Separation kit for blood culture positive bacterium liquid

Abstract

The invention provides a separation kit for blood culture positive bacterium liquid. The separation kit comprises the following components in percentage by weight: 60%-85% of dimethyl silicone oil, 6%-18% of ethylene propylene diene monomer, 5%-20% of silicon dioxide, 0.5%-2% of quaternary ammonium salt, 0.5%-3% of glycerol and 0.03%-0.1% of methyl phthalate. According to the kit provided by the invention, a blood culture positive specimen is directly connected to a drug sensitive experiment, purified bacteria are rapidly obtained, and the time of the whole detection process is greatly shortened; the colored blood cells and colored components in blood culture positive can be distinguished, the accuracy of drug sensitivity experiments conducted through optical methodology in the market is met, and cell particles and resin particles can be distinguished to meet the problem that holes are blocked through the Coulter counting principle in the market.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine, and more specifically, it is a pretreatment kit applied after positive blood culture and before drug sensitivity test in microbial inspection. Background Art

[0002] Bloodstream infection (BSI) is a disease with systemic infectious symptoms caused by pathogenic microorganisms invading the blood circulation and multiplying in the body. It is the most serious clinical infection, damaging all organs of the patient's body. In severe cases, it can even lead to septic shock, disseminated intravascular coagulation (DIC), and multiple organ failure. In recent years, with the extensive development of invasive diagnostic and treatment technologies and the unreasonable use of broad-spectrum antibiotics, the incidence of bloodstream infection has been increasing year by year, and its mortality rate is also at a relatively high level. Some studies have shown that the case fatality rate caused by bloodstream infection is about 20%-50%. In clinical work, blood culture is generally considered the gold standard for diagnosing bloodstream infection. For each hour of delay in drug administration for patients with positive blood culture, the mortality rate increases by 7.6%.

[0003] The process from the start of patient examination to clinical drug use is as follows: First, culture on a blood culture device to determine whether it is a bloodstream infection. After the positive alarm of the blood culture device, the positive blood culture specimen needs to be transferred to a blood plate for culturing for 18-24 hours, waiting for the formation of single colonies, and then bacterial identification and drug sensitivity tests are performed on the pure colonies. The whole process takes 3-5 days. With the wide application of the fully automated blood culture system in clinical microbiology laboratories, the sensitivity and timeliness of blood culture have been greatly improved. The establishment of the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) rapid microbial identification platform provides a strong guarantee for early empirical anti-infective treatment in clinical practice, and the subsequent development of rapid drug sensitivity has also greatly shortened the time of clinical drug use for positive blood culture. However, the time required for pure culture (18-24 hours) from positive blood culture specimens to drug sensitivity tests is still the most critical factor restricting the entire inspection process.

[0004] Therefore, in view of the above problems in the prior art, there is a need to propose a new kit to at least solve the technical problem of the too long time required for pure culture from positive blood culture specimens to drug sensitivity tests in the prior art. Summary of the Invention

[0005] The present invention provides a kit for separating positive blood culture bacteria to at least solve the technical problem of the too long time required for pure culture from positive blood culture specimens to drug sensitivity tests in the prior art.

[0006] The present invention provides a separation kit for blood culture positive bacterial liquid. The above separation kit includes dimethyl silicone oil accounting for 60% - 85% by weight percentage, ethylene propylene diene monomer rubber accounting for 6% - 18%, silicon dioxide accounting for 5% - 20%, quaternary ammonium salt accounting for 0.5% - 2%, glycerol accounting for 0.5% - 3%, and methyl phthalate accounting for 0.03% - 0.1%.

[0007] Optionally, the above separation kit includes dimethyl silicone oil accounting for 65% - 77% by weight percentage, ethylene propylene diene monomer rubber accounting for 10% - 16%, silicon dioxide accounting for 10% - 16%, quaternary ammonium salt accounting for 0.8% - 1.8%, glycerol accounting for 1% - 2%, and methyl phthalate accounting for 0.04% - 0.08%.

