CN115747113B - Method for separating and culturing brucella ovis - Google Patents
Method for separating and culturing brucella ovis Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for separating and culturing brucella ovis. The method relates to a selective biphasic culture flask or a selective aerobic blood culture flask, and the components comprise casein pancreatin digest, soybean papain hydrolysate, sodium chloride, glucose, dipotassium hydrogen phosphate, sodium polyanisole sulfonate, pyridoxine hydrochloride, compound vitamins, agar, erythromycin, ampicillin sodium and the like. According to the invention, the selective biphasic culture bottle and the selective aerobic blood culture bottle are used for selectively culturing the brucella ovis of suspected brucella colonies, so that the common brucella ovis strains in China can be identified visually, the operation is simple, high professional skills are not needed, whether the brucella ovis infection is detected by observing the existence of the colonies, and the method can be applied to the preliminary identification of the brucella ovis strains, related researches and the like.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a method for separating and culturing brucella ovis.
Background
Brucellosis (Brucella) can cause epidemic disease, brucellosis, in the world of people and animals. At present, brucella has been found to have 12 species, the major causes of human infection are ovine species (b.melititis), bovine species (b.abortus), porcine species (b.suis) and canine species (b.canis). Brucella melitensis is classified into 1-3 biological types, wherein the 3 type of the Brucella melitensis is a dominant epidemic strain in China. Isolation of Brucella is a gold standard for diagnosis of Brucella. In appendix D of "diagnosis of Brucella (WