the traditional method is to determine the minimum inhibitory concentration by changing the turbidity of the broth after the bacteria grow. This takes a long time, usually 18 hours.
  • some commercial companies have optimized it to use a more sensitive turbidity meter or add Redox indicators try to detect the growth or inhibition of bacteria early.
  • the resistance counting method of the present invention can quantitatively count bacterial cells in a short time, and can quickly determine the sensitivity of antibacterial drugs. , Through the analysis and comparison of changes in the number of bacteria, to determine the inhibitory effect of antibiotics on bacteria, and quickly determine the sensitivity of antibiotics. This method is very suitable for rapid drug susceptibility testing, with stable and reliable results.
  • a bacteria counting device which can be used to obtain the current number of bacteria of the bacterial drug mixture and the current number of bacteria of the positive control in the method for detecting the inhibition of bacteria by antibacterial drugs. Quantity.
  • This is a bacteria counting device that uses resistance counting to measure the number of bacteria.
  • a bacteria counting device that uses resistance counting to measure the number of bacteria, that is, design sampling components, counting pool components, and circuit control systems, and combine them
  • the technical solution of the present invention is exactly Regarding the improvement of the size of the bacteria to the gem hole aperture, adjust the gem hole aperture.
  • the punching assembly is used to backflush to eliminate the clogging of the gem hole when the gem hole is blocked, or the burning assembly is used to burn and eliminate the blockage of the gem hole when the gem hole is blocked.
  • the overall schematic diagram of the above-mentioned bacteria counting device is shown in FIG. 1.
  • the above-mentioned bacteria counting device includes a counting pool assembly 1, a sampling assembly 2, a signal conditioning circuit 3 and a housing 4.
  • the counting cell assembly 1 and the sampling assembly 2 are fixedly connected, the signal conditioning circuit 3 is shown in FIG. 5, and the signal conditioning circuit 3 is placed under the ropeway 41 as shown in FIG. 1-2, that is, placed in the bacteria counting device.
  • the signal conditioning circuit 3 is connected to the inner electrode 141 and the outer electrode 142 in the counting cell assembly 1.
  • the signal conditioning circuit 3 includes a signal acquisition board, a main control board, etc., and the housing 4 is located in the counting cell assembly 1, the above The sampling component 2 and the outside of the signal conditioning circuit 3, wherein the sampling component 2 includes a movement mechanism, and the sampling component 2 takes the bacterial liquid to be tested and puts it into the counting pool assembly 1 through the movement mechanism.
  • the sampling assembly 2 drives the cableway 41 to slide.
  • the structure diagram of the counting cell assembly 1 is shown in FIG. 2, and the partial cross-sectional structure diagram of the counting cell assembly 1 is shown in FIG. 2-1.
  • the counting cell assembly 1 includes a gem hole 11, a front pool 12, a rear pool 13, and The inner electrode 141 and the outer electrode 142 connecting the front and rear tanks, the jewel hole 11 is located between the front tank 12 and the back tank 13, and the inner electrode 141 and the outer electrode 142 are connected between the front tank 12 and the back tank 13 .
  • the back tank 13 includes an upper liquid port 131, a middle liquid port 132, and a lower liquid port 133.
  • the liquid in the back tank 13 is under negative pressure, which enables the bacteria entering the front tank 12 to be tested.
  • the liquid completely flows through the jewel hole 11 and completely enters the back tank 13.
  • the effect is most obvious when the middle liquid port 132 of the back tank 13 is under negative pressure, and the upper liquid port 131 and the lower liquid port in the back tank 13 133 are two flushing ports.
  • the wire of the outer electrode 142 is screwed on the metal of the outer wall of the middle liquid port 132.
  • the inner electrode 141 is platinum, which is used to count bacteria in the bacterial sample to be counted.
  • the sample of the liquid to be tested passes through the jewel hole, and the front and rear pool electrodes sense the resistance change, thereby generating a pulse signal in the circuit, and the number of bacteria is measured according to the number of pulses.
  • the measured signal intensity of the inner electrode 141 and the outer electrode 142 is a sensor for counting bacteria. Because the diluent is conductive, when a certain voltage is applied between the two electrodes, there is a certain resistance between the micropores of the jewel hole 11, and the cells are non-conductive. When a cell enters the small hole, it will Change the resistance between the small holes to generate a pulse signal in the circuit. The pulse signal is processed and transmitted to the PC for analysis. According to the number of pulses and pulse amplitude characteristics, the number, size and other parameters of the cells can be measured and performed Statistics, the working principle diagram is shown in Figure 4, through the resistance count of the bacteria counting device, the number of bacteria in the above-mentioned bacteria test solution is obtained and sent to the PC (computer) terminal.
  • the structure diagram of the above-mentioned sampling assembly 2 is shown in FIG. 3, as shown in FIGS. 1, 1-1 and 3.
  • the above-mentioned sampling assembly 2 includes a three-dimensionally movable mechanical arm, a sampling needle 22, a swab 23, and a reagent plate 24. And plunger pump 25 and so on.
  • the movement mechanism included in the sampling assembly 2 includes the mechanical arm and the sampling needle 22.
  • the mechanical arm includes an X-axis moving mechanical arm 21-1, a Y-axis moving mechanical arm 21-2, and a Z-axis moving mechanical arm 21 -3.
  • One end of the sampling needle 22 passes through the swab 23.
  • FIG. 3-1 A partial cross-sectional view of the schematic diagram of the cooperative relationship between the sampling needle 22 and the swab 23 is shown in Figure 3-1.
  • the water pipe 231 enters water and the water outlet pipe 232 exits.
  • the other end of the sampling needle 22 is fixedly connected to the mechanical arm 21-2, and moves with the movement of the mechanical arm 21-2, thereby achieving the target sampling function, that is, the sampling needle 22
  • the bacterial test solution is collected from the reagent plate 24.
  • the plunger pump 25 is connected to the sampling needle 22, and the plunger pump 25 controls the suction and discharge of the bacterial test from the sampling needle 22. Test fluid.
  • the two sampling needles 22 are fixed on the support of the three-dimensional motion mechanical arm, and each sampling needle 22 has one swab 23, or four sampling needles 22 may be used.
  • multiple probes can not only be relatively static, but also can move independently.
  • FIG. 5 is a signal conditioning circuit on a signal processing board that collects tiny signals, and then uploads the number of bacteria through amplification and filtering, signal collection, and so on.
  • FIG. 1 The working process schematic diagram of the sampling component of the above-mentioned bacteria counting device is shown in Figure 1, Figure 1-1 and Figure 1-2.
  • the above-mentioned sampling component 2 quickly moves to the designated position, and the above-mentioned plunger pump 25. Control the initial mixing of the sampling needle 22 on the bacterial test liquid in the reagent plate 24 and the suction of the bacterial test liquid, and then spit the bacterial test liquid into a moving device that follows the sampling assembly 2 In the front pool 12 of the counting pool assembly 1.
  • the schematic diagram of the structure of the gem hole 11 is shown in Figure 2-2.
  • the gem hole diameter 111 (the diameter of the gem hole) of the gem hole 11 is set at 30 microns to 70 microns.
  • the length 112 of the aforementioned gem hole 11 is 30 micrometers to 100 micrometers, preferably 40 micrometers to 70 microns, the aforementioned gem hole diameter 111 is 50 micrometers, and the aforementioned gem hole length 112 is The 50 microns is most suitable for the measurement of bacteria. If the hole of the jewel hole 11 is blocked, the high-pressure recoil design added to the back tank 13 can eliminate the blocking phenomenon.
  • test result data of the resistance count of different gem hole diameters with this design is as follows:
  • the above-mentioned jewel hole diameter 111 is 50 microns
  • the above-mentioned jewel hole length 112 is the best when the jewel hole length 112 is 50 microns, that is, the test result data of resistance counting is the best.
  • the effect is not as good as the effect of 50 microns, but it can still be counted.
  • the inaccuracy caused by the count is relatively speaking. That is, the same counting standard is for different bacterial liquids to be tested, and the trend judgment of the bacterial count of the bacterial liquid to be tested is still accurate. , It shows that other gem hole length and aperture specifications can still measure the magnitude of bacteria count.
  • the high-pressure recoil design is to close the negative pressure of the middle liquid port 132 of the rear tank 13 to allow the upper liquid port 131 and the lower liquid port 133 of the rear tank 13 to enter liquid, and the rear tank 13 generates pressure to backwash the above
  • the jewel hole 11 can eliminate the complete blockage of the jewel hole 11 or the incomplete blockage of the jewel hole 11. If the high pressure recoil fails, the burning function can also be used to eliminate the blockage, that is, to ensure that the hole diameter becomes smaller. The phenomenon of complete blockage of the above-mentioned jewel hole or incomplete blockage of the above-mentioned jewel hole is not easy to occur.
  • the front pool 12 is a four-channel integrated structure, using polyoxymethylene or other plastic materials, and the distance between the two front pool channel openings 121 of the front pool 12 is 18 mm.
  • the internal liquid volume of the tank 12 is greater than 2.5 ml. Using polyoxymethylene materials or other plastic materials can easily ensure that the dimensions of the front pool 12 and the rear pool 13 are accurate and the structure is more stable.
  • the embodiment of the present invention also provides a detection scheme and a solution for eliminating the blockage of the gemstone hole, as shown in the optional complete machine diagram of Figure 1-1.
  • a plunger pump beside the above-mentioned bacteria counting device, which is used for high-pressure recoil to eliminate clogging, or/and high-frequency counting voltage is applied to the electrodes at both ends of the small hole for high-pressure burning to eliminate clogging. See the exemplary method for specific operations.
  • the diluent has conductivity, when a certain voltage is applied between the two electrodes, there is a certain resistance between the micropores.
  • the cell is non-conductive. When a cell enters the small hole, it will change the resistance between the small holes, thereby generating a pulse signal in the circuit, processing the pulse signal and sending it to the PC for analysis, according to the number of pulses and Characteristics such as pulse amplitude can measure the number and size of cells and perform statistics.
  • Targeted design of bacteria counting signal conditioning circuit and acquisition algorithm by amplifying the signal, the effective signal is completely retained, and the effective signal is adjusted to the most beneficial amplification factor for algorithm identification by adjusting the gain; low-pass filtering will filter out high-frequency noise, The over-limit amplitude is filtered out by buffering and limiting.
  • pulse recognition, slope recognition, wave peak detection, wave valley detection, broadband detection and other algorithms accurately identify the signal of bacterial characteristics, so as to obtain the number of bacteria from the pulse signal.
  • the circuit control system or processing device determines the number of the group of pulse signals of the first type as a first number, wherein the first type
  • Each pulse signal in a group of pulse signals is a pulse signal generated by one of the above-mentioned bacteria triggered by the above-mentioned jewel hole; in the case that the above-mentioned pulse signal includes a group of pulse signals of the second type, the above-mentioned circuit control system or processing device will The product of the number of pulse signals of the second type and the predetermined number is determined as the second number, wherein each pulse signal in the pulse signal of the second type is composed of the predetermined number of bacteria At the same time, the pulse signal generated by the above-mentioned gem hole is triggered.
  • the number of bacteria in the bacterial sample to be counted is determined as the first number; where the pulse signal only includes a set of pulses of the second type In the case of a signal, the number of bacteria in the bacterial sample to be counted is determined as the second number; in the case where the pulse signal includes a group of pulse signals of the first type and a group of pulse signals of the second type, The number of bacteria in the bacterial sample to be counted is determined as the sum of the first number and the second number.
  • the circuit control system or processing device in the embodiment of the present invention may determine whether the pulse signal includes a group of pulse signals of the second type through the following steps:
  • the above-mentioned second type of pulse signal is in error with the above-mentioned first type of pulse signal When it is within the range, it is recorded as a valid count, otherwise an error is reported to re-count or the count result is converted according to the error value.
