Detection method and application of hemolytic streptococcus

Abstract

The invention provides a hemolytic streptococcus detection method and application, the detection method uses phage lyase PlyC specificity to rapidly identify, combine and split cell walls of host bacteria-hemolytic streptococcus, so as to release ATP in host bacteria cells; as the ATP level and the concentration of the bacterial liquid have positive correlation, a linear curve of the concentration of the bacterial liquid and the luminous intensity is obtained through an ATP bioluminescence method, and hemolytic streptococcus can be quantitatively detected in real time; the hemolytic streptococcus is rapidly and specifically detected in real time, so that medical equipment products with unqualified microbiological indexes can be rapidly screened, and a new technical support is provided for scientific supervision and industrial development of the medical equipment products.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a detection method and application of hemolytic streptococci. Background Art

[0002] All medical device products that come into contact with human tissue or blood must be tested for microbial indicators. For example, disposable medical protective masks are divided into ordinary and sterilization grades. The sterilization grade requires the product to be sterile, and the ordinary grade requires that the total bacterial colony count shall not exceed 100 cfu / g, and the total fungal colony count shall not exceed 100 cfu / g. In addition, some control bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and hemolytic Streptococcus shall not be detected in the ordinary grade. The traditional microbial detection method is the culture method, which is cumbersome and time-consuming for several days or even half a month. In order to improve the efficiency of inspection and testing, there is an urgent need for a rapid method for detecting microorganisms to test the microbial indicators in medical device products.

[0003] ATP (Adenosine triphosphate) bioluminescence method uses the reaction between ATP and luciferin-luciferase complex to determine whether ATP exists. During the bioluminescence reaction, luciferin is oxidized and emits fluorescence. The number of photons can be measured by ATP fluorescence meter. The number of photons is proportional to the ATP content. ATP is an energy carrier used for metabolism in all living organisms. Microorganisms in a certain growth period have a relatively constant level of ATP, which makes the ATP concentration and the content of live microorganisms have a good linear relationship. Therefore, the total number of live microorganisms in the sample can be indirectly detected by detecting the fluorescence intensity produced by the ATP bioluminescence reaction.

[0004] When using commonly used broad-spectrum chemical reagents for bacterial lysis, hemolytic streptococci are usually not effectively lysed. And because such bacterial lysis solutions have the effect of inhibiting luciferase activity, the solution after lysis needs to be further diluted to eliminate this inhibition, so this method will indirectly reduce the sensitivity of the ATP biological method for detecting the total number of microorganisms.

[0005] Therefore, it is necessary to propose a rapid and specific detection method to realize the instant detection of hemolytic streptococci and provide new technical support for the scientific supervision and industrial development of medical device products. Summary of the invention

[0006] The present invention provides a method for detecting hemolytic streptococci, which is used for specifically lysing hemolytic streptococci and combining with ATP bioluminescence method to quickly detect the total colony count of hemolytic streptococci, so as to meet the needs of quickly detecting microbial indicators in medical device products and screening unqualified medical device products.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The first aspect of the present invention is to provide a method for detecting hemolytic streptococci, comprising the steps of:

[0009] The test samples of different gradients were cleaved by lyase PlyC, and the ATP bioluminescence value of each test sample was detected by bioluminescence method to construct a standard curve of total colony count-ATP bioluminescence value;

[0010] The lytic enzyme PlyC is added to the sample to be tested for lysis, and the standard ATP curve is read based on the ATP bioluminescence value of the sample after lysis to obtain the total number of colonies of hemolytic streptococci in the sample to be tested.

[0011] Furthermore, the ATP bioluminescence value detection process is to freeze-dry and solidify the lytic enzyme PlyC and the ATP detection kit into the wells of the bioluminescence plate, and use an ELISA reader to dynamically detect the bioluminescence peak value of the sample solution to be tested.

[0012] The second aspect of the present invention is to propose the application of the detection method described in the first aspect in the quantitative detection of hemolytic streptococci in medical devices.

