CN112189674A

CN112189674A - Biological sample safety protective agent and application thereof

Info

Publication number: CN112189674A
Application number: CN202011079961.8A
Authority: CN
Inventors: 农高惠; 何林声
Original assignee: Zhuhai Dehao Biotechnology Co ltd
Current assignee: Zhuhai Dehao Biotechnology Co ltd
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2021-01-08

Abstract

The invention discloses a biological sample safety protective agent, which comprises a component A and a component B, wherein the component A comprises oxidase and an enzyme stabilizer; the component B comprises a substrate corresponding to the oxidase in the component A, a long-chain quaternary ammonium salt and a specific enzyme inhibitor, wherein the oxidase and the corresponding substrate can generate hydrogen peroxide after meeting; the specific enzyme inhibitor can specifically inhibit tricarboxylic acid cycle and electron transfer chain associated enzyme, and does not interfere the activity of key enzyme in diagnostic reagent for detecting biological samples. Also discloses the application of the biological sample safety protective agent in the sterilization and disinfection treatment of biological samples containing pathogens, and a method for performing the sterilization and disinfection treatment on the biological samples by using the safety protective agent. The biological sample safety protective agent provided by the invention can eliminate the safety risk of a biological sample, does not influence the subsequent inspection result, is environment-friendly, does not increase the hardware investment, and does not increase the burden of personnel.

Description

Biological sample safety protective agent and application thereof

Technical Field

The invention relates to the technical field of biology and medicine, in particular to a biological sample safety protective agent and application thereof.

Background

In medicine, clinical specimens pose a significant biological safety risk, for example: respiratory tract samples (such as sputum, throat swab, tracheal lavage fluid, etc.) can carry mycobacterium tuberculosis, influenza virus, SARS virus, novel coronavirus, rubella virus, mumps virus, respiratory syncytial virus, mycoplasma pneumoniae, meningococcus, etc.; the blood sample can carry HIV, HBV, HCV, Salmonella typhi, etc.; the genitourinary tract sample may carry gonococci, treponema pallidum, ureaplasma urealyticum, chlamydia trachomatis, papilloma virus, herpes simplex virus, etc.; the fecal sample can carry salmonella typhi, shigella dysenteriae, campylobacter jejuni, rotavirus, enterovirus and the like; the skin lesion tissue sample often carries yeast-like fungi and filamentous fungi, and the gastric mucosa sample can carry helicobacter pylori and the like.

The sample inspection comprises the links of pretreatment, inspection, post-treatment and the like, and all the links have biological safety risks, which are specifically shown as follows: sample pretreatment: the centrifugation of a body fluid sample, the homogenization of a luminal secretion (such as sputum, leucorrhea and the like) sample, the picking of pathological change samples of feces and tissue samples and the like have the risks of causing infection of operators and pollution to laboratories; and (3) sample inspection: the operations of specimen flaking, wet film observation, culture inoculation and the like have the risks of causing infection of operators and pollution to laboratories. The risk of smear film making and wet film observation is high, and the probability of sample splashing or aerosol formation is high if the smear, drying and fixing processes are not operated in a biological safety cabinet, so that the infection of an operator and the pollution of a laboratory are easily caused, and the risk is higher when respiratory tract samples are treated; sample post-treatment: at present, the residual samples and cultures are stored in a centralized way and processed in a unified way. If the container is not tightly sealed or the container is broken and leaked, the sample overflows and the like in the process of temporarily storing and processing the sample, the operator infection and the laboratory pollution are caused at a high opportunity.