[0008] Optionally, the above separation kit includes dimethyl silicone oil accounting for 75% by weight percentage, ethylene propylene diene monomer rubber accounting for 11%, silicon dioxide accounting for 11%, quaternary ammonium salt accounting for 1.45%, glycerol accounting for 1.5%, and methyl phthalate accounting for 0.05%.

[0009] Optionally, the above separation kit includes dimethyl silicone oil accounting for 73% by weight percentage, ethylene propylene diene monomer rubber accounting for 12%, silicon dioxide accounting for 12%, quaternary ammonium salt accounting for 1.45%, glycerol accounting for 1.5%, and methyl phthalate accounting for 0.05%.

[0010] Optionally, the above separation kit includes 75 grams of dimethyl silicone oil, 11 grams of ethylene propylene diene monomer rubber, 11 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol, and 0.05 grams of methyl phthalate.

[0011] Optionally, the above separation kit includes 77 grams of dimethyl silicone oil, 10 grams of ethylene propylene diene monomer rubber, 10 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol, and 0.05 grams of methyl phthalate.

[0012] Optionally, the above separation kit includes 73 grams of dimethyl silicone oil, 12 grams of ethylene propylene diene monomer rubber, 12 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol, and 0.05 grams of methyl phthalate.

[0013] Optionally, the above preparation method includes:

[0014] Weigh the reagents of the above separation kit, mix the weighed reagents evenly and then package them into finished products, sterilize the above finished products, and conduct quality inspection on the above sterilized finished products.

[0015] Optionally, the above finished product is a solid.

[0016] The application of the above separation kit in drug sensitivity tests after separating blood culture positive bacterial liquid.

[0017] The present invention provides a kit, the main components of which are dimethyl silicone oil, ethylene propylene diene monomer rubber, silicon dioxide, quaternary ammonium salt, glycerol, and methyl phthalate. It is mainly used to separate broken blood cells, resin particles and other macromolecular impurities from bacteria in a blood culture positive specimen after blood culture is positive.

[0018] The kit provided by the present invention directly connects a blood culture positive specimen to a drug sensitivity test, quickly obtains purified bacteria, and greatly shortens the time of the entire inspection process; it can distinguish colored blood cells and colored components in the blood culture positive, meet the accuracy of the drug sensitivity test by optical methodology in the market, and can distinguish cell particles and resin particles to solve the problem of blocking holes by the Coulter counting principle in the market.

[0019] The kit provided by the present invention is not used for culturing bacteria. After blood culture is positive, the concentration of bacteria is 10 8 cfu / mL. The traditional operation is carried out by inoculating a plate. The main purpose of this kit is to isolate enough bacteria for the drug sensitivity test, and the number of bacteria for the drug sensitivity test is 10 5 cfu / mL. The present invention can obtain a bacteria concentration greater than 10 6 cfu / mL under the action of a centrifuge, and the entire experimental operation process takes 20 minutes.

[0020] The kit finally obtained by the present invention is not a culture medium but a separation gel. This separation gel forms a high molecular polymer under the combination of various substances and does not need to be dissolved in water. Dimethyl silicone oil / ethylene propylene diene monomer rubber and silicon dioxide are the main components of the polysiloxane separation gel. After mixing, the formed separation gel has a good separation effect.

[0021] The role of the quaternary ammonium salt in the present invention is to adjust the charge property of the gel, improve the separation selectivity and increase the separation efficiency. It does not come into direct contact with bacteria in the separation gel, so it has no bactericidal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features and advantages of the exemplary embodiments of the present invention will become readily understood. In the drawings, several embodiments of the present invention are shown in an exemplary rather than restrictive manner, wherein:

[0023] Figure 1 It is a process flow diagram of the preparation process of an optional separation kit for blood culture positive bacterial liquid provided by an embodiment of the present invention.