[0013] The third aspect of the present invention is to provide a quantitative detection method for hemolytic streptococci in masks, including the detection method described in the first aspect; the preparation process of the test sample includes:

[0014] S1. Take the mask for sterilization, apply hemolytic streptococcus, cut the infected mask into several pieces, inoculate them in culture medium for culture and positive identification;

[0015] S2. Take the diluent for the contaminated mask to prepare the sample solution, and dilute it 10 times to prepare 10 -1 ~10 -6 Several gradient bacterial solutions were prepared and the total number of colonies in each gradient bacterial solution was determined.

[0016] Furthermore, in step S1, the culture medium is TSB culture medium, and the culture conditions are culturing at 33° C. for 18 to 24 hours; the positive identification process is to take a sample from the TSB culture medium and streak it on a sheep blood agar plate, culture it at 33° C. for 18 to 24 hours, and observe whether the colonies on the sheep blood agar plate are single and whether they all have hemolytic rings.

[0017] Furthermore, in step S2, the total colony count of the test sample is determined by a plate colony counting method.

[0018] Furthermore, the preparation process of the sample to be tested or the sample solution is to cut the mask to be tested or the contaminated mask into several pieces, place them in a sterile homogenizing bag containing a diluent, and wash them with an oscillator to obtain the washing liquid.

[0019] Preferably, the composition and content of the diluent are: 5mM Tris-HCl, 5mM MgCl 2 6H 2 O, pH 7.4~7.6.

[0020] Preferably, the amount of diluent used in the preparation of the sample to be tested is 200 μL; the amount of lyase PlyC used in the detection of the sample to be tested is 0.4 mg, and the lysis condition is lysis at room temperature for 60 minutes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The detection method of the present invention utilizes the bacteriophage lytic enzyme PlyC to specifically and rapidly identify, bind to and lyse the cell wall of the host bacteria - hemolytic streptococci, thereby releasing ATP in the host bacteria cell; since the ATP level is positively correlated with the bacterial solution concentration, a linear curve of the bacterial solution concentration and the luminescence intensity is obtained by the ATP bioluminescence method, that is, the hemolytic streptococci can be quantitatively detected in real time.

[0023] The detection method of the present invention uses low-temperature freeze drying to solidify PlyC and ATP bioluminescent detection reagents at the bottom of the bioluminescent plate detection plate, and then adds the sample solution to be tested to lyse at room temperature. During this period, an ELISA instrument is used to dynamically detect the bioluminescent peak value of the sample solution to be tested, and the live bacterial concentration of hemolytic streptococci in the sample solution to be tested is calculated using a fitting equation of the luminescent value and the bacterial solution concentration. The present invention can detect hemolytic streptococci in real time, quickly and specifically, which is conducive to the rapid screening of medical device products with unqualified microbial indicators, and provides new technical support for scientific supervision of medical device products and industrial development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 It is a fitting standard curve of the total number of hemolytic streptococci on the surface of the contaminated sample of the present invention and the ATP bioluminescence value. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In one embodiment, a method for real-time and rapid detection of hemolytic streptococci is provided, by specifically utilizing the bacteriophage lytic enzyme PlyC to quickly identify, bind and lyse the cell wall of the host bacteria-hemolytic streptococci, thereby releasing the ATP in the host bacterial cell. Since the ATP level is positively correlated with the bacterial liquid concentration, a linear standard ATP curve of the bacterial liquid concentration and the luminescence intensity is obtained by the ATP bioluminescence method, which can quantitatively detect hemolytic streptococci in real time. The bacteriophage lytic enzyme PlyC is a highly active, stable and specific biological cell wall lysing agent. The use of PlyC not only solves the problem of the difficulty of hemolytic streptococci in breaking the cell wall, but also has the advantage of specific cell wall breaking. In addition, the product after the lytic enzyme PlyC can be directly used for ATP bioluminescence detection without affecting the luciferase activity. In addition, the PlyC and ATP bioluminescent detection reagents were freeze-dried and solidified at the bottom of a 96-well bioluminescent plate detection plate, and 200 μL of the test sample solution was added and lysed at room temperature for 60 minutes. During this period, an ELISA instrument was used to dynamically detect the bioluminescent peak value of the test sample solution, and the live bacterial concentration of hemolytic streptococci in the test sample solution was calculated using a fitting equation of the luminescent value and the bacterial solution concentration.