  • the embodiment of the present invention also provides a detection scheme and a solution for the blockage of the gem hole.
  • the embodiment of the present invention further includes: in the case where it is detected that the voltage before the front pool and the back tank exceeds a predetermined threshold, it is determined that the gemstone hole is blocked, wherein: The front pool is the anode, and the back pool is the cathode.
  • the more serious the blockage of the jewel hole the greater the resistance between the two sides of the jewel hole, the greater the voltage between the front pool and the back pool, and the predetermined threshold It can be set according to different measurement requirements (for example, different measurement accuracy) of the number of bacteria.
  • the embodiment of the present invention further includes:
  • the embodiment of the present invention further includes: high pressure recoil and high pressure burning.
  • the middle part is facing the position of the jewel hole, the positive pressure backwash liquid from the middle flushing port, the upper and lower flushing ports are discharged, the combination of the plunger pump and the valve is controlled, and the positive pressure is applied to the back pool through the upper and lower liquid ports.
  • the waste liquid is discharged from the waste liquid port of the front tank, and the waste liquid is drawn through the cooperation of the battery valve and the waste liquid pump.
  • the burning process is to apply a high-frequency counting voltage to the electrodes at both ends of the small hole under a voltage of 110V.
  • the counting voltage is a continuous DC voltage, and when high-voltage burning, it will be designed as Power-on and power-off at a short interval of time will form a high frequency.
  • an arc discharge will occur between the two electrodes.
  • the point where the spark is emitted is the small hole, which will easily
  • the protein and debris are removed, and some instruments are designed to be supplied with a separate AC power supply, which is controlled by a relay or SCR at the front end of the electrode line.
  • high-pressure boiling and heating can also be used to melt the protein to achieve burning to eliminate pore blocking.
  • multi-probe design new probe structure, 2 probes move at the same time, achieving the effect of 4 probes, saving costs; this design is applied to the count of bacteria, and two are driven by a 3-dimensional motion robot arm Sampling a stainless steel probe, as shown in Figure 3, the two above-mentioned sampling needles 22 are separated by 18mm, go to the target plate to aspirate the sample, and spit the liquid to be tested into two of the counting cells, a total of 4 counting cells, then these two The counting cell channel starts to work; then the 3D motion robotic arm drives the two sampling stainless steel probes to the target plate to aspirate the sample, and spit the liquid to be tested into the other two counting cells.
  • the two counting cell channels also start to work until After detecting a channel, go to aspirate the sample and repeat the same action as above.
  • Two probes (the aforementioned sampling needle 22) move at the same time, and multi-channel detection can save waiting time and the cost of adding probe components.
  • the three-dimensional arm moves together with the counting cell to shorten the time from sample suction to sample delivery.
  • the three-dimensional arm moves together with the counting cell and is relatively static.
  • the X, Y, and Z axes of the three-dimensional arm are directly used to stake out to the counting cell after aspiration without excessive 3D actions.
  • the sampling needle 22 is separated by 18mm.
  • the distance between the two test holes 241 of the reagent plate 24 is 9mm, and the two The interval between the sampling needles 22 is exactly twice the distance between the test holes of the above reagent plate, and the stroke is small, which saves time and avoids excessively large distances, which causes the movement stroke to become longer, which increases the running time and reduces the efficiency.
  • the negative pressure will bring the front pool liquid to the back pool, so that through the jewel hole, when there is bacteria passing through the jewel hole, a pulse will be generated
  • the signal (provided is a constant current source, the bacteria changes through the representative resistance and then the voltage changes), the circuit is filtered, the signal is amplified, and then filtered, the signal reaches the single-chip microcomputer, and the single-chip microcomputer performs AD sampling.
  • the single-chip program also has a pulse recognition algorithm for processing After that, it will be uploaded to the PC software.
  • the working flow chart of the liquid flow direction is shown in Figure 4-1.
  • the above-mentioned bacteria counting device is a 4-channel counting liquid path bacteria counting device, because the liquid of each channel The two channels are the same. Take two channels 1 and 2 (CH1 and CH2) as an example to illustrate their working principle: as shown in Figure 4-1, the fluid circuit diagram, before the sample addition and counting, the above Counting cell assembly 1 is cleaned.
  • V1 solenoid valve is combined with a 10ML pump to absorb the diluent (bacteria reagent to be tested), and then the liquid is removed through the cooperation of the V1 solenoid valve, V2 solenoid valve, V3 solenoid valve, and V4 solenoid valve with the pump.
  • the sample addition and counting process is as follows: add liquid to the front pool 12, and then add the diluent through the combination of solenoid valve and pump to dilute, the swab 23 is lifted, and the swab 23 is cleaned: the sampling needle 22 is lifted, The lower bottom surface of the sampling needle 22 is wrapped in the swab 23.
  • the V5 solenoid valve and P1 pump cooperate to discharge the diluent from the V4 solenoid valve channel after flushing the outer wall of the sampling needle 22 to the waste liquid pool, and then pass the V5 solenoid valve Cooperate with the P1 pump to discharge the diluent flushing the inner wall of the sampling needle 22 from the V4 solenoid valve channel to the waste liquid pool.
  • the V4 solenoid valve has 3 ports, one inlet, two outlets, and two outlets (assuming 1 and 2). At least one outlet is connected to the inlet at the same time, so the diluent can be controlled to clean the inner and outer walls of the sampling needle 22. When finished, go to aspirate the liquid to be tested. And the two channels that are first loaded are counted for a certain period of time through the negative pressure of the V6 solenoid valve and the P3 pump. When the scheduled time is reached, the front pool 12 and the back pool 13 are performed through the cooperation of the respective solenoid valves and pumps. Clean and wait for the next injection.
  • the prepared strains are ground on the bottle wall of the AST (antibiotic susceptibility test) broth, mixed well, and the cap is covered and the turbidity is measured with a turbidity meter (BD PhoenixSpec Nephelomter).
  • the turbidity is 0.5 McDonnell units for use. Inoculate the bacteria to be tested according to the inoculation concentration of the CLSI (American Clinical Laboratory Standards Organization) broth dilution method drug susceptibility test, and incubate in an incubator at 37°C (degrees Celsius).
  • Figure 6 shows the changes in the amount of bacteria in Escherichia coli broth culture at different times
  • the bacterial counting method can detect significant differences within 30 minutes of broth culture, which demonstrates the feasibility of the bacterial drug sensitivity detection method.
  • the 10 of the drug susceptibility test tubes (or cups) contain the required antibiotics at the double dilution concentration (different drug concentrations refer to the US CLSI standard); the eleventh tube contains no antibiotics and serves as a positive control (PC); there is no bacterial suspension The twelfth tube was used as a negative control (NC).
  • the prepared strains are ground on the bottle wall of MH (antibiotic susceptibility) broth, mixed well, and the cap is covered and the turbidity is measured with a turbidity meter (BD PhoenixSpec Nephelomter). The turbidity is 0.5 McDonnell units for use.
  • Bacterial liquid preparation pick the spare colonies to prepare a bacterial suspension, the concentration of the bacterial suspension is 0.5 McDonnell units. Add the colony suspension to the broth containing various antibiotics of different concentrations (MH). After each tube of inoculation, the bacterial content is from 1X10 ⁇ 4cfu/ml to 5X10 ⁇ 7cfu/ml, and the optimum is 5X10 ⁇ 6cfu/ml (colony formation Units/ml), that is, the optimum is 5X10 ⁇ 6 colony forming units per ml.
  • the results of observing the turbidity and number of bacteria in broth culture are shown in Figure 8.
  • the abscissa is the concentration of antibiotics, corresponding to the first horizontal value in Table 4, the unit is ⁇ g/ml (micrograms per milliliter), vertical
  • the coordinates are the number of bacteria, and the unit is number/ ⁇ l (number per microliter).
  • the eleventh tube was used as a positive control (PC)
  • the twelfth tube was used as a negative control (NC)
  • the other 10 tubes were test tubes. 12 tubes were tested at each detection time point.
  • the result of the resistance method drug susceptibility test is shown in Figure 8, that is, the inhibition of ampicillin on the bacteria ATCC 25922 Escherichia coli in this example is detected, and the detection result is a minimum inhibitory concentration of 4 ⁇ g/ml.
  • the bacteria in this example were also subjected to three other methods of drug susceptibility tests, namely, according to the French Mérieux VITEK microbial identification drug susceptibility system operation manual to detect the inhibition of ampicillin on the bacteria ATCC 25922 Escherichia coli in this example ,
  • the test result is the minimum inhibitory concentration of 4 ⁇ g/ml (micrograms per milliliter); according to the Etest method, please refer to the operation manual of the Etest drug susceptibility kit of Thermo Fisher Scientific in the United States.
  • the detection of ampicillin against the bacteria ATCC 25922 in this example is a minimum inhibitory concentration of 2 ⁇ g/ml; according to the broth dilution method drug susceptibility test, the method is detailed in the broth dilution method drug susceptibility test standard of the American Association for Clinical Laboratory Standards, and the detection of ampicillin is effective
  • the test result was a minimum inhibitory concentration of 4 ⁇ g/ml, which shows that their results are consistent, and these results are all sensitive.
  • the MIC Minimum Inhibitory Concentration
  • Example 1 and Example 2 it can be seen that the inhibition results of the antibacterial drug of the present invention on the detection method of bacteria inhibition are compared with the results of conventional methods: the results are compared with the results of VITEK (Mérieux drug sensitivity test method), Etest and BMD (broth) Dilution method) results are compared, and the comparison standard is determined according to FDA (U.S. Food and Drug Administration) regulations.
  • VITEK Mérieux drug sensitivity test method
  • Etest and BMD broth Dilution method
  • Inoculate six common clinical strains (ATCC29212, ATCC29213, ATCC27853, ATCC25922, Klebsiella pneumoniae ATCC700603, and Acinetobacter baumannii) in accordance with the requirements of CLSI standards, and record their growth trends to determine whether there is a possibility of determining their growth within 2 hours Sex.
  • Negative control test the uninoculated culture medium, test with a resistance bacteria counter, and record the number of particles;
  • a group of class A drugs were selected to compare the results of standard strains commonly used in clinical practice, and the consistency results of 2h and 24h were investigated. Strains and corresponding antibiotics are shown in Table 5.
  • a reagent board is used to record the results of the 24h experiment and determine the coincidence rate of the sensitivity between 2h and 24h.
  • the result is shown in Figure 11, indicating that the commonly used antibiotics in clinical use resistance counting method for drug sensitivity of 11 common clinical enterobacteria Compared with traditional CLSI results, the test has a higher coincidence rate.
  • a group of class A drugs were selected for comparison of clinically commonly used standard bacterial strains, and the results were checked for consistency between 2h and 24h. The growth trend was recorded to determine whether the growth possibility was confirmed within 2h.
  • the strains and corresponding antibiotics are shown in Table 7.
