Rapid identification method for blood culture positive sample pathogenic bacteria based on mass spectrometer

Abstract

The invention relates to the technical field of microbiological detection, and discloses a rapid identification method for pathogenic bacteria in a blood culture positive sample based on a mass spectrometer, and the method comprises the following steps: injecting a positive blood culture blood sample into a blood collection tube containing separation gel with the specific gravity of 1.0451.055 g / cm < 3 >; a bacteria enrichment layer is formed above the separation gel through centrifugation; sucking the bacterial layer, and washing the bacterial layer with a normal saline solution with the concentration of 0.40%-0.50% for multiple times to obtain a purified bacterial precipitate; and finally, directly applying the precipitate to a mass spectrometer for identification. According to the method, the traditional time-consuming seed transfer culture step is replaced by physical separation and enrichment, so that the identification time of pathogenic bacteria is remarkably shortened; meanwhile, the sample purity and the identification accuracy are ensured through the washing step with specific parameters. The method only needs conventional consumables, the detection cost is effectively reduced, the operation is simplified, and the method is suitable for clinical rapid diagnosis popularization and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism detection, and particularly to a method for rapidly identifying pathogenic bacteria in a positive blood culture sample based on a mass spectrometer. BACKGROUND

[0002] Bloodstream infection (BSI) is a serious infectious disease caused by pathogenic bacteria invading the blood circulation system, and its morbidity and mortality are at a high level. For the diagnosis and treatment of bloodstream infection, early, rapid and accurate identification of pathogenic bacteria, and selection of effective antimicrobial drugs accordingly, are the key to control infection and save patients' lives. Delaying effective treatment can significantly increase the risk of death in patients.

[0003] Currently, the gold standard for diagnosing bloodstream infection in clinic is still automated blood culture. After the blood culture instrument prompts positive alarm, the traditional inspection process usually needs to transfer the positive blood culture liquid to a suitable solid culture medium, incubate for 18 to 24 hours, and then pick the colonies from the culture medium for subsequent pathogenic bacteria identification and drug sensitivity test. This necessary in vitro culture step is the most time-consuming link in the whole identification process, which directly leads to a significant time delay between the discovery of infection and the acquisition of accurate etiological evidence, and cannot fully meet the urgent needs of clinical rapid drug guidance.

[0004] In order to shorten this time window, a variety of technical solutions for enriching pathogenic bacteria directly from positive blood culture liquid have appeared in the prior art. Some commercial kits can achieve relatively rapid identification, but their high price makes it difficult to be widely applied in clinic, especially for primary medical units. At the same time, researchers have also tried a variety of non-kit purification and separation methods. However, these methods often rely on organic solvents such as saponin and sodium dodecyl sulfate to lyse blood cells, or combined with complex centrifugation and membrane washing technology. This not only makes the operation process tend to be complicated and time-consuming, increases the technical burden of the operator, but also introduces the risk of residual and sample contamination of chemical lysis agents or separation reagents, which may interfere with the highly sensitive mass spectrometry detection signal and affect the accuracy of the identification result.

[0005] Therefore, it is a current technical problem to be solved in the field to develop a method for rapidly and accurately separating and purifying pathogenic bacteria from positive blood culture samples for mass spectrometry identification, which is simple to operate, low in cost, and not easy to introduce pollution. SUMMARY

[0006] The present application provides a method for rapidly identifying pathogenic bacteria in a positive blood culture sample based on a mass spectrometer, which aims to solve the technical problems of long time-consuming, complicated operation or dependence on high-cost special reagent kits in the prior art.

[0007] To achieve the above object, the present application provides the following technical solutions. A rapid identification method of pathogenic bacteria in blood culture positive samples based on mass spectrometer, comprising the following steps: Firstly, a blood collection tube containing separation gel is provided, wherein the specific gravity of the separation gel is set to be 1.045-1.055 g / cm 3 , and the separation gel is composed of hydrophobic organic compounds and silica powder.

[0008] Subsequently, 8-10 mL of positive blood culture blood is injected into the blood collection tube.