In clinical testing, the test items for a sample may include: bacterial culture, physiological examination (such as volume, appearance, smell, pH, specific gravity, etc.), biochemical examination (such as enzyme, protein, nucleic acid, saccharide, lipid, vitamin, metabolite, etc.), immunological examination (such as antigen, antibody detection, etc.), molecular examination (such as genome sequence, pathogenic gene, specific gene, mutation site, etc.), morphological examination items (also called microscopic examination, such as examination of cell, bacterium, fungus, protozoan, ovum, cast, crystal, etc.). Wherein, any pathogen killing operation can not be carried out before the bacteria culture so as to avoid influencing the separation of target bacteria. On the premise of ensuring that the physiology, biochemistry, immunology, nucleic acid and form of the object to be detected are unchanged, other projects firstly kill pathogens and then carry out detection so as to eliminate the biosafety risk of the sample and ensure the safety of personnel and experimental environment.

At present, all inspection projects are carried out before the biological safety risk of a sample is not eliminated, operators and experimental environments are in the biological safety risk in the whole inspection process, and the root cause is the lack of a biological sample safety protective agent which can eliminate the biological safety risk of the sample and does not influence the subsequent detection result.

The pathogen is killed by adopting the traditional killing preparation and the traditional method, although the biological safety risk of a sample can be eliminated, the subsequent detection result is greatly influenced, and the method has no practical significance: 1) killing with chemical disinfectant: the strong disinfectant comprises aldehydes and strong oxidants, and the aldehydes are easy to generate pungent odor to cause new chemical pollution; strong oxidants have obvious interference on dyeing, particularly fluorescent dyeing, and often cause fluorescent quenching. Moderate and low-efficiency disinfectants such as quaternary ammonium salts, surfactants and the like have slow effect and cannot eliminate the biological safety risk of the sample in a short time. 2) Killing by a thermal sterilization method: leading to the denaturation and the cleavage of visible components in the sample, and the disintegration and the inactivation of proteins, enzymes, antigens, antibodies, nucleic acids and the like, and the subsequent tests of physiology, biochemistry, immunology, nucleic acids, morphology and the like can not be carried out. 3) Temporary storage of the rest samples and cultures for centralized treatment: the risk is difficult to control during temporary storage, and the existing measures are all passive measures.

Current biosafety safeguards require significant resource investment: a biological safety laboratory needs to be established to ensure the environmental safety; personal protection appliances need to be equipped to ensure the safety of personnel; all operations are required to be operated in the biological safety cabinet as much as possible so as to ensure the safety of the inspection process; the above requirements may even lead to changes in the functional layout of the laboratory.

Disclosure of Invention

The invention aims to provide a biological sample safety protective agent, which can eliminate the safety risk of a biological sample, does not influence the subsequent inspection result, is environment-friendly, does not increase the hardware investment, and does not increase the personnel burden.

In order to achieve the purpose, the invention adopts the technical scheme that:

a biological sample safety protective agent comprises a component A and a component B, wherein the component A comprises oxidase and an enzyme stabilizer; the component B comprises a substrate corresponding to the oxidase in the component A, a long-chain quaternary ammonium salt and a specific enzyme inhibitor, wherein the oxidase and the corresponding substrate can generate hydrogen peroxide after meeting;

the specific enzyme inhibitor can specifically inhibit tricarboxylic acid cycle and electron transfer chain associated enzymes, and does not interfere the activity of key enzymes (such as horseradish peroxidase, alkaline phosphatase and the like) in a diagnostic reagent for detecting biological samples.

In the above technical scheme, the oxidase is one of glucose oxidase, alcohol oxidase, urate oxidase or cholesterol oxidase; the substrate corresponding to the oxidase is glucose, ethanol, uric acid or cholesterol;

the long-chain quaternary ammonium salt is a double-long-chain quaternary ammonium salt, preferably didecyl dimethyl ammonium chloride or didecyl dimethyl ammonium bromide;

the specific enzyme inhibitor is 2-methyl-4-isothiazolin-3-one or 5-chloro-2-methyl-4-isothiazolin-3-one or a mixture of the two.