  • Table 8 (The value in the first row is the concentration of antibiotic drugs, in ⁇ g/ml (micrograms per milliliter), and the value in the second row is the number of bacteria, in units/ ⁇ l (units per microliter))
  • Table 9 (The first row of values is the concentration of antibiotic drugs, in ⁇ g/ml (micrograms per milliliter), and the second row of values is the number of bacteria, in units/ ⁇ l (each per microliter))
  • Table 10 (The first row of values is the antibiotic drug concentration value, in ⁇ g/ml (micrograms per milliliter), and the second row of values is the number of bacteria, in units/ ⁇ l (each per microliter))
  • Table 11 (The first row of values is the concentration of antibiotic drugs, in ⁇ g/ml (micrograms per milliliter), and the second row of values is the number of bacteria, in units/ ⁇ l (each per microliter))
  • Table 12 (The value in the first row is the concentration of antibiotic drugs, in ⁇ g/ml (micrograms per milliliter), and the value in the second row is the number of bacteria, in units/ ⁇ l (units per microliter))

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Abstract

本发明公开了一种抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法。其中，该抑制检测方法包括：在待检测的细菌中加入预定浓度的抗菌药物后设为细菌药物混合物，同时，不加入上述抗菌药物的待检测的细菌设为阳性对照；在与上述抗菌药物的加入时刻相隔到达第一预定时长时，获取上述细菌药物混合物的上述细菌的当前数量以及上述阳性对照的上述细菌的当前数量；根据上述细菌药物混合物的上述细菌的当前数量和上述阳性对照的上述细菌的当前数量的比值确定上述预定浓度的上述抗菌药物对上述细菌的抑制或部分抑制或不抑制。这种方法适合快速药敏试验。

Description

抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法 技术领域
本发明涉及生物医药领域，具体而言，涉及一种抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法。
背景技术
细菌耐药问题越来越严重，并快速在全球范围内广泛传播，各国政府都非常重视，我国也出台了大量的管理规定，合理使用抗生素是应对细菌耐药的最核心工作，快速抗生素敏感试验是重中之重。
鉴于质谱技术和核酸技术的发展，细菌鉴定快速化已经基本实现(当天出结果，1-2小时内完成)，因此研发新的快速抗生素敏感试验就更加急迫和有现实意义。尽快将经验性广谱抗生素治疗切换为靶向治疗是抗生素管理的基石，但目前的药敏试验报告时间制约了临床实践，传统手工方法报告时间太长，因此目前临床上多采用全自动药敏试验方法，其中法国梅里埃公司VITEK系统和美国BD公司Phoenix系统是最快的检测系统，两个系统的可靠性和准确性已被证明，但平均时间Phoenix为12.1小时，Vitek2为9.8小时，这种报告时间，考虑到日常工作流程和作息时间，实际上医生只能第二天针对性选择用药。
目前，为缩短药敏试验报告时间，国内外进行了大量的研究，已经开发了多种方法，如质谱法、流式细胞仪法、振动悬臂微生物细胞称重法、等温微量产热法、磁珠旋转法、微滴检测法、实时PCR法、微阵列法、电导率法、表面等离子体共振法、RNA测序法、噬菌体法、实时显微镜法、微观声波法，但这些技术只是研究阶段，仅进行小样本分析，并且都需要专业技术人员操作，设备昂贵，并且是非传统的专用设备，操作复杂，性能不稳定，成本很高，使用不方便，实用前景不好判断。
快速药敏试验可分为表型法和非表型法两大类。非表型法主要是基于核酸的分子生物学方法，如实时PCR法、微阵列法、RNA测序法、转录组和全基因组测序等，其优点是：1.时间短，如直接阳性血培养物的多重PCR可检测多种耐药基因；2.数字PCR可以实现定量分析；3.明确对应耐药机理。缺点为：1.细菌的耐药机理复杂庞大，如果全面用于临床，工作量过大而影响经济性和快速性；2.耐药基因检测，由于遗传异质性的问题，其基因检测结果同表型的一致性问题还需要大量的验证工作；3.不能检测新的耐药机理，而敏锐发现新的耐药机理是临床迫切需要的；4.尚未在临床实践中应用，不成熟，需进一步的临床观察，要在全球专家认可和标准化后，才可实际临床应用。
表型法药敏试验，直接观察细菌在体外对药物的反应，可直接观察细菌对抗生素的敏感和耐受，传统的表型法药敏试验都已经充分地开发、检验、验证并在临床实践中得到了充分的证明，成为AST方法的参考标准。在基因基础的非表型法药敏试验未完善和明确定论前，这种“中间技术”即那些在传统培养法基础上发展的技术可以更早实施，并有很大的可行性，也正在引起人们期待。
另外，药敏试验中大多需要用到细胞计数仪。细胞计数仪大多是指测量血小板、白细胞、红细胞等个数的仪器。全自动细胞计数仪应用广泛，技术方案库尔特原理分析法一直是国际上认可的测量细胞及颗粒大小的标准对照方法，在血液学分析中一直占有重要位置。
现有技术中应用于细菌的计数装置及方法，还存在以下问题：①目前在市场上还不存在利用电阻计数法测量细菌个数的设备。②现有细胞计数仪宝石孔孔径适宜测量红细胞、白细胞等尺寸较大的，保证细胞能一个一个通过宝石孔；细菌个头较小，不能正常的一个一个通过宝石孔，测量细菌同一时间可能同时经过宝石孔的个数大于等于2个，造成计数不准确。③测量红细胞、白细胞等时，宝石孔如果小于50um的条件下，又会造成现有计数仪器的宝石孔堵孔现象，所以现有技术宝石孔孔径限制在50um以上。④手工操作：现有技术中细菌个数的判定采用显微镜能较容易、准确的测量他们的个数，也可以通过染色法、投影法或者照相制成图片，再按照放大倍数测算，耗费人力物力，用时较多，所以这些计数方法均没有在临床应用中进行大面积的推广。⑤手工操作：细菌的大小和形态随种类变化，有分支状、丝状、纺锤状、链状等，以上几种方法一旦遇到细菌重叠较多的时候利用现有技术便会很难判定个数。
因此，提出一种经济、快速的药敏试验方案显得尤为重要，设计一种快速准确且方便的针对细菌的自动化细菌计数装置也非常重要。
发明内容
本发明旨在提供一种抗菌药物对细菌的抑制检测方法、细菌计数装置及其方法，以至少解决现有技术中抗菌药物对细菌的抑制检测方法出结果时间长的技术问题，医生只能第二天针对性选择用药。
根据本发明的一个方面，提供了一种抗菌药物对细菌的抑制检测方法。该抑制检测方法包括：在待检测的细菌中加入预定浓度的抗菌药物后设为细菌药物混合物，同时，不加入上述抗菌药物的待检测的细菌设为阳性对照；在与上述抗菌药物的加入时刻相隔到达第一预定时长时，获取上述细菌药物混合物的上述细菌的当前数量以及上述阳性对照的上述细菌的当前数量；根据上述细菌药物混合物的上述细菌 的当前数量和上述阳性对照的上述细菌的当前数量的比值确定上述预定浓度的上述抗菌药物对上述细菌的抑制或部分抑制或不抑制。
可选的，在上述细菌药物混合物的上述细菌的当前数量和上述阳性对照的上述细菌的当前数量的比值等于第一预定阈值的情况下，确定出上述预定浓度的上述抗菌药物对上述细菌存在抑制。
可选的，上述第一预定阈值为0至0.6中任一个取值。
可选的，所述第一预定阈值为0至0.4中任一个取值。
可选的，在上述细菌药物混合物的上述细菌的当前数量和上述阳性对照的上述细菌的当前数量的比值等于第二预定阈值的情况下，确定出上述预定浓度的上述抗菌药物对上述细菌存在部分抑制、但未达到抑制；
在与上述抗菌药物的加入时刻相隔到达第二预定时长时，获取上述细菌药物混合物的上述细菌的第二当前数量以及上述阳性对照的上述细菌的第二当前数量，其中，上述第二预定时长大于上述第一预定时长；
在上述细菌药物混合物的上述细菌的第二当前数量与上述阳性对照的上述细菌的第二当前数量的比值等于上述第一预定阈值的情况下，确定出上述预定浓度的上述抗菌药物对上述细菌存在抑制。
可选的，在上述细菌药物混合物的上述细菌的当前数量和上述阳性对照的上述细菌的当前数量的比值大于第二预定阈值的情况下，确定出上述预定浓度的上述抗菌药物对上述细菌不存在抑制。
可选的，上述第一预定时长为0至1.5小时中任一个取值，且上述第一预定时长不等于0小时。
可选的，上述第二预定阈值为0.4至0.8中任一个取值。
可选的，采用电阻计数法获取上述细菌药物混合物的上述细菌的当前数量以及上述阳性对照的上述细菌的当前数量。
可选的，上述抗菌药物对细菌的抑制检测方法包括如下检测步骤：
a.准备上述细菌菌种：将上述细菌菌株接种在培养基上，并在20摄氏度(℃)至40摄氏度(℃)温度下孵育15小时至24小时，备用；
b.制备上述细菌药物混合物以及上述阳性对照，20℃至40℃温度下孵育；
c.经过上述第一预定时长或上述第二预定时长后用上述电阻计数法获取上述细菌药物混合物的上述细菌的当前数量或第二当前数量以及上述阳性对照的上述细菌的当前数量或第二当前数量；
d.在上述细菌药物混合物的上述细菌的当前数量或第二当前数量和上述阳性对照的上述细菌的当前数量或第二当前数量的比值等于0至0.4中任一个取值的情况下，确定出上述预定浓度的上述抗菌药物对上述细菌存在抑制。
可选的，上述步骤a中，将上述细菌菌株接种在血琼脂培养基上，并在37摄氏度(℃)温度下孵育18小时；和/或
上述步骤b中，制备上述细菌药物混合物以及上述阳性对照，37℃温度下孵育；和/或
上述步骤c中，上述第一预定时长为0.5小时或1小时或1.5小时；上述第二预定时长为2小时或2.5小时或3小时。
可选的，采用流式细菌计数法或显微镜细菌计数方法或计数器测定法或电子计数器计数法或活细胞计数法或测定细胞重量法获取上述细菌药物混合物的上述细菌的当前数量以及上述阳性对照的上述细菌的当前数量。
