CN112266909B

CN112266909B - Viscous biological sample digestant and preparation method thereof

Info

Publication number: CN112266909B
Application number: CN202011099347.8A
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: 2020-10-14
Filing date: 2020-10-14
Publication date: 2023-03-03
Anticipated expiration: 2040-10-14
Also published as: CN112266909A

Abstract

The invention discloses a digestant composition of a viscous biological sample, which comprises protease, a stabilizer and a synergist; the protease is papain, cathepsin, subtilisin or proteinase K; the stabilizer comprises a saccharide, cyclodextrin and a biological buffer; the synergist is sodium pyrophosphate, triethanolamine, sodium ethylene diamine tetracetate, tartaric acid or sodium citrate. The invention also provides a digestant for viscous biological samples, which comprises the digestant composition and deionized water. The invention also provides a preparation method of the digestant for the viscous biological sample, which comprises the following steps: dissolving stabilizer, synergist and hyperosmotic agent in deionized water, adjusting pH to 7.2-7.4, adding protease, stirring and dissolving to obtain the final product. The digestive solution of the invention has stable liquid state, does not need to be compounded before use by users, does not damage pathogenic bacteria, can be stored at normal temperature, takes effect within the optimal pH range of the pathogenic bacteria, and can be universally used for different detection items.

Description

Viscous biological sample digestant and preparation method thereof

Technical Field

The invention relates to the field of medical examination, in particular to a viscous biological sample digestant and a preparation method thereof.

Background

Many clinical specimens, such as sputum, cervical specimens, etc., are sticky. In these samples, clinically insignificant normal flora was distributed on the surface of the mucus cake, while clinically significant pathogenic bacteria were mostly encapsulated inside the mucus cake. When the sample is smeared directly, visible components such as bacteria, cells and the like are overlapped in a plurality of layers and are difficult to stain and observe; when direct streaking inoculation is performed, the pathogenic bacteria inside the mucous membrane mass cannot be dispersed, and it is difficult to obtain a satisfactory separation effect. Therefore, before smear examination and isolation culture examination of such specimens, it is necessary to liquefy and homogenize the specimens, which is called specimen digestion in clinical practice.

There are both reducing agent and enzymatic methods for digestion of viscous samples, both currently accomplished by commercial reagents.

The reducing agent method uses reagents such as dithiothreitol and N-acetylcysteine, and exerts a de-binding action by breaking a disulfide bond (-S-S-). Dithiothreitol is often used for sample digestion in the isolation and culture of general bacteria, and N-acetylcysteine is used for sample digestion in the isolation and culture of mycobacteria. Because the reducing agent is unstable after being dissolved, the agent is provided in two components, wherein the reducing agent is provided in a dry powder form, the solvent is provided in a liquid form, and a user needs to mix and dissolve the two components (compound) before using; the reagent is unstable at normal temperature and needs to be frozen (-20 ℃) for storage; after compounding, the components need to be used up within 48 hours, otherwise, the components are oxidized and failed; when the method is used for sample digestion, pathogenic bacteria (particularly fastidious bacteria such as haemophilus influenzae and neisseria meningitidis) are easy to damage thalli, and culture detection fails.

The enzymatic method is also called pancreatin method, the used reagent is trypsin, belongs to endopeptidase, and plays a role in debonding by selectively cutting peptide bonds formed by lysine or arginine carboxyl groups. Because the trypsin is stable but inactive in an acid environment and active but unstable in an alkaline environment, the reagents are all provided in two components, wherein the trypsin is provided in a dry powder form, the solvent is provided in a liquid form, and the trypsin needs to be automatically compounded by a user before use; the reagent is unstable at normal temperature and needs to be stored by freezing (-20 ℃); after compounding, the compound needs to be used up within 48 hours, otherwise, the enzyme activity is lost; the optimum pH value of the trypsin is 7.8-8.5, the trypsin is alkaline and can cause slight damage to pathogenic bacteria, and the culture detection is influenced when the digestion time is too long.

In conclusion, the currently common sample digestive reagent needs to be stored at low temperature, needs to be compounded by users, has unstable liquid state, damages pathogenic bacteria and is not universal among different detection projects.

Disclosure of Invention

The present invention has been made in view of the above problems, and an object of the present invention is to provide a stable viscous biological sample digesting agent composition.