In the above technical solution, the enzyme stabilizer comprises an enzyme protective agent, a biological buffer, a complexing agent and a hyperosmotic agent;

the enzyme protective agent comprises proteins and non-reducing sugar, wherein the proteins are bovine serum albumin, peptone or serum, and preferably bovine serum albumin; the non-reducing sugar is trehalose, sucrose, soluble starch or cellulose, preferably trehalose;

the biological buffer is trihydroxymethyl aminomethane, 3-morpholine propanesulfonic acid, hydroxyethyl piperazine ethanesulfonic acid, piperazine-N, N' -di (2-ethanesulfonic acid) or 2- (N-morpholino) ethanesulfonic acid; preferably hydroxyethyl piperazine ethanesulfonic acid;

the complexing agent is sodium pyrophosphate, triethanolamine, sodium ethylene diamine tetracetate, tartaric acid or sodium citrate; preferably sodium ethylene diamine tetracetate;

the hypertonic agent is neutral salt selected from one of sodium chloride, potassium nitrate and sodium sulfate; sodium chloride is preferred.

In the technical scheme, in the aqueous solution of the component A, the concentration of oxidase is 100-500U/ml, the concentration of proteins is 1-10g/L, the concentration of non-reducing sugar is 10-50g/L, the concentration of biological buffer is 10-50mmol/L, the concentration of complexing agent is 0.1-1g/L, and the concentration of hyperosmotic agent is 30-50 g/L;

in the aqueous solution of the component B, the concentration of a substrate corresponding to oxidase is 10-100g/L, the concentration of long-chain quaternary ammonium salt is 100-1000mg/L, and the concentration of a specific enzyme inhibitor is 75-450 mg/L;

preferably, component A is mixed with component B in equal volumes.

Preferably, the specific enzyme inhibitor is 2-methyl-4-isothiazolin-3-ketone, and the concentration is 90mg-450 mg/L; or the specific enzyme inhibitor is 5-chloro-2-methyl-4-isothiazoline-3-ketone, and the concentration is 75mg-375 mg/L.

The invention also provides the application of the biological sample safety protective agent in the sterilization and disinfection treatment of biological samples containing pathogens; preferably the pathogen is a enveloped virus, bacterial propagule or fungus.

The cytovirus is influenza virus or new coronavirus, the bacteria comprise gram-positive bacteria and gram-negative bacteria, the gram-positive bacteria are staphylococcus aureus, streptococcus or pneumococcus, the gram-negative bacteria are haemophilus influenzae, salmonella typhi, dysentery bacillus or legionella pneumophila, and the fungi are candida albicans, cryptococcus neoformans or dermatophytes.

The invention also provides a method for sterilizing and disinfecting a biological sample, which is implemented by respectively adding the component A and the component B of any one of the biological sample safety protective agents into the biological sample to be treated and uniformly mixing, wherein the preferable treatment time is more than 10 minutes.

The biological sample is a clinical biological sample, preferably sputum and bronchial secretions, nasopharyngeal swabs, suppurative and wound infection samples, urethral and vaginal swab samples, fecal samples, cerebrospinal fluid samples, conjunctival secretion samples, ear and papilla infection samples and blood samples.

The using amount of the safety protection agent is equal to the volume of the biological sample to be detected, and the component A and the component B in the safety protection agent are mixed in equal volume.

The biological sample safety protective agent consists of a hydrogen peroxide generation system, long-chain quaternary ammonium salt and a specific enzyme inhibitor. The hydrogen peroxide can rapidly kill pathogen propagules through strong oxidation; the long-chain quaternary ammonium salt can destroy the cell membrane of a pathogen, so that intracellular substances leak to sterilize, and the sterilization effect of hydrogen peroxide can be enhanced; the specific enzyme inhibitor can inactivate tricarboxylic acid cycle and electron transfer chain associated enzymes, block energy metabolism of pathogens and play a role in long-acting preservation; the enzyme activity is maintained by enzyme protective agent, enzyme action auxiliary agent, hypertonicity and the like, and the component A is prevented from being polluted by bacteria falling in the operation environment carelessly after unsealing.