根据本发明的另一个方面，提供了一种细菌计数装置。该细菌计数装置可用于上述抗菌药物对细菌的抑制检测方法中获取细菌药物混合物的细菌的当前数量以及阳性对照的细菌的当前数量。该细菌计数装置包括：采样组件，用于获取待计数细菌样品；计数池组件，包括：宝石孔、前池、后池及电极，其中，上述前池和上述后池通过上述宝石孔相连通，在上述宝石孔两侧各有一个上述电极，上述前池和上述后池之间的液体压力为负压，上述负压用于使得上述待计数细菌样品从上述前池经过上述宝石孔进入上述后池；电路控制系统，用于在检测到上述宝石孔两侧产生的脉冲信号的情况下，根据上述脉冲信号确定上述待计数细菌样品中细菌的数量，其中，上述脉冲信号用于表示上述待计数细菌样品中的细菌通过了上述宝石孔。
可选的，上述电路控制系统包括：第一处理器，用于检测上述脉冲信号，将上述脉冲信号传输给处理设备，并获取上述处理设备发送的上述待计数细菌样品中细菌的数量，其中，上述待计数细菌样品中细菌的数量根据上述脉冲信号所表示的细菌特征数据确定得到；或者
第二处理器，用于检测上述脉冲信号，并根据上述脉冲信号所表示的细菌特征数据确定上述待计数细菌样品中细菌的数量。
可选的，上述电路控制系统包括：第一电源电路，用于通过上述电极向上述宝石孔提供恒定电流，其中，上述脉冲信号是在向上述宝石孔提供上述恒定电流的情况下由一个或多个上述细菌通过上述宝石孔触发产生的脉冲信号；或者
第二电源电路，用于通过上述电极向上述宝石孔提供恒定电压，其中，上述脉冲信号是在向上述宝石孔提供上述恒定电压的情况下由一个或多个上述细菌通过上述宝石孔触发产生的脉冲信号。
可选的，上述宝石孔的直径为第一目标直径范围内的直径，其中，上述第一目标直径范围用于在上述待计数细菌样品中的细菌通过上述宝石孔时一次仅允许一个细菌通过上述宝石孔；或
上述宝石孔的直径为第二目标直径范围内的直径，其中，上述第二目标直径范围用于在上述待计数细菌样品中的细菌通过上述宝石孔时一次允许多个细菌通过上述宝石孔。
可选的，在上述宝石孔的直径为第一目标直径范围内的直径的情况下，上述宝石孔的直径为30微米至70微米，和/或，上述宝石孔的长度为30微米至100微米。
可选的，在上述宝石孔的直径为第一目标直径范围内的直径的情况下，上述宝石孔的直径为40微米至60微米，和/或，上述宝石孔的长度为40微米至70微米。
可选的，在上述宝石孔的直径为第一目标直径范围内的直径的情况下，上述宝石孔的直径为50微米，和/或，上述宝石孔的长度为50微米。
根据本发明的再一个方面，提供了一种细菌计数方法。该细菌计数方法可用于上述抗菌药物对细菌的抑制检测方法中获取细菌药物混合物的细菌的当前数量以及阳性对照的细菌的当前数量。该细菌计数方法包括：将待计数细菌样品加入到计数池组件，其中，上述计数池组件包括：宝石孔、前池、后池及电极，上述前池和上述后池通过上述宝石孔相连通，上述前池和上述后池之间的液体压力为负压，上述负压用于使得上述待计数细菌样品从上述前池经过上述宝石孔进入上述后池，在上述宝石孔两侧各有一个上述电极，在上述电极通电的情况下，上述宝石孔两侧之间具有预定电阻；
检测上述宝石孔两侧是否存在由于上述宝石孔两侧之间的电阻发生变化而产生的脉冲信号，其中，上述脉冲信号用于表示上述待计数细菌样品中的细菌通过了上述宝石孔；
在检测到上述宝石孔两侧产生的脉冲信号的情况下，获取根据上述脉冲信号确定出的上述待计数细菌样品中细菌的数量。
可选的，上述获取根据上述脉冲信号确定出的上述待计数细菌样品中细菌的数量，包括：
将上述脉冲信号传输给处理设备，并获取上述处理设备发送的上述待计数细菌样品中的细菌数量，其中，上述待计数细菌样品中的细菌数量根据上述脉冲信号所表示的细菌特征数据确定得到；或者
根据上述脉冲信号所表示的细菌特征数据确定出上述待计数细菌样品中的细菌数量。
可选的，上述宝石孔的直径为第一目标直径范围内的直径，其中，上述第一目标直径范围用于在上述待计数细菌样品中的细菌通过上述宝石孔时一次仅允许一个细菌通过上述宝石孔；或
上述宝石孔的直径为第二目标直径范围内的直径，其中，上述第二目标直径范围用于在上述待计数细菌样品中的细菌通过上述宝石孔时一次允许多个细菌通过上述宝石孔。
在上述宝石孔两侧各有一个上述电极，因为宝石孔两侧的上述电极形成了恒流源，而上述细菌为非传导性质，故上述细菌在通过上述宝石孔时产生电压脉冲信号，故能由上述方案根据上述脉冲信号确定上述待计数细菌样品中细菌的数量，“脉冲信号”可选为“电压脉冲信号”。
所述脉冲信号所表示的细菌特征数据，包括：通过调理电路放大、增益所述脉冲信号，通过低通滤波滤除噪声，通过缓冲限幅滤除超限幅值；通过脉冲识别、斜率识别、波峰检测、波谷检测、宽带检测等算法识别所述脉冲信号中具有所述细菌特征数据的信号。
上述宝石孔出现堵塞现象(堵孔)，分为完全堵孔与不完全堵孔，即上述宝石孔出现完全堵塞现象及上述宝石孔出现不完全堵塞现象。
如果发生完全堵孔，则计数量会异常而不能计数出正确结果，则采用上述反冲组件或上述灼烧组件以消除堵孔现象；但如果发生不完全堵孔，能显示数据，则直接影响测试结果，从观察计数时间能分辨出是否出现了不完全堵孔现象，即观察计数时间有一个参考值，如果上述细菌计数装置正常工作，微孔通畅，吸入上述待计数细菌样品的时间是固定的，当计数时间延长，则表示上述细菌计数装置的检测器发生了不完全堵孔现象，可选的，另一种方案或者发生堵孔时会有一个算法判断超限的数量，从而判断此次数据不准确，判定为堵孔现象或者受到外界干扰。
由于在装置正常工作状态下，已经设好了计数时间固定值，计数时间是均匀的，由于正常工作状态下，小孔电压基本稳定在一定范围内，如发生小孔电压升高或者计数量异常的情况，则证明上述宝石孔出现堵孔或者杂质干扰现象，堵孔原因有很多，大多数情况是因为多种细菌混合不均匀，或者不经常清 洗宝石孔，则可能会出现非计数物质的堆积，从而产生堵孔。
可选的，还有一种通过电压区间来判断是否堵孔的方式，即电压分3个等级，分别为正常、偏高或异常，当电压变高时，则表示上述细菌计数装置的检测器发生了堵孔现象，偏高是微堵孔现象(即不完全堵孔现象)，异常是完全堵孔，正常即为不堵孔状态；如发生小孔电压升高或者计数量异常的情况，或者判断为基线异常，则证明上述宝石孔出现堵孔或者杂质或者干扰现象。
正常情况下：上述后池的中间液体口为负压，后池有三个通道，上下两个通道通过阀接通稀释液的，里面通过的可称作未被污染的液体；中间口直通阀然后通往泵，然后排出成为废液，中间口这里也会有电极(此电极为外电极不锈钢材质，内电极在前池为铂金)，正常情况下中间液体为负压，保证上述待计数细菌样品的液体能从上述前池进入上述后池，通过上述宝石孔的过程中完成计数，对上述待计数细菌样品完成计数后，通过使液体进入上述后池的上下端进液口及从上述后池的另一端出液口流出的方式清洗上述后池，例如通过上述后池的进出液口进入并流出液体的方式清洗上述后池，例如，进出液的方式中，进入后池的是稀释液，出液的是废液，也可以包含样本与稀释液，上下通道相互连接，为1分2，1为通往稀释液的主通道，2分别接后池上下通道口，中间通道，即有电极的通道。
堵孔情况下：上述后池的中间液体口关闭负压，可选择的一种方式是，开始通过加压泵施加正压，使上述后池产生压力，反冲上述宝石孔，消除上述宝石孔出现完全堵塞现象或上述宝石孔出现不完全堵塞现象。另一种可选择的方式是，通过上述后池的上下两个液体口进液，使上述后池产生压力，反冲上述宝石孔，消除上述宝石孔出现完全堵塞现象或上述宝石孔出现不完全堵塞现象。
进一步地，上述计数池组件还可选的包括：
反冲组件，用于在上述宝石孔出现堵塞现象时反冲而消除上述宝石孔的堵塞。进一步地，上述后池的液体的负压关闭，分别从上述后池的两个液体口进液，使上述后池产生压力，反冲上述宝石孔，而消除上述宝石孔的堵塞。或者，通过加压泵施加正压，使上述后池产生压力，反冲上述宝石孔，而消除上述宝石孔的堵塞。
进一步地，上述计数池组件还可选的包括：
灼烧组件，用于在上述宝石孔出现堵塞现象时灼烧消除上述宝石孔的堵塞。
进一步地，上述灼烧组件用于在上述宝石孔出现堵塞现象时通过上述电极向上述宝石孔提供高于预定电压值的电压，以融化上述宝石孔中的堵孔物质。
与上述细菌计数装置相连接的计算机检测到堵孔之后，即计算机的报警或提示信息，可以人为的去执行高压灼烧去消除堵孔，即人为的点击计算机(PC端)上的操作按钮，启动高压灼烧电路，即正常计数的是直流电压(相对低压部分)，灼烧的时候是直流高压，灼烧的方式是高低压快速切换，高压灼烧过程中会形成高频，在通断电的瞬间，宝石孔两侧会产生拉弧放电，产生的电火花正好灼烧掉宝石孔孔中的堵孔物质。另一种可选的灼烧消除堵孔的方式为，当正常计数是通过开关电路提供稳定的低压成分时，灼烧用直流高压，灼烧时因为是高压，对待测液加热煮沸，将蛋白质成分融化消除以达到灼烧消除堵孔的效果。
进一步地，上述预定电压值的电压为90伏至110伏电压。
进一步地，上述预定电压值的电压为110伏电压。
进一步地，上述前池为塑胶材料。
进一步地，上述前池为聚甲醛材料。
进一步地，上述后池为塑胶材料。
进一步地，上述后池为聚甲醛材料。
塑胶材料，尤其聚甲醛材料机加工性能好，容易保证上述前池和上述后池的尺寸，结构更稳固。
本发明实施例的抗菌药物对细菌的抑制检测方法最突出的有益效果在于：
(1)报告时间的突破。可在获得纯培养物后1-2小时内发报告。
(2)实用性突破。但真正的优势在于，配合细菌鉴定快速化(1-2小时内完成)，考虑到日常工作流程和作息时间，可在获得细菌纯培养的当天实现靶向抗生素治疗，这不仅可以降低病人死亡率和医疗成本，而且还可能减缓细菌耐药性的上升。
(3)技术成熟，稳定，结果可靠。
(4)原理接近国际标准化方案(肉汤稀释法药敏试验)，转化为临床实用性强。
(5)成本低。
(6)易于自动化。
本发明实施例提供的细菌计数装置及方法技术方案具有以下有益效果：
本发明实施例实现了利用电阻计数法测量细菌个数的装置自动化的应用，解决了现今细菌计数的时间慢，效率低的问题，实现了细菌计数速度快且准确的效果。
本发明实施例改进后的宝石孔，保证细菌能一个一个的通过微孔，防止重叠现象影响细菌个数的计量，实现了采用电阻计数法测量细菌个数，准确而高效；增设高压反冲与灼烧功能预防堵孔，如有堵孔现象，后池部分增添了高压反冲设计，消除上述宝石孔出现完全堵塞现象或上述宝石孔出现不完全堵塞现象，如高压反冲失效，还可选用灼烧功能消除堵孔，即确保在孔径变小的情况下，不容易出现上述宝石孔完全堵塞现象或上述宝石孔不完全堵塞现象。
针对性的设计细菌计数信号调理电路；增加信号调理电路，滤除了非细菌信号，精确的将细菌特征的信号识别出来，减少了误判断的情况。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示意性地示出了根据本发明实施方式的一种细菌计数装置整机示意图；
图1-1示意性地示出了根据本发明实施方式的一种细菌计数装置整机的运动吸样过程示意图；
图1-2示意性地示出了根据本发明实施方式的一种细菌计数装置整机的将吸取的待测样本加入到计数池组件中的示意图；
图2示意性地示出了根据本发明实施方式的一种计数池组件的结构示意图；
图2-1示意性地示出了根据本发明实施方式的一种图2的部分对称的剖面结构示意图；
图2-2示意性地示出了根据本发明实施方式的一种宝石孔的剖面结构示意图；
图3示意性地示出了根据本发明实施方式的一种采样组件的结构示意图；
图3-1示意性地示出了根据本发明实施方式的一种采样针和拭子的配合关系的结构示意剖面图；
图3-2示意性地示出了根据本发明实施方式的一种试剂板的结构示意图；
