Trace pathogen enrichment device and method

Abstract

According to the trace pathogen enrichment device and the detection method provided by the invention, pathogen products separated from uncultured samples extracted by using the device can be subjected to drug sensitivity test after simple culture and bacteria collection treatment, so that the culture process is shortened compared with pathogen diagnosis and identification of clinical infected samples, a drug sensitivity result can be obtained more quickly, and the detection efficiency is improved. And a more timely diagnosis and treatment reference is provided for clinicians.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to devices and methods for enriching trace pathogens. Background Technology

[0002] Infectious diseases are common in clinical practice. Globally, 31.5 million people suffer from bloodstream infections and 5 million from meningitis each year. Half of all women and 1 in 20 men worldwide suffer from urinary tract infections. In addition, chest and abdominal infections are also very common. Pathogen diagnosis is a crucial step in their treatment, and the routine method for pathogen detection is microbial culture. However, this method is not the ideal gold standard because it is time-consuming (1-5 days), results are often unavailable, the positive rate is low (detection rate less than 15%), the results are incomplete or not sensitive enough, and it can be misleading. Furthermore, it is relatively labor-intensive, thus failing to meet clinical needs.

[0003] Bloodstream infections, in particular, often require a 1-5 day diagnostic time between patient sampling and receiving infection results due to the low pathogen concentration in samples (1-10 CFU / mL). Consequently, 19.4 million people worldwide experience severe bloodstream infections annually, with an average mortality rate of 16-40%. It is estimated that 3 million cases of neonatal sepsis occur globally each year, resulting in 1.2 million child deaths annually, a mortality rate of 11-19%. Furthermore, over 75,000 women die annually from postpartum sepsis. In US hospitals, sepsis is not only the most expensive disease to treat but also a leading cause of death. Some reports estimate up to 3.1 million cases of sepsis annually in the US, costing $24 billion, with a mortality rate between 20% and 50%. In Europe, the probability of death from sepsis is even higher, approximately 10-15% higher than in the US. Recent studies indicate that patients with severe or shock-type bloodstream infections have a 7.6% increased risk of death per hour without antibiotic treatment. Survival rates are significantly reduced within the first six hours of identifying hypotension if antibiotics are not administered. Therefore, shortening the diagnosis time can greatly improve patient survival rates. Rapid early diagnosis is thus the most reliable hope for improving the survival rate of patients with infectious diseases and limiting survivor disability. Highly sensitive products that can provide diagnostic information rapidly within a few hours, with small sample sizes, are urgently needed in clinical practice.

[0004] Antimicrobial susceptibility testing is a method used to detect the resistance and sensitivity of bacteria or other microorganisms to antibiotics or other drugs. It is an important means to guide rational drug use in clinical practice, reduce drug abuse, and improve treatment efficacy. Traditional antimicrobial susceptibility testing mainly includes disk diffusion, dilution, and automated antimicrobial susceptibility testing systems. To meet the needs of rapid clinical diagnosis, antimicrobial susceptibility testing needs to develop towards faster and simpler methods. By adopting new technologies and methods, the time cycle of antimicrobial susceptibility testing can be shortened, providing clinicians with more timely medication guidance. However, antimicrobial susceptibility test results are often affected by various factors such as culture conditions and microbial species. Infected samples often need to be cultured before antimicrobial susceptibility testing, and the sample enrichment and isolation culture time is relatively long, making it difficult to quickly select appropriate antimicrobial drugs and reasonable antimicrobial dosages.

[0005] Meanwhile, MALDI-TOF MS is a novel soft ionization biomolecular mass spectrometry technique developed in recent years. Clinical microbiology laboratories have begun to acquire this equipment and widely use it to identify large numbers of bacteria and fungi after culture. The identification results are automatically interpreted, analyzed, and reported, and the specimen is automatically unloaded. The detection cost is low; the instrument only requires sample plates and a dedicated mass spectrometry matrix as consumables, without any other additional reagents, and the technical requirements for staff are not high. Due to its high efficiency and accuracy, and the low cost per experiment, it has become a popular method for microbial identification.

[0006] Currently, MALDI-TOF MS mass spectrometry cannot be used for direct detection of clinical specimens and mixed cultures. The analyte must be a single, pure microorganism. Its high accuracy and reproducibility in identifying microorganisms are based on the detection of microbial ribosomal proteins (relative molecular mass 2000–20000), which are highly abundant proteins immobilized and expressed intracellularly in microorganisms. Therefore, theoretically, different culture conditions should not lead to significant differences in MALDI-TOF MS identification results. However, to achieve more ideal identification results, laboratories currently need to culture the bacteria twice and then pick single-clone colonies for MALDI-TOF MS identification, which is time-consuming and labor-intensive. Furthermore, MALDI-TOF MS identification of bacteria typically requires 10... 5 ~10 7 The bacterial count is measured in colony-forming units (CFU). Therefore, if a direct test is performed, samples from early stages of bloodstream infections or meningitis may not accumulate a sufficient bacterial count for detection, or an excessive number of samples may be required. Furthermore, if the sample contains two or more mixed infections, the test results will usually fail or be erroneous.

[0007] Pathogen culture remains the gold standard for detecting infectious samples. However, pathogen culture has several drawbacks. First, the diagnostic time is long, typically 1-5 days from patient sampling to obtaining infection results. Further time is required for purified pathogens to undergo drug susceptibility testing. During this period, treatment usually involves broad-spectrum, non-targeted antibiotics. While this may be effective in treating the disease, it can lead to multidrug resistance in many microorganisms. Second, the variability of pathogens in a sample results in significant differences in their culture requirements, meaning the dominant bacteria cultured may not be the original state found in the sample. Third, the sample pretreatment process for infectious samples used in drug susceptibility testing or MALDI-TOF MS detection is overly complex. Pathogen culture requires substantial investment of manpower, resources, time, and money, resulting in high costs and demanding skilled operators, hindering its widespread adoption. Finally, bacteria cultured over extended periods may be contaminated, leading to inaccurate results for clinicians. Summary of the Invention

[0008] To improve the above-mentioned technical problems, the present invention provides a new reagent and method for the isolation and enrichment of infectious samples. This method can effectively remove most of the human cells, proteins, sugars, salts and other impurities from uncultured or cultured clinical samples such as blood, cerebrospinal fluid, urine, pleural effusion and peritoneal fluid, to obtain relatively pure live pathogens, which can be used for drug sensitivity testing.

[0009] Therefore, in a first aspect, this disclosure provides a method for obtaining pathogens from a sample, comprising the following steps:

[0010] (1) Provide infectious samples and treatment solutions;

[0011] (2) Provides an apparatus for obtaining pathogens from a sample containing pathogens;

[0012] (3) Inject the infectious sample and the treatment solution into their respective collection tubes;

[0013] (4) Using the device, separate and obtain a suspension containing pathogens; and

[0014] (5) Perform drug sensitivity testing using the suspension containing the pathogen or the culture after enrichment culture of the suspension.

[0015] In one embodiment, the apparatus for obtaining pathogens from a sample containing pathogens includes:

[0016] A centrifugal unit includes a centrifuge and one or more liquid-containing elements, said liquid-containing elements including a plurality of liquid-containing tubes that are fluidly connected to each other and valves between the liquid-containing tubes;

[0017] Gas supply / suction unit, used to supply gas to components containing liquid or generate negative pressure;

[0018] Interface component unit, including fluid connection component and valve mating component; and

[0019] Control unit, used to control the connection between the gas supply / suction unit and the liquid-containing components;

[0020] Its features are,

[0021] The liquid-containing component and the gas supply / suction unit can be connected through the fluid connection component of the interface component unit, and the control unit can establish at least two different fluid connection methods between the liquid-containing pipe and the gas supply / suction unit by controlling the valve engagement component of the interface component unit and the valve engagement between the liquid-containing pipe.