The performance of the safety protective agent is evaluated by the killing effect on staphylococcus aureus, legionella, candida albicans and bacillus subtilis, and the reason is that: staphylococcus aureus is an important pyogenic coccus, is easy to mutate, is one of gram-positive bacteria with the strongest resistance to physical and chemical environments, and is inactivated, which means that other gram-positive bacteria propagules are killed; legionella is an important respiratory infectious bacterium, is one of gram-negative bacteria with the strongest resistance to physical and chemical environment, is acid-resistant and heat-resistant, and is inactivated, so that other gram-negative bacteria are killed; candida albicans is the most common mucosal infectious fungus in clinic, has stronger resistance to physicochemical environment than the fungus, and is killed, which means that other fungi are also killed. The bacillus subtilis can form spores, has low nutritional requirement and fast reproduction, is a representative of bacillus, can kill propagules of the bacillus subtilis and inhibit the gemmation and reproduction of the spores of the bacillus subtilis, and means that other pathogens cannot survive. The virus is less resistant to the environment, and the above conditions under which pathogenic bacteria are killed mean that the virus is inactivated.

The invention has the beneficial effects that: 1) the use of the biological safety protective agent can timely eliminate the biological safety risk of clinical samples; the biological safety of personnel and environment in the inspection process is guaranteed; the biological safety protection level can be improved, the defects of the existing biological safety protection measures are overcome, the environmental safety of inspectors and laboratories is guaranteed, other resource investment is not increased, and the current situation of function layout and personnel requirements of the existing laboratories is not changed. 2) The use of the biological safety protective agent has no interference on the subsequent test results. 3) The requirement on the operation of personnel is not high. 4) Is environment-friendly and does not cause environmental pollution. 5) The biological safety protective agent can also be used as a harmless treatment article for accidental leakage of clinical samples, microbial cultures and clinical samples.

Drawings

FIG. 1 is a graph showing the results of the inhibition of the growth of pathogenic bacteria by the safeners of the invention.

FIG. 2 is a graph showing the effect of the safener of the present invention on staining a treated biological sample.

FIG. 3 is a graph showing the effect of staining a clinical leucorrhea specimen after treatment with a safener according to the present invention.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are conventional unless otherwise specified.

Main reagent and experimental material sources:

glucose oxidase (CAS number: 9001-37-0), trehalose (CAS number: 99-20-7)

Hydroxyethyl piperazine Ethanesulfonic acid (CAS number: 7365-45-9), bovine serum Albumin (CAS number: 17879-45-7)

Bisdecyl dimethyl ammonium chloride (CAS number: 7173-51-5), soluble starch (CAS number: 9005-84-9)

2-methyl-4-isothiazolin-3-one (MIT) (CAS No.: 2682-20-4), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) (CAS No.: 26172-55-4)

The above reagents are conventional in the art and are commercially available.

The strains used in the examples were: staphylococcus aureus strains: ATCC6538, legionella pneumophila strain: ATCC33152, candida albicans strain: ATCC60193, bacillus subtilis strain: ATCC6633, which are conventional in the art, are commercially available.

The following media are commonly used in the art and are commercially available:

nutrient agar: contains peptone, beef extract, sodium chloride, agar, etc.;

BCYE agar: comprises N-carbamoylmethyl ethanesulfonic acid, yeast extract, active carbon, L-cysteine, soluble ferric pyrophosphate and agar;

samboluo agar: comprises glucose, peptone and agar;

the remaining reagents in the examples, if not indicated, were conventional in the art and were also commercially available.