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

a digestant composition for a viscous biological sample comprising a protease, a stabilizer, and a potentiator;

the protease is papain, cathepsin, subtilisin or proteinase K;

the stabilizer comprises a saccharide, cyclodextrin and a biological buffer;

the synergist is sodium pyrophosphate, triethanolamine, sodium ethylene diamine tetracetate, tartaric acid or sodium citrate, preferably sodium ethylene diamine tetracetate.

The cathepsin is cathepsin B; the saccharide is selected from one or more of sucrose, trehalose, and mannitol, preferably trehalose.

The cyclodextrin is selected from one or more of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, preferably beta-cyclodextrin.

The biological buffer is selected from one or more of 2- (N-morpholine) ethanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, 2- [ (2-aminoethyl) amino ] ethanesulfonic acid sodium salt, piperazine-N, N ' -bis (2-ethanesulfonic acid), 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, 3- (N-morpholine) propanesulfonic acid, N- (2-hydroxyethyl) piperazine-N ' - (2-ethanesulfonic acid), preferably N- (2-hydroxyethyl) piperazine-N ' - (2-ethanesulfonic acid).

The digester composition also includes a hyperosmotic agent.

Preferably, the hyperosmotic agent is a neutral salt, optionally sodium sulfate, sodium chloride or magnesium sulfate, preferably sodium chloride.

The invention also provides a digestant for viscous biological samples, which comprises the digestant composition as described in any one of the preceding items, and also comprises deionized water.

The concentration of each component in the digestant is as follows: the mass volume ratio concentration of the protease is 0.1-1%, the mass volume ratio concentration of the saccharides is 0.5-1.5%, the mass volume ratio concentration of the cyclodextrin is 0.5-1.5%, the concentration of the biological buffer is 10-50mmol/L, the mass volume ratio concentration of the synergist is 0.1-0.5%, and the mass volume ratio concentration of the hypertonic agent in the digestive agent aqueous solution is 0-6%.

Preferably, the mass volume ratio concentration of the hypertonic agent in the digestant is 3-6%. The high permeability agent is not an essential component of the digestant, and can be further added to ensure that the inactivated bacteria in the air do not fall into the digestive system to cause pollution and failure.

The invention also provides a preparation method of the digestant for the viscous biological sample, which comprises the following steps: dissolving stabilizer, synergist and hyperosmotic agent in deionized water, adjusting pH to 7.2-7.4, adding protease, and stirring to dissolve completely.

The beneficial effects of the invention are:

the invention adopts the new protease to replace the trypsin in the traditional digestant, the action site of the adopted new enzyme is similar to that of the trypsin, the pH adaptation range is wider, the protease is stable in liquid at normal temperature, and the protease can be directly obtained commercially and has acceptable cost; the stability of the enzyme solution is enhanced by a stabilizing agent; the digestion effect is enhanced by the synergist which can complex metal ions among mucins, and the excellent digestion effect of the digestant is ensured. Furthermore, a hyperosmotic agent is added to ensure that the sealing is not polluted and failed due to the falling of sundry bacteria in the air after the sealing is started.

After the digestant is prepared into the aqueous solution, the liquid state is stable, the complex formulation before the use of a user is not needed, pathogenic bacteria are not damaged, the digestant can be stored at normal temperature, the digestant can take effect within the optimal pH range of the pathogenic bacteria, can be universally used for different detection items, and can resist the pollution of environmental microorganisms to the reagents after being unsealed.

Drawings

FIG. 1 is a visual inspection of the digestion of a viscous sample with a digestant solution of the invention, wherein a is normal saline treated sputum mass and b is digestant solution digested sputum mass.

FIG. 2 is a microscopic view of the digestion effect of the digestive agent solution of the present invention on a viscous sample, wherein a is an undigested sputum smear and b is a digested sputum smear.

FIG. 3 shows the results of the culture test of the viscous specimen treated with the digestive solution of the present invention on fastidious bacteria, wherein a is the normal saline control group culture and b is the enzyme co-culture test group culture.

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 all conventional ones unless otherwise specified.

Main source of reagents

Papain: CAS number 9001-73-4

Trypsin: CAS number 9002-07-7

Cathepsin B: CAS number 9047-22-7

Subtilisin: CAS number 9014-01-1

Proteinase K CAS number: 39450-01-6

Trehalose: CAS number 99-20-7

Beta-cyclodextrin: CAS number 7585-39-9

α -cyclodextrin: CAS number 10016-20-3

Gamma-cyclodextrin: CAS number 17465-86-0

N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid): CAS number 7365-45-9

2- (N-morpholine) ethanesulfonic acid: CAS number 145224-94-8

Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane: CAS number 6976-37-0

3- (N-morpholine) propanesulfonic acid: CAS number 1132-61-2

Disodium ethylene diamine tetraacetate: CAS number 139-33-3

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

Haemophilus influenzae: ATCC10211, commercially available.