[0022] In one embodiment, the apparatus for extracting pathogens from a sample containing pathogens further includes a positioning mechanism. The positioning mechanism allows the interface component unit to connect to one or more liquid-containing elements at a predetermined position.

[0023] In one embodiment, the liquid-containing element includes a plurality of liquid-containing tubes, each of which is used to contain a liquid, such as a sample or different processing solutions.

[0024] In one embodiment, the apparatus for removing pathogens from a sample containing pathogens further includes a waste liquid tank. The waste liquid tank can be located on the liquid-containing element or separately and in fluid communication with the interface component unit to receive liquid discharged from the interface component unit as needed.

[0025] In one embodiment, the liquid-containing element itself may be fixedly connected to the centrifuge or connected to the centrifuge via a fixing mechanism. Preferably, the liquid-containing element and / or fixing mechanism may further include elements that can cooperate with a positioning mechanism, such that the positioning mechanism enables the liquid-containing element to establish a fluid connection with the interface component unit according to instructions from the control unit.

[0026] In one embodiment, the liquid-containing element is an integrated fluid detection box, comprising: a chip, a liquid-containing tube fixed to the upper surface of the chip with an upper opening and a sealed bottom, a flow channel disposed on the lower surface of the chip and communicating with the bottom of the liquid-containing tube, a valve disposed on the flow channel, and a sealing plate 5 covering the back of the chip; the sealing plate 5 seals the flow channel by tightly covering the lower surface of the chip; the valve has a rotatable flow groove at its bottom, and the valve is used to connect or disconnect the flow channel by rotating the flow groove; the liquid-containing tube includes a first liquid-containing tube, a second liquid-containing tube, a third liquid-containing tube, a fourth liquid-containing tube, a fifth liquid-containing tube, and a sixth liquid-containing tube arranged sequentially; the flow channel includes a first flow channel with both ends communicating with the bottom of the first liquid-containing tube and the upper part of the third liquid-containing tube, a second flow channel with both ends communicating with the bottom of the second liquid-containing tube and the upper part of the third liquid-containing tube, a third flow channel with both ends communicating with the bottom of the fourth liquid-containing tube and the upper part of the third liquid-containing tube, and a ... The system comprises a fourth flow channel connected to the bottom of the fifth liquid-containing pipe and the upper part of the third liquid-containing pipe, and a fifth flow channel whose two ends are respectively connected to the bottom of the sixth liquid-containing pipe and the upper part of the third liquid-containing pipe; the valve includes a first valve disposed on the first flow channel, a second valve disposed on the second flow channel, a third valve disposed on the third flow channel, a fourth valve disposed on the fourth flow channel, and a sixth valve disposed on the fifth flow channel, wherein the bottom of the first valve, the second valve, the third valve, the fourth valve, and the sixth valve are all provided with flow grooves; the first valve is used to connect or disconnect the first flow channel by rotating the flow groove, the second valve is used to connect or disconnect the second flow channel by rotating the flow groove, the third valve is used to connect or disconnect the third flow channel by rotating the flow groove, the fourth valve is used to connect or disconnect the fourth flow channel by rotating the flow groove, and the sixth valve is used to connect or disconnect the fifth flow channel by rotating the flow groove.

[0027] In one embodiment, the first selective lysis buffer and the microbial sedimentation agent are mixed and then placed into the first liquid-holding tube of the detection box; the second selective lysis buffer is placed into the second liquid-holding tube of the detection box; the third selective lysis buffer is placed into the fourth liquid-holding tube of the detection box; the first washing solution is placed into the fifth liquid-holding tube of the detection box; and the second washing solution is placed into the sixth liquid-holding tube of the detection box.

[0028] In one embodiment, the selective lysis buffer is a phosphate (sodium or potassium) buffer or Tris hydrochloride buffer containing 0.01% to 0.045% of one or more of sodium chloride, potassium chloride, potassium bicarbonate, ammonium sulfate, ammonium sulfide, magnesium sulfate, magnesium chloride, disodium EDTA, sodium citrate, PEG4000-8000, PVP, glucose, mannitol, sorbitol, betaine, and BSA, at pH 6-9.

[0029] In one embodiment, the washing solution is a 0.1-10 mM phosphate (sodium or potassium salt) or Tris hydrochloric acid buffer containing 0.001% to 0.045% of one or more of sodium chloride, potassium chloride, magnesium sulfate, magnesium chloride, disodium ethylenediaminetetraacetate, and sodium citrate, with a pH of 6-9.

[0030] In one embodiment, the first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.01% sodium chloride, 0.01% ammonium sulfate, 0.01% sodium citrate, and 0.01% sorbitol; the microbial flocculant is Novec 7500; the second selective lysis buffer is an aqueous solution containing the following components: 5 mM The first wash buffer is an aqueous solution containing the following components: Tris hydrochloric acid buffer (pH 7), 0.01% potassium chloride, 0.01% magnesium sulfate, 0.01% glucose, and 0.01% betaine; the second wash buffer is an aqueous solution containing the following components: Tris hydrochloric acid buffer (pH 7), 0.01% potassium chloride, 0.01% magnesium chloride, 0.01% mannitol, and 0.01% BSA; the third selective lysis buffer is an aqueous solution containing the following components: 5mM potassium phosphate buffer (pH 7.5), 0.01% sodium chloride, 0.01% magnesium sulfate, and 0.01% sodium citrate; the fourth wash buffer is an aqueous solution containing the following components: 9mM potassium phosphate buffer (pH 8), 0.01% potassium chloride, 0.01% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

[0031] In one embodiment, the first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.045% sodium chloride, 0.045% ammonium sulfate, 0.045% sodium citrate, and 0.045% sorbitol; the microbial flocculant is Novec 7500; the second selective lysis buffer is an aqueous solution containing the following components: 5 mM The first wash buffer is an aqueous solution containing the following components: Tris hydrochloric acid buffer (pH 7), 0.045% potassium chloride, 0.045% magnesium sulfate, 0.045% glucose, and 0.045% betaine; the second wash buffer is an aqueous solution containing the following components: Tris hydrochloric acid buffer (pH 7), 0.045% potassium chloride, 0.045% magnesium chloride, 0.045% mannitol, and 0.045% BSA; the third selective lysis buffer is an aqueous solution containing the following components: 5mM sodium phosphate buffer (pH 7.5), 0.045% sodium chloride, 0.045% magnesium sulfate, and 0.045% sodium citrate; the fourth wash buffer is an aqueous solution containing the following components: 9mM potassium phosphate buffer (pH 8), 0.045% potassium chloride, 0.045% magnesium chloride, and 0.045% disodium ethylenediaminetetraacetate.

[0032] In one embodiment, the first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol; the microbial flocculant is Novec 7500; the second selective lysis buffer is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine; the third selective lysis buffer contains the following components. The aqueous solution of the following components is: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA; the first washing solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 7.5), 0.02% sodium chloride, 0.03% magnesium sulfate, and 0.01% sodium citrate; the second washing solution is an aqueous solution containing the following components: 9 mM potassium phosphate buffer (pH 8), 0.03% potassium chloride, 0.03% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

[0033] In one embodiment, the centrifuge unit may also include a counterweight. Alternatively, in one embodiment, the centrifuge unit includes multiple (e.g., two, three, or four) liquid-containing elements. Preferably, these liquid-containing elements are arranged rotationally symmetrically about the centrifuge's axis of rotation.

[0034] In one embodiment, the gas supply / vacuum unit may include an air pump and / or a gas storage tank. Controlled by a control unit, the gas supply / vacuum unit supplies gas to a liquid-containing component or generates negative pressure. In one embodiment, the gas supply / vacuum unit may meterly supply air to a liquid-containing component or generate negative pressure using an air pump. When supplying gas, the gas supply / vacuum unit may include a gas storage tank, thereby providing a gas other than air, such as nitrogen. In this case, the gas supplied by the gas supply unit is used, for example, to purge supernatant in a liquid-containing pipe or to transfer liquid between multiple fluidly connected liquid-containing pipes.