Example 1

The experimental research method comprises the following specific steps:

first, sterilization test method

1. Preparation of a test bacterial liquid: selecting Staphylococcus aureus, Bacillus subtilis, Legionella pneumophila and Candida albicans solid plate culture in logarithmic growth phase, respectively scraping bacterial colonies, placing into sterile physiological saline, adjusting bacterial concentration to 0.5 McLeod turbidity, and diluting at a ratio of 1:1000 to make bacterial liquid concentration about 5 x 10⁵CFU/ml, preparing 4 test bacteria liquids;

2. each bacterial liquid is divided into two parts, one part is used for a sterilization test, and the other part is used for a control test;

3. and (3) sterilization: adding an equal volume of bactericide to be detected into the test tube/hole, and adding an equal volume of sterile normal saline into the control tube/hole;

4. inoculation: respectively blowing and beating the liquid in the sterilizing tube/hole uniformly 10min, 20min, 30min and 45min after mixing, sucking 10ul of the liquid and uniformly coating the liquid on a special culture plate for the strain; the culture media corresponding to the strains are shown in table 1:

TABLE 1

Bacterial strains	Staphylococcus aureus	Bacillus subtilis	Legionella pneumophila	Candida albicans
					Culture medium	Nutrient agar plate	Nutrient agar plate	BCYE agar plate	Sapaul agar plate

5. Culturing: directly putting staphylococcus aureus and bacillus subtilis into a constant-temperature incubator at 37 ℃ for culture; placing legionella pneumophila into a carbon dioxide culture bag, and placing the bag in a constant-temperature incubator at 37 ℃ for culture; putting Candida albicans into a wet box, and culturing in a constant-temperature incubator at 28 ℃;

6. after the culture, the culture plate is taken out, and the total number of colonies on the plate is calculated.

Second, method for testing influence of sterilization on bacterial morphology stainability

1. Preparation of a test bacterial liquid: selecting Staphylococcus aureus, Bacillus subtilis, Legionella pneumophila and Candida albicans solid in logarithmic growth phasePlating the culture, scraping the colonies into sterile physiological saline, adjusting the bacterial concentration to 0.5 McClure turbidity, the bacterial concentration is about 1.5 x 10⁸CFU/ml；

2. Each inoculum was divided into two portions, one portion for bactericidal test and the other portion for control test;

3. and (3) sterilization: adding an equal volume of bactericide to be detected into the test tube, and adding an equal volume of sterile normal saline into the control tube;

4. smearing: after 10min, 30min and 60min, respectively taking 20ul of bacterial liquid from the test tube and the control tube, adding the bacterial liquid onto a clean glass slide, coating an inoculating loop, naturally drying, and fixing by conventional flame;

5. dyeing: the gram stain, the acid-resistant stain and the acid-resistant fluorescent stain are purchased from Kyoto Biotechnology Limited, dyed according to the instruction of product use and dried. Carrying out experiment by self-prepared fluorescent staining solution (acridine orange) according to a conventional fluorescent staining method, dropwise adding the solution to cover all smear areas, standing for 2min, washing with water, and drying;

6. reading the film: observing with a biological fluorescence microscope under a high power microscope.

Third, test method for influence of sterilization on dyeability of leucorrhea sample

1. Preparation of leucorrhea sample liquid for test: fully eluting a clinical leucorrhea swab by using 1ml of physiological saline to prepare leucorrhea sample liquid;

2. each sample liquid is divided into two parts, one part is used for a sterilization test, and the other part is used for a control test;

4. smearing: after 10min, 30min and 60min, respectively taking 20ul of sample liquid from the test tube and the control tube, adding the sample liquid onto a clean glass slide, coating an inoculating loop, naturally drying, and fixing by conventional flame;

Example 2 minimum inhibitory concentration test

First, minimum inhibitory concentration test of hydrogen peroxide system

Hydrogen peroxide is produced by the decomposition of an oxidase-catalyzed substrate, and when the amount of substrate is sufficiently large, the production of hydrogen peroxide is controlled by the amount of enzyme used.