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

EXAMPLE 1 preparation of test samples

1. The digestive agent solution is prepared by taking the following components by mass or volume:

dissolving solute except papain to constant volume, adjusting pH to 7.2-7.4, adding papain, stirring to dissolve completely, filtering with 0.22um microporous membrane for sterilization, and refrigerating at 2-8 deg.C for use in the same day.

2. The preparation of the trypsin solution of the control sample comprises the following components in percentage by weight:

dissolving solute except trypsin to constant volume, measuring pH value to 7.8 + -0.1, adding trypsin, stirring to dissolve completely, filtering with 0.22um microporous membrane for sterilization, and refrigerating at 2-8 deg.C for use on the day.

3. Preparation of control sample physiological saline

Sodium chloride 8.5g

Deionized Water 1000ml

Dissolving sodium chloride, diluting to desired volume, filtering with 0.22um microporous membrane for sterilization, and daily using or refrigerating at 2-8 deg.C.

Example 2 test sample digestion Performance on viscous samples

1. Sputum digestion test

1) Taking a clinical sputum specimen, adding 5ml of sterile normal saline, shaking for several times, and sucking away all liquid around the sputum block;

2) Repeating the step 1) twice to obtain experimental phlegm;

3) The sputum was divided into 3 equal portions, and digestion experiments were performed with 3 experimental samples (digestant of the invention, control sample trypsin solution, control sample physiological saline) of example 1, respectively, with the amounts of experimental samples as shown in table 1 (equal volumes of sputum and digestion solution added);

TABLE 1

4) Digesting, continuously shaking, and visually observing the digestion effect of the sputum block 10min, 20min and 30min after the test solution is added.

As a result: the digestion effect of the 1-2 tubes is gradually obvious along with the prolongation of the co-cultivation time, and the No. 3 tube is not changed all the time. Table 2 shows the results of digestion by co-cultivation for 30min, and the treatment effect is shown in FIG. 1. The results show that the digestion effect of the digestive agent solution is the same as that of the trypsin solution, and the digestion effect of the physiological saline is not.

TABLE 2

Pipe number	1	2	3
				Digestive effect	Good taste	Good taste	Is free of

The digestion effect judgment standard is as follows:

well: the liquid is evenly turbid after stirring, and no sputum lumps can be seen by naked eyes

The method comprises the following steps: the liquid is turbid after stirring, and a small amount of phlegm lumps are seen

Difference: the liquid is slightly turbid after stirring, and a large amount of phlegm lumps appear

None: the liquid is clear after stirring, and the sputum lumps have no signs of dispersion

2. Smear microscopic observation test before and after sputum digestion

The method comprises the following steps:

1) The inoculating loop picks up sputum subjected to digestion treatment for 30min and smears the sputum on a glass slide into a region with the size of a coin;

2) Naturally drying

3) Flame holding

4) Dyeing the acridine orange dye liquor for 2min, washing with water, and naturally drying;

5) Observing with a biological fluorescence microscope under a high power microscope.

As a result: as shown in fig. 2, a large number of reticuloendothelial filaments were visible in the saline control group before digestion, and cells were overlapped; neither the trypsin solution control group nor the digestant solution group of the present invention had reticuloendothelial filaments, and cells were singly dispersed.

Example 3 Effect test on the culture detection of fastidious bacteria

The method comprises the following steps:

1. the haemophilus influenzae strains are recovered and grow to the logarithmic growth phase;

2. adjusting to 0.5MCF turbidity with sterile normal saline;

3. then sterile normal saline is used for dilution according to the proportion of 1;

4. respectively at the time points of co-cultivation of 10min, 30min and 60min, sampling, diluting with sterile normal saline solution by 1 percent (10), adding 10ul of the diluted solution onto a chocolate plate culture medium, marking the inoculating loop without division so as to uniformly disperse the inoculating loop on the surface of the whole plate, and repeatedly making 3 plates at each time point of each group;

5. colony number for culture observation: culturing at 37 deg.C for 48-72 hr, and observing to count colonies.

The above test was performed in triplicate.