[0035] In one embodiment, the interface assembly unit includes a fluid connection assembly and a valve mating assembly. When the centrifuge stops rotating, the fluid connection assembly of the interface assembly unit connects to the liquid-containing element, thereby establishing fluid communication; the valve mating assembly of the interface assembly unit mats with one or more valves. Preferably, in this case, a positioning mechanism can position the fluid connection assembly and the liquid-containing element and / or the valve mating assembly and the individual valves to each other, thereby establishing a reliable connection. As an example, the positioning mechanism may include mating (electro)magnets, positioning pins / holes, guide plates / rails, etc. One end of the fluid connection assembly of the interface assembly can be fixedly connected to a gas supply / suction unit, and the other end can be detachably or removably connected to the liquid-containing element. This allows gas to be supplied to the liquid-containing element or negative pressure to be generated through suction, according to instructions from the control unit, after the fluid connection assembly and the liquid-containing element are relatively stationary and a connection is established. During this process, the valve mating assembly controls the opening and closing of one or more valves in the liquid-containing element according to instructions from the control unit, thereby transferring fluid between different liquid-containing pipes or transferring it out of the liquid-containing element.

[0036] In one embodiment, the control unit may be electrically connected to the centrifuge and the gas supply / vacuum unit for sending commands. In another embodiment, the control unit may also be electrically connected to the interface component unit and / or the positioning mechanism. The control unit also includes elements for storing commands, which may store commands associated with sample type and directed to the centrifuge unit, the gas supply / vacuum unit, the interface component unit, and / or the positioning mechanism, thereby enabling the user to pre-select the appropriate operation based on the sample type.

[0037] In one embodiment, the control unit is electrically connected to the liquid supply / aspiration unit, the cap opening / closing unit, and the centrifugation unit in order to send commands. The control unit also includes an element for storing commands, which can store commands associated with sample type and the number of operations of the liquid supply / aspiration unit, the cap opening / closing unit, and the centrifugation unit, thereby enabling the user to pre-select the corresponding operation according to the sample type.

[0038] In one embodiment, the liquid-containing element includes a plurality of liquid-containing tubes that are fluidly connected to each other, such as six liquid-containing tubes: a first liquid-containing tube, a second liquid-containing tube, a third liquid-containing tube, a fourth liquid-containing tube, a fifth liquid-containing tube, and a sixth liquid-containing tube. Optionally, the liquid-containing element also includes a waste liquid tank. Alternatively, the waste liquid tank may also be in separate fluid communication with the interface component unit.

[0039] In one implementation, the control unit commands the centrifuge to rotate to a position engaging with the positioning mechanism, aligning the interface assembly unit. This establishes fluid and mechanical connections between the liquid-containing element, the gas supply / vacuum unit, and the interface assembly. Different valves are opened sequentially according to stored instructions in the element's storage, establishing or closing fluid connections between sample tubes. The control unit controls the centrifuge rotation according to pre-stored instructions, and establishes fluid and mechanical connections between the liquid-containing element, the gas supply / vacuum unit, and the interface assembly when the centrifuge is stationary. After opening different valves to establish different fluid connection channels and performing a liquid transfer action in the gas supply / vacuum unit, the interface assembly is disconnected from the liquid-containing element, and the centrifugation process continues. Depending on the needs and pre-stored instructions, any operation establishing a fluid connection or performing a liquid transfer action in the gas supply / vacuum unit can be repeated at least once, for example, two, three, or four times.

[0040] As an example, before the device operates, the control unit mixes the first selective lysis buffer and an optional microbial flocculant and fills the first liquid collection tube. The collected sample is then placed into the third liquid collection tube of the detection cartridge. The second selective lysis buffer is added to the second liquid collection tube, the third selective lysis buffer to the fourth liquid collection tube, the first washing solution to the fifth liquid collection tube, and the second washing solution to the sixth liquid collection tube. The liquid-containing components are then fixed to the centrifuge. The control unit is then activated and a pre-stored instruction is selected based on the sample type to automatically operate the device. For example, when blood, pleural effusion, peritoneal effusion, urine, or cerebrospinal fluid is selected as the sample, the control unit sends an instruction to execute the following steps:

[0041] (1) Rotate the centrifuge to the position where the positioning mechanism is engaged, so that the fluid connection component and valve engagement component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively;

[0042] (2) The valve assembly opens the first valve, starts the air pump to generate negative pressure, thereby drawing the reagent in the first liquid-containing tube into the third liquid-containing tube, then closes the first valve, and then the positioning mechanism and interface assembly unit disengage from the liquid-containing element, and starts the centrifuge for vortex oscillation and first centrifugation; after the first centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve assembly in the interface assembly unit establish fluid connection and mechanical connection with the sample tube and valve respectively, and the fifth valve is opened, the air pump generates positive pressure, and transfers the supernatant of the third liquid-containing tube to the waste liquid pool;

[0043] (3) Close the fifth valve, open the second valve, start the air pump to generate negative pressure, and draw the second selective lysis liquid in the second liquid-containing tube of a predetermined volume into the third liquid-containing tube; close the second valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, and start the centrifuge for vortex oscillation and second centrifugation; after the second centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve connection component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, and the air pump generates positive pressure to transfer the supernatant of the third liquid-containing tube to the waste liquid pool;

[0044] (4) Close the fifth valve, open the third valve, start the air pump to generate negative pressure, and draw the third selective lysis liquid in the fourth liquid collection tube of the predetermined volume into the third liquid collection tube; close the third valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and third centrifugation; after the third centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, the air pump generates positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0045] (5) Close the fifth valve, open the fourth valve, start the air pump to generate negative pressure, and draw the first washing liquid in the fifth liquid collection tube of a predetermined volume into the third liquid collection tube; close the fourth valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and fourth centrifugation; after the fourth centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, start the air pump to generate positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0046] (6) Close the fifth valve, open the sixth valve, start the air pump to generate negative pressure, and draw the second washing liquid in the sixth liquid collection tube of a predetermined volume into the third liquid collection tube; close the fourth valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and fifth centrifugation; after the fifth centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, start the air pump to generate positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0047] (7) Obtain a sample of the extracted pathogen in the third liquid collection tube.

[0048] In one implementation, steps (2), (3), (4), (5), and / or (6) above may be performed independently of each other once or multiple times, such as twice, three times, four times, or five times. For example, for a blood sample, step (5) above may be performed twice and step (6) above may be performed twice.

[0049] In a second aspect, this disclosure provides a method for obtaining pathogens from a sample, comprising the following steps:

[0050] (1) Provide sample tubes and processing solutions, including infectious samples;

[0051] (2) Provide an apparatus for obtaining pathogens from a sample containing pathogens according to the second aspect of this disclosure;

[0052] (3) Place one or more sample tubes in the element that houses the sample tubes and fill the respective processing solution reservoirs with the processing solution;

[0053] (4) Using the device, separate and obtain a suspension containing pathogens; and

[0054] (5) Perform drug sensitivity testing using the suspension containing the pathogen or the culture after enrichment culture of the suspension.

[0055] In one embodiment, the apparatus for obtaining pathogens from a sample containing pathogens includes:

[0056] A centrifugation unit includes a centrifuge and one or more components that house sample tubes;

[0057] The mixing unit is used to invert and mix the sample tube.

[0058] Oscillating unit for vortex oscillation of sample tubes;

[0059] A liquid supply / absorption unit for supplying liquid to and / or aspirating liquid from a sample tube;

[0060] The cap opening and closing unit is used to open and close the cap of the sample tube;

[0061] A treatment fluid reservoir for holding the treatment fluid; and

[0062] The control unit is used to control the working sequence of the liquid supply / suction unit, the lid opening / closing unit, and the centrifugation unit.