[0028] In the above examples, the streptococcal phage lytic enzyme PlyC can be expressed and purified through a prokaryotic expression system to obtain a high-purity PlyC protein, which can specifically lyse β-hemolytic streptococci. 600 The sterilization rate of the bacterial solution with a concentration of 0.56 reached 99.6% after 1 hour. The streptococcal cells treated with PlyC were lysed and fragmented under an electron microscope, indicating that PlyC can effectively hydrolyze the cell wall of hemolytic streptococci. PlyC can be used to identify and hydrolyze hemolytic streptococci in the sample solution by taking advantage of its exclusive lysis function for streptococci.

[0029] Specifically, taking the disposable medical protective mask of Class II medical device as an example, the method and steps for detecting hemolytic streptococci are as follows:

[0030] 1. Preparation of ATP bioluminescence detection plate

[0031] 0.4 mg PlyC and 100 μL ATP detection reagent (enhanced ATP detection kit, Biolight) were mixed and solidified at the bottom of a 96-well bioluminescent plate using low-temperature freeze-drying technology to prepare an ATP bioluminescent detection plate for later use.

[0032] 2. Preparation of Linear Standard ATP Curve

[0033] 1) Obtain the contaminated sample: sterilize several disposable medical protective masks, and evenly apply different concentrations of hemolytic streptococci on the masks. Place the contaminated mask in a biosafety cabinet for about 20 minutes. When there are no droplets on the surface of the mask, cut it into equal sizes (0.04-0.08g) for use. Inoculate the contaminated mask in trypticase soy broth (TSB), culture it at 33°C for 18-24h, take a sample from the TSB culture medium and streak it on a sheep blood agar plate, and culture it at 33°C for 18-24h. If the colonies grown on the blood plate are single and have hemolytic rings, and the colonies are Gram-positive streptococci after Gram staining, it means that the contaminated sample is ready;

[0034] 2) Determine the total number of colonies in the contaminated sample: Take the contaminated mask (10 g) and put it into 200 mL of bacterial dilution solution (5 mM Tris-HCl, 5 mM MgCl 2 6H 2 O, pH 7.4-7.6) in a sterile homogenizing bag, use an oscillator to fully wash the contaminated mask for 10 minutes to prepare the test sample solution. Use the bacterial solution diluent to dilute the test sample solution 10 times in a gradient to prepare 10 -1 ~10 -6 Gradient dilution solution. Pipette 1 mL of appropriate dilution gradient bacterial solution into an empty dish and pour 15-20 mL of 50°C nutrient agar. After solidification, invert and incubate at 33°C for 18-24 hours. Count the number of colonies in the dish to estimate the total number of colonies in each contaminated sample.

[0035] 3) Detecting the luminescence value of the contaminated sample: Pipette 200 μL of the test sample solution in step 1)-2) into the ATP bioluminescence detection well plate prepared in step 1, and lyse at room temperature for 60 minutes. During this period, a microplate reader ((Bio-Tek multifunctional microplate reader)) is used to dynamically detect the bioluminescence value of the test sample solution, and the maximum luminescence value of each well is taken as the ATP bioluminescence value of each contaminated sample;

[0036] 4) Determine the constant value: According to the ATP bioluminescence value and the total number of colonies corresponding to each contaminated sample, the following linear relationship function is constructed:

[0037] Y=mX+k (1)

[0038] Wherein, Y is the common logarithm of ATP bioluminescence value, X is the common logarithm of total colony count; m and k are constants.

[0039] The above linear relationship function is the constructed standard curve, such as Figure 1 As shown, where: m = 0.8580, k = -0.8654. 2 =0.9984, indicating a very high degree of fit.