图4示意性地示出了根据本发明实施方式的一种电阻计数的工作原理示意图；
图4-1示意性地示出了根据本发明实施方式的一种细菌计数装置的液路图工作原理示意图；
图5示意性地示出了根据本发明实施方式的一种信号调理电路的流程示意图；
图6为本发明实施例的一种可选的大肠埃希氏菌肉汤培养不同时间菌量变化示意图；
图7为本发明实施例的一种可选的大肠埃希氏菌肉汤培养不同时间浊度变化示意图；
图8为本发明实施例的一种可选的肉汤细菌培养浊度与菌数变化观察的结果示意图；
图9为本发明实施例3中生长实验的结果示意图；
图10为本发明实施例3中2h和24h对比的结果示意图；
图11为本发明实施例3中敏感度符合率的结果示意图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明药敏试验(抗菌药物对细菌的抑制检测方法)的本质就是观察抗生素对细菌生长、新陈代谢和繁殖的影响，根据体外试验观察到的药物对细菌生长、新陈代谢和繁殖的影响的情况(即对细菌的抑制情况)，结合临床和药代学的情况推断未来用药的有效性。传统方法通过液体或固体培养基中细菌数量的变化来监测抗生素对细菌的杀灭作用，其观察的是细菌群体，如果能对每个细菌个体进行准确的数量监测，而非细菌群体数量总和的变化趋势检测，就可早期快速地检测到其药物对细菌的影响，因此药敏试验的时间上必将有重大突破，但由于现有技术缺乏准确、实用和可自动化的个体细菌微观检测技术，因此本发明技术方案从另一个角度创造性的发明了用细菌计数的方法快速进行细菌药物敏感性检测。
具体而言，本发明其中之一的技术方案是将人体血细胞计数最成熟、最可靠、最快速、最经济的电阻计数法(库尔特原理)创造性的运用到抗菌药物对细菌的抑制检测方法中，实现了快速抗生素敏感试验的检测，在细菌生长时加入不同浓度的抗菌药物，可发现一定浓度以上的抗菌药物对细菌的生长繁殖产生抑制，从而测定出最小抑菌浓度。传统的方法是通过细菌生长后肉汤的浊度发生变化来测定最小抑菌浓度，这需要较长时间，通常为18小时，近年一些商业化公司进行优化，用更敏感的比浊仪或加入氧化还原指示剂试图早期发现细菌生长或抑制情况，这些方法也需要10小时才能报告，因为抗菌药物同细菌接触短时间就会产生影响，因此寻找最快地明确药物对细菌作用和影响的方法或方案就非常有实际意义，就可在很短的时间测定细菌对药物的敏感性，本发明用电阻计数法在短时间内可对细菌细胞进行定量的计数，能快速地测定出抗菌药物敏感性，通过细菌数量变化的分析比较，来判断抗生素对细菌 的抑制作用，快速地测定出抗菌药物敏感性。这种方法非常适合快速药敏试验，结果稳定可靠。
在本发明一具体的实施方式中，提供了一种细菌计数装置，该细菌计数装置可用于上述抗菌药物对细菌的抑制检测方法中获取细菌药物混合物的细菌的当前数量以及阳性对照的细菌的当前数量。这是一种用电阻计数法测量细菌个数的细菌计数装置，首先需要发明用电阻计数法测量细菌个数的细菌计数装置，即设计采样组件、计数池组件以及电路控制系统，并将其组合以适合用电阻计数法测量细菌个数的完整的细菌计数装置，在此基础上，由于现有细菌计数装置的宝石孔孔径适宜测量红细胞、白细胞等尺寸较大的细胞，本发明技术方案正是针对细菌尺寸对宝石孔孔径的改进，调整宝石孔孔径，在待计数细菌样品中的细菌通过宝石孔时一次仅允许一个细菌或多个细菌通过宝石孔，当出现宝石孔堵塞异常现象时，反冲组件，用于在上述宝石孔出现堵塞现象时反冲而消除上述宝石孔的堵塞，或灼烧组件，用于在上述宝石孔出现堵塞现象时灼烧消除上述宝石孔的堵塞。
示例性装置
上述细菌计数装置整机示意图如图1所示，上述细菌计数装置包括计数池组件1、采样组件2、信号调理电路3以及外壳4。上述计数池组件1与上述采样组件2固定连接，上述信号调理电路3如图5所示，上述信号调理电路3置于如图1-2所示的索道41下，即置于上述细菌计数装置内，上述信号调理电路3与上述计数池组件1内的内电极141与外电极142连接，上述信号调理电路3包括信号采集板，主控板等，上述外壳4位于上述计数池组件1、上述采样组件2以及上述信号调理电路3的外侧，其中上述采样组件2包括运动机构，上述采样组件2通过上述运动机构采取细菌待测液放入上述计数池组件1中，上述计数池组件1由上述采样组件2带动在上述索道41上滑行。
上述计数池组件1的结构示意图如图2所示，上述计数池组件1的局部剖面结构示意图如图2-1所示，上述计数池组件1包括宝石孔11、前池12、后池13、连通前后池的内电极141以及外电极142，上述宝石孔11位于上述前池12与上述后池13中间，上述内电极141以及上述外电极142连接于上述前池12与上述后池13之间。
如图2-1所示，上述后池13包括上液体口131、中间液体口132以及下液体口133，上述后池13的液体为负压，能使进入上述前池12的上述细菌待测液全部流经上述宝石孔11而完全进入上述后池13，其中上述后池13的上述中间液体口132为负压时效果最为明显，上述后池13中的上述上液体口131及下液体口133为两个冲洗口，上述外电极142的导线拧在上述中间液体口132的外壁金属上，可选的，上述内电极141为铂金，用于对待计数细菌样本中的细菌进行计数。检测时待测液样本通过宝石孔微孔，前后池电极感受电阻变化，从而在电路中产生一个脉冲信号，根据脉冲的个数计量细菌的个数。
上述内电极141以及上述外电极142的测量信号强度为对细菌进行计数的感受器。因为稀释液具有导电性，当在两电极之间加一定的电压时，上述宝石孔11的微孔之间有一定的电阻，而细胞具有非导电性，当有细胞进入小孔时，就会改变小孔间的电阻，从而在电路中产生一个脉冲信号，将脉冲信号进行处理传到PC端分析，根据脉冲的个数以及脉冲幅度等特性就能测出细胞的数量、大小等参数并进行统计，工作原理图如图4所示，通过细菌计数装置的电阻计数，得到上述细菌待测液的细菌数量，传送给PC(电脑)端。
上述采样组件2的结构示意图如图3所示，如图1、图1-1及图3所示，上述采样组件2包括可以三维运动的机械臂、采样针22、拭子23、试剂板24及柱塞泵25等。上述采样组件2包括的运动机构即包括上述机械臂以及上述采样针22，上述机械臂包括X轴运动的机械臂21-1，Y轴运动的机械臂21-2，Z轴运动的机械臂21-3，上述采样针22的一端穿过上述拭子23，上述采样针22和上述拭子23的配合关系的结构示意图的部分剖面图如图3-1所示，需要清洗的时候，由进水管231进水然后出水管232出水，上述采样针22的另一端与上述机械臂21-2固定连接，随上述机械臂21-2的运动而运动，从而实现目标采样功能，即上述采样针22从上述试剂板24中采取到上述细菌待测液，如图1-1所示，上述柱塞泵25与上述采样针22连接，上述柱塞泵25控制上述采样针22的吸吐上述细菌待测液。两个上述采样针22固定于3维运动机械臂的支架上，每一个上述采样针22上分别有一个上述拭子23，也可以采用4个上述采样针22。
而且多个探针间不仅仅能相对静止，也可以独立运动。
上述信号调理电路3的结构示意图如图5所示，为信号处理板上的信号调理电路，采集微小信号，然后经过放大滤波，信号采集等将细菌个数上传。
上述细菌计数装置的采样组件的工作过程示意图如图1、图1-1及图1-2所示，当上述细菌计数装置工作时，上述采样组件2快速移动到指定位置，由上述柱塞泵25控制上述采样针22对上述试剂板24中的上述细菌待测液的初始混匀以及对上述细菌待测液的吸样，然后将上述细菌待测液吐入跟随上 述采样组件2一起运动的上述计数池组件1的上述前池12中。
可选的，上述宝石孔11的结构示意图如图2-2所示，为兼顾细菌的测试信号强度及计数时间，上述宝石孔11的宝石孔孔径111(宝石孔直径)设置在30微米至70微米的范围内，优选40微米至60微米，上述宝石孔11的长度112为30微米至100微米，优选40微米至70微米，上述宝石孔孔径111为50微米，且上述宝石孔的长度112为50微米时最适合细菌的测量，如上述宝石孔11的孔堵塞，则上述后池13增添的高压反冲设计可消除堵孔现象。
对同一待测标准菌液(大概标准菌数2500个/mL)用本设计测试不同宝石孔孔径的电阻计数的测试效果数据如下：
1)当宝石孔长度定为50微米时，不同孔径大小测得的每毫升细菌个数(换算之后的)数据对比如表1所示
表1
序号/孔径 30um 40um 50um 60um 70um
1 1035 2132 2501 1142 936
2 1056 2200 2488 1132 921
3 1034 2150 2493 1135 902
4 1026 2140 2487 1136 910
5 1019 2157 2495 1156 930
6 1034 2169 2510 1158 940
分析证明，宝石孔孔径太小，会发生堵孔现象，造成测得的粒子数下降，如果宝石孔孔径越大，同一时间经过的粒子数增加，造成计数不准确，测得的粒子数也会下降。由上表1可知，上述宝石孔孔径111为50微米时最优，即电阻计数的测试效果数据最好。
2)当宝石孔孔径定为50微米，不同宝石孔长度测得每毫升细菌个数(换算之后的)数据对比如表2所示。
表2
序号/长度 30um 40um 50um 60um 70um 80um 90um 100um
1 1700 1800 2501 1650 1025 951 850 725
2 1750 1800 2488 1645 1030 988 845 730
3 1705 1805 2493 1651 1050 993 851 750
4 1725 1795 2487 1648 1064 987 848 764
5 1736 1796 2495 1646 1036 995 846 736
6 1710 1810 2510 1652 1037 910 852 737
分析证明，宝石孔孔径不变的情况下，如果宝石孔长度越长，同一时间经过的粒子数增加，造成计数不准确，测得的粒子数会下降；如果宝石孔长度过短因为流速很快，也会造成很多粒子测试不到，使得测得的粒子数会下降；由上表2可知，上述宝石孔长度为50微米时最优，即电阻计数的测试效果数据最好。
由上表2可知，上述宝石孔孔径111为50微米，且上述宝石孔长度112为50微米时最优，即电阻计数的测试效果数据最好，其他范围的宝石孔孔径以及宝石孔长度虽然计数效果没有50微米的效果好，但仍然可以计数，造成的计数不准确是相对而言，即同一个计数标准针对不同待测菌液，则对待测菌液的细菌数的趋势判断仍然是准确的，则表明其他宝石孔长度和孔径的规格依然能测量细菌计数的量级。
高压反冲设计具体而言，关闭上述后池13的中间液体口132的负压，使上述后池13的上液体口131及下液体口133进液，上述后池13产生压力，反冲上述宝石孔11，消除上述宝石孔11出现完全堵塞现象或上述宝石孔11出现不完全堵塞现象，如高压反冲失效，还可选用灼烧功能消除堵孔，即确保在孔径变小的情况下，不容易出现上述宝石孔完全堵塞现象或上述宝石孔不完全堵塞现象。
可选的，如图2所示，上述前池12为四通道一体化结构，采用聚甲醛材料或其他塑胶材料，上述前池12的两个前池通道口121的间距为18毫米，上述前池12的内部液体容积大于2.5毫升。采用聚甲醛材料或其他塑胶材料，容易保证上述前池12和上述后池13的尺寸准确，结构更稳固。