[0063] Its features are,

[0064] The control unit controls the working sequence of the liquid supply / suction unit, the cap opening / closing unit, and the centrifugation unit in a working cycle according to the sample type.

[0065] In one embodiment, a sample tube receiving element can accommodate multiple independent sample tubes, such as two, three, or four sample tubes. Preferably, the sample tube receiving element may further include a sample tube securing mechanism. For example, the sample tube can be clamped in the sample tube receiving element by the sample tube securing mechanism, or secured in the sample tube receiving element by a threaded engagement, provided that the sample tube does not rotate axially when the cap opening and closing unit opens and closes the cap of the sample tube.

[0066] In one embodiment, the apparatus for extracting pathogens from a sample containing pathogens further includes a positioning mechanism. The positioning mechanism can align the liquid supply / aspiration unit at a predetermined position with an element that houses a sample tube or one or more sample tubes thereon.

[0067] In one embodiment, the apparatus for removing pathogens from a sample containing pathogens further includes a waste liquid tank. The waste liquid tank may be in fluid communication with a liquid supply / suction unit to receive liquid discharged from the liquid supply / suction unit as needed.

[0068] In one embodiment, the liquid supply / suction unit may include a first liquid filling gun, a second liquid filling gun, a first liquid dispensing gun, and a second liquid dispensing gun, wherein the first liquid filling gun is connected to a first treatment liquid storage tank, a second treatment liquid storage tank, a third treatment liquid storage tank, a first washing liquid storage tank, and a second washing liquid storage tank, and the second liquid filling gun is connected to a microbial sedimentation agent storage tank.

[0069] In one embodiment, the liquid supply / aspiration unit may include an array of dispensing nozzles and an array of dispensing nozzles, wherein the array comprises at least two rows, such as three, four, five, or six rows of first dispensing nozzles, second dispensing nozzles, first dispensing nozzles, and second dispensing nozzles. In one embodiment, the first dispensing nozzle, second dispensing nozzle, first dispensing nozzle, and second dispensing nozzle are arranged sequentially. This arrangement provides the possibility of processing multiple batches of samples simultaneously. For example, for the same patient, multiple samples (including but not limited to: blood, pleural effusion, peritoneal fluid, urine, or cerebrospinal fluid) can be collected, with each sample placed in a separate sample tube. The dispensing nozzle array is individually controlled by a control unit to process each sample separately, thereby simultaneously obtaining a refined sample for analysis at the end of processing.

[0070] In one embodiment, the apparatus for obtaining pathogens from a sample containing pathogens further includes a processing solution reservoir for containing processing solutions. The processing solution reservoir includes a microbial flocculant reservoir, a first processing solution reservoir, a second processing solution reservoir, a third processing solution reservoir, a first washing solution reservoir, and a second washing solution reservoir. Therefore, the processing solution may include a microbial flocculant, a first processing solution, a second processing solution, a third processing solution, a first washing solution, and a second washing solution. The microbial flocculant reservoir can contain a microbial flocculant, the first processing solution reservoir can contain a first selective lysis buffer, the second processing solution reservoir can contain a second selective lysis buffer, the third processing solution reservoir can contain a third selective lysis buffer, the first washing solution reservoir can contain a first washing solution, and the second washing solution reservoir can contain a second washing solution.

[0071] In one embodiment, the centrifugation unit may also include a counterweight. Alternatively, in one embodiment, the centrifugation unit includes multiple (e.g., two, three, or four) elements for receiving sample tubes. Preferably, these elements for receiving sample tubes are arranged rotationally symmetrically about the centrifuge's axis of rotation.

[0072] In one embodiment, the element for accommodating sample tubes may be a holder. In this case, the holder has at least one receiving portion capable of holding an individual sample tube. The holder may be, for example, a centrifuge basket. The centrifuge basket may be a disposable centrifuge basket or a reusable centrifuge basket. There are no particular limitations on the individual sample tubes; they may be clinically commonly used laboratory sample tubes, as long as they can be inserted into and secured in the holder. Similarly, there are no particular limitations on the holder, as long as it can be reliably connected to the centrifuge and secure the sample tube inserted therein.

[0073] In one implementation, the sample tube may or may not have a cap. As an example, the sample tube may be a routine clinical sampling tube. For handling capped sample tubes, the device for extracting pathogens from samples containing pathogens may also include a cap-opening / closing unit that cooperates in opening and closing the cap. As an example, the cap-opening / closing unit may include a single-head, double-head, or multi-head capping mechanism. Upon command from a control unit, the capping mechanism opens or closes the cap of the sample tube, thereby protecting the sample within the tube from leakage or contamination.

[0074] In one embodiment, the liquid supply / aspiration unit may include a pipette arm, such as a pipette arm comprising a pipette tip. In one embodiment, the pipette arm includes a pipette tip having a tip. According to instructions from the control unit, the pipette arm may insert its pipette tip into a sample tube to a predetermined depth. In one embodiment, the pipette arm includes multiple pipettes, each for aspirating different liquids, thereby avoiding cross-contamination. In one embodiment, the pipette tip may be detachable or replaceable. In one embodiment, the liquid supply / aspiration unit may include a first dispensing pipette, a second dispensing pipette, a first scavenging pipette, and a second scavenging pipette, wherein the first dispensing pipette is communicatable to a first treatment liquid reservoir, a second treatment liquid reservoir, a third treatment liquid reservoir, a first washing liquid reservoir, and a second washing liquid reservoir, and the second dispensing pipette is communicatable to a microbial flocculant reservoir.

[0075] In one implementation, a positioning mechanism can position the liquid supply / aspiration unit and one or more sample tubes relatively stationary relative to each other. As an example, the positioning mechanism may include cooperating (electro)magnets, positioning pins / holes, guide plates / rails, etc. The liquid supply / aspiration unit can be connected to a processing fluid reservoir, thereby allowing, according to instructions from a control unit, the same or different liquids from the processing fluid reservoir to the same or different sample tubes, respectively.

[0076] In one embodiment, a capped sample tube is placed in and secured within an element that houses the sample tube. The capped sample tube may contain only the collected sample or may already contain a first selective lysis buffer mixed into the sample. In the case of containing only the collected sample, the processing fluid reservoir includes a first processing fluid reservoir, and the first processing fluid reservoir is in fluid communication with the pipette of the liquid supply / aspiration unit.

[0077] In one implementation, the control unit controls the centrifuge to oscillate or rotate according to pre-stored instructions, and alternately operates the cap-opening / closing unit and the liquid supply / absorption unit when the centrifuge is stationary, thereby opening the sample tube cap, aspirating the supernatant and adding new processing liquid, closing the sample tube cap, and then continuing centrifugation. Depending on the needs and pre-stored instructions, any step of opening the sample tube cap, aspirating the supernatant and adding new processing liquid, closing the sample tube cap, and then continuing centrifugation can be repeated at least once, for example, two, three, or four times.

[0078] In one embodiment, the treatment solution may include a microbial flocculant, a first treatment solution, a second treatment solution, a third treatment solution, a first washing solution, and a second washing solution. As an example, before the device is operated, the microbial flocculant, the first selective lysis buffer, the second selective lysis buffer, the third selective lysis buffer, the first washing solution, and the second washing solution can be respectively placed into a microbial flocculant reservoir, a first treatment solution reservoir, a second treatment solution reservoir, a third treatment solution reservoir, a first washing solution reservoir, and a second washing solution reservoir. Then, the sample tube is fixed in a fixed position, and the number of times liquid is added and the supernatant is aspirated is set according to the sample type and pre-stored instructions in the control unit. The control unit is then activated to allow the device to operate automatically.

