Identification method for detecting MGIT960 liquid culture medium based on MALDI-TOFMS

Abstract

The invention discloses an identification method for detecting an MGIT960 liquid culture medium based on MALDI-TOFMS (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry). The identification method comprises the following steps: selecting 60 samples reported by a # imgabs0 # 960 mycobacterium rapid liquid culture system, and carrying out test operation; the optimal detection time, namely the turbidity of a liquid culture strain, is determined by continuously culturing after liquid culture positive reporting and detecting in different time periods, meanwhile, the operation of optimally obtaining the ribosomal protein of the strain is determined by selecting magnetic beads with different diameters and a wall breaking method, and the accuracy of a # imgabs1 # 960 liquid culture MALDI-TO0F-MS identification method is compared with an identification result of a traditional scheme, so that the optimal detection time, namely the turbidity of the liquid culture strain is determined. And the detection rates of different processing methods are recorded to find an optimal processing scheme, and the whole scheme is analyzed and evaluated to determine the optimal processing method, so that the target of improving the detection rate is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of liquid culture medium identification, and in particular to an identification method for MGIT™960 liquid culture medium based on MALDI-TOFMS detection. Background Art

[0002] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a new technology for rapid detection and identification of microorganisms, which is of great significance for the rapid, accurate and real-time identification of bacteria. However, mycobacteria have tough cell walls and high lipid content, so they grow slowly and It is not easy to lyse and obtain sufficient protein, which makes MALDI-TOFMS identification face many difficulties. Although there are relevant research reports on the application of MALDI-TOFMS to detect mycobacteria at home and abroad, the pretreatment methods are mostly limited to solid culture. Saleeb et al. first applied MALDI-TOFMS to solid culture identification. Using their self-designed pretreatment method, the accuracy of identification to species (or complex groups) can reach 100%. But it is limited to solid culture. For liquid culture identification, due to the potential interference of samples and liquid media, the number of positive bacteria is low, etc., it is still difficult to accurately identify mycobacteria from liquid culture medium. Balada-Llasat et al. first systematically evaluated the direct MALDI-TOFMS identification of MGIT liquid culture positive specimens in 2013. Their pretreatment method performed steps such as collection of bacteria and washing before protein extraction and purification. The results showed that the accuracy of direct identification of liquid culture was comparable to that of solid culture, but there were still cases of identification failure.

[0003] The main difficulties in using MALDI-TOF MS to identify liquid cultures are as follows: (I) How to obtain sufficient bacterial quantity for identification? The sensitivity of the automatic detection system is designed to detect the growth of mycobacteria as quickly as possible. Therefore, the positive liquid sample contains less bacterial quantity. After the liquid culture medium is reported positive, it is continued to be incubated to obtain sufficient bacterial quantity for identification. Then, after the liquid sample is reported positive, how many days should it be cultured and what is the culture concentration?

[0004] (II) Removing the influence of liquid culture medium components on bacterial protein. The main components of MCIT™960 liquid culture medium are fluorescent indicator (fixed at the bottom of the tube) and culture medium. If the liquid culture medium is directly heated and inactivated, the protein components in the culture medium may interfere with the extraction of bacterial protein. Many literatures have mentioned that the influence of liquid culture medium on bacterial protein can be removed by taking the precipitate after centrifugation.

[0005] (III) Obtaining ribosomal proteins from bacteria. Mycobacterium cell walls are tough and have high lipid content. To extract ribosomal proteins from mycobacteria, the cell walls must be fully destroyed. The methods provided in the literature generally use physical lysis methods such as mechanical grinding with micro glass beads and ultrasonic vibration to promote the lysis of mycobacteria with tough cell walls. However, the diameter of the beads and the vibration time vary from literature to literature.

[0006] In summary, this topic aims to explore different experimental conditions, that is, continue to culture after the liquid culture is positive, and detect in different time periods to determine the best detection time, that is, the turbidity of liquid culture bacteria. At the same time, different diameter magnetic beads and cell wall breaking methods are used to determine the best operation to obtain the ribosomal protein of the bacteria. In this way, the experimental goal of improving the detection rate is achieved.

