Method for directly preparing bacterial liquid for drug sensitivity experiment from blood culture positive specimen
By centrifuging with separating glue in blood culture positive specimens, the target concentration of bacterial fluid was directly obtained, which solved the problem of too long time in traditional methods, achieved rapid drug sensitivity experiments, and improved the efficiency of clinical treatment.
Patent Information
- Application Number
- CN202510133801.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-02-06
- Filing Date
- 2025-02-06
- Publication Date
- 2025-05-30
AI Technical Summary
After the blood culture is positive, the sample needs to be transferred to the blood plate for 18-24 hours, waiting for a single colony to form, and then drug sensitivity experiments are carried out. The entire process takes 2-3 days, which cannot meet the urgently needed clinical rescue needs.
By absorbing blood culture positive specimens into a centrifuge tube containing separation glue, centrifugation allows the target bacteria to remain in the supernatant to form the first supernatant, and then counting and diluting them until the target concentration of the bacterial solution is reached, the drug sensitivity experiment is performed directly.
This method can significantly shorten the time for obtaining the target concentration of bacterial fluid based on traditional methods, thereby shortening the cycle of drug sensitivity experiments and improving the efficiency of clinical treatment.
Smart Images
Figure CN120060430A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and particularly to a method for directly preparing a bacterial solution for a drug sensitivity test from a positive blood culture specimen in microbial inspection. Background Art
[0002] Blood stream infections (BSI) refer to a systemic infectious disease caused by the invasion of pathogens such as bacteria and fungi into the bloodstream. The pathogenic bacteria that enter the blood multiply and grow at an alarming rate of once every 20 minutes, spreading rapidly. They can trigger a strong reaction in the body within a few hours and quickly develop into septic shock, disseminated intravascular coagulation (DIC), and multiple organ failure, with a mortality rate of 25 - 46%. For every hour of delay in rescue, the mortality rate of the patient increases by 5%. Therefore, shortening the time required to determine the blood-borne bacteria species and antibiotic sensitivity will greatly improve the success rate of rescue.
[0003] Blood culture is the simplest, most accurate, and most commonly used method for detecting blood stream infections and is the etiological basis for confirming blood stream infections in the body. Early blood culture detection and early and correct antibacterial treatment are the primary measures to control blood stream infections. In addition, as the gold standard for diagnosing blood stream infections, blood culture can accurately isolate the infectious pathogen, and combined with the drug sensitivity results, a correct and precise treatment plan can be given.
[0004] However, after the positive alarm of blood culture by the traditional method, the positive blood culture specimen needs to be transferred to a blood plate for culturing for 18 - 24 hours, waiting for single colonies to form, and then the drug sensitivity test is performed on the pure colonies. The whole process takes 2 - 3 days, and the development of the patient's condition often cannot wait for such a long time.
[0005] Therefore, there is an urgent need to develop a method for directly performing a drug sensitivity test on a positive blood culture specimen to shorten the time. Summary of the Invention
[0006] The present invention provides a method for directly preparing a bacterial solution for a drug sensitivity test from a positive blood culture specimen, so as to at least solve the technical problem of too long time for preparing the bacterial solution for the drug sensitivity test in the prior art.
[0007] The present invention provides a method for directly preparing a bacterial solution for drug sensitivity testing from a blood culture positive specimen. The above-mentioned blood culture positive specimen is aspirated into a centrifuge tube containing a separating gel. The above-mentioned blood culture positive specimen and the above-mentioned separating gel are mixed into a liquid to be centrifuged. The liquid to be centrifuged of the above-mentioned blood culture positive specimen is centrifuged, so that the target bacteria of the above-mentioned blood culture positive specimen remain in the supernatant after centrifuging the above-mentioned liquid to be centrifuged, forming a first supernatant. The target bacteria in the above-mentioned first supernatant are counted to obtain the first number of bacteria in the above-mentioned first supernatant. The first concentration of the above-mentioned first supernatant is deduced according to the above-mentioned first number of bacteria. The above-mentioned first supernatant is diluted according to the above-mentioned first concentration to obtain a bacterial solution with a target concentration. The above-mentioned bacterial solution with a target concentration is directly subjected to drug sensitivity testing.