[0079] For example, when blood is selected as the sample, the control unit sends instructions to perform the following steps:

[0080] (1) Use the cap opening and closing unit to place the sample tube in a fixed position, then use the identification unit to scan the identification code on the sample tube, and then use the cap opening and closing unit to put the sample tube into the shaking unit and shake the sample tube inverted. Start the cap opening and closing unit to open the cap of the sample tube, use the liquid supply / suction unit to draw about 3 mL of sample from the sample tube and supply it to the centrifuge tube through the first liquid dispensing gun, then use the positioning mechanism to position the liquid supply / suction unit to the first processing liquid storage tank and supply the first selective lysis solution to the centrifuge tube through the first liquid dispensing gun, then use the positioning mechanism to position the liquid supply / suction unit to the microbial flocculant storage tank and supply the microbial flocculant to the sample tube through the second liquid dispensing gun, start the cap opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, then move the centrifuge tube to the centrifuge unit and start the centrifuge for the first centrifugation, after the first centrifugation is completed, start the cap opening and closing unit to open the cap of the centrifuge tube, use the first liquid dispensing gun to draw the supernatant in the centrifuge tube and discard it to the waste liquid tank;

[0081] (2) Position the liquid supply / suction unit to the second processing liquid storage tank through the positioning mechanism and supply the second selective lysis liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube. Move the centrifuge tube to the oscillation unit for vortex oscillation. Then move the centrifuge tube to the centrifuge unit and start the centrifuge for the second centrifugation. After the second centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube. Use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0082] (3) Position the liquid supply / suction unit to the third processing liquid storage tank through the positioning mechanism and supply the third selective lysis liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube. Move the centrifuge tube to the oscillation unit for vortex oscillation. Then move the centrifuge tube to the centrifuge unit and start the centrifuge for the third centrifugation. After the third centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube. Use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0083] (4) Position the liquid supply / suction unit to the first washing liquid storage tank through the positioning mechanism, and supply the first washing liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, and then move the centrifuge tube to the centrifuge unit. Start the centrifuge for the fourth centrifugation. After the fourth centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube, and use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0084] (5) Position the liquid supply / suction unit to the second washing liquid reservoir through the positioning mechanism, and supply the second washing liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, and then move the centrifuge tube to the centrifuge unit. Start the centrifuge for the fifth centrifugation. After the fifth centrifugation is completed, start the opening and closing unit to open the cap of the sample tube, and use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it into the waste liquid pool.

[0085] (6) Use the cap opening and closing unit to close the cap of the centrifuge tube and obtain the pathogen analysis sample.

[0086] In one implementation, step (4) and / or step (5) may be performed once, twice, three times, or four times. For example, for a blood sample, step (4) may be performed twice and step (5) may be performed twice.

[0087] In various aspects and embodiments herein, the centrifuge may have an angular rotor oscillating centrifugation module, or the element that houses the sample tubes may be an angular rotor oscillating centrifugation module.

[0088] Beneficial effects

[0089] The technical solution of this invention allows for the extraction of pathogen products isolated from uncultured samples, followed by simple culturing (only one step of liquid culture) and bacterial collection for drug susceptibility testing. Cultured samples such as blood, cerebrospinal fluid, and pleural / peritoneal fluid can be directly used for drug susceptibility testing after simple concentration. Compared to pathogen diagnosis and identification of clinical infection samples, the technical solution of this invention shortens the culture process, enabling faster acquisition of drug susceptibility results and providing clinicians with more timely diagnostic and treatment references.

[0090] The automated sample separation and processing system provided by this invention requires no manual operation, no complicated personnel training or practice, saving time, effort and manpower. Attached Figure Description

[0091] Figure 1 A schematic diagram of some structures in the device used in Example 2 is shown; 1 is the identification unit, 2 is the shaking unit, 3 is the oscillation unit, 4 is the centrifugation unit, 5 is the treatment liquid storage tank, 6 is the waste liquid tank, and 7 is the heating unit.

[0092] Figure 2 The inhibition zones of various drugs in the drug susceptibility test are shown for a sample (Kpn isolation group E3) containing an initial concentration of 1000 Klebsiella pneumoniae / μL and a positive control. Among them, A and C are the inhibition zones of various drugs against the sample group, and B and D are the inhibition zones of various drugs against the positive control group.

[0093] Figure 3 The inhibition zones of various drugs in the drug susceptibility test are shown for a sample (Kpn isolation group E2) containing an initial concentration of 100 Klebsiella pneumoniae / μL and a positive control. Among them, A and C are the inhibition zones of various drugs against the sample group, and B and D are the inhibition zones of various drugs against the positive control group.

[0094] Figure 4 The inhibition zones of various drugs in the drug susceptibility test are shown for a sample (Efm isolation group E3) containing an initial concentration of 1000 CFU / μL of Enterococcus faecalis and a positive control. In the figure, A represents the inhibition zones of various drugs against the sample group, and B represents the inhibition zones of various drugs against the positive control group.

[0095] Figure 5 The inhibition zones of various drugs in the drug susceptibility test are shown for a sample (Efm isolation group E2) containing an initial concentration of 100 CFU / μL of Enterococcus faecalis and a positive control. In the figure, A represents the inhibition zones of various drugs against the sample group, and B represents the inhibition zones of various drugs against the positive control group.

[0096] Figure 6 The inhibition zones of various drugs in the drug susceptibility test are shown for the sample (Sau isolation group E3) containing an initial concentration of 1000 Staphylococcus aureus / μL and the positive control. Among them, A and C are the inhibition zones of various drugs against the sample group, and B and D are the inhibition zones of various drugs against the positive control group.

[0097] Figure 7 The inhibition zones of various drugs in the drug susceptibility test are shown for the sample (Sau isolation group E2) containing an initial concentration of 100 Staphylococcus aureus / μL and the positive control. Among them, A and C are the inhibition zones of various drugs against the sample group, and B and D are the inhibition zones of various drugs against the positive control group. Detailed Implementation

[0098] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0099] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0100] Sample preparation

[0101] First, count Klebsiella pneumoniae, Enterococcus faecalis, or Staphylococcus aureus, then dilute with sterile water to 100,000 (E5) CFU / μL, and then serially dilute 10 times to 10,000 (E4) CFU / μL, 1,000 (E3) CFU / μL, 100 (E2) CFU / μL, and 10 (E1) CFU / μL.

[0102] Experimental and control groups were set up for the isolation, enrichment, extraction, and detection of bloodstream infection samples:

[0103] Experimental group: Each sample tube was filled with 6 mL of pathogen-free negative blood. The three sample tubes were divided into three groups of one tube each. 6 μL of Klebsiella pneumoniae (or Enterococcus faecalis / Staphylococcus aureus) at a concentration of 1000 (E3) CFU / μL, 100 (E2) CFU / μL, and 10 (E1) CFU / μL were added to each group, respectively. The mixture was then used as a simulated positive sample for later use. At the same time, a sample tube containing 6 mL of pathogen-free negative blood was prepared as a negative extraction control group (BNTC).

[0104] Control group: Six centrifuge tubes containing 250 μL of pathogen lysis buffer and 50 μL of microbial sedimentation agent were divided into three groups of two. 3 μL of Escherichia coli (or Enterococcus faecalis / Staphylococcus aureus) at concentrations of 1000 (E3) CFU / μL, 100 (E2) CFU / μL, and 10 (E1) CFU / μL were added to each group, respectively. The mixture was then used as the control sample for later use.

[0105] Example 1: Automated process for isolating and extracting pathogens 1

[0106] 1. Prepare the corresponding treatment solution according to the following composition:

[0107] The first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol.

[0108] Microbial settling aid: Novec7500.

[0109] The second selective lysis buffer is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine.

[0110] The third selective lysis buffer is an aqueous solution containing the following components: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA.

[0111] The first washing solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 7.5), 0.02% sodium chloride, 0.03% magnesium sulfate, and 0.01% sodium citrate.

[0112] The second washing solution is an aqueous solution containing the following components: 9 mM potassium phosphate buffer (pH 8), 0.03% potassium chloride, 0.03% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

[0113] 2. Mix the first selective lysis buffer and the microbial sedimentation agent and place them into the first collection tube of the detection kit. Place the second selective lysis buffer into the second collection tube of the detection kit, and the third selective lysis buffer into the fourth collection tube of the detection kit. Place the first washing buffer into the fifth collection tube of the detection kit, and the second washing buffer into the sixth collection tube of the detection kit. Place 3 mL of sample into the third collection tube of the detection kit. Then fix the detection kit to the centrifuge spindle.

[0114] 3. Turn on the control unit and automatically execute the following procedure:

[0115] (1) Rotate the centrifuge to the position where the positioning mechanism is engaged, so that the fluid connection component and valve engagement component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively;

[0116] (2) The valve assembly opens the first valve, starts the air pump to generate negative pressure, thereby drawing the reagent in the first liquid-containing tube into the third liquid-containing tube, then closes the first valve, and then the positioning mechanism and interface assembly unit disengage from the liquid-containing element, and starts the centrifuge for vortex oscillation and first centrifugation; after the first centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve assembly in the interface assembly unit establish fluid connection and mechanical connection with the sample tube and valve respectively, and the fifth valve is opened, the air pump generates positive pressure, and transfers the supernatant of the third liquid-containing tube to the waste liquid pool;

[0117] (3) Close the fifth valve, open the second valve, start the air pump to generate negative pressure, and draw the second selective lysis liquid in the second liquid-containing tube of a predetermined volume into the third liquid-containing tube; close the second valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, and start the centrifuge for vortex oscillation and second centrifugation; after the second centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve connection component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, and the air pump generates positive pressure to transfer the supernatant of the third liquid-containing tube to the waste liquid pool;

[0118] (4) Close the fifth valve, open the third valve, start the air pump to generate negative pressure, and draw the third selective lysis liquid in the fourth liquid collection tube of the predetermined volume into the third liquid collection tube; close the third valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and third centrifugation; after the third centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, the air pump generates positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0119] (5) Close the fifth valve, open the fourth valve, start the air pump to generate negative pressure, and draw the first washing liquid in the fifth liquid collection tube of a predetermined volume into the third liquid collection tube; close the fourth valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and fourth centrifugation; after the fourth centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, start the air pump to generate positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0120] (6) Close the fifth valve, open the sixth valve, start the air pump to generate negative pressure, and draw the second washing liquid in the sixth liquid collection tube of a predetermined volume into the third liquid collection tube; close the fourth valve to disengage the positioning mechanism and interface component unit from the liquid-containing element, start the centrifuge for vortex oscillation and fifth centrifugation; after the fifth centrifugation is completed, the liquid-containing element returns to the position of the positioning mechanism, so that the fluid connection component and valve matching component in the interface component unit establish fluid connection and mechanical connection with the sample tube and valve respectively, open the fifth valve, start the air pump to generate positive pressure, and transfer the supernatant of the third liquid collection tube to the waste liquid pool;

[0121] (7) Obtain a sample of the extracted pathogen in the third liquid collection tube.

[0122] The specific extraction steps are shown in Table 1 below:

[0123]

[0124] Example 2: Automated process for separating and extracting pathogens 2

[0125] 1. Prepare the corresponding treatment solution according to the following composition:

[0126] The first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol.

[0127] Microbial settling aid: Novec7500.

[0128] The second selective lysis buffer is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine.

[0129] The third selective lysis buffer is an aqueous solution containing the following components: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA.

[0130] The first washing solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 7.5), 0.02% sodium chloride, 0.03% magnesium sulfate, and 0.01% sodium citrate.

[0131] The second washing solution is an aqueous solution containing the following components: 9 mM potassium phosphate buffer (pH 8), 0.03% potassium chloride, 0.03% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

[0132] 2. Transfer approximately 6 mL of the collected sample into a sample tube, cap the sample tube, and prepare two empty 5 mL centrifuge tubes, capping them as well.

[0133] 3. Turn on the control unit and automatically execute the following procedure:

[0134] (1) Use the cap opening and closing unit to place the sample tube in a fixed position, then use the identification unit to scan the identification code on the sample tube, and then use the cap opening and closing unit to put the sample tube into the shaking unit and shake the sample tube inverted. Start the cap opening and closing unit to open the cap of the sample tube, use the liquid supply / suction unit to draw about 3 mL of sample from the sample tube and supply it to the centrifuge tube through the first liquid dispensing gun, then use the positioning mechanism to position the liquid supply / suction unit to the first processing liquid storage tank and supply the first selective lysis solution to the centrifuge tube through the first liquid dispensing gun, then use the positioning mechanism to position the liquid supply / suction unit to the microbial flocculant storage tank and supply the microbial flocculant to the sample tube through the second liquid dispensing gun, start the cap opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, then move the centrifuge tube to the centrifuge unit and start the centrifuge for the first centrifugation, after the first centrifugation is completed, start the cap opening and closing unit to open the cap of the centrifuge tube, use the first liquid dispensing gun to draw the supernatant in the centrifuge tube and discard it to the waste liquid tank;

[0135] (2) Position the liquid supply / suction unit to the second processing liquid storage tank through the positioning mechanism and supply the second selective lysis liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube. Move the centrifuge tube to the oscillation unit for vortex oscillation. Then move the centrifuge tube to the centrifuge unit and start the centrifuge for the second centrifugation. After the second centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube. Use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0136] (3) Position the liquid supply / suction unit to the third processing liquid storage tank through the positioning mechanism and supply the third selective lysis liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube. Move the centrifuge tube to the oscillation unit for vortex oscillation. Then move the centrifuge tube to the centrifuge unit and start the centrifuge for the third centrifugation. After the third centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube. Use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0137] (4) Position the liquid supply / suction unit to the first washing liquid storage tank through the positioning mechanism, and supply the first washing liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, and then move the centrifuge tube to the centrifuge unit. Start the centrifuge for the fourth centrifugation. After the fourth centrifugation is completed, start the opening and closing unit to open the cap of the centrifuge tube, and use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it to the waste liquid tank.

[0138] (5) Position the liquid supply / suction unit to the second washing liquid reservoir through the positioning mechanism, and supply the second washing liquid to the centrifuge tube through the first liquid addition gun. Start the opening and closing unit again to close the cap of the centrifuge tube, move the centrifuge tube to the oscillation unit for vortex oscillation, and then move the centrifuge tube to the centrifuge unit. Start the centrifuge for the fifth centrifugation. After the fifth centrifugation is completed, start the opening and closing unit to open the cap of the sample tube, and use the first liquid extraction gun to extract the supernatant in the centrifuge tube and discard it into the waste liquid pool.

[0139] (6) Use the cap opening and closing unit to close the cap of the centrifuge tube and obtain the pathogen analysis sample.

[0140] The specific extraction steps are shown in Table 2 below:

[0141]

[0142] Test Example 1: Bloodstream infection sample obtained in Example 2

[0143] Add 1 mL of MH broth to centrifuge tubes containing 6 μL of extracts of Klebsiella pneumoniae (or Enterococcus faecalis / Staphylococcus aureus) at 1000 (E3) CFU / μL or 100 (E2) CFU / μL. Seal the tubes and incubate at 37°C for 6–24 hours. Then, perform drug susceptibility testing on the corresponding bacterial suspensions as Klebsiella pneumoniae (or Enterococcus faecalis / Staphylococcus aureus) isolated and cultured samples and the corresponding positive control groups. The test steps are as follows.