[0040] 3. Detection of hemolytic streptococci in mask samples

[0041] 1) Cut the mask to be tested into several 1cm×3cm small pieces and put them into 200mL diluent (5mM Tris-HCl, 5mMMgCl 2 6H 2 O, pH 7.4-7.6) in a sterile homogenizing bag, use an oscillator to fully wash the contaminated mask for 10 minutes to prepare the sample solution to be tested;

[0042] 2) Add 200 μL of the sample solution to be tested into the 96-well plate for ATP bioluminescence detection prepared in step 1, and lyse at room temperature for 60 minutes, during which the bioluminescence peak value of the sample solution to be tested is dynamically detected using an ELISA reader (Bio-Tek multi-function ELISA reader).

[0043] 3) According to the bioluminescence peak value, substitute it into the standard curve obtained in step 2, calculate the corresponding X value, and obtain the viable count of hemolytic streptococci in the sample solution by conversion.

[0044] Example

[0045] For masks from different batches and manufacturers, the above detection method was used to detect hemolytic streptococci, and the conventional PCR molecular detection method (fluorescence quantitative PCR detection kit YR020012) was used for comparison. The total number of hemolytic streptococci determined by the conventional PCR molecular detection method was used as a benchmark to calculate the deviation percentage of the total number of hemolytic streptococci determined in the embodiment to verify the accuracy of the ATP bioluminescence method. The results are shown in Table 1.

[0046] Table 1:

[0047]

[0048] It is not difficult to see from Table 1 that when the detection method provided in this embodiment is used to detect hemolytic streptococci, the results are close to those of the PCR molecular detection method, so it can be used as a better choice for real-time rapid detection of hemolytic streptococci.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting hemolytic streptococci, characterized in that the steps include: The test samples of different gradients were cleaved by lyase PlyC, and the ATP bioluminescence value of each test sample was detected by bioluminescence method to construct a standard curve of total colony count-ATP bioluminescence value; The lytic enzyme PlyC is added to the sample to be tested for lysis, and the standard ATP curve is read based on the ATP bioluminescence value of the sample after lysis to obtain the total number of colonies of hemolytic streptococci in the sample to be tested.

2. The detection method according to claim 1, characterized in that: The ATP bioluminescence value detection process is to freeze-dry and solidify the lyase PlyC and the ATP detection kit into the wells of the bioluminescence plate, and dynamically detect the bioluminescence peak value of the sample solution to be tested using an ELISA reader.

3. Application of the detection method according to claim 1 or 2 in the quantitative detection of hemolytic streptococci in medical devices.

4. A quantitative detection method for hemolytic streptococci in masks, characterized in that: The detection method comprising claim 1 or 2; The preparation process of the test sample includes: S1. Take the mask for sterilization, apply hemolytic streptococcus, cut the infected mask into several pieces, inoculate them in culture medium for culture and positive identification; S2. Take the diluent for the contaminated mask to prepare the sample solution, and dilute it 10 times to prepare 10 -1 ~10 -6 Several gradient bacterial solutions were prepared and the total number of colonies in each gradient bacterial solution was determined.

5. The quantitative detection method according to claim 4, characterized in that: In step S1, the culture medium is TSB culture medium, and the culture condition is culturing at 33° C. for 18 to 24 hours; the positive identification process is to take a sample from the TSB culture medium and inoculate it on a sheep blood agar plate, culture it at 33° C. for 18 to 24 hours, and observe whether the colonies on the sheep blood agar plate are single and whether they all have hemolytic rings.

6. The detection method according to claim 4, characterized in that: In step S2, the total colony count of the test sample is determined by a plate colony counting method.

7. The quantitative detection method according to claim 4, characterized in that: The preparation process of the sample to be tested or the sample solution is as follows: the mask to be tested or the contaminated mask is cut into several pieces, and the pieces are placed in a sterile homogenizing bag containing a diluent, and the pieces are washed by an oscillator and then the washing liquid is obtained.

8. The quantitative detection method according to claim 7, characterized in that: The composition and content of the diluent are: 5mM Tris-HCl, 5mM MgCl2·6H2O, pH 7.4-7.

6.

9. The quantitative detection method according to claim 7, characterized in that: The amount of diluent used in the preparation of the sample to be tested is 200 μL; the amount of lyase PlyC used in the detection of the sample to be tested is 0.4 mg, and the lysis condition is lysis at room temperature for 60 minutes.

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