[0009] Then, the blood collection tube is centrifuged at a speed of 3000-4000 rpm for 8-12 minutes, until the blood plasma and culture liquid layer, the bacterial layer, the separation gel layer and the blood cell layer are formed in the tube in a clear stratification from top to bottom. Before the bacterial layer is sucked, the blood plasma and culture liquid layer on the top are removed.

[0010] Then, the bacterial layer above the separation gel layer is collected. This step can be performed by direct suction; or, especially when the amount of bacteria is small or not easy to suck, a small amount of liquid (such as the sample diluent used in the subsequent washing step) can be added to the bacterial layer, the bacteria are resuspended by gentle blowing, and then the suspension containing the bacteria is sucked. During the whole collection process, attention should be paid to the operation to maximize the recovery of bacteria while avoiding disturbance and inhaling the separation gel below. To improve the accuracy of the operation, a pipette tip with a smaller tip can be selected. The sucked bacterial layer is placed in a sharp EP tube and washed, and then centrifuged to obtain a bacterial precipitate. The washing step includes using a physiological saline solution with a concentration of 0.40%-0.50% as a sample diluent, and repeating the washing 2-3 times, and each time must be fully mixed. After each washing, centrifugation is performed at a speed of 13000-15000 rpm for 1-3 minutes.

[0011] Finally, the obtained bacterial precipitate is identified by mass spectrometry. The mass spectrometry identification step includes uniformly spreading the bacterial precipitate on a mass spectrometry target plate, adding a matrix solution, and detecting by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

[0012] The working mechanism of the present application is based on the difference in specific gravity between each component of blood and pathogenic bacteria, and separation is achieved through the physical properties of the separation gel. The separation gel is a high-viscosity gel in a static state. Under the action of centrifugal force, the internal hydrogen bond network structure is destroyed, and it is converted into a low-viscosity fluid. Since the specific gravity of the separation gel is set to be between the liquid components and the solid components of the blood, the separation gel undergoes a position reversal during centrifugation, forming a stable physical isolation layer, which intercepts the bacterial layer with smaller specific gravity above it, and the blood cells with larger specific gravity sink below it. After centrifugation, the separation gel returns to a high-viscosity gel state, stably fixing the separated layers.

[0013] The present application provides a rapid identification method for pathogenic bacteria in blood culture positive samples based on mass spectrometer. It has the following beneficial effects: 1. The present application realizes direct physical separation of positive blood culture samples by using a separation gel with a specific gravity range of 1.045-1.055 g / cm 3 The process design can effectively enrich the amount of bacteria sufficient for subsequent identification through one centrifugation step, thus eliminating the time-consuming incubation step of transferring the medium in the traditional method, and allowing direct mass spectrometric identification without waiting for colony formation, thereby significantly shortening the time from sample reception to output of the identification report.

[0014] 2. The present application improves the purity of the bacterial sample used for identification, thereby improving the accuracy of the identification results. In the washing step, a physiological saline solution with a specific concentration range is used, which can effectively destroy the red blood cells that may remain in the sample while having little effect on the target bacteria. Combined with 2-3 repeated washing operations and centrifugation in a sharp EP tube to facilitate the aggregation of the precipitate, the final obtained bacterial precipitate has high purity, reducing the interference of host cells and other impurities on mass spectrometric detection.

[0015] 3. The present application reduces the cost of single detection and simplifies the operation. The implementation of the present application does not require the use of special commercial separation kits with complex structure or high price. Only the separation gel blood collection tube, standard centrifuge and conventional physiological saline and other laboratory general consumables and reagents are needed. The entire operation process consists of clear physical steps such as centrifugation, aspiration and washing. The process is fixed and easy to master, which not only reduces the cost of materials, but also reduces the complexity of operation and the dependence on specific reagents, making it more suitable for development in primary medical units.

[0016] Reference signs Figure 1 Flow chart for comparison between the method of the present application and the traditional method. DETAILED DESCRIPTION

[0017] With reference to the drawings of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of the present application.