作为一种示例，在细菌通过宝石孔的过程中，可能会使得宝石孔产生堵塞，从而导致细菌的计数不准确。为了解决因宝石孔产生堵塞而导致的细菌的计数不准确的问题，本发明实施例还提供了宝石孔堵塞的检测方案和消除方案，如图1-1的可选的整机图所示，其中在上述细菌计数装置旁侧设有一柱塞泵， 用于高压反冲来消除堵孔，或/和在小孔两端的电极上加上高频计数电压用于高压灼烧来消除堵孔，具体的操作见示例性方法。
示例性方法
可选的，激光成型的宝石孔两侧各有一个铂电极，因为稀释液具有导电性，当在两电极之间加一定的电压时，微孔之间有一定的电阻。而细胞具有非导电性，当有细胞进入小孔时，就会改变小孔间的电阻，从而在电路中产生一个脉冲信号，将脉冲信号进行处理传到PC端分析，根据脉冲的个数以及脉冲幅度等特性就能测出细胞的数量、大小等参数并进行统计。
针对性的设计细菌计数信号调理电路与采集算法，通过放大信号，将有效信号完整保留，通过调节增益将有效信号调节到最有利于算法识别的放大倍数；低通滤波将高频噪声滤除，通过缓冲限幅将超限的幅值滤除。通过脉冲识别，斜率识别、波峰检测、波谷检测、宽带检测等算法精确的将细菌特征的信号识别出来，从而从脉冲信号获得细菌数量。
在上述脉冲信号包括第一类型的一组脉冲信号的情况下，上述电路控制系统或处理设备将上述第一类型的一组脉冲信号的个数确定为第一数量，其中，上述第一类型的一组脉冲信号中的每个脉冲信号是由一个上述细菌通过上述宝石孔触发产生的脉冲信号；在上述脉冲信号包括第二类型的一组脉冲信号的情况下，上述电路控制系统或处理设备将上述第二类型的一组脉冲信号的个数与预定数量之间的乘积确定为第二数量，其中，上述第二类型的一组脉冲信号中的每个脉冲信号是由上述预定数量的上述细菌同时通过上述宝石孔触发产生的脉冲信号。
在上述脉冲信号仅包括上述第一类型的一组脉冲信号的情况下，将上述待计数细菌样品中细菌的数量确定为上述第一数量；在上述脉冲信号仅包括上述第二类型的一组脉冲信号的情况下，将上述待计数细菌样品中细菌的数量确定为上述第二数量；在上述脉冲信号包括上述第一类型的一组脉冲信号和上述第二类型的一组脉冲信号的情况下，将上述待计数细菌样品中细菌的数量确定为上述第一数量与上述第二数量之和。
作为一种示例，本发明实施例中的所述电路控制系统或处理设备可以通过以下步骤确定所述脉冲信号是否包括第二类型的一组脉冲信号：
只能通过1个细菌时，是准确计数，2个细菌或3个细菌同时通过宝石孔所产生的第二类型的脉冲信号，上述第二类型的脉冲信号与上述第一类型的脉冲信号在误差范围内时，记为有效计数，否则报错重新计数或按误差值换算计数结果。
作为一种示例，在细菌通过宝石孔的过程中，可能会使得宝石孔产生堵塞，从而导致细菌的计数不准确。为了解决因宝石孔产生堵塞而导致的细菌的计数不准确的问题，本发明实施例还提供了宝石孔堵塞的检测方案和消除方案。
作为一种示例性的宝石孔堵塞的检测方案，本发明实施例还包括：在检测到上述前池和上述后池之前的电压超过预定阈值的情况下，确定出上述宝石孔存在堵塞，其中，上述前池为阳极，上述后池为阴极，上述宝石孔的堵塞越严重，上述宝石孔两侧之间的电阻就越大，上述前池和上述后池之间的电压就越大，上述预定阈值可以根据细菌数量的不同测量要求(例如，不同的测量精度)进行设置。
对于反冲消除堵孔的方案，作为一种示例性的宝石孔堵塞的消除方案，本发明实施例还包括：
对于灼烧消除堵孔的方案，作为一种示例性的宝石孔堵塞的消除方案，本发明实施例还包括：高压反冲和高压灼烧。
如遇堵孔，中间部分正对宝石孔位置，从中间冲洗口正压反冲液体，上下两个冲洗口排出，柱塞泵与阀的组合控制，通过上下液体口对后池施加正压，废液从前池废液口放出，废液通过电池阀与废液泵的配合来抽取。
灼烧的过程是在110V电压下，在小孔两端的电极上加上高频计数电压，正常计数的时候，计数电压是持续提供的直流电压，而在高压灼烧的时候，就会设计成间隔很短时间的通电断电，这样就形成高频，在通断电的瞬间，两个电极之间就会产生拉弧放电，电火花的发出点就是小孔，这样就会很轻易的将蛋白、碎片清除掉，也有的仪器设计成单独交流供电，在电极线的前端通过继电器或者可控硅来控制。可选的，也可通过高压煮沸加热融化蛋白质的方式来实现灼烧消除堵孔现象。
可选的，多探头设计，新型的探针结构，2个探针同时运动，达到了4个探针的效果，节省成本；本设计应用于细菌的计数，由3维运动机械臂带动2个采样不锈钢探头，如图3所示，两个上述采样针22的间隔为18mm，去目标板吸样，将待测液体吐入其中两个计数池，总共4个计数池，这时这两个计数池通道开始工作；然后3维运动机械臂带动2个采样不锈钢探头再去目标板吸样，将待测液体吐入另外两个计数池，这时这两个计数池通道也开始工作，直到有通道检测完毕，再去吸样，重复上述相同 动作。2个探头(上述采样针22)同时运动，多通道检测，既可以节省等待时间又可以节省增加探头组件的成本。
可选的，三维臂与计数池一起运动，缩短吸样到送样的时间。
三维臂与计数池一起运动，相对静止，需要检测时，吸样后直接利用三维臂X、Y、Z轴就近放样至计数池，而不需要过多的3维动作。
可选的，如图3所示，上述采样针22间隔18mm，如图3-2所示的试剂板24结构示意图，上述试剂板24的两个测试孔位241的间距为9mm，两个上述采样针22间隔正好为上述试剂板的测试孔位间距的2倍，行程少，节省时间，避免间距过大，造成运动行程变长而增加运行时间，降低效率。
可选的，如图4所示，加样针将液体注入前池后，负压作用将前池液体带往后池，这样通过宝石孔，当有细菌通过宝石孔电压时，便会产生脉冲信号(提供的是恒流源，细菌通过代表电阻变化然后电压产生变化)，电路经过滤波，信号放大，然后在滤波，信号达到单片机，单片机进行AD采样的过程，单片机程序也有脉冲识别算法，处理好之后，便会上传到PC软件。处理信号的过程：AD采样大概采样10M量级，然后算法处理为若干K，这若干K，即总数、直方图信息。然后上传PC机。
可选的，上述细菌计数装置在工作过程中，液体流向的工作流程图如图4-1液路图所示，上述细菌计数装置为4通道计数液路细菌计数装置，因为每一个通道的液路一致，以其中两路通道1及通道2(CH1及CH2)为例阐述其工作原理：如图4-1液路图所示，在加样计数之前，每一次计数之前，先会对上述计数池组件1进行清洗，V1电磁阀与10ML泵的配合，吸取稀释液(待测细菌试剂)，然后再通过V1电磁阀、V2电磁阀、V3电磁阀、V4电磁阀与泵的配合将液体注入上述前池12，然后通过10毫升泵与V1电磁阀、V2电磁阀及V3电磁阀的配合，对液路施加正压发冲上述宝石孔11，然后通过V8电磁阀、V9电磁阀与P1泵将上述前池12的废液排出完毕，P3泵与V6电磁阀、V7电磁阀将上述后池13的废液排出。其中加样计数过程如下：将液体加入上述前池12，然后通过电磁阀与泵的组合加入稀释液稀释，上述拭子23抬起，上述拭子23清洗过程：上述采样针22抬起，上述采样针22的下底面包裹在上述拭子23内，V5电磁阀与P1泵配合会将来自V4电磁阀通道的冲洗上述采样针22外壁后的稀释液排出到废液池，然后通过V5电磁阀与P1泵配合将来自V4电磁阀通道的冲洗上述采样针22内壁的稀释液排出到废液池。V4电磁阀为3通，一个进口，两个出口，两个出口(假设为1和2)，同一时间至少有一个出口与进口相通，所以可以控制稀释液清洗上述采样针22内壁与外壁，清洗完毕，再去吸取待测液。而先加样的两个通道通过V6电磁阀与P3泵的负压开始进行一定时间的计数，当到达预订时间时，通过各自电磁阀与泵的配合将上述前池12与上述后池13进行清洗，等待下一次的进样。
应当注意，尽管在上文详细描述中提及了装置的若干单元/模块或子单元/模块，但是这种划分仅仅是示例性的并非强制性的。实际上，根据本发明的实施方式，上文描述的两个或更多单元/模块的特征和功能可以在一个单元/模块中具体化。反之，上文描述的一个单元/模块的特征和功能可以进一步划分为由多个单元/模块来具体化。
此外，尽管在附图中以特定顺序描述了本发明方法的操作，但是，这并非要求或者暗示必须按照该特定顺序来执行这些操作，或是必须执行全部所示的操作才能实现期望的结果。附加地或备选地，可以省略某些步骤，将多个步骤合并为一个步骤执行，和/或将一个步骤分解为多个步骤执行。
下面将结合具体实施例进一步描述本发明的抗菌药物对细菌的抑制检测方法的有益效果。
实施例1
目的：寻找肉汤细菌培养时，细菌变化敏感指标，为快速药敏试验确定理论依据，建立细菌药物敏感性检测方法。
材料和方法：
1.菌种制备：
将三种标准菌株(ATCC 25922大肠埃希氏菌、ATCC 25923金黄色葡萄球菌，ATCC 27853铜绿假单胞菌)转种备用、37℃温度下孵育18小时。
2.肉汤制备：
准备好菌株在AST(抗生素药敏试验)肉汤的瓶壁上研磨，混匀，盖好瓶盖用比浊仪(BD公司PhoenixSpec Nephelomter)测浊度，浊度为0.5麦氏单位，备用。按CLSI(美国临床实验室标准组织)肉汤稀释法药敏试验的接种浓度接种待测细菌，放置37℃(摄氏度)温度下培养箱孵育。
3.细菌计数
37℃培养箱孵育，在0分钟，10分钟，30分钟，60分钟，90分钟，120分钟时，用电阻计数法(RC-3000型电阻法(库尔特)颗粒计数器珠海欧美克)进行细胞计数。测2次取平均值，记录数据。
4.浊度测定
37℃培养箱孵育，在0分钟，10分钟，30分钟，60分钟，90分钟，120分钟时，用比浊仪(BD公司PhoenixSpec Nephelomter)测2次浊度，取平均值，记录数据。结果如下表3：
表3肉汤细菌培养浊度与菌数变化观察：
Figure PCTCN2020085275-appb-000001
大肠埃希氏菌肉汤培养不同时间菌量变化如图6所示；
大肠埃希氏菌肉汤培养不同时间浊度变化如图7所示。
结论
1.细菌肉汤培养时，不同方法观察细菌变化差异很大，目前常用的群体细菌生长比浊法敏感性差，在120分钟内不能测定出差异。
2.细菌计数法可在肉汤培养30分钟内测定出显著差异，论证了细菌药物敏感性检测方法的可行性。
实施例2
氨苄西林对ATCC 25922大肠埃希氏菌的抑制检测方法(用电阻计数法)：
材料和方法：
1.菌种制备：
将标准菌株ATCC 25922大肠埃希氏菌转种备用、37℃孵育18小时。
2.肉汤制备：
药敏试验管(或杯)中的10个含有对倍稀释浓度(不同药物浓度参考美国CLSI标准)的所需抗生素；第十一管不含抗生素，作为阳性对照(PC)；没有细菌悬浮液的第十二管作为阴性对照(NC)。
准备好菌株在MH(抗生素药敏)肉汤的瓶壁上研磨，混匀，盖好瓶盖用比浊仪(BD公司PhoenixSpec Nephelomter)测浊度，浊度为0.5麦氏单位，备用。
按CLSI(美国临床实验室标准组织)肉汤稀释法药敏试验的接种浓度接种待测细菌，放置37℃培养箱孵育。
菌液制备：挑取备用菌落制成菌悬液，菌悬液浓度为0.5个麦氏单位。将菌落悬浮液加入含有各种抗生素不同浓度(MH)肉汤中，每管接种后，含菌量为1X10^4cfu/ml至5X10^7cfu/ml，最适为5X10^6cfu/ml(菌落形成单位/毫升)，即最适为5X10^6个菌落形成单位每毫升。
3.电阻计数法(库尔特原理)
37℃温度下培养箱孵育，在0分钟，10分钟，30分钟，60分钟，90分钟，120分钟时，分别用电阻计数法(RC-3000型电阻法(库尔特)颗粒计数器，珠海欧美克)进行细胞计数。测2次取平均值，记录数据。