[0144] 1.1 Culture samples of Klebsiella pneumoniae (Kpn) after isolation and positive control drug susceptibility test

[0145] (1) After isolating Klebsiella pneumoniae (Kpn), the cultured sample was first vortexed and mixed, centrifuged at 200×g for 30s, the supernatant was aspirated, the middle precipitate and the bottom microbial sedimentation agent were discarded, and then the upper bacterial suspension left in the sample tube was mixed with the positive control and centrifuged for 2min, the supernatant was discarded, and then washed twice with sterile water and resuspended in 1mL of physiological saline.

[0146] (2) Add 2 mL of physiological saline to the sample tube and calibrate it on a bacterial turbidimeter;

[0147] (3) Dilute the bacterial resuspension with physiological saline to a bacterial suspension of 0.5 McF;

[0148] (4) Use a sterile cotton swab to take bacterial solution, gently squeeze out the excess bacterial solution in the tube wall on the liquid surface, and spread it on MH plate (three times on the entire surface of the culture medium, rotating the plate 60° each time and making a circle along the plate to make the bacterial solution evenly spread on the entire plate). Let the plate dry for 3-5 minutes.

[0149] (5) Take the drug sensitivity tablets Amikacin AK, Ampicillin / Sulbactam SAM, Piperacillin PRL, Cefuroxime CXM, Cefepime FEP, Ceftazidime CAZ, Imipenem IPM, Cefotaxime CTX, Augmentin AMC, Meropenem MEM, Ciprofloxacin CIP, Cefoperazone / Sulbactam SCF, Piperacillin / Tazobactam TZP, and Compound Sulfamethoxazole SXT. Use tweezers to place the drug sensitivity tablets onto the coated agar plates as required and gently press the paper tablets to make them adhere tightly to the agar plane.

[0150] (6) Incubate at 37°C for 18-24 hours;

[0151] (7) Measure the diameter of the inhibition zone with calipers and determine the result according to the standard. For the sample containing Klebsiella pneumoniae at an initial concentration of 1000 CFU / μL (Kpn isolation group E3), the diameter of the inhibition zone and the determination results for various drugs are shown in Table 2 and... Figure 2 As shown in Table 3, for samples containing Klebsiella pneumoniae at an initial concentration of 100 CFU / μL (Kpn isolation group E2), the diameters of the inhibition zones of various drugs and the determination results are shown in Table 3. Figure 3 As shown in the figure. The results showed that the drug susceptibility test results of the cultured samples after Klebsiella pneumoniae (Kpn) isolation were consistent with those of the positive control.

[0152] Table 2. Results of drug susceptibility testing of Klebsiella pneumoniae (Kpn) isolate E3 and positive control.

[0153]

[0154]

[0155] Table 3. Results of drug susceptibility testing of Klebsiella pneumoniae (Kpn) isolate E2 and positive control.

[0156]

[0157]

[0158] 1.2 Culture samples of Enterococcus faecalis (Efm) after isolation and positive control drug susceptibility test

[0159] (1) After separating the Efm culture sample, first vortex it to mix well, centrifuge at 200×g for 30s, aspirate the supernatant, discard the middle precipitate and the microbial sedimentation agent at the bottom, then mix the upper bacterial suspension left in the sample tube with the positive control separately, centrifuge for 2min, discard the supernatant, wash twice with sterile water, and then resuspend in 1mL of physiological saline.

[0160] (2) Add 2 mL of physiological saline to the test tube and calibrate it on a bacterial turbidimeter;

[0161] (3) Dilute the bacterial resuspension with physiological saline to a bacterial suspension of 0.5 McF;

[0162] (4) Use a sterile cotton swab to take bacterial solution, gently squeeze out the excess bacterial solution in the tube wall on the liquid surface, and spread it on MH plate (three times on the entire surface of the culture medium, rotating the plate 60° each time and making a circle along the plate to make the bacterial solution evenly spread on the entire plate). Let the plate dry for 3-5 minutes.

[0163] (5) Take the drug sensitivity tablets ampicillin AMP, penicillin PEN, teicoplanin TEC, gentamicin CN (120mg), vancomycin VAN, and linezolid LZD. Use tweezers to place the drug sensitivity tablets on the plate with bacteria as required and gently press the paper to make it adhere tightly to the agar plane.

[0164] (6) Incubate at 37℃ for 18-24 hours;

[0165] (7) Measure the diameter of the inhibition zone with calipers and determine the result according to the standard. For samples containing an initial concentration of 1000 CFU / μL of Enterococcus faecalis (Efm isolation group E3), the diameters of the inhibition zones of various drugs and the determination results are shown in Table 4 and... Figure 4 As shown in Table 5, for samples containing an initial concentration of 100 CFU / μL of Enterococcus faecalis (Efm isolation group E2), the diameters of the inhibition zones of various drugs and the determination results are shown in Table 5. Figure 5 As shown in the figure. The results showed that the drug susceptibility test results of the cultured samples after isolation of Enterococcus faecalis (Efm) were consistent with those of the positive control.

[0166] Table 4. Results of antimicrobial susceptibility testing of Enterococcus faecalis (Efm) isolate E3 and positive control.

[0167]

[0168] Table 5. Results of antimicrobial susceptibility testing of Enterococcus faecalis (Efm) isolate E2 and positive control.

[0169]

[0170]

[0171] 1.3 Staphylococcus aureus (Sau) isolation and culture samples and positive control drug susceptibility test

[0172] (1) After separating Sau, the cultured sample was first vortexed and mixed, centrifuged at 200×g for 30s, the supernatant was aspirated, the middle precipitate and the bottom microbial sedimentation agent were discarded, and then the upper bacterial suspension left in the sample tube was mixed with the positive control and centrifuged for 2min, the supernatant was discarded, and then washed twice with sterile water and resuspended in 1mL of physiological saline.

[0173] (2) Add 2 mL of physiological saline to the test tube and calibrate it on a bacterial turbidimeter;

[0174] (3) Dilute the bacterial resuspension with physiological saline to a bacterial suspension of 0.5 McF;

[0175] (4) Use a sterile cotton swab to take bacterial solution, gently squeeze out the excess bacterial solution in the tube wall on the liquid surface, and spread it on MH plate (three times on the entire surface of the culture medium, rotating the plate 60° each time and making a circle along the plate to make the bacterial solution evenly spread on the entire plate). Let the plate dry for 3-5 minutes.

[0176] (5) Take the drug sensitivity tablets gentamicin CN (10mg), penicillin P, cefoxitin FOX, ampicillin / sulbactam SAM, levofloxacin LEV, cefuroxime CXM, cefazolin KZ, erythromycin E, clindamycin CC, compound sulfamethoxazole SXT, linezolid LZD, rifampin RA, and fosfomycin FOS. Use tweezers to place the drug sensitivity tablets on the plate with bacteria as required and gently press the paper to make them adhere tightly to the agar plane.

[0177] (6) Incubate at 37℃ for 18-24 hours;

[0178] (7) Measure the diameter of the inhibition zone with calipers and judge the result according to the standard.

[0179] For samples containing an initial concentration of 1000 Staphylococcus aureus (Sau) at μL (Sau isolation group E3), the diameters of the inhibition zones of various drugs and the determination results are shown in Table 6. Figure 6 As shown in Table 7, for samples containing an initial concentration of 100 CFU / μL of Enterococcus faecalis (Sau isolation group E2), the diameters of the inhibition zones of various drugs and the determination results are shown in Table 7. Figure 7 As shown in the figure. The results showed that the drug susceptibility test results of the cultured samples after Staphylococcus aureus (Sau) isolation were consistent with those of the positive control.

[0180] Table 6. Results of antimicrobial susceptibility testing of Staphylococcus aureus (Sau) isolate E3 and positive control.

[0181]

[0182] Table 7 Results of antimicrobial susceptibility testing for Staphylococcus aureus (Sau) isolate E2 and positive control.