[0024] The following specific embodiments are used to further illustrate but not limit the present invention. The following examples are only a preferred embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] The principles and spirit of the present invention will be described below with reference to several exemplary embodiments. It should be understood that these embodiments are provided only to enable those skilled in the art to better understand and then implement the present invention, and do not limit the scope of the present invention in any way. On the contrary, these embodiments are provided to make the disclosure of this application more thorough and complete, and to convey the scope of the disclosure of this application fully to those skilled in the art.

[0026] Example 1

[0027] 1. Determination of the formula

[0028] 1.1 Experimental purpose: By adjusting various components, determine a formula that is more suitable for the instrument and reagents with better repeatability and stability.

[0029] 1.2 Experimental requirements

[0030] Detect the performance indicators of the finished product, and the requirements are shown in Table 1:

[0031] Table 1

[0032] Index Required Index Capacity Not less than 10% of the required specification Sterile The sterilized kit should be sterile Number of centrifuged bacteria <![CDATA[The amount of bacteria contained after centrifugation is (1 - 10) * 10 4 -10 6 cfu / mL]]> Coulter counter equipment test No clogging of holes Color of supernatant Visually observed to be clear and transparent without red residue

[0033] The formula list is shown in Table 2:

[0034] Table 2 (unit in g in the formulation)

[0035]

[0036]

[0037] The preparation process of the above blood culture positive bacterial liquid separation kit:

[0038] Reagent preparation: Prepare the reagent items required for production.

[0039] Reagent weighing: Accurately weigh the reagents into a beaker in sequence according to the formula requirements.

[0040] Canning: Mix the weighed reagents evenly according to the specification requirements and divide them into bottles.

[0041] Packaging: Paste labels on the canned buffer solution and put it into a packaging box.

[0042] Sterilization: Sterilize the packaged buffer solution finished product according to the process requirements.

[0043] Quality inspection: Inspect according to the corresponding finished product inspection regulations;

[0044] Finished product warehousing: Handle the warehousing procedures for the qualified buffer solution finished product and indicate the person in charge.

[0045] As shown in the above process flow chart Figure 1 This is a schematic process flow diagram of the preparation process of an optional isolation kit for blood culture positive bacterial liquid provided by an embodiment of the present invention.

[0046] 1.3 Experimental steps

[0047] 1.3.1 Prepare finished products according to the above preparation process.

[0048] 1.3.1 Volume: Randomly select 10 person - portions of kits with different formulations, inject water of the required specification with a syringe, weigh the mass before and after injection, and calculate the volume from the obtained mass difference, which should be not less than 10% of the required specification. 1.3.2 Sterility: Randomly select 10 person - portions of kits with different formulations, add corresponding sterile blood to the buffer, directly place it in an environment of 35°C - 37°C for 72 hours, then take an appropriate amount of blood to smear on a plate, and after placing the plate in an environment of 35°C - 37°C for 24 hours, it should be sterile.

[0049] 1.3.3 Number of centrifuged bacteria: Randomly select 5 person - portions of kits with different formulations, disinfect the septum of the positive blood culture bottle with 75% ethanol, collect sufficient blood culture, aspirate 3 ml - 5 ml of the supernatant and inject it into the kits with different formulations under sterile conditions, tighten the lid, centrifuge at a speed of 3000 for 10 minutes, and dilute the obtained supernatant for plate counting.

[0050] 1.3.4 Randomly select 5 person - portions of kits with different formulations, disinfect the septum of the positive blood culture bottle with 75% ethanol, collect sufficient blood culture, aspirate 3 ml - 5 ml of the supernatant and inject it into the kits with different formulations under sterile conditions, tighten the lid, centrifuge at a speed of 3000 for 10 minutes, count the obtained supernatant on a Coulter device, observe the change in quantity, and record whether there is a phenomenon of clogging holes.