As a result: as shown in fig. 3 and table 3. The growth of the bacillus influenzae and the haemophilus influenzae is up to 60min, the colony number of a test group is not obviously different from that of a normal saline control group, and the fact that the digestive agent solution has no influence on the growth activity of the haemophilus influenzae is proved.

TABLE 3

Example 4 stability test

1. 1000ml of each of the digestion agent solution of the present invention and the control trypsin solution were prepared as in example 1;

2. aseptically dividing the digestive agent solution and the control trypsin solution into 2 parts respectively;

the digestion agent solution of the present invention and the control trypsin solution were each collected at 3.4 ℃ and left at room temperature (25 ℃) and tested as in example 2 after 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, and 45 days.

The results are shown in Table 4, and the digestion effect of the digestant solution is the same as that of the new formulation when the digestant solution is placed at 4 ℃ and room temperature for 45 days. After being placed at 4 ℃ for 72 hours in contrast with the trypsin solution, the digestion effect begins to decline, and no digestion effect exists after being placed for 5 days; after the trypsin solution is placed at 25 ℃ for 48 hours, the digestion effect begins to decline, and the digestion effect is completely absent after the trypsin solution is placed for 4 days.

TABLE 4

The digestion effect judgment standard is as follows:

well: the result shows that no sputum lumps can be seen by naked eyes after stirring, the liquid is uniform and turbid, no mucus thread is seen in smear observation, and cells are dispersed;

the method comprises the following steps: the result shows that the sputum lumps and the liquid are turbid, the mucous membrane is partially seen in the smear observation, most cells are dispersed, and the cells are partially overlapped.

Difference: the sputum lump is clear from the liquid, the liquid is clear, a large amount of mucus threads are seen in the smear, and the cells are overlapped.

Example 5

Digestant solution samples 1-4 were prepared, each sample having the specific composition shown in table 5:

TABLE 5

The samples 1 to 4 were used to perform the "digestion performance test on a viscous sample" in the same manner as in example 2, and as a result, it was found that: after the samples 1-4 digest the sputum specimen for 30min, the sputum digestion effect is good, no mucus thread is seen in smear dyeing observation, cells are scattered into single cells, the staining is clear, and the result is not different. Indicating that the digestant solution has consistent digestion performance on viscous samples within the effective concentration range.

The results of the "test for influence on fastidious bacteria detection" carried out using samples 1-4 in accordance with example 3 show that, when co-cultivation with Haemophilus influenzae is up to 60min, no significant difference is observed between the colony count and the normal saline control group, indicating that the growth activity of Haemophilus influenzae is not influenced by the digestant solution within the effective concentration range.

The stability test was performed according to example 4 using samples 1-4, and left at room temperature for 45 days, with no difference in the digestion performance of the viscous sample from the new formulation. The results show that the stability of the samples of different combinations is excellent over the effective concentration range.

Claims

1. A digestant for a viscous biological sample, comprising: consists of protease, a stabilizing agent, a synergist, a hyperosmotic agent and deionized water;

the protease is papain, cathepsin, subtilisin or proteinase K;

the stabilizing agent consists of saccharides, cyclodextrin and biological buffer, the saccharides are selected from one or more of sucrose, trehalose and mannitol, the cyclodextrin is selected from one or more of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, and the biological buffer is selected from one or more of 2- (N-morpholine) ethanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, 3- (N-morpholine) propanesulfonic acid and N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid);

the synergist is sodium pyrophosphate, triethanolamine, sodium ethylene diamine tetracetate or tartaric acid;

the hypertonic agent is sodium sulfate, sodium chloride or magnesium sulfate;

the concentration of each component in the digestant is as follows: the mass volume ratio concentration of protease is 0.1-1%, the mass volume ratio concentration of saccharide is 0.5-1.5%, the mass volume ratio concentration of cyclodextrin is 0.5-1.5%, the concentration of biological buffer is 10-50mmol/L, the mass volume ratio concentration of synergist is 0.1-0.5%, and the mass volume ratio concentration of hypertonic agent in digestive solution is 3-6%;

the digestant is prepared by the following method: dissolving stabilizer, synergist and hyperosmotic agent in deionized water, adjusting pH to 7.2-7.4, adding protease, and stirring to dissolve completely.

2. The viscous biological sample digestant of claim 1, wherein: the cathepsin is cathepsin B.

3. The method for producing a digesting agent for a viscous biological sample according to claim 1 or 2, wherein: dissolving stabilizer, synergist and hyperosmotic agent in deionized water, adjusting pH to 7.2-7.4, adding protease, and stirring to dissolve completely.