[0008] Optionally, counting the target bacteria in the above-mentioned first supernatant means detecting the quantity of the above-mentioned target bacteria.
[0009] Optionally, the above-mentioned separating gel separates blood into plasma and blood cells.
[0010] Optionally, the above-mentioned separating gel retains the above-mentioned target bacteria in the above-mentioned first supernatant under the action of centrifugation.
[0011] Optionally, the Coulter counting method is used to count the above-mentioned target bacteria.
[0012] Optionally, the second concentration of the above-mentioned bacterial solution with a target concentration is 10 4 to 10 6 colony-forming units per milliliter (cfu / mL), and the above-mentioned bacterial solution with a target concentration is directly subjected to drug sensitivity testing.
[0013] Optionally, 2 to 8 milliliters of the above-mentioned blood culture positive specimen is aspirated into the above-mentioned centrifuge tube. The liquid to be centrifuged containing the above-mentioned blood culture positive specimen is centrifuged. The rotation speed of the above-mentioned centrifugation is 1000 to 6000 revolutions per minute (r / min), and the time of the above-mentioned centrifugation is 6 to 14 minutes, so that the target bacteria of the above-mentioned blood culture positive specimen remain in the supernatant after centrifuging the above-mentioned liquid to be centrifuged, forming the above-mentioned first supernatant. The above-mentioned first supernatant containing the above-mentioned target bacteria is diluted 25 - 100 times to obtain the above-mentioned bacterial solution with a target concentration.
[0014] Optionally, 3 to 6 milliliters of the above-mentioned blood culture positive specimen is aspirated into the above-mentioned centrifuge tube. The liquid to be centrifuged containing the above-mentioned blood culture positive specimen is centrifuged. The rotation speed of the above-mentioned centrifugation is 2000 to 4000 revolutions per minute, and the time of the above-mentioned centrifugation is 8 to 12 minutes, so that the target bacteria of the above-mentioned blood culture positive specimen remain in the supernatant after centrifuging the above-mentioned liquid to be centrifuged, forming the above-mentioned first supernatant. The above-mentioned first supernatant containing the above-mentioned target bacteria is diluted 40 to 60 times to obtain the above-mentioned bacterial solution with a target concentration.
[0015] Optionally, the target bacteria in the positive blood culture specimen remain in the supernatant after centrifugation of the liquid to be centrifuged above, forming the above-mentioned first supernatant, and other impurities in the positive blood culture specimen sink to the lower end of the centrifuge tube.
[0016] Optionally, transfer the above-mentioned target concentration bacterial solution into the kit of the drug susceptibility analyzer for drug susceptibility testing.
[0017] A method for directly preparing a bacterial solution from a positive blood culture specimen for drug susceptibility testing provided by the present invention obtains target bacteria directly from the positive blood culture specimen by centrifugation, saving the time of plate culture.
[0018] The present invention provides a method for preparing a bacterial solution for directly performing drug susceptibility testing on a positive blood culture specimen, providing a reference for the formulation of clinical treatment plans.
[0019] The main steps of this method are as follows:
[0020] Draw 5 ml of blood into a blood culture bottle, inoculate 50 cfu / mL of ATCC25922 Escherichia coli and place it in a commercially available blood culture device for culture; after the blood culture sample shows positive, aspirate a volume of a positive specimen and add it to a centrifuge tube containing separation gel, and centrifuge to make the target bacteria in the positive blood culture specimen remain in the supernatant, and other impurities sink to the lower end of the centrifuge tube; dilute the supernatant by b times and transfer it into the plate supporting the fully automatic rapid drug susceptibility analyzer, and count the target bacteria on the machine; then dilute the target bacteria and perform subsequent rapid drug susceptibility testing. The schematic flow chart of directly preparing a target concentration bacterial solution from a positive blood culture specimen and then performing drug susceptibility testing is as Figure 1 shown.
[0021] Therefore, the method of the present invention can be applied to almost all drug susceptibility tests, because other impurities sink to the lower end of the centrifuge tube, and the target concentration bacterial solution does not contain other impurities, which is more conducive to the counting of target bacteria and the determination of turbidity.
