Method and kit for rapidly testing sensitivity of salmonella to ceftriaxone

Abstract

The invention discloses a method for rapidly testing sensitivity of salmonella to ceftriaxone. The method comprises the following steps: S1, preparing a salmonella solution; s2, centrifuging the bacterial liquid and removing supernate of the centrifuged bacterial liquid; s3, providing a ceftriaxone solution, wherein the ceftriaxone solution contains ceftriaxone and water; s4, mixing the ceftriaxone solution with the bacterial solution to form a mixed solution, placing the mixed solution in an incubator, and standing for a first preset time; s5, centrifuging the mixed solution after standing for a first preset time, and extracting supernate of the mixed solution; and S6, identifying the ceftriaxone in the supernatant of the mixed solution by using a time-flight mass spectrometer, and judging the sensitivity of the salmonella to the ceftriaxone according to whether a characteristic peak corresponding to the ceftriaxone disappears or not. Compared with PCR (polymerase chain reaction) and LAMP (loop-mediated isothermal amplification) methods, the method has the advantages that the detection result is more reliable, the time consumed by the method is less than that consumed by manual drug sensitivity and full-automatic drug sensitivity identification instruments, and the method can be used for conventional detection in laboratories to timely feed back drug sensitivity results to clinics.

Description

Technical Field

[0001] The present invention belongs to the technical field of Salmonella drug resistance detection, and particularly relates to a method and kit for rapidly testing the sensitivity of Salmonella to ceftriaxone. Background Art

[0002] Salmonella is the main pathogenic bacterium causing diarrhea. However, the time required for the isolation, culture, and drug sensitivity identification of Salmonella is often 3 - 4 days. During this period, in order to prevent the deterioration of the patient's condition, doctors often choose empirical medication and use ampicillin for treatment. However, with the increasing frequency of ampicillin use, the drug resistance rate of Salmonella to it is also gradually increasing; thus, doctors may choose ceftriaxone for treatment, but with the increasing ceftriaxone resistance rate, it often leads to the failure of empirical medication. In order to avoid the increase in the ceftriaxone resistance rate and avoid blindly using high - level drugs, it is extremely urgent to quickly and accurately detect the drug sensitivity of Salmonella.

[0003] Currently, the main methods for detecting Salmonella drug - resistant genes include the PCR method, the LAMP method, manual drug sensitivity testing, and fully automated drug sensitivity identification instruments, etc. The PCR and LAMP methods can only detect a limited number of drug - resistant genes and cannot detect all drug - resistant genes, resulting in their reports being only for reference and unable to ensure that Salmonella definitely does not carry drug - resistant genes. Although manual drug sensitivity testing and fully automated drug sensitivity identification instruments can accurately obtain the drug resistance situation of Salmonella, they require 10 - 14 hours, and from inoculation, sub - purification, drug sensitivity testing to issuing a report, it takes 3 - 4 days, resulting in doctors being unable to treat patients in a timely manner and delaying the treatment opportunity.

[0004] The present invention combines a mass spectrometer and a detection kit, and only requires a small amount of Salmonella to quickly detect its sensitivity to ceftriaxone. It not only shortens the time for issuing a report, improves the detection efficiency, but also helps doctors select the correct antibiotics to treat patients in a timely manner. Summary of the Invention

[0005] The purpose of the present invention is to provide a method and kit for rapidly testing the sensitivity of Salmonella to ceftriaxone, so as to solve the problems of long detection time and unreliable detection results of existing detection methods.

[0006] To achieve the above object, a method for rapidly testing the sensitivity of Salmonella to ceftriaxone provided by the present invention is characterized by comprising the following steps: S1, preparing a Salmonella bacterial solution; S2, centrifuging the bacterial solution and removing the supernatant of the centrifuged bacterial solution; S3, providing a ceftriaxone solution, which contains ceftriaxone and water; S4, mixing the ceftriaxone solution with the bacterial solution to form a mixed solution, placing the mixed solution in an incubator and standing for a first preset time, the temperature of the incubator being greater than or equal to 35 degrees and less than or equal to 37 degrees, and the first preset time being greater than or equal to 45 minutes and less than or equal to 60 minutes; S5, centrifuging the mixed solution after standing for the first preset time and extracting the supernatant of the centrifuged mixed solution; S6, identifying ceftriaxone in the supernatant of the mixed solution using a time-of-flight mass spectrometer and determining the sensitivity of Salmonella to ceftriaxone by whether the characteristic peak corresponding to ceftriaxone disappears.

[0007] Preferably, the McFarland turbidity of the bacterial solution is greater than or equal to 1.5 and less than or equal to 2.0, the volume of the ceftriaxone solution is 20 microliters, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L.

[0008] Preferably, step S2 includes: centrifuging the bacterial solution at a first rotation speed for a second preset time, the first rotation speed being greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the second preset time being greater than or equal to 1 minute and less than or equal to 2 minutes; removing the supernatant of the centrifuged bacterial solution to obtain a concentrated solution of Salmonella.

[0009] Preferably, step S5 includes: centrifuging the mixed solution after standing for the first preset time at a second rotation speed for a third preset time, the second rotation speed being greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the third preset time being greater than or equal to 1 minute and less than or equal to 2 minutes; extracting the supernatant of the centrifuged mixed solution.

[0010] Preferably, the mass-to-charge ratio of the characteristic peak corresponding to ceftriaxone is greater than or equal to 554 and less than or equal to 556.

[0011] An embodiment of the present invention also provides a kit for rapidly testing the sensitivity of Salmonella to ceftriaxone, which includes a plurality of centrifuge tubes arranged in parallel, each centrifuge tube containing a ceftriaxone solution, the ceftriaxone solution containing ceftriaxone and water, and each centrifuge tube having a sealing part at the top, and all the sealing parts of the plurality of centrifuge tubes are connected in sequence to form a row.

[0012] Preferably, the centrifuge tube is integrally connected to the sealing part and can be separated under an external force.

[0013] Preferably, the volume of the centrifuge tube is 0.5 ml, the volume of the ceftriaxone solution is 20 μl, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L.

[0014] Compared with the prior art, in the present invention, Salmonella is mixed with ceftriaxone and left to stand in an incubator for a first preset time, and then detected by a time-of-flight mass spectrometer. Thus, whether Salmonella is resistant to ceftriaxone due to carrying a drug-resistant gene is determined by whether the characteristic peak corresponding to ceftriaxone disappears. This method is not only more reliable than the detection results of PCR and LAMP methods, but also takes less time than manual drug sensitivity and fully automatic drug sensitivity identification instruments. It can be used for routine laboratory tests, timely feedback drug sensitivity results to the clinic, and guide rational clinical drug use. Description of the Drawings

[0015] Figure 1 It is a front view structural schematic diagram of a kit for rapidly testing the sensitivity of Salmonella to ceftriaxone according to an embodiment of the present invention.

[0016] Figure 2 It is a top view structural schematic diagram of a kit for rapidly testing the sensitivity of Salmonella to ceftriaxone according to an embodiment of the present invention. Detailed Embodiments

[0017] To describe in detail the technical content, structural features, and achieved effects of the present invention, the following is described in detail in conjunction with the embodiments and with reference to the accompanying drawings.

[0018] As Figures 1 to 2 shown, an embodiment of the present invention provides a method for rapidly testing the sensitivity of Salmonella to ceftriaxone, including the following steps:

[0019] S1. Prepare a Salmonella bacterial solution; specifically, the McFarland turbidity of the bacterial solution can be greater than or equal to 1.5 and less than or equal to 2.0.

[0020] S2. Centrifuge the bacterial solution and remove the supernatant of the centrifuged bacterial solution; specifically, by centrifuging the bacterial solution, a Salmonella bacterial solution with a higher concentration is obtained, which is convenient for subsequent detection.

[0021] S3. Provide a ceftriaxone solution 10, which contains ceftriaxone and water; specifically, the amount of ceftriaxone can be set according to actual needs for consumption. For example, the volume of the ceftriaxone solution 10 is 20 μl, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L. In addition, as Figures 1 to 2 shown, the ceftriaxone solution 10 can be obtained from a pre-prepared kit 100.

[0022] S4. Mix the ceftriaxone solution 10 with the bacterial solution to form a mixed solution, place the mixed solution in an incubator and let it stand for a first preset time. The temperature of the incubator is greater than or equal to 35°C and less than or equal to 37°C, and the first preset time is greater than or equal to 45 minutes and less than or equal to 60 minutes. Specifically, mix the ceftriaxone solution 10 with the bacterial solution thoroughly, for example, shake it well, and then place the mixed solution in the incubator and incubate it for the first preset time, so that ceftriaxone has sufficient time to act on Salmonella. If Salmonella is sensitive to the drug, the ceftriaxone will be consumed completely. If Salmonella is not sensitive to the drug, the ceftriaxone will still be retained.