Preparing hydrogen peroxide sterilizing liquid with different concentrations: the prepared glucose aqueous solution has the mass percentage concentration of 5 percent and the concentration of the glucose oxidase stock solution of 12800u/ml, and hydrogen peroxide can be continuously generated after the glucose oxidase is contacted with glucose to form the sterilization solution. The sterilizing solutions with different concentrations are prepared by a multiple dilution method through a sterile plastic micropore plate, wherein the 1 st to 8 th holes contain hydrogen peroxide, and the 9 th hole is a no hydrogen peroxide contrast, and is specifically shown in table 2.

TABLE 2

And (3) sterilization test: the hydrogen peroxide sterilization solutions prepared in Table 2 were used in accordance with the method "one, sterilization test method" of example 1, and the final enzyme concentrations of each test well of the sterilization test are shown in Table 3:

TABLE 3

Order of test wells	1	2	3	4	5	6	7	8	9
										Final enzyme concentration (u/ml)	3200	1600	800	400	200	100	50	25	0

As shown in Table 4, the results of colony counting after completion of bacterial culture showed that the amount of hydrogen peroxide produced was sufficient to achieve bactericidal effect at an enzyme solution concentration of 50 u/ml.

TABLE 4

Final enzyme concentration (u/ml)	3200	1600	800	400	200	100	50	25	0
										Staphylococcus aureus (CFU)	0	0	0	0	0	0	0	0	531
Legionella pneumophila (CFU)	0	0	0	0	0	0	0	0	493
										Bacillus subtilis (CFU)	0	0	0	0	0	0	0	20	542
Candida albicans (CFU)	0	0	0	0	0	0	0	10	250

Second, minimum inhibitory concentration test of long chain quaternary ammonium salt

Preparing quaternary ammonium salt sterilizing liquid with different concentrations: the initial mass percentage concentration of the aqueous solution of the bisdecyl dimethyl ammonium chloride is 10%, the sterilization solutions with different concentrations are prepared by a double ratio dilution method through a sterile plastic micropore plate, the 1 st to 8 th holes contain the bisdecyl dimethyl ammonium chloride, the 9 th holes are contrasted by the non-bisdecyl dimethyl ammonium chloride, and the specific formula is shown in table 5:

TABLE 5

And (3) sterilization test: the quaternary ammonium salt sterilizing solutions with different concentrations prepared in table 5 were used according to the method "the first and sterilization test method" of example 1, and the final concentration of the bisdecyl dimethyl ammonium chloride in each test hole is shown in table 6:

TABLE 6

Order of test wells	1	2	3	4	5	6	7	8	9
										Final concentration of quaternary ammonium salt (mg/L)	3200	1600	800	400	200	100	50	25	0

As a result: as shown in Table 7, the minimum inhibitory concentration of bis-decyl dimethyl ammonium chloride is 50 mg/L.

TABLE 7

Final concentration of quaternary ammonium salt (mg/L)	3200	1600	800	400	200	100	50	25	0
										Staphylococcus aureus (CFU)	0	0	0	0	0	0	0	0	563
Legionella pneumophila (CFU)	0	0	0	0	0	0	0	0	485
										Bacillus subtilis (CFU)	0	0	0	0	0	0	0	21	502
Candida albicans (CFU)	0	0	0	0	0	0	0	8	242

Third, minimum inhibitory concentration test of specific enzyme inhibitor

Preparing specific enzyme inhibitor sterilizing liquid with different concentrations: the initial mass percentage concentration of MIT is 10%, the sterilization solution with different concentrations is prepared by a sterile plastic microporous plate by adopting a multiple dilution method, the 1 st to 8 th holes contain MIT, the 9 th hole is a non-MIT control, and the specific formula is shown in Table 8:

TABLE 8

And (3) sterilization test: the sterilization solutions of specific enzyme inhibitors at different concentrations prepared in Table 8 were used in accordance with the method "one, sterilization test method" of example 1, and the final MIT concentrations of the test wells are shown in Table 9:

TABLE 9

Order of test wells	1	2	3	4	5	6	7	8	9
										MIT Final concentration (mg/L)	2400	1200	600	300	150	75	32.5	16.25	0

As a result: as shown in Table 10, the minimum inhibitory concentration of MIT was 32.5 mg/L.