[0022] In a preferred example, the volume a of the positive blood culture sample aspirated is 2 - 8 ml, the sub-optimal is 3 ml, and the optimal is 4 ml. In another preferred example, the centrifugation speed is 1000 - 6000 revolutions, the sub-optimal is 4000 revolutions, and the optimal is 3000 revolutions. In another preferred example, the centrifugation time is 6 - 14 minutes, the sub-optimal is 12 minutes, and the optimal is 10 minutes. In another preferred example, the dilution multiple b of the supernatant after centrifugation is 25 - 100 times, the sub-optimal is 60 times, and the optimal is 50 times.
[0023] The separating gel tube is a reagent for serum separation, which can separate blood into serum and blood cells. The function of the separating gel is to separate serum and blood cells through a special colloid. This can not only reduce cross-infection between serum and blood cells and improve the accuracy of experiments, but also quickly count bacteria and perform drug sensitivity tests on the bacteria in the serum. The characteristic of the separating gel is that it can separate blood into serum and blood cells, and does not mix pathogenic bacteria with blood cells, but can leave the pathogenic bacteria in the serum for subsequent direct counting and drug sensitivity tests. The main components of this separating gel are polyolefin, polyester and propylene.
[0024] There are many existing techniques for the operation methods of bacterial identification or agar dilution drug sensitivity experiments, but they have not been widely applied in clinical laboratories. The biggest reason is that the most common method used in existing clinical laboratories is the broth dilution method to obtain the MIC drug sensitivity experiment; and taking the grayish-white precipitate in the edge layer of the separating gel after centrifugation requires a series of operations such as washing and centrifugation again. This repeated centrifugation and washing is not acceptable in the busy clinical work. The key point solved by the present invention is just to meet the requirements of the broth dilution method drug sensitivity experiment with the least manual operation: the concentration of the bacterial liquid after positive blood culture is 10 8 CFU / mL, while the concentration required for the broth dilution method drug sensitivity experiment is 5*10 5 CFU / mL. Although most bacteria in the existing techniques are taken from the grayish-white precipitate in the edge layer of the separating gel after centrifugation, and indeed most bacteria are concentrated here in daily experiments, there will still be about 10% - 20% of the bacteria in the supernatant that are not completely centrifuged down. And this part of the bacteria has fully met the requirements for the broth dilution method drug sensitivity experiment. The experiments conducted after selecting bacteria in the existing techniques are identification experiments and agar dilution method experiments, which have relatively high requirements for the initial value of the bacterial liquid concentration. Therefore, the supernatant is abandoned. In addition, the key point for the success of the present invention is also to use the technology of Coulter to count bacteria and dilute them to the target concentration. The micro broth dilution method for drug sensitivity experiments has clear requirements for the initial concentration of bacteria, and it is not the higher the better. A high concentration is likely to lead to a high MIC and a drug resistance-biased drug sensitivity experiment result, while a too low bacterial liquid concentration is likely to lead to a low MIC and a sensitivity-biased drug sensitivity experiment. Therefore, the present invention solves this problem through the Coulter counting method. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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 but non-limiting manner, wherein:
[0026] Figure 1 It is a schematic flow chart of a drug sensitivity experiment after directly preparing a bacterial liquid with a target concentration from a positive blood culture specimen provided by the technical solution of the present invention.
[0027] 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. Specific embodiments
[0028] 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 given 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 be able to fully convey the scope of the disclosure of this application to those skilled in the art.
[0029] Example 1
[0030] Comparison of the time required for the traditional blood culture method and the method of the present invention to obtain a target concentration of bacterial solution
[0031] The experimental method is as follows:
[0032] 1.1 Experimental purpose: To compare the time required to obtain the target bacteria by the traditional blood culture method and the method of the present invention.
[0033] 1.2 Preparation of experimental drugs
[0034] Syringe, incubator, inoculation loop, alcohol lamp, turbidimeter, pipettor, blood plate 1.3 Experimental steps
[0035] 1.3.1 Draw 5 ml of blood into a blood culture bottle, inoculate with 50 cfu / mL of ATCC25922 Escherichia coli, and place it in a commercially available blood culture device for culture. After the blood culture sample reports positive, divide the specimen into two equal parts for subsequent use in the two methods.
[0036] 1.3.2 One part is processed according to the traditional method:
[0037] (1) In a laminar flow hood, use a pipettor to aspirate 200 μl of the blood culture positive sample and inoculate it onto a blood plate, smear it evenly, then place it in an incubator and incubate overnight at 37 °C. Observe the colonies after 24 hours to obtain pure bacterial colonies;
[0038] (2) Use an inoculation loop to pick up the pure colonies into a bacterial diluent (5 ml), and adjust the cell concentration therein with a McFarland turbidimeter to 0.5 McFarland for standby;
[0039] (3) Use a micropipette to aspirate 260 μL of the 0.5 McFarland bacterial suspension prepared in (1) and inject it into 26 mL of the enrichment culture medium, and mix well. At the same time, aspirate 50 μL of the 0.5 McFarland bacterial suspension in (1) and inject it into 5 mL of the dilution solution, mix well as a negative control, and the bacterial suspension for the fully automatic rapid drug sensitivity analyzer can be obtained.
[0040] 1.3.3 One portion is processed according to the method of the present invention:
[0041] (1) Aspirate 4 mL of the positive specimen and add it to a serum separation gel tube, and mix well;
[0042] (2) After mixing, place it in a centrifuge and centrifuge at 3000 rpm for 10 minutes;
[0043] (3) Dilute the supernatant 50-fold with the sample dilution solution and transfer it to the plate supporting the fully automatic rapid drug sensitivity analyzer, and count the target bacterial value on the machine;
[0044] (4) Determine the target bacterial value counted on the machine, and then dilute it to the amount for the fully automatic rapid drug sensitivity analyzer with the enrichment culture medium.
[0045] 1.4 The experimental results are shown in Table 1
[0046] Upper machine counting / cfu Plate counting / cfu Time used This method <![CDATA[6.4*10 5 > <![CDATA[7.6*10 5 > 20 min Traditional method <![CDATA[6.5*10 5 > <![CDATA[7.8*10 6 > 24h
[0047] Table 1
[0048] 1.5 Experimental conclusion
[0049] It can be seen from Table 1 of the experimental results that there is no obvious difference in the number of bacteria obtained by the traditional plate counting method and the number of bacteria obtained by this method, and the time used in this method is 24 h less than that of the traditional method.
[0050] Example 2 Comparison of traditional blood culture method and the method of the present invention on rapid drug sensitivity results
[0051] The experimental method is as follows:
[0052] 2.1 Experimental purpose: Compare the rapid drug sensitivity results obtained by the traditional blood culture method and the method of the present invention.
[0053] 2.2 Experimental supplies: syringe, incubator, inoculation loop, alcohol lamp, turbidimeter, pipette, blood plate, rapid drug sensitivity analyzer.
[0054] 2.3 Experimental steps
[0055] 2.3.1 Put a clinically identified positive blood culture bottle, identified as a single bacterium Escherichia coli, with the strain source from the Clinical Microbiology Laboratory of Peking Union Medical College Hospital, into a commercially available blood culture device for culture. After the blood culture sample reports positive, divide the specimen into two equal parts for the subsequent two methods.
[0056] 2.3.2 One is processed by traditional method:
[0057] (1) Place 200 μL of the blood culture sample on a clean bench and use a pipette to inoculate it onto a blood plate. Spread it evenly and then place it in an incubator. Incubate it at 37°C overnight. After 24 hours, observe the colonies to obtain pure bacterial colonies.
[0058] (2) Use an inoculating loop to pick up pure colonies and place them in a bacterial dilution solution (5 ml). Use a McFarland turbidimeter to measure the bacterial concentration to 0.5 McFarland and set aside for later use.
[0059] (3) Use a micropipette to draw 260 μl of the 0.5% Melvin solution prepared in (1) and inject it into 26 ml of the enrichment culture medium, and mix thoroughly. At the same time, draw 50 μl of the 0.5% Melvin solution in (1) and inject it into 5 ml of the diluent, and mix well as a negative control, and you can get the bacterial solution for the drug sensitivity analyzer.