[0018] Embodiment 1-3: Embodiment 1: The embodiment provides a rapid identification method of pathogenic bacteria of a blood culture positive sample based on a mass spectrometer, and specific steps are as follows: (1) a coagulation blood collection tube containing a separation gel with a specific gravity of 1.045 g / cm 3 a coagulation blood collection tube containing a separation gel; (2) 8 mL of blood of a positive blood culture is extracted aseptically and injected into the blood collection tube; (3) the blood collection tube is centrifuged at a speed of 3000 rpm for 8 minutes; (4) after centrifugation, the upper plasma and culture solution layers are discarded. The bacterial layer above the separation gel is collected by direct suction or suction after resuspension by adding a liquid into the bacterial layer; (5) the suctioned bacterial layer is transferred into a sharp EP tube containing 1 mL of a 0.40% physiological saline solution, and is uniformly mixed by oscillation. Centrifugation is performed at a speed of 13000 rpm for 1 minute. After the supernatant is discarded, 1 mL of the 0.40% physiological saline solution is added again, and the washing and centrifugation operation is repeated once, and a total of two times of washing is completed; (6) the finally obtained bacterial precipitate is uniformly smeared on a mass spectrometry target plate, a matrix solution is added, and after natural crystallization, MALDI-TOF MS is used for detection to obtain an identification result.

[0019] Embodiment 2: The embodiment provides a rapid identification method of pathogenic bacteria of a blood culture positive sample based on a mass spectrometer, and specific steps are as follows: (1) a coagulation blood collection tube containing a separation gel with a specific gravity of 1.050 g / cm 3 a coagulation blood collection tube containing a separation gel; (2) 9 mL of blood of a positive blood culture is extracted aseptically and injected into the blood collection tube; (3) the blood collection tube is centrifuged at a speed of 3500 rpm for 10 minutes; (4) after centrifugation, the upper plasma and culture solution layers are discarded. The bacterial layer above the separation gel is collected by direct suction or suction after resuspension by adding a liquid into the bacterial layer; (5) The extracted bacterial layer is transferred to a sharp EP tube containing 1 mL of 0.45% physiological saline solution, and mixed uniformly by shaking. Centrifugation is performed at 14000 rpm for 2 minutes. After discarding the supernatant, 1 mL of 0.45% physiological saline solution is added again, and the washing and centrifugation operation is repeated once, for a total of two washes; (6) The final bacterial precipitate is evenly smeared on a mass spectrometry target plate, and a matrix solution is added. After natural crystallization, MALDI-TOF MS is used for detection to obtain the identification result.

[0020] Example 3: The present embodiment provides a rapid identification method for pathogenic bacteria in blood culture positive samples based on a mass spectrometer, and the specific steps are as follows: (1) A coagulation blood collection tube containing a separation gel with a specific gravity of 1.055 g / cm 3 a separation gel coagulation blood collection tube; (2) 10 mL of positive blood culture blood is aseptically extracted and injected into the blood collection tube; (3) The blood collection tube is centrifuged at 4000 rpm for 12 minutes; (4) After centrifugation, the upper plasma and culture liquid layers are discarded. The bacterial layer above the separation gel is collected by direct extraction or by adding a liquid to resuspend the bacterial layer and then extracting it; (5) The extracted bacterial layer is transferred to a sharp EP tube containing 1 mL of 0.50% physiological saline solution, and mixed uniformly by shaking. Centrifugation is performed at 15000 rpm for 3 minutes. After discarding the supernatant, the washing and centrifugation operation is repeated twice, for a total of three washes; (6) The final bacterial precipitate is evenly smeared on a mass spectrometry target plate, and a matrix solution is added. After natural crystallization, MALDI-TOF MS is used for detection to obtain the identification result.

[0021] Comparative Example 1-2: Comparative Example 1: Please refer to the attached Figure 1 The traditional subculture identification method mainly includes the following steps: aseptically extracting positive blood culture blood and subculturing it in a culture medium, and picking colonies for mass spectrometry identification after 18-24 hours of incubation.

[0022] Comparative Example 2: Compared with Example 2, the difference lies in that the specific gravity of the separation gel in the coagulation blood collection tube used in step (1) is 1.035 g / cm 3 . The remaining steps are the same.