Watch 10

MIT Final concentration (mg/L)	2400	1200	600	300	150	75	32.5	16.25	0
										Staphylococcus aureus (CFU)	0	0	0	0	0	0	0	0	498
Legionella pneumophila (CFU)	0	0	0	0	0	0	0	0	460
										Bacillus subtilis (CFU)	0	0	0	0	0	0	0	18	487
Candida albicans (CFU)	0	0	0	0	0	0	0	6	232

Example 3

Firstly, the formulation of the safety protection agent of the invention is shown in table 11:

TABLE 11

The bisdecyl dimethyl ammonium chloride solution, the MIT solution and the CMIT solution are prepared by adopting deionized water.

Preparing the component A and the component B according to the proportion respectively, and then filtering and sterilizing the components respectively by using 0.22um microporous filter membranes for later use. When the component A and the component B are used, equal volume is added into a biological sample to be detected, and the sum of the volumes of the component A and the component B is the same as the volume of the biological sample to be detected.

Secondly, the killing effect of the safety protective agent on pathogenic bacteria

(1) The test was carried out by referring to the "first and sterilization test method" described above, and the amounts of substances added to the test tube and the control tube are shown in Table 12, taking a bacterial solution of each test bacterium:

TABLE 12

Item	Test tube	Control tube
			5*10⁵CFU/ml bacterial liquid	1ml	1ml
Component A of the safety protection agents according to the invention	0.5ml	-
			Component B of the inventive safety agents	0.5ml	-
Sterile normal saline	-	1ml
			Total volume	2ml	2ml

(2) Test results

As shown in figure 1, after 10min of co-cultivation, the safety protective agent of the invention can kill staphylococcus aureus, legionella pneumophila and candida albicans. Co-culturing for 30min to kill Bacillus subtilis propagules; after the bacillus subtilis is cultured for 45min, the bacillus subtilis still has no killing effect on bacillus subtilis spores.

Thirdly, the influence of the safety protective agent of the invention on the dyeing effect

(1) Effect of bacterial culture staining Effect the method "second, the method for testing the effect of Sterilization on staining of bacterial morphology" of example 1 was used. The result is shown in figure 2, which is the staining result of the slide after co-cultivation for 60min, the staining results of the test group and the control group are consistent, and the result shows that the safety protective agent of the invention has no interference to the conventional staining and fluorescent staining results of the strain.

(2) Effect on basic staining effect of leucorrhea the test method of influence of bactericidal activity on staining of leucorrhea samples was performed in the same manner as in the third method of example 1. The results are shown in FIG. 3. The figure shows the staining result of the sheet after co-cultivation for 60min, and the staining results of the test group and the control group are consistent, which shows that the safety protective agent of the invention has no interference on the conventional staining and fluorescent staining of bacteria and cells in secretions.

Example 4

The following safety protective agent samples 1 to 4 were prepared, and the specific composition of each sample is shown in table 13 (the bis-decyl dimethyl ammonium chloride solution, the MIT solution, and the CMIT solution in the table are all aqueous solutions, and the concentrations are mass percent concentrations):

watch 13

The samples 1 to 4 were tested according to the "one, sterilization test method" in example 1, and as a result, it was found that: after all samples 1-4 are co-cultured for 10min, the staphylococcus aureus, legionella pneumophila and candida albicans can be killed. Co-culturing for 30min to kill Bacillus subtilis propagules; after the bacillus subtilis is cultured for 45min, the bacillus subtilis still has no killing effect on bacillus subtilis spores. The safety protective agent of the invention has consistent effect of killing pathogens within the effective concentration range.