ATCC 25922大肠埃希氏菌对氨苄西林快速药敏试验的细菌数量变化如下表4所示:
表4
Figure PCTCN2020085275-appb-000002
肉汤细菌培养浊度与菌数变化观察的结果如图8所示，其中横坐标为抗生素药物浓度值，对应附表4中第一横行数值，单位为μg/ml(微克每毫升)，纵坐标为细菌数量值，单位为个/μl(个每微升)。
共12个管进行试验，第十一管作为阳性对照(PC)，第十二管作为阴性对照(NC)，另10管为测试管，每个检测时间点12个管都进行测定。
电阻法药敏试验的结果如图8所示，即检测氨苄西林对本实施例中细菌ATCC 25922大肠埃希氏菌的抑制，检测结果为4μg/ml的最小抑菌浓度。
此外，本实施例中细菌同时还进行另外三种方法的药敏试验，即按照法国梅里埃公司VITEK微生物鉴定药敏系统操作手册检测氨苄西林对本实施例中细菌ATCC 25922大肠埃希氏菌的抑制，检测结果为4μg/ml(微克每毫升)的最小抑菌浓度；按照Etest法，方法详见美国赛默飞公司Etest药敏试剂盒操作手册，检测氨苄西林对本实施例中细菌ATCC 25922大肠埃希氏菌的抑制，检测结果为2μg/ml的最小抑菌浓度；按照肉汤稀释法药敏试验，方法详见美国临床实验室标准协会肉汤稀释法药敏试验标准，检测氨苄西林对本实施例中细菌ATCC 25922大肠埃希氏菌的抑制，检测结果为4μg/ml的最小抑菌浓度，可见它们的结果都一致，这些结果都是敏感的。
结论
1. 60分钟内测定出MIC(最小抑菌浓度)。
2.本发明实施例60分钟内测定出MIC(最小抑菌浓度)与VITEK(梅里埃公司药敏试验法)法，Etest法和肉汤稀释法药敏试验结果一致，即再次论证了本发明细菌药物敏感性检测方法的可行性，并有显著的快速获得细菌药物敏感性结果的有益效果。
由实施例1和实施例2可知，本发明抗菌药物对细菌的抑制检测方法的抑制结果与常规方法结果对比分析:将结果与VITEK(梅里埃公司药敏试验法)，Etest和BMD(肉汤稀释法)结果进行比较，比较标准根据FDA(美国食品药品监督管理局)规定进行确定。
与阳性对照相比，观测细胞计数降低20％、40％、60％或80％时作为药敏结果判断点，并与传统方法一致性进行比较，其结论是当细胞计数降低了40％至60％以上时其正确率达到100％，其中当细胞计数降低了60％以上时最优，不同细菌到达细胞计数降低了40％至60％的时间不同，临床常见菌都可在90至120分钟内完成，大多临床常见菌都可在90分钟内完成。
实施例3
1.生长实验
1.1实验目的
按照CLSI标准要求对临床常用的六种常见菌株(ATCC29212、ATCC29213、ATCC27853、ATCC25922、肺炎克雷伯菌ATCC700603和鲍曼不动杆菌)进行接种，记录其生长趋势确定2h有无确定其生长的可能性。
1.2实验方法
1.2.1阴性对照：对未接菌的培养基进行检测，用电阻细菌计数仪进行检测，记录粒子数；
1.2.2菌株准备：按照CLSI标准要求，挑取0.5麦氏24h内新鲜菌株，取100ul加入到10ml的培养基中；
1.2.3分别在0h/0.5h/1h/1.5h记录结果，同时去掉阴性本底，并进行结果分析。
2.3实验结果见图9，通过生长实验结果可以看到，细菌在2h已经发生了明显的变化，通过电阻计数的方法能够很清晰的捕捉到这种变化，从而在观察检测的角度表明了电阻计数的可行性。
2. 2h和24h药敏对比结果(从两个时间段药敏的结果上进行对比确定电阻法进行细菌药敏的可行性)。
2.1实验目的
针对临床常用标准菌株的分别选取一组A类药物进行结果对比，考察2h及24h一致性结果。菌株及对应抗生素见表5。
表5
菌株名称 抗生素
铜绿假单胞菌(ATCC27853) 头孢他啶
金黄色葡萄球菌(ATCC29213) 红霉素
粪肠球菌(ATCC29212) 青霉素
肺炎克雷伯菌 庆大霉素
鲍曼不动杆菌 美罗培南
2.2实验方法
2.2.1菌株准备：按照CLSI标准要求，按照菌株名称列表5分别挑取0.5麦氏24h内新鲜菌株；
2.2.2接种：分别取100ul 0.5麦氏菌悬液，加入到提前配置好的梯度抗生素48孔板中，每孔加入500ul；
2.2.3孵育及检测：在37℃孵育2h用电阻细菌计数仪进行检测，调整仪器到最佳灵敏状态，细菌计数，并记录24h常规药敏结果，进行对比分析。
2.3实验结果分析
表6
Figure PCTCN2020085275-appb-000003
从图10和表6的结果可以看到，针对于临床常见的五种菌株选取CLSI要求上的首选药物进行抗生素药敏实验，通过电阻计数法(按照2h阳性计数值的60％作为临界点，确定MIC)得到的MIC，与常规药敏实验方法，通过肉眼观察24h的药敏结果进行对比，其差别处于可接受范围，从而证明电阻计数法在细菌药敏实验上的可行性。
3.敏感度符合率
选用临床常见11株肠杆菌(大肠埃希菌2株、摩根摩根菌、志贺氏菌、产气肠杆菌、弗劳地枸橼酸杆菌、肺炎克雷伯菌2株、奇异变形杆菌、阴沟肠杆菌、沙门菌属)山东鑫科生产的96孔肠杆菌药敏试剂板，按照CLSI要求接种试剂板，在37℃下孵育2h，接种两个试剂板，一个试剂板在2h进行细菌计数结果判读，并与阳性对照进行结果对比，初步选用结果值不小于60％的阳性结果值作为阳性值，进行结果记录。一个试剂板用于记录24h的实验结果，并判断2h与24h的敏感度符合率，其结果如图11所示，说明临床常用抗生素针对于临床常见11种肠杆菌用电阻计数法进行药物敏感度测试与传统CLSI结果对比具有较高的符合率。
实施例4
活细菌药敏试验结果及与传统方法的一致性情况
1.1实验目的
针对临床常用标准菌株的分别选取一组A类药物进行结果对比，考察2h及24h一致性结果，记录其生长趋势确定2h有无确定其生长的可能性，菌株及对应抗生素见表7。
表7
菌株名称 抗生素
铜绿假单胞菌(ATCC27853) 头孢他啶
金黄色葡萄球菌(ATCC29213) 红霉素
粪肠球菌(ATCC29212) 青霉素
肺炎克雷伯菌 庆大霉素
鲍曼不动杆菌 美罗培南
1.2实验方法
1.2.1菌株准备：按照CLSI标准要求，按照表7菌株名称列表分别挑取0.5麦氏24h内新鲜菌株。
1.2.2接种：分别取100ul 0.5麦氏菌悬液，加入到提前配置好的梯度抗生素48孔板中，每孔加入500ul。
1.2.3孵育及检测：在37℃孵育2h进行活细菌计数，并记录24h常规药敏结果，进行对比分析。
1.3实验结果分析,
1.3.1实验结果
铜绿假单胞菌对头孢他啶的药敏结果：
活细胞快速药敏结果：MIC＝2；传统药敏结果：MIC＝2。
活细胞快速药敏具体数据见表8。
表8(其中第一横行数值为抗生素药物浓度值，单位为μg/ml(微克每毫升)，第二横行数值为细菌数量值，单位为个/μl(个每微升))
Figure PCTCN2020085275-appb-000004
金黄色葡萄球菌对红霉素的药敏结果：
活细胞快速药敏结果：MIC＝0.25；传统药敏结果：MIC＝0.25。
活细胞快速药敏具体数据见表9。
表9(其中第一横行数值为抗生素药物浓度值，单位为μg/ml(微克每毫升)，第二横行数值为细菌数量值，单位为个/μl(个每微升))
Figure PCTCN2020085275-appb-000005
粪肠球菌对青霉素的药敏结果：
活细胞快速药敏结果：MIC＝1；传统药敏结果：MIC＝2。
活细胞快速药敏具体数据见表10。
表10(其中第一横行数值为抗生素药物浓度值，单位为μg/ml(微克每毫升)，第二横行数值为细菌数量值，单位为个/μl(个每微升))
Figure PCTCN2020085275-appb-000006
肺炎克雷伯菌对庆大霉素的药敏结果：
活细胞快速药敏结果：MIC＝8；传统药敏结果：MIC＝8。
活细胞快速药敏具体数据见表11。
表11(其中第一横行数值为抗生素药物浓度值，单位为μg/ml(微克每毫升)，第二横行数值为细菌数量值，单位为个/μl(个每微升))
Figure PCTCN2020085275-appb-000007
鲍曼不动杆菌对美罗培南的药敏结果：
活细胞快速药敏结果：MIC＝0.12；传统药敏结果：MIC＝0.12。
活细胞快速药敏具体数据表12。
表12(其中第一横行数值为抗生素药物浓度值，单位为μg/ml(微克每毫升)，第二横行数值为细菌数量值，单位为个/μl(个每微升))
Figure PCTCN2020085275-appb-000008
从表7至表12的结果可以看到，针对于临床常见的五种菌株选取CLSI要求上的首选药物进行抗生素药敏实验，通过活细胞计数法(按照2h阳性计数值的60％作为临界点，确定MIC)得到的MIC，与传统药敏实验方法，通过肉眼观察24h的药敏结果进行对比，其差别处于可接受范围，从而证明活细胞计数法在细菌药敏实验上的可行性，与传统方法具有一致性。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (32)

  1. 一种抗菌药物对细菌的抑制检测方法，其特征在于，包括：
    在待检测的细菌中加入预定浓度的抗菌药物后设为细菌药物混合物，同时，不加入所述抗菌药物的待检测的细菌设为阳性对照；
    在与所述抗菌药物的加入时刻相隔到达第一预定时长时，获取所述细菌药物混合物的所述细菌的当前数量以及所述阳性对照的所述细菌的当前数量；
    根据所述细菌药物混合物的所述细菌的当前数量和所述阳性对照的所述细菌的当前数量的比值确定所述预定浓度的所述抗菌药物对所述细菌的抑制或部分抑制或不抑制。
  2. 根据权利要求1所述的方法，其特征在于，在所述细菌药物混合物的所述细菌的当前数量和所述阳性对照的所述细菌的当前数量的比值等于第一预定阈值的情况下，确定出所述预定浓度的所述抗菌药物对所述细菌存在抑制。
  3. 根据权利要求2所述的方法，其特征在于，所述第一预定阈值为0至0.6中任一个取值。
  4. 根据权利要求3所述的方法，其特征在于，所述第一预定阈值为0至0.4中任一个取值。
  5. 根据权利要求2所述的方法，其特征在于，在所述细菌药物混合物的所述细菌的当前数量和所述阳性对照的所述细菌的当前数量的比值等于第二预定阈值的情况下，确定出所述预定浓度的所述抗菌药物对所述细菌存在部分抑制、但未达到抑制；
    在与所述抗菌药物的加入时刻相隔到达第二预定时长时，获取所述细菌药物混合物的所述细菌的第二当前数量以及所述阳性对照的所述细菌的第二当前数量，其中，所述第二预定时长大于所述第一预定时长；
    在所述细菌药物混合物的所述细菌的第二当前数量与所述阳性对照的所述细菌的第二当前数量的比值等于所述第一预定阈值的情况下，确定出所述预定浓度的所述抗菌药物对所述细菌存在抑制。
  6. 根据权利要求5所述的方法，其特征在于，在所述细菌药物混合物的所述细菌的当前数量和所述阳性对照的所述细菌的当前数量的比值大于第二预定阈值的情况下，确定出所述预定浓度的所述抗菌药物对所述细菌不存在抑制。
  7. 根据权利要求1所述的方法，其特征在于，所述第一预定时长为0至1.5小时中任一个取值，且所述第一预定时长不等于0小时。
  8. 根据权利要求5所述的方法，其特征在于，所述第二预定阈值为0.6至0.8中任一个取值。
  9. 根据权利要求1至8任一所述的方法，其特征在于，采用电阻计数法获取所述细菌药物混合物的所述细菌的当前数量以及所述阳性对照的所述细菌的当前数量。