[0023] Test Example 1-2: Test Example 1: Identification accuracy and sample purity test Experimental description: Sample grouping: 100 positive blood culture blood samples confirmed as single pathogenic bacteria infection by traditional culture method of Comparative Example 1 were prepared. The 100 samples were randomly divided into four groups: group A (25 samples), group B (25 samples), group C (25 samples) and group D (25 samples).

[0024] Sample processing: The samples in group A were processed according to the method described in Example 1; The samples in group B were processed according to the method described in Example 2; The samples in group C were processed according to the method described in Example 3; The samples in group D were processed according to the method described in Comparative Example 2.

[0025] Mass spectrometry detection and data analysis: The final bacterial precipitate obtained after processing of each group was subjected to MALDI-TOF MS detection.

[0026] Record the identification results output by the mass spectrometer: only one identification result option and the reliability is 60%-99.9%, record as "identification success", 2-4 options and the reliability is >60% or no identification options, record as "identification failure".

[0027] Calculate the identification accuracy of each group, which is calculated as: (number of samples identified successfully / total number of samples in the group) x 100%.

[0028] For samples with identification failure, analyze their mass spectra and record whether there are significant non-bacterial source impurity peaks.

[0029] Experimental data: The test data of each group was collated and recorded in the following table.

[0030] Table 1. Comparison of identification accuracy achieved by different methods Summary: The test results above show that when the specific gravity of the separation gel is controlled in the range of 1.045-1.055 g / cm 3 , the present technical solution can achieve high-accuracy identification results. The mechanism is that this specific gravity range is located between the blood liquid component (specific gravity about 1.02 g / cm 3between 1.005 and 1.015. Under the action of centrifugal force, the separation gel can form a physical isolation layer, intercepting the bacterial layer with smaller specific gravity above it, while allowing blood cells with larger specific gravity to settle below it, thereby achieving preliminary and effective enrichment of bacteria. The specific gravity in Comparative Example 2 is lower than this range, which causes the separation gel to fail to effectively separate bacteria from supernatant components such as plasma, resulting in a large amount of interfering proteins in the subsequent sample, thus significantly reducing the identification accuracy.

[0031] On the basis of preliminary separation and enrichment by the separation gel, the subsequent washing step further improves the purity of the bacterial sample. A physiological saline solution with a concentration range of 0.40%-0.50% is used. The osmotic pressure of this concentration solution helps to destroy a small amount of red blood cells that may remain during the operation, while having little effect on the structure of the bacteria itself. Through repeated 2-3 times of washing and centrifugation operation, soluble impurities and cell lysates can be removed, and bacterial precipitates can be effectively aggregated at the bottom of the sharp EP tube. This step ensures that the sample used for mass spectrometry is a high-purity bacterial precipitate, which is another key factor for obtaining high-confidence identification results.

[0032] Therefore, the technical effect of the technical solution is achieved through a specific combination of process parameters. First, the separation gel with an accurate specific gravity range is used for physical separation to achieve targeted enrichment of bacteria. Then, a specific concentration of washing solution is used for multiple purifications to remove interfering substances. The entire process does not rely on the specific binding of biological or chemical reagents and bacteria, but rather combines physical separation and purification steps to directly obtain a bacterial sample with the required purity and quantity for mass spectrometer detection from positive blood culture blood, thereby eliminating the subculture step and achieving the technical effect of rapid identification.

[0033] Test Example 2: Comparison Test of Identification Report Timeliness Experimental Explanation: Test Object: The 134 positive blood culture blood samples described in Test Example 1 are used as the test objects in this test.

[0034] Grouping Processing and Experimental Design: To ensure the scientificity and accuracy of the comparison, this test uses a paired sample self-control design to eliminate individual differences between samples. The specific operation is as follows: Sample Homogenization and Halving: Each of the 134 positive blood culture samples is thoroughly mixed, and then equally divided into two sterile samples labeled Sample A and Sample B. Through this operation, it is ensured that the two sub-samples A and B are completely consistent in terms of bacterial species, bacterial concentration, and sample matrix.

[0035] Parallel test: sample A is processed by the method of the present application (follow the steps of Example 2); sample B is processed by the traditional method (follow the steps of Comparative Example 1). The two methods start operating simultaneously on A and B tubes from the same original sample.