[0051] 1.4 Experimental results:

[0052] 1.4.1 The experimental results of volume are shown in Table 3

[0053] Table 3

[0054] Formula 1 2 3 4 5 6 7 8 9 10 Average Component A 95％ 98％ 94％ 98％ 95％ 96％ 95％ 95％ 96％ 94％ 96％ Component B 92％ 94％ 97％ 93％ 96％ 98％ 96％ 93％ 96％ 96％ 95％ Component C 95％ 96％ 95％ 96％ 96％ 95％ 96％ 95％ 96％ 96％ 96％ Component D 96％ 98％ 96％ 98％ 98％ 93％ 96％ 96％ 98％ 98％ 97％ Component E 95％ 96％ 96％ 98％ 95％ 95％ 96％ 95％ 96％ 98％ 96％ Component F 96％ 98％ 95％ 96％ 98％ 96％ 98％ 96％ 98％ 94％ 97％ Component G 96％ 98％ 96％ 98％ 93％ 96％ 98％ 96％ 98％ 96％ 97％ Component H 95％ 96％ 96％ 98％ 93％ 93％ 96％ 98％ 96％ 98％ 96％ Component I 95％ 96％ 96％ 98％ 96％ 95％ 93％ 93％ 98％ 96％ 96％ Component J 96％ 98％ 93％ 93％ 98％ 96％ 98％ 93％ 93％ 98％ 96％

[0055] 1.4.2 The experimental results of sterility are shown in Table 4

[0056] Table 4

[0057]

[0058]

[0059] 1.4.3 The experimental results of the number of centrifuged bacteria are shown in Table 5

[0060] Table 5

[0061] Formula 1 2 3 4 5 Average Component A <![CDATA[4.7*10 5 > <![CDATA[4.8*10 5 > <![CDATA[5.8*10 5 > <![CDATA[4.4*10 5 > <![CDATA[4.5*10 5 > <![CDATA[4*10 5 > Component B <![CDATA[5.6*10 5 > <![CDATA[4.3*10 5 > <![CDATA[4.8*10 5 > <![CDATA[4.4*10 5 > <![CDATA[4.5*10 5 > <![CDATA[5.8*10 5 > Component C <![CDATA[4.1*10 5 > <![CDATA[4.2*10 5 > <![CDATA[4.7*10 5 > <![CDATA[5.9*10 5 > <![CDATA[5*10 5 > <![CDATA[5.2*10 5 > Component D <![CDATA[8.5*10 4 > <![CDATA[8.1*10 4 > <![CDATA[9.2*10 4 > <![CDATA[8.2*10 4 > <![CDATA[8.9*10 4 > <![CDATA[8.3*10 4 > Component E <![CDATA[9.7*10 4 > <![CDATA[8.1*10 4 > <![CDATA[9.8*10 4 > <![CDATA[8.4*10 4 > <![CDATA[9.3*10 4 > <![CDATA[8.6*10 4 > Component F <![CDATA[9*10 4 > <![CDATA[8.9*10 4 > <![CDATA[9.7*10 4 > <![CDATA[9.2*10 4 > <![CDATA[8.1*10 4 > <![CDATA[8*10 4 > Component G <![CDATA[8.9*10 4 > <![CDATA[8.6*10 4 > <![CDATA[8.4*10 4 > <![CDATA[8.6*10 4 > <![CDATA[8.8*10 4 > <![CDATA[8.1*10 4 > Component H <![CDATA[1.0*10 6 > <![CDATA[1.5*10 6 > <![CDATA[1.9*10 6 > <![CDATA[1.6*10 6 > <![CDATA[1.1*10 6 > <![CDATA[1*10 6 > Component I <![CDATA[1.2*10 6 > <![CDATA[1.1*10 6 > <![CDATA[1.8*10 6 > <![CDATA[1.5*10 6 > <![CDATA[1*10 6 > <![CDATA[1.4*10 6 > Component J <![CDATA[1.5*10 6 > <![CDATA[2*10 6 > <![CDATA[1*10 6 > <![CDATA[1.1*10 6 > <![CDATA[1.2*10 6 > <![CDATA[1.4*10 6 >

[0062] 1.4.4 Kurt Count Plugging Situation

[0063] Table 6

[0064]

[0065]

[0066] 1.5 Experimental Conclusions

[0067] The above results show that the content of each component has no effect on the volume and sterility index of the finished product, and has no effect on the Kurt count and color. From the colony count values, it can be observed that the number of bacteria in the ABC formula is closer to 5*10 5 CFU / mL required by CLSI. This bacterial liquid content is better for the subsequent microbial susceptibility test and is within the detection range of the conventional Kurt principle.