[0007] Therefore, an identification method based on MALDI-TOFMS detection of MGIT™960 liquid culture medium was proposed, and this method was used to optimize the pre-treatment process of the detection target in order to achieve a satisfactory detection rate in clinical practice. Summary of the invention

[0008] The purpose of the present invention is to solve the shortcomings of the prior art mentioned in the background technology, and to propose an identification method based on MALDI-TOFMS detection of MGIT™960 liquid culture medium.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions: An identification method for MGIT™960 liquid culture medium based on MALDI-TOFMS detection comprises the following steps: S1. Select 60 cases 960 Mycobacterium Rapid Liquid Culture System reports positive specimens and conducts test operations; S2. Record the turbidity of the positive liquid culture and transfer 1.2 ml of the culture medium into a 1.5 ml Eppendorf tube; S3, centrifuging the culture solution in step S2, and carefully aspirating and discarding the supernatant with a pipette; S4, after aspirating the supernatant, add 300 μL of deionized water and place in a 100°C metal bath for 30 min; S5. After the sample has cooled, add 900 μL of 75% ethanol and mix thoroughly; S6, centrifuging the sample liquid to which ethanol was added in step S5 again, and carefully aspirating and discarding the supernatant with a pipette; S7, precipitate the sample liquid from which the supernatant was removed in step S6, and add about 10 μL of iodine oxide / silica gel beads to the precipitate, and add 15-30 μL of pure acetonitrile depending on the amount of precipitate; S8, add 70% formic acid solution with the same volume as acetonitrile, vortex for 30 seconds and then centrifuge; S9, after centrifugation in step S8, take 1 μL of the supernatant and add it dropwise to the target plate of the mass spectrometer, and dry it naturally at room temperature; S10, covering the above sample points with 1 μL of HCCA matrix solution, and allowing to dry naturally before mass spectrometry identification; S11. Continue to culture the original tube for 1 day, 3 days, and 5 days, repeat the above records and operations, record the test results respectively, and transfer 60 cultures to solid culture medium, use traditional methods for identification, use PNB solid culture, MPB64 antigen detection and gene chip technology to identify mycobacterium species, and record the identification results as a control for the mass spectrometry test results to evaluate the accuracy of the MALDI-TOF-MS test results.

[0010] As a further feature of the method of the present invention, in steps S3 and S5, the parameters of the centrifugal treatment are: centrifugal time is 2 min, and centrifugal speed is 13000 rpm.

[0011] As a further step in the method of the present invention, in step S7, the pure acetonitrile added must be sufficient to cover the precipitate.

[0012] As a further step in the method of the present invention, in step S7, 0.1 mm silica beads are taken from each culture and processed in four ways: vortex shaking for 2 min: 0.1 mm silica beads, vortex shaking for 2 min: 0.5 mm silica beads, ultrasonic disruption for 2 min: 0.5 mm silica beads, and ultrasonic disruption for 2 min.

[0013] As a further step in the method of the present invention, in step S8, the parameters of the centrifugal treatment are: centrifugal speed of 13000 r / min, and centrifugal time of 2 min.

[0014] As a further step in the method of the present invention, in step S11, S111, when the MALDI-TOF-MS identification result is inconsistent with the traditional result, the strain to be tested is subjected to single / multiple gene detection to determine the strain.

[0015] S112, result processing: 1) Comparison of identification results with traditional solutions The accuracy of the 960 liquid culture MALDI-TOF-MS identification method; 2) Record the detection rates of different treatment methods to find the best treatment plan, and analyze and evaluate the overall plan to establish the optimal treatment method.

[0016] Beneficial effects of the present invention This application compares the identification results with those of traditional solutions The accuracy of the 960 liquid culture MALDI-TOF-MS identification method and the detection rate of different treatment methods can be recorded to find the best treatment plan, analyze and evaluate the overall plan, and better establish the optimal treatment method.