[0036] Timing and data recording: For each original sample, the timing starts when processing its A and B sub-samples simultaneously.

[0037] “Secondary reporting time” is defined as the total time consumed from the start of timing to obtaining the pathogen identification result.

[0038] “Tertiary reporting time” is defined as the total time consumed from the start of timing to completing the subsequent drug sensitivity test and obtaining the final report.

[0039] The secondary and tertiary reporting times of each original sample obtained by the two methods are recorded respectively, and the average value of 134 groups of paired data is finally calculated.

[0040] Experimental data: The average time statistics of the reports processed by the two methods are recorded in the following table.

[0041] Table 2. Comparison of average report processing time by different methods Summary: The test results of the above data show that there is a significant difference in the timeliness of report output in the technical solution. Compared with the traditional method, the average output time of the secondary report is shortened from 40.63 hours to 19.84 hours, and the average output time of the tertiary report is shortened from 56.11 hours to 43.29 hours. This time shortening is a direct result of the process design of the technical solution, that is, the time-consuming biological culture step is replaced by the physical separation step.

[0042] The core mechanism of the technical effect is that the present application omits the transfer medium incubation step required by the traditional method. The traditional method requires inoculating positive blood culture liquid on solid medium and incubating for 18-24 hours to obtain a sufficient number and purity of colonies for subsequent detection. The present application uses a separation gel with a specific specific gravity range to centrifuge, directly separates bacteria from cells and other liquid components in blood through physical separation, and obtains bacterial enrichment for mass spectrometry analysis in a short time, thereby shortening the time to obtain identifiable samples from 18-24 hours to less than 1 hour, which directly constitutes the basis for the significant shortening of the secondary reporting time.

[0043] The shortening of the secondary reporting time wins time for the subsequent steps, thereby leading to the corresponding advance of the tertiary reporting time. After obtaining the accurate pathogen identification result, targeted drug sensitivity test can be carried out immediately. Since the pathogen identification result is obtained nearly 24 hours in advance, the entire drug sensitivity test process is also advanced. Finally, by integrating a series of steps such as separation enrichment, washing and purification, and mass spectrometry detection, the technical scheme changes the traditional serial workflow, and realizes the time compression of the whole process from sample processing to final report output.

Claims

1. A method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer, characterized in that, The method comprises the following steps: S1, providing a blood collection tube containing separation gel, the specific gravity of the separation gel ranging from 1.045 to 1.055 g / cm 3 ; S2, injecting 8-10 mL of positive blood culture blood into the blood collection tube; S3, centrifuging the blood collection tube at a speed of 3000-4000 rpm for 8-12 minutes until a top-down plasma and culture liquid layer, a bacteria layer, a separation gel layer and a blood cell layer are formed in the tube; S4, sucking the bacteria layer above the separation gel layer; S5, washing the sucked bacteria layer and centrifuging to obtain a bacterial precipitate; S6, mass spectrometric identification of the bacterial precipitate.

2. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The separation gel is composed of a hydrophobic organic compound and silica powder.

3. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The washing in the S5 step uses a physiological saline solution with a concentration of 0.40%-0.50% as a sample diluent.

4. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The centrifugation in the S5 step is performed at a speed of 13000-15000 rpm for 1-3 minutes.

5. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The washing step in the S5 step is repeated 2-3 times, and each time needs to be fully mixed.

6. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The S5 step is performed in a sharp EP tube to make the bacterial precipitate aggregate after centrifugation.

7. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer according to claim 1, characterized in that, The S6 step comprises: uniformly smearing the bacterial precipitate on a mass spectrometry target plate, adding a matrix solution, and detecting by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

8. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer of claim 1, wherein, Before the mass spectrometric identification in the S6 step, the positive blood culture blood does not need to be incubated in a transfer medium.

9. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer of claim 1, wherein, Before the S4 step, further comprising a step of removing the plasma and culture liquid layer.

10. The method for rapid identification of pathogenic bacteria from blood culture positive samples based on mass spectrometer of claim 1, wherein, The S4 step comprises: collecting the bacteria layer above the separation gel by directly sucking, or resuspending by adding liquid into the bacteria layer and then sucking.

Images