[0068] Therefore, the formulation composition and the optimal ratio of the above-mentioned blood culture positive bacterial liquid separation kit are shown in Table 7.

[0069] Table 7 (unit in g in the formulation)

[0070]

[0071] Example 2. Consistency Analysis of On-Machine Comparative Experiment and Traditional Method

[0072] 2.1 Experimental Purpose: The above-mentioned blood culture positive bacterial liquid separation kit provided by the embodiments of the present invention is to replace the traditional purification of colonies (plate streaking method), so as to shorten the time from blood culture positive specimens to susceptibility tests. Therefore, it is necessary to compare with the plate streaking method.

[0073] 2.2 Experimental Requirements:

[0074] The number of bacteria required for the susceptibility test is 10 4 to 10 5 CFU / mL.

[0075] 2.3 Experimental Steps

[0076] 2.3.1 Four clinically identified positive blood culture bottles, identified as single bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus, with the strain source from the Clinical Microbiology Laboratory of Peking Union Medical College Hospital. After 18-24 hours of subculture by the plate streaking method from the positive blood culture bottles, pick the bacteria and prepare a 0.5 McFarland suspension for standby, and dilute it to 10 4—10 5 Add cfu / mL to the Gram-negative bacillus drug sensitivity plate according to the drug sensitivity test procedure, and then put it into the drug sensitivity analyzer for detection, and record the MIC respectively.

[0077] 2.3.2 Draw 3 ml - 5 ml of supernatant from the positive blood culture bottle and inject it into the isolation kit of the above-mentioned positive blood culture bacterial liquid under sterile conditions. After centrifugation (3000 revolutions for 10 minutes), obtain the final supernatant for standby.

[0078] 2.3.3 Perform Coulter counting on the above-obtained standby centrifuged liquid and dilute it into the drug sensitivity culture medium. According to the drug sensitivity test procedure, add it to the Gram-negative bacillus drug sensitivity plate detected by the Coulter method and the optical detection drug sensitivity reagent plate respectively, and then put it into the drug sensitivity analyzer for detection;

[0079] 2.3.4 Record the Coulter principle counting drug sensitivity MIC value and the conventional drug sensitivity MIC value respectively. 2.4 The experimental results are shown in Table 8.

[0080] Table 8 Comparison of drug sensitivity results between pure colonies and this kit

[0081]

[0082]

[0083]

[0084]

[0085] 2.5 Experimental conclusion

[0086] It can be seen from the experimental results that the positive blood culture results using the kit are consistent with those of the conventional drug sensitivity test, but the sample processing time is shortened from 18 - 24 hours to 20 minutes.

[0087] Example 3 Influence of the blood culture positive kit on the optical drug sensitivity detection equipment and the Coulter detection principle equipment before and after treatment.

[0088] 3.1 Experimental purpose: The isolation kit of the positive blood culture bacterial liquid provided in this embodiment of the present invention can distinguish the colored blood cells and colored components in the positive blood culture, meet the accuracy of the drug sensitivity test by the optical methodology in the market, and can distinguish cell particles and resin particles to solve the problem of pore blockage by the Coulter counting principle in the market.

[0089] 3.2 Experimental requirements

[0090] Provide detectable experimental conditions for the drug sensitivity detection instrument;

[0091] 3.3 Experimental steps

[0092] 3.3.1 Untreated sample dilution, inoculation and on-machine testing

[0093] Directly take 100 μL of the liquid from the positive blood culture bottle and dilute it into 10 mL of diluent, and mix well. Respectively take 10 aliquots of 100 μL and add them to the optical reagent plate without culturing, and observe the turbidity differentiation. Take 10 aliquots of 500 μL and add them to the Coulter test reagent plate, and then perform on-machine testing, and record the experimental situation.