[0017] The present application continues to culture after the liquid culture is positive, and detects in different time periods to determine the optimal detection time, that is, the turbidity of the liquid culture bacteria. At the same time, magnetic beads of different diameters and wall breaking methods are selected to determine the best operation for obtaining the ribosomal proteins of the bacteria, thereby achieving the experimental goal of improving the detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of an identification method for MGIT™960 liquid culture medium based on MALDI-TOFMS proposed in the present invention. DETAILED DESCRIPTION

[0019] The content of the present invention can be more easily understood by selecting the following detailed description of the preferred implementation method of the present invention and the embodiments included. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. When there is a conflict, the definition in this specification shall prevail.

[0020] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0021] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0022] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0023] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0024] Approximate terms in the specification and claims are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes acceptable and modified parts close to the quantity without causing changes in the relevant basic functions. Accordingly, the use of "about", "approximately", etc. to modify a numerical value means that the present invention is not limited to the exact numerical value. In some examples, the approximate terms may correspond to the accuracy of the instrument for measuring the numerical value. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0025] In addition, the indefinite articles "a" and "an" before the elements or components of the present invention have no restrictions on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the number is obviously intended to be in the singular form.

[0026] In the description of the present invention, “plurality” means two or more than two, unless otherwise clearly and specifically defined.

[0027] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0028] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "provided with", "mounted / connected", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0030] Reference Figure 1 , an identification method based on MALDI-TOFMS detection of MGIT™960 liquid culture medium, comprising the following steps: 60 cases were selected 960 Mycobacterium Rapid Liquid Culture System reports positive specimens and conducts test operations; 1) Record the turbidity of the positive liquid culture and take 1.2 ml of culture medium into a 1.5 ml Eppendorf tube; 2) The culture solution in step S2 was centrifuged for 2 min at a speed of 13000 rpm, and then the supernatant was carefully aspirated and discarded with a pipette; 3) After aspirating the supernatant, add 300 μL of deionized water and place in a 100°C metal bath for 30 minutes; 4) After the sample has cooled, add 900 μL of 75% ethanol and mix thoroughly; 5) The sample liquid to which ethanol was added in step S5 was centrifuged again, the centrifugation time was 2 min, the centrifugation speed was 13000 rpm, and then the supernatant was carefully aspirated and discarded with a pipette; 6) Precipitate the sample liquid from which the supernatant was removed in step S6, and add about 10 μL of oxidized chlorine / silica gel beads to the precipitate, and add 15-30 μL of pure acetonitrile depending on the amount of precipitate, wherein the added pure acetonitrile should cover the precipitate; 0.1 mm silica gel beads were used for each culture, and four treatment methods were used: vortex shaking for 2 min: 0.1 mm silica gel beads, vortex shaking for 2 min: 0.5 mm silica gel beads, ultrasonic disruption for 2 min: 0.5 mm silica gel beads, ultrasonic disruption for 2 min; 7) Add 70% formic acid solution with the same volume as acetonitrile, vortex for 30 seconds and then centrifuge; 8) In step S8, centrifuge at a speed of 13000 r / min for 2 min, then take 1 μL of the supernatant and drip it onto the target plate of the mass spectrometer, and dry it naturally at room temperature; 9) Cover the above sample points with 1 μL of HCCA matrix solution and allow to dry naturally before mass spectrometry identification; 10) Continue to culture the original tube for 1 day, 3 days, and 5 days, repeat the above records and operations, record the test results respectively, and transfer 60 cultures to solid medium for identification using traditional methods, use PNB solid culture, MPB64 antigen detection and gene chip technology to identify mycobacterium species, and record the identification results as a control for mass spectrometry test results to evaluate the accuracy of MALDI-TOF-MS test results; 11) When the MALDI-TOF-MS identification results are inconsistent with the traditional results, the strain to be tested is subjected to single / multiple gene detection to determine the strain.

[0031] 12) Result processing: ①Comparison of the results with those of traditional methods The accuracy of the 960 liquid culture MALDI-TOF-MS identification method; ② Record the detection rates of different treatment methods to find the best treatment plan, and analyze and evaluate the overall plan to establish the optimal treatment method.