[0023] S5. Centrifuge the mixed solution after standing for the first preset time and extract the supernatant of the centrifuged mixed solution. Specifically, after step S4, centrifuge the mixed solution so that both live Salmonella and dead Salmonella are at the bottom, avoiding the influence of Salmonella on the test results. Just extract the supernatant of the mixed solution for testing.

[0024] S6. Use a time-of-flight mass spectrometer to identify the ceftriaxone in the supernatant of the mixed solution and judge the sensitivity of Salmonella to ceftriaxone by whether the characteristic peak corresponding to ceftriaxone disappears. Specifically, when the characteristic peak of ceftriaxone in the test result of the time-of-flight mass spectrometer disappears, it means that Salmonella is sensitive to ceftriaxone, that is, not drug-resistant. When the characteristic peak of ceftriaxone in the test result of the time-of-flight mass spectrometer does not disappear, it means that Salmonella is not sensitive to ceftriaxone, that is, drug-resistant.

[0025] In the embodiment of the present invention, by mixing Salmonella with ceftriaxone and placing it in an incubator to stand for a first preset time, and then detecting it with a time-of-flight mass spectrometer, the content of ceftriaxone is judged by whether the characteristic peak corresponding to ceftriaxone disappears, so as to judge whether Salmonella is drug-resistant to ceftriaxone due to carrying a drug-resistant gene. This method is not only more reliable than the detection results of PCR and LAMP methods, but also takes less time than manual drug sensitivity and fully automatic drug sensitivity identification instruments. It can be used for routine laboratory tests, timely feedback drug sensitivity results to the clinic, and guide clinical rational drug use.

[0026] In the embodiment of the present invention, step S2 of centrifuging the bacterial solution and removing the supernatant of the centrifuged bacterial solution includes:

[0027] S21. Centrifuge the bacterial solution at a first rotation speed for a second preset time. The first rotation speed is greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the second preset time is greater than or equal to 1 minute and less than or equal to 2 minutes. The centrifugation time is short and the efficiency is high. Of course, the first rotation speed and the second preset time can be selected according to actual needs as long as the bacterial solution can be centrifuged and stratified.

[0028] S22. Remove the supernatant of the bacterial liquid after centrifugation to obtain a concentrated solution of Salmonella.

[0029] In the embodiment of the present invention, step S5 of centrifuging the mixture after standing for the first preset time and extracting the supernatant of the centrifuged mixture includes:

[0030] S51. Centrifuge the mixture after standing for the first preset time at a second rotation speed for a third preset time. The second rotation speed is greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the third preset time is greater than or equal to 1 minute and less than or equal to 2 minutes. The centrifugation time is short and the efficiency is high. Of course, the second rotation speed and the third preset time can be selected according to actual needs as long as the mixture can be centrifuged and stratified.

[0031] S52. Extract the supernatant of the centrifuged mixture.

[0032] In the embodiment of the present invention, the mass-to-charge ratio of the characteristic peak corresponding to ceftriaxone is greater than or equal to 554 and less than or equal to 556. Specifically, since the molecular weight of ceftriaxone is 554.58, a proton will be added when the time-of-flight mass spectrometer identifies ceftriaxone. Therefore, the mass-to-charge ratio (m / z) of the characteristic peak of ceftriaxone is greater than or equal to 554 and less than or equal to 556. Any characteristic peak within this range can be identified as the characteristic peak of ceftriaxone.

[0033] As Figures 1 to 2 shown, the embodiment of the present invention also provides a kit 100 for quickly testing the sensitivity of Salmonella to ceftriaxone, including a plurality of centrifuge tubes 1 arranged in parallel. Each centrifuge tube 1 contains a ceftriaxone solution 10. The ceftriaxone solution 10 contains ceftriaxone and water. Each centrifuge tube 1 is provided with a sealing part 2 at the top. All the sealing parts 2 of the plurality of centrifuge tubes 1 are connected in sequence to form a row. Specifically, the volume of the centrifuge tube 1 is 0.5 ml, the volume of the ceftriaxone solution 10 is 20 μl, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L. The kit 100 is used to provide the ceftriaxone solution 10 in step S2 of the above embodiment. The plurality of centrifuge tubes 1 can be nine for example. The nine sealing parts 2 are connected in sequence to form a row, which is convenient for production and packaging. When not in use, the kit 100 is stored in a refrigerator at -20 °C. When needed, it is used after returning to room temperature, which can further reduce the time for detecting the sensitivity of Salmonella to ceftriaxone. The design is ingenious.