  10. 根据权利要求9所述的方法，其特征在于，所述抗菌药物对细菌的抑制检测方法包括如下检测步骤：
    a.准备所述细菌菌种：将所述细菌菌株接种在培养基上，并在20摄氏度(℃)至40摄氏度(℃)温度下孵育15小时至24小时，备用；
    b.制备所述细菌药物混合物以及所述阳性对照，20℃至40℃温度下孵育；
    c.经过所述第一预定时长或所述第二预定时长后用所述电阻计数法获取所述细菌药物混合物的所述细菌的当前数量或第二当前数量以及所述阳性对照的所述细菌的当前数量或第二当前数量；
    d.在所述细菌药物混合物的所述细菌的当前数量或第二当前数量和所述阳性对照的所述细菌的当前数量或第二当前数量的比值等于0至0.4中任一个取值的情况下，确定出所述预定浓度的所述抗菌药物对所述细菌存在抑制。
  11. 根据权利要求10所述的方法，其特征在于，
    所述步骤a中，将所述细菌菌株接种在血琼脂培养基上，并在37摄氏度(℃)温度下孵育18小时；和/或
    所述步骤b中，制备所述细菌药物混合物以及所述阳性对照，37℃温度下孵育；和/或
    所述步骤c中，所述第一预定时长为0.5小时或1小时或1.5小时；所述第二预定时长为2小时或2.5小时或3小时。
  12. 根据权利要求1至8任一所述的方法，其特征在于，采用流式细菌计数法或显微镜细菌计数方法或计数器测定法或电子计数器计数法或活细胞计数法或测定细胞重量法获取所述细菌药物混合物的所述细菌的当前数量以及所述阳性对照的所述细菌的当前数量。
  13. 根据权利要求12所述的方法，其特征在于，所述细菌药物混合物的所述细菌的当前数量以及所述阳性对照的所述细菌的当前数量通过细菌计数装置获取，所述细菌计数装置包括：
    采样组件，用于获取待计数细菌样品；
    计数池组件，包括：宝石孔、前池、后池及电极，其中，所述前池和所述后池通过所述宝石孔相连通，在所述宝石孔两侧各有一个所述电极，所述前池和所述后池之间的液体压力为负压，所述负压用于使得所述待计数细菌样品从所述前池经过所述宝石孔进入所述后池；
    电路控制系统，用于在检测到所述宝石孔两侧产生的脉冲信号的情况下，根据所述脉冲信号确定所述待计数细菌样品中细菌的数量，其中，所述脉冲信号用于表示所述待计数细菌样品中的细菌通过了所述宝石孔。
  14. 根据权利要求13所述的方法，其特征在于，所述电路控制系统包括：
    第一处理器，用于检测所述脉冲信号，将所述脉冲信号传输给处理设备，并获取所述处理设备发送的所述待计数细菌样品中细菌的数量，其中，所述待计数细菌样品中细菌的数量根据所述脉冲信号所表示的细菌特征数据确定得到；或者
    第二处理器，用于检测所述脉冲信号，并根据所述脉冲信号所表示的细菌特征数据确定所述待计数细菌样品中细菌的数量。
  15. 根据权利要求13所述的方法，其特征在于，所述电路控制系统包括：
    第一电源电路，用于通过所述电极向所述宝石孔提供恒定电流，其中，所述脉冲信号是在向所述宝石孔提供所述恒定电流的情况下由一个或多个所述细菌通过所述宝石孔触发产生的脉冲信号；或者
    第二电源电路，用于通过所述电极向所述宝石孔提供恒定电压，其中，所述脉冲信号是在向所述宝石孔提供所述恒定电压的情况下由一个或多个所述细菌通过所述宝石孔触发产生的脉冲信号。
  16. 根据权利要求13至15中任一项所述的方法，其特征在于，所述宝石孔的直径为第一目标直径范围内的直径，其中，所述第一目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次仅允许一个细菌通过所述宝石孔；或
    所述宝石孔的直径为第二目标直径范围内的直径，其中，所述第二目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次允许多个细菌通过所述宝石孔。
  17. 根据权利要求16所述的方法，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为30微米至70微米，和/或，所述宝石孔的长度为30微米至100微米。
  18. 根据权利要求17所述的方法，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为40微米至60微米，和/或，所述宝石孔的长度为40微米至70微米。
  19. 根据权利要求18所述的方法，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为50微米，和/或，所述宝石孔的长度为50微米。
  20. 根据权利要求13所述的方法，其特征在于，所述细菌药物混合物的所述细菌的当前数量以及所述阳性对照的所述细菌的当前数量通过以下细菌计数方法获取，其特征在于，包括：
    将待计数细菌样品加入到计数池组件，其中，所述计数池组件包括：宝石孔、前池、后池及电极，所述前池和所述后池通过所述宝石孔相连通，所述前池和所述后池之间的液体压力为负压，所述负压用于使得所述待计数细菌样品从所述前池经过所述宝石孔进入所述后池，在所述宝石孔两侧各有一个所述电极，在所述电极通电的情况下，所述宝石孔两侧之间具有预定电阻；
    检测所述宝石孔两侧是否存在由于所述宝石孔两侧之间的电阻发生变化而产生的脉冲信号，其中，所述脉冲信号用于表示所述待计数细菌样品中的细菌通过了所述宝石孔；
    在检测到所述宝石孔两侧产生的脉冲信号的情况下，获取根据所述脉冲信号确定出的所述待计数细菌样品中细菌的数量。
  21. 根据权利要求20所述的方法，其特征在于，所述获取根据所述脉冲信号确定出的所述待计数细菌样品中细菌的数量，包括：
    将所述脉冲信号传输给处理设备，并获取所述处理设备发送的所述待计数细菌样品中的细菌数量，其中，所述待计数细菌样品中的细菌数量根据所述脉冲信号所表示的细菌特征数据确定得到；或者
    根据所述脉冲信号所表示的细菌特征数据确定出所述待计数细菌样品中的细菌数量。
  22. 根据权利要求19或20所述的方法，其特征在于，其特征在于，所述宝石孔的直径为第一目标直径范围内的直径，其中，所述第一目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次仅允许一个细菌通过所述宝石孔；或
    所述宝石孔的直径为第二目标直径范围内的直径，其中，所述第二目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次允许多个细菌通过所述宝石孔。
  23. 一种细菌计数装置，其特征在于，包括：
    采样组件，用于获取待计数细菌样品；
    计数池组件，包括：宝石孔、前池、后池及电极，其中，所述前池和所述后池通过所述宝石孔相连通，在所述宝石孔两侧各有一个所述电极，所述前池和所述后池之间的液体压力为负压，所述负压用于使得所述待计数细菌样品从所述前池经过所述宝石孔进入所述后池；
    电路控制系统，用于在检测到所述宝石孔两侧产生的脉冲信号的情况下，根据所述脉冲信号确定所述待计数细菌样品中细菌的数量，其中，所述脉冲信号用于表示所述待计数细菌样品中的细菌通过了所述宝石孔。
  24. 根据权利要求23所述的装置，其特征在于，所述电路控制系统包括：
    第一处理器，用于检测所述脉冲信号，将所述脉冲信号传输给处理设备，并获取所述处理设备发送的所述待计数细菌样品中细菌的数量，其中，所述待计数细菌样品中细菌的数量根据所述脉冲信号所表示的细菌特征数据确定得到；或者
    第二处理器，用于检测所述脉冲信号，并根据所述脉冲信号所表示的细菌特征数据确定所述待计数细菌样品中细菌的数量。
  25. 根据权利要求23所述的装置，其特征在于，所述电路控制系统包括：
    第一电源电路，用于通过所述电极向所述宝石孔提供恒定电流，其中，所述脉冲信号是在向所述宝石孔提供所述恒定电流的情况下由一个或多个所述细菌通过所述宝石孔触发产生的脉冲信号；或者
    第二电源电路，用于通过所述电极向所述宝石孔提供恒定电压，其中，所述脉冲信号是在向所述宝石孔提供所述恒定电压的情况下由一个或多个所述细菌通过所述宝石孔触发产生的脉冲信号。
  26. 根据权利要求23至25中任一项所述的装置，其特征在于，所述宝石孔的直径为第一目标直径范围内的直径，其中，所述第一目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次仅允许一个细菌通过所述宝石孔；或
    所述宝石孔的直径为第二目标直径范围内的直径，其中，所述第二目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次允许多个细菌通过所述宝石孔。
  27. 根据权利要求26所述的装置，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为30微米至70微米，和/或，所述宝石孔的长度为30微米至100微米。
  28. 根据权利要求27所述的装置，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为40微米至60微米，和/或，所述宝石孔的长度为40微米至70微米。
  29. 根据权利要求28所述的装置，其特征在于，在所述宝石孔的直径为第一目标直径范围内的直径的情况下，所述宝石孔的直径为50微米，和/或，所述宝石孔的长度为50微米。
  30. 一种细菌计数方法，其特征在于，包括：
    将待计数细菌样品加入到计数池组件，其中，所述计数池组件包括：宝石孔、前池、后池及电极，所述前池和所述后池通过所述宝石孔相连通，所述前池和所述后池之间的液体压力为负压，所述负压用于使得所述待计数细菌样品从所述前池经过所述宝石孔进入所述后池，在所述宝石孔两侧各有一个所述电极，在所述电极通电的情况下，所述宝石孔两侧之间具有预定电阻；
    检测所述宝石孔两侧是否存在由于所述宝石孔两侧之间的电阻发生变化而产生的脉冲信号，其中，所述脉冲信号用于表示所述待计数细菌样品中的细菌通过了所述宝石孔；
    在检测到所述宝石孔两侧产生的脉冲信号的情况下，获取根据所述脉冲信号确定出的所述待计数细菌样品中细菌的数量。
  31. 根据权利要求30所述的方法，其特征在于，所述获取根据所述脉冲信号确定出的所述待计数细菌样品中细菌的数量，包括：
    将所述脉冲信号传输给处理设备，并获取所述处理设备发送的所述待计数细菌样品中的细菌数量，其中，所述待计数细菌样品中的细菌数量根据所述脉冲信号所表示的细菌特征数据确定得到；或者
    根据所述脉冲信号所表示的细菌特征数据确定出所述待计数细菌样品中的细菌数量。
  32. 根据权利要求30或31所述的方法，其特征在于，所述宝石孔的直径为第一目标直径范围内的直径，其中，所述第一目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次仅允许一个细菌通过所述宝石孔；或
    所述宝石孔的直径为第二目标直径范围内的直径，其中，所述第二目标直径范围用于在所述待计数细菌样品中的细菌通过所述宝石孔时一次允许多个细菌通过所述宝石孔。
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