[0060] 2.3.3 One portion is processed according to the method of the present invention:
[0061] (1) Pipette 4 ml of positive specimen into a centrifuge tube containing separation gel (disposable vacuum blood collection tube containing separation gel) and mix thoroughly;
[0062] (2) After mixing, place the mixture in a centrifuge and centrifuge at 3000 rpm for 10 minutes;
[0063] (3) Dilute the supernatant 50 times with sample diluent and transfer it to the plate of the antimicrobial susceptibility analyzer to count the target bacteria;
[0064] (4) Determine the target bacterial count value, and then dilute it with enrichment culture medium to the amount that can be counted on the antimicrobial sensitivity analyzer.
[0065] 2.4 Experimental Results
[0066] The results are shown in Table 2. The MIC results obtained by the rapid drug sensitivity test of the two bacterial suspensions were consistent.
[0067] Table 2 Rapid drug sensitivity results of traditional method and method of the present invention
[0068]
[0069]
[0070] 2.5 Experimental Conclusion
[0071] The MIC results obtained by the rapid drug sensitivity test of the two bacterial suspensions were consistent, proving that the method of the present invention is less time-consuming (about 24 hours).
[0072] Example 3
[0073] Comparative Study on the Effect of Obtaining Target Bacterial Liquid from the Supernatant and Precipitate after Centrifugation of Serum Separation Gel Tubes 3.1 Experimental Purpose: By comparing the content and drug sensitivity test data of the supernatant bacterial liquid and precipitate bacterial liquid after centrifugation with those of the positive blood culture bottle bacteria liquid that has been clinically identified, explore the accuracy of the supernatant bacterial liquid and an effective way to reduce the difficulty of experimental operation.
[0074] 3.2 Preparation of Experimental Drugs
[0075] Syringe, incubator, inoculation loop, alcohol lamp, turbidimeter, pipettor, blood plate, rapid drug sensitivity analyzer
[0076] 3.3 Experimental Steps
[0077] 3.3.1 The positive blood culture bottle that has been clinically identified is identified as a single bacterium Escherichia coli, and the strain source is the Clinical Microbiology Laboratory of Peking Union Medical College Hospital.
[0078] 3.3.2 Process according to the method of the present invention:
[0079] (1) Control group:
[0080] After streaking the positive blood culture bottle on the plate for 18 - 24 hours, pick the bacteria to prepare a 0.5 McFarland suspension for standby, dilute it to 10 4 —10 5 cfu / mL, add it to the Gram-negative bacillus drug sensitivity plate according to the drug sensitivity test steps, and then put it into the drug sensitivity analyzer for detection, and record the MIC respectively.
[0081] (2) Experimental group:
[0082] 1) Respectively suck 4 mL of the positive specimen and add it to 2 tubes containing serum separation gel tubes, mix well, label them as tube 1 and tube 2; at the same time, after mixing, put them into the centrifuge and centrifuge at 3000 rpm for 10 minutes;
[0083] 2) Suck out the supernatant of tube 1, measure the turbidity with a turbidimeter and dilute it to the required target bacterial liquid concentration with the sample diluent, then transfer it into the plate supporting the automatic rapid drug sensitivity analyzer, count the target bacterial value and drug sensitivity results on the machine, and at the same time perform blood plate counting.
[0084] 3) Remove the supernatant of tube 2, dilute the precipitate with 4 mL of sample diluent, blow and suck repeatedly, measure the turbidity with a turbidimeter and dilute it to the required target bacterial liquid concentration with the sample diluent, then transfer it into the plate supporting the automatic rapid drug sensitivity analyzer, count the target bacterial value and drug sensitivity results on the machine, and at the same time perform blood plate counting.
[0085] 4) Determine the target bacterial value counted on the machine, and then dilute it to the amount for the automatic rapid drug sensitivity analyzer to be put on the machine with the enrichment culture medium.