[0183]

[0184]

[0185] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for obtaining pathogens from samples for drug susceptibility testing, comprising the following steps: (1) Provide infectious samples and treatment solutions; (2) Provides an apparatus for extracting pathogens from a sample containing pathogens; (3) Inject the infectious sample and the treatment solution into their respective collection tubes; (4) Using the device, separate and obtain a suspension containing pathogens; and (5) Perform drug sensitivity testing using the suspension containing the pathogen or the culture after enrichment culture of the suspension.

2. The method of claim 1, wherein the apparatus for obtaining the pathogen from a sample containing the pathogen comprises: A centrifugal unit includes a centrifuge and one or more liquid-containing elements, said liquid-containing elements including a plurality of liquid-containing tubes that are fluidly connected to each other and valves between the liquid-containing tubes; Gas supply / suction unit, used to supply gas to components containing liquid or generate negative pressure; The interface component unit includes a fluid connection component and a valve mating component; Positioning mechanism, multiple liquid collection pipes and waste liquid tank; and Control unit, used to control the connection between the gas supply / suction unit and the liquid-containing components; Its features are, The liquid-containing component and the gas supply / suction unit can be connected through the fluid connection component of the interface component unit, and the control unit can establish at least two different fluid connection methods between the liquid-containing pipe and the gas supply / suction unit by controlling the valve cooperation component of the interface component unit and the valve cooperation between the liquid-containing pipe. The collection tubes contain a microbial settling agent, a selective lysis buffer, and a washing buffer, respectively. The selective lysis buffer is a phosphate (sodium or potassium) buffer or Tris hydrochloric acid buffer containing 0.01% to 0.045% of sodium chloride, potassium chloride, potassium bicarbonate, ammonium sulfate, ammonium sulfide, magnesium sulfate, magnesium chloride, disodium EDTA, sodium citrate, PEG4000-8000, PVP, glucose, mannitol, sorbitol, betaine, and BSA, with a pH of 6-9. The washing solution is a 0.1-10 mM phosphate (sodium or potassium salt) or Tris hydrochloric acid buffer containing one or more of sodium chloride, potassium chloride, magnesium sulfate, magnesium chloride, disodium ethylenediaminetetraacetate, or sodium citrate, with a pH of 6-9.

3. The method according to claim 2, wherein the liquid-containing element itself may be fixedly connected to the centrifuge or connected to the centrifuge via a fixing mechanism; preferably, the liquid-containing element and / or the fixing mechanism may further include an element that can cooperate with a positioning mechanism, such that the positioning mechanism enables the liquid-containing element to establish a fluid connection with the interface component unit according to the instructions of the control unit.

4. The method according to claim 2 or 3, wherein the first liquid-containing tube contains a mixture of a first selective lysis buffer and a microbial flocculant, the second liquid-containing tube contains a second selective lysis buffer, the fourth liquid-containing tube contains a third selective lysis buffer, the fifth liquid-containing tube contains a first washing solution, and the sixth liquid-containing tube contains a second washing solution, wherein, The first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol; The microbial settling agent is Novec 7500; The second selective lysis buffer is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine. The third selective lysis buffer is an aqueous solution containing the following components: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA; The first washing solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 7.5), 0.02% sodium chloride, 0.03% magnesium sulfate, and 0.01% sodium citrate; The second washing solution is an aqueous solution containing the following components: 9 mM potassium phosphate buffer (pH 8), 0.03% potassium chloride, 0.03% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

5. A method for obtaining pathogens from samples for drug susceptibility testing, comprising the following steps: (1) Provide infectious samples and treatment solutions; (2) Provides an apparatus for extracting pathogens from a sample containing pathogens; (3) Inject the infectious sample and the treatment solution into their respective treatment solution storage units; (4) Using the device, separate and obtain a suspension containing pathogens; and (5) Perform drug sensitivity testing using the suspension containing the pathogen or the culture after enrichment culture of the suspension.

6. The method of claim 5, wherein the apparatus for obtaining the pathogen from a sample containing the pathogen comprises: A centrifugation unit includes a centrifuge and one or more components that house sample tubes; The mixing unit is used to invert and mix the sample tube. Oscillating unit for vortex oscillation of sample tubes; A liquid supply / absorption unit for supplying liquid to and / or aspirating liquid from a sample tube; The cap opening and closing unit is used to open and close the cap of the sample tube; A treatment fluid reservoir for holding the treatment fluid; Positioning mechanism and waste liquid tank; and The control unit is used to control the working sequence of the liquid supply / suction unit, the lid opening / closing unit, and the centrifugation unit. Its features are, The control unit controls the working sequence of the liquid supply / suction unit, the cap opening / closing unit, and the centrifugation unit in one working cycle according to the sample type; The processing liquid storage mechanism includes a microbial settling agent storage tank containing a microbial settling agent, a first processing liquid storage tank containing a first selective lysis liquid, a second processing liquid storage tank containing a second selective lysis liquid, a third processing liquid storage tank containing a third selective lysis liquid, a first washing liquid storage tank containing a first washing liquid, and a second washing liquid storage tank containing a second washing liquid, wherein; The selective lysis buffer is a phosphate (sodium or potassium) buffer or Tris hydrochloric acid buffer containing 0.01% to 0.045% of sodium chloride, potassium chloride, potassium bicarbonate, ammonium sulfate, ammonium sulfide, magnesium sulfate, magnesium chloride, disodium ethylenediaminetetraacetate, sodium citrate, PEG4000-8000, PVP, glucose, mannitol, sorbitol, betaine, and BSA, at pH 6-9. The washing solution is a 0.1-10 mM phosphate (sodium or potassium salt) or Tris hydrochloric acid buffer containing one or more of sodium chloride, potassium chloride, magnesium sulfate, magnesium chloride, disodium ethylenediaminetetraacetate, or sodium citrate, with a pH of 6-9.

7. The method according to claim 6, wherein the first selective lysis buffer is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 8), 0.03% sodium chloride, 0.035% ammonium sulfate, 0.02% sodium citrate, and 0.025% sorbitol; The microbial settling agent is Novec 7500; The second selective lysis buffer is an aqueous solution containing the following components: 5 mM Tris hydrochloric acid buffer (pH 7), 0.03% potassium chloride, 0.035% magnesium sulfate, 0.02% glucose, and 0.025% betaine. The third selective lysis buffer is an aqueous solution containing the following components: 5 mM potassium phosphate buffer (pH 8), 0.03% potassium bicarbonate, 0.035% magnesium chloride, 0.02% mannitol, and 0.025% BSA; The first washing solution is an aqueous solution containing the following components: 5 mM sodium phosphate buffer (pH 7.5), 0.02% sodium chloride, 0.03% magnesium sulfate, and 0.01% sodium citrate; The second washing solution is an aqueous solution containing the following components: 9 mM potassium phosphate buffer (pH 8), 0.03% potassium chloride, 0.03% magnesium chloride, and 0.01% disodium ethylenediaminetetraacetate.

8. The method according to claim 6 or 7, wherein the element accommodating the sample tubes may accommodate a plurality of independent sample tubes and / or include a mechanism for fixing the sample tubes.

9. The method according to claim 6 or 7, characterized in that, The liquid supply / suction unit may include a first liquid filling gun, a second liquid filling gun, a first liquid dispensing gun, and a second liquid dispensing gun. The first liquid filling gun can be connected to a first treatment liquid storage tank, a second treatment liquid storage tank, a third treatment liquid storage tank, a first washing liquid storage tank, and a second washing liquid storage tank. The second liquid filling gun can be connected to a microbial sedimentation agent storage tank.

10. The method according to claim 6, characterized in that, The liquid supply / suction unit includes a liquid dispensing gun array and a liquid dispensing gun array, wherein the array includes at least two rows, such as three, four, five or six rows of first liquid dispensing guns, second liquid dispensing guns, first liquid dispensing guns and second liquid dispensing guns.

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