The samples 1 to 4 were used to perform the experiment according to the test method for the influence of sterilization on the stainability of bacteria morphology in example 1, and the results show that: the staining results of the test group and the control group of the samples 1-4 are consistent, which shows that the safety protective agent of the invention has no interference to the conventional staining and fluorescent staining results of the strains.

The samples 1 to 4 are used to carry out experiments according to the test method for the influence of sterilization on the dyeing property of the leucorrhea sample in the example 1, and the results show that: the staining results of the test group and the control group of the samples 1-4 are consistent, which shows that the protective agent of the invention has no interference on the conventional staining and fluorescent staining results of the leucorrhea specimen.

Claims

1. A biological sample safety protection agent, characterized in that: the enzyme inhibitor comprises a component A and a component B, wherein the component A comprises oxidase and an enzyme stabilizer; the component B comprises a substrate corresponding to the oxidase in the component A, a long-chain quaternary ammonium salt and a specific enzyme inhibitor, wherein the oxidase and the corresponding substrate can generate hydrogen peroxide after meeting;

the specific enzyme inhibitor can specifically inhibit tricarboxylic acid cycle and electron transfer chain associated enzyme, and does not interfere the activity of key enzyme in diagnostic reagent for detecting biological samples.

2. The biological sample safety agent of claim 1, wherein: the oxidase is one of glucose oxidase, alcohol oxidase, urate oxidase or cholesterol oxidase; the substrate corresponding to the oxidase is glucose, ethanol, uric acid or cholesterol;

3. The biological sample safety agent according to claim 1 or 2, wherein: the enzyme stabilizer comprises an enzyme protective agent, a biological buffering agent, a complexing agent and a hypertonic agent;

4. The biological sample safety agent of claim 3, wherein: in the aqueous solution of the component A, the concentration of oxidase is 100-500U/ml, the concentration of proteins is 1-10g/L, the concentration of non-reducing sugar is 10-50g/L, the concentration of biological buffer is 10-50mmol/L, the concentration of complexing agent is 0.1-1g/L, and the concentration of hypertonic agent is 30-50 g/L;

preferably, component A is mixed with component B in equal volumes.

5. The biological sample safety agent of claim 4, wherein: the specific enzyme inhibitor is 2-methyl-4-isothiazoline-3-ketone, and the concentration is 90mg-450 mg/L; or the specific enzyme inhibitor is 5-chloro-2-methyl-4-isothiazoline-3-ketone, and the concentration is 75mg-375 mg/L.

6. Use of the biological sample safety protection agent of any one of claims 1 to 5 in a sterilization process for a biological sample containing a pathogen; preferably the pathogen is a enveloped virus, bacterial propagule or fungus.

7. The use of claim 6, wherein: the cytovirus is influenza virus or new coronavirus, the bacteria comprise gram-positive bacteria and gram-negative bacteria, the gram-positive bacteria are staphylococcus aureus, streptococcus or pneumococcus, the gram-negative bacteria are haemophilus influenzae, salmonella typhi, dysentery bacillus or legionella pneumophila, and the fungi are candida albicans, cryptococcus neoformans or dermatophytes.

8. A method for sterilizing and disinfecting a biological sample, which is characterized in that the component A and the component B of the biological sample safety protective agent according to any one of claims 1 to 5 are respectively added into the biological sample to be treated and mixed uniformly, and the treatment time is preferably more than 10 minutes.

9. The method according to claim 8, wherein the biological sample is a clinical biological sample, preferably a sputum and bronchial secretions, nasopharyngeal swabs, purulent and wound infected samples, urethral and vaginal swab samples, fecal samples, cerebrospinal fluid samples, conjunctival secretions samples, ear and papillary infected samples, blood samples.

10. The method of claim 8 or 9, wherein the safener is used in an amount equal to the volume of the biological sample to be tested and wherein component a and component B of the safener are mixed in equal volumes.