[0032] A review of 60 cases The specimens reported positive by the 960 Mycobacterium Rapid Liquid Culture System were tested in the above steps. The best detection time was determined by continuing to culture after the liquid culture was positive and testing in different time periods, i.e., the turbidity of the liquid culture strain. Magnetic beads of different diameters and cell wall breaking methods were used to determine the best operation for obtaining the ribosomal protein of the bacteria. The results were compared with those of the traditional scheme. The accuracy of the 960 liquid culture MALDI-TOF-MS identification method was measured, the detection rates of different treatment methods were recorded to find the best treatment plan, and the overall plan was analyzed and evaluated to establish the optimal treatment method and achieve the goal of improving the detection rate.

[0033] The examples referred to herein are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to claim the widest possible range that can be imagined, and the embodiments presented herein are only illustrations of selected implementations according to the combination of all possible embodiments. Therefore, it is the applicant's intention that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges within them, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A method for identifying MGIT™960 liquid culture medium based on MALDI-TOFMS, characterized in that: The following steps are involved: S1. Select 60 cases 960 Mycobacterium Rapid Liquid Culture System reports positive specimens and conducts test operations; S2. Record the turbidity of the positive liquid culture and transfer 1.2 ml of the culture medium into a 1.5 ml Eppendorf tube; S3, centrifuging the culture solution in step S2, and carefully aspirating and discarding the supernatant with a pipette; S4, after aspirating the supernatant, add 300 μL of deionized water and place in a 100°C metal bath for 30 min; S5. After the sample has cooled, add 900 μL of 75% ethanol and mix thoroughly; S6, centrifuging the sample liquid to which ethanol was added in step S5 again, and carefully aspirating and discarding the supernatant with a pipette; S7, precipitate the sample liquid from which the supernatant was removed in step S6, and add about 10 μL of iodine oxide / silica gel beads to the precipitate, and add 15-30 μL of pure acetonitrile depending on the amount of precipitate; S8, add 70% formic acid solution with the same volume as acetonitrile, vortex for 30 seconds and then centrifuge; S9, after centrifugation in step S8, take 1 μL of the supernatant and add it dropwise to the target plate of the mass spectrometer, and dry it naturally at room temperature; S10, covering the above sample points with 1 μL of HCCA matrix solution, and allowing to dry naturally before mass spectrometry identification; S11. Continue to culture the original tube for 1 day, 3 days, and 5 days, repeat the above records and operations, record the test results respectively, and transfer 60 cultures to solid culture medium, use traditional methods for identification, use PNB solid culture, MPB64 antigen detection and gene chip technology to identify mycobacterium species, and record the identification results as a control for the mass spectrometry test results to evaluate the accuracy of the MALDI-TOF-MS test results.

2. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: In steps S3 and S5, the parameters of the centrifugal treatment are: centrifugal time is 2 minutes, and centrifugal speed is 13000 rpm.

3. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: In step S7, pure acetonitrile is added to cover the precipitate.

4. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: In step S7, 0.1 mm silica beads were taken from each culture and processed in four ways: vortex shaking for 2 min: 0.1 mm silica beads, vortex shaking for 2 min: 0.5 mm silica beads, ultrasonic disruption for 2 min: 0.5 mm silica beads, and ultrasonic disruption for 2 min.

5. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: In step S8, the parameters of the centrifugal treatment are: centrifugal speed 13000r / min, centrifugal time 2min.

6. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: In step S11, S111, when the MALDI-TOF-MS identification result is inconsistent with the traditional result, the strain to be tested is subjected to single / multiple gene detection to determine the strain.

7. The identification method of MGIT™960 liquid culture medium based on MALDI-TOFMS detection according to claim 1, characterized in that: S112, result processing: By comparing the identification results with those of traditional methods The accuracy of the 960 liquid culture MALDI-TOF-MS identification method; Record the detection rates of different treatment methods to find the best treatment plan, and analyze and evaluate the overall plan to establish the optimal treatment method.

Images