[0086] 3.4 Experimental Results
[0087] Table 3 Comparison of Counting Target Bacteria Values
[0088]
[0089] Table 4 Comparison of Rapid Drug Sensitivity Results of Supernatant and Precipitate
[0090]
[0091] 3.5 Experimental Conclusions
[0092] The experimental results in Table 3 and Table 4 show that the number of bacteria obtained from the supernatant is of the same order of magnitude as the clinical results and meets the requirements; the number of bacteria obtained from the precipitate is lower than the clinical standard. The reason is that different types of resins are added to the blood culture bottle to reduce the risk of false negatives caused by patients using antibiotics in advance during use. Some resins will be distributed together with bacteria in the upper layer of the separation gel. This has no impact on bacterial identification and traditional drug sensitivity results, but it will cause the Coulter counting method to misidentify the resin as bacteria. In rapid drug sensitivity analysis, the drug sensitivity results of the supernatant are consistent with the clinical ones and meet the requirements, while the precipitate shows abnormal values (resistant to imipenem, tigecycline, ertapenem, and high count values), which do not meet the requirements. This is because the precipitate contains resin, which affects bacterial counting and causes the analyzer to clog the holes. In addition, using the supernatant operation can meet the clinical bacterial quantity requirements, and there is no need to discard the supernatant and perform repeated pipetting operations, making the operation simpler and providing a better basis for the later automation of the whole method.
Claims
1. A method for directly preparing bacterial liquid for drug sensitivity test from blood culture positive specimens, characterized in that: The blood culture-positive specimen is drawn into a centrifuge tube, wherein the centrifuge tube contains a separation gel, the blood culture-positive specimen is mixed with the separation gel to form a liquid to be centrifuged, the liquid to be centrifuged of the blood culture-positive specimen is centrifuged, so that the target bacteria of the blood culture-positive specimen remain in the supernatant after the liquid to be centrifuged is centrifuged to form a first supernatant, the target bacteria in the first supernatant are counted to obtain a first bacterial number of the first supernatant, a first concentration of the first supernatant is inferred based on the first bacterial number, the first supernatant is diluted based on the first concentration to obtain a bacterial solution of a target concentration, and the bacterial solution of the target concentration is directly subjected to a drug sensitivity test.
2. The method according to claim 1, characterized in that Counting the target bacteria in the first supernatant refers to detecting the number of the target bacteria.
3. The method according to claim 1, characterized in that The separation gel separates the blood into plasma and blood cells.
4. The method according to claim 1, characterized in that: The separation gel retains the target bacteria in the first supernatant under the action of centrifugation.
5. The method according to claim 2, characterized in that: The target bacteria were counted using the Coulter counting method.
6. The method according to claim 1, characterized in that The second concentration of the target concentration bacterial solution is 10 4 Up to 10 6 The target concentration of bacterial solution was directly used for drug sensitivity test.
7. The method according to claim 1, characterized in that 2 to 8 ml of the blood culture positive specimen is drawn into the centrifuge tube, and the liquid to be centrifuged containing the blood culture positive specimen is centrifuged at a speed of 1000 to 6000 revolutions per minute (r / min) for 6 to 14 minutes, so that the target bacteria of the blood culture positive specimen remain in the supernatant after the liquid to be centrifuged is centrifuged to form the first supernatant, and the first supernatant including the target bacteria is diluted 25-100 times to obtain the bacterial liquid of the target concentration.
8. The method according to claim 7, characterized in that 3 to 6 ml of the blood culture positive specimen is drawn into the centrifuge tube, and the liquid to be centrifuged containing the blood culture positive specimen is centrifuged at a speed of 2000 to 4000 rpm for 8 to 12 minutes, so that the target bacteria of the blood culture positive specimen remain in the supernatant after the liquid to be centrifuged is centrifuged to form the first supernatant, and the first supernatant including the target bacteria is diluted 40 to 60 times to obtain the bacterial liquid of the target concentration.
9. The method according to claim 1, characterized in that: The target bacteria of the blood culture positive specimen remain in the supernatant after the liquid to be centrifuged is centrifuged to form the first supernatant, and other impurities of the blood culture positive specimen sink to the lower end of the centrifuge tube.
10. The method according to claim 1, characterized in that The bacterial solution of the target concentration is transferred into the test kit of the drug sensitivity analyzer to perform a drug sensitivity test.