[0094] 3.3.2 On-machine testing after kit treatment

[0095] Absorb 3 - 5 mL of the supernatant from the positive blood culture bottle and inject it into the isolation kit for positive blood culture bacteria liquid under sterile conditions. After centrifugation (3000 rpm for 10 min), obtain the final supernatant for standby. Perform Coulter counting on the obtained standby centrifuged liquid and dilute it into the drug sensitivity culture medium. According to the drug sensitivity experiment steps, add it to the drug sensitivity plate for detecting Gram-negative bacilli by the Coulter method and the optical detection drug sensitivity reagent plate respectively, and then put it into the drug sensitivity analyzer for detection; and record the experimental results.

[0096] 3.4 The experimental results are shown in Table 9

[0097] Table 9 Comparison between treated and untreated kits

[0098]

[0099] 3.5 Experimental conclusion

[0100] It can be seen from the experimental results that in the optical test, turbidity represents that the bacterial liquid has grown, and clarity represents that the bacterial liquid has not grown. However, the bacteria that have not been cultured will not grow. If the kit is not treated, the optical test equipment will judge the non-growth as growth, resulting in misinterpretation of the MIC results; and not treating the kit will cause blockage of the Coulter equipment, while no blockage will occur after kit treatment.

Claims

1. A kit for separating blood culture positive bacterial liquid, characterized in that: The separation kit comprises 60% to 85% by weight of dimethyl silicone oil, 6% to 18% of ethylene propylene rubber, 5% to 20% of silicon dioxide, 0.5% to 2% of quaternary ammonium salt, 0.5% to 3% of glycerol and 0.03% to 0.1% of methyl phthalate.

2. The separation kit according to claim 1, characterized in that The separation kit comprises 65% to 77% by weight of dimethyl silicone oil, 10% to 16% of ethylene propylene rubber, 10% to 16% of silicon dioxide, 0.8% to 1.8% of quaternary ammonium salt, 1% to 2% of glycerol and 0.04% to 0.08% of methyl phthalate.

3. The separation kit according to claim 1, characterized in that The separation kit comprises 75% by weight of dimethyl silicone oil, 11% of ethylene propylene rubber, 11% of silicon dioxide, 1.45% of quaternary ammonium salt, 1.5% of glycerol and 0.05% of methyl phthalate.

4. The separation kit according to claim 1, characterized in that The separation kit comprises 73% by weight of dimethyl silicone oil, 12% of ethylene propylene rubber, 12% of silicon dioxide, 1.45% of quaternary ammonium salt, 1.5% of glycerol and 0.05% of methyl phthalate.

5. The separation kit according to claim 1, characterized in that The separation kit includes 75 grams of dimethyl silicone oil, 11 grams of ethylene propylene rubber, 11 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol and 0.05 grams of methyl phthalate.

6. The separation kit according to claim 1, characterized in that The separation kit includes 77 grams of dimethyl silicone oil, 10 grams of ethylene propylene rubber, 10 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol and 0.05 grams of methyl phthalate.

7. The separation kit according to claim 1, characterized in that The separation kit includes 73 grams of dimethyl silicone oil, 12 grams of ethylene propylene rubber, 12 grams of silicon dioxide, 1.45 grams of quaternary ammonium salt, 1.5 grams of glycerol and 0.05 grams of methyl phthalate.

8. The method for preparing the separation kit according to any one of claims 1 to 7, characterized in that: The preparation method comprises: Weigh the reagents of the separation kit according to any one of claims 1 to 7, mix the weighed reagents evenly and then can them into finished products, sterilize the finished products, and perform quality inspection on the sterilized finished products.

9. The method for preparing the separation kit according to claim 8, characterized in that: The finished product is solid.

10. Use of the separation kit according to any one of claims 1 to 7 in drug sensitivity testing after separating positive bacterial liquid from blood culture.

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