[0034] Furthermore, the centrifuge tube 1 and the sealing portion 2 are connected as one body and can be separated under the action of an external force. Specifically, in actual use, the centrifuge tube 1 and the sealing portion 2 can be sealed as one body. For example, the centrifuge tube 1 is a brown centrifuge tube 1, and the centrifuge tube 1 and the sealing portion 2 are a glass structure as one body. When the ceftriaxone solution 10 needs to be used, it is only necessary to break off one of the centrifuge tubes 1 from the connection with the corresponding sealing portion 2, and the remaining test kit 100 is returned to the refrigerator for storage. Of course, in some other embodiments, the centrifuge tube 1 and the sealing portion 2 can also be threaded, etc. The specific connection form is not limited, as long as it is convenient to actually take the ceftriaxone solution 10.

[0035] The procedure for using the kit 100 according to an embodiment of the present invention is as follows: the operator takes the kit 100 out of the refrigerator, returns it to room temperature, draws 0.5 ml of the ceftriaxone solution 10 in the centrifuge tube 1, mixes it thoroughly with the Salmonella precipitate, and then places it in an incubator at a temperature greater than or equal to 35 degrees Celsius and less than or equal to 37 degrees Celsius for incubation for a time greater than or equal to 45 minutes and less than or equal to 60 minutes. The kit is then centrifuged at a speed greater than or equal to 4000 rpm and less than or equal to 10000 rpm for 1-2 minutes. The supernatant after centrifugation is then analyzed using a time-of-flight mass spectrometer to determine whether the Salmonella is resistant to ceftriaxone due to carrying a resistance gene by observing whether the characteristic peak at 554-556 m / z disappears.

[0036] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the scope of the patent application of the present invention still fall within the scope covered by the present invention.

Claims

1. A method for rapidly testing the sensitivity of Salmonella to ceftriaxone, characterized in that, It includes the following steps: S1. Configure a Salmonella bacterial solution; S2. Centrifuge the bacterial solution and remove the supernatant of the centrifuged bacterial solution; S3. Provide a ceftriaxone solution, which contains ceftriaxone and water; S4. Mix the ceftriaxone solution with the bacterial solution to form a mixed solution, place the mixed solution in an incubator and let it stand for a first preset time. The temperature of the incubator is greater than or equal to 35°C and less than or equal to 37°C, and the first preset time is greater than or equal to 45 minutes and less than or equal to 60 minutes; S5. Centrifuge the mixed solution after standing for the first preset time and extract the supernatant of the centrifuged mixed solution; S6. Use a time-of-flight mass spectrometer to identify the ceftriaxone in the supernatant of the mixed solution and judge the sensitivity of Salmonella to ceftriaxone by whether the characteristic peak corresponding to ceftriaxone disappears.

2. The method for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 1, characterized in that, The McFarland turbidity of the bacterial solution is greater than or equal to 1.5 and less than or equal to 2.0, the volume of the ceftriaxone solution is 20 μL, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L.

3. The method for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 1, wherein Step S2 includes: Centrifuge the bacterial solution at a first rotation speed for a second preset time. The first rotation speed is greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the second preset time is greater than or equal to 1 minute and less than or equal to 2 minutes; Remove the supernatant of the centrifuged bacterial solution to obtain a concentrated solution of Salmonella.

4. The method for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 1, characterized in that, Step S5 includes: Centrifuge the mixed solution after standing for the first preset time at a second rotation speed for a third preset time. The second rotation speed is greater than or equal to 4000 revolutions per minute and less than or equal to 10000 revolutions per minute, and the third preset time is greater than or equal to 1 minute and less than or equal to 2 minutes; Extract the supernatant of the centrifuged mixed solution.

5. The method for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 1, wherein The mass-to-charge ratio of the characteristic peak corresponding to ceftriaxone is greater than or equal to 554 and less than or equal to 556.

6. A kit for rapidly testing the sensitivity of Salmonella to ceftriaxone, characterized in that, It includes a number of centrifugal tubes arranged in parallel. Each centrifugal tube contains a ceftriaxone solution, which contains ceftriaxone and water. Each centrifugal tube is provided with a sealing part at the top, and all the sealing parts of the several centrifugal tubes are connected in sequence to form a row.

7. The kit for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 6, wherein The centrifugal tube is integrally connected to the sealing part and can be separated under an external force.

8. The kit for rapidly testing the sensitivity of Salmonella to ceftriaxone according to claim 6, wherein The volume of the centrifugal tube is 0.5 mL, the volume of the ceftriaxone solution is 20 μL, and the content of ceftriaxone is greater than or equal to 70 mg / L and less than or equal to 80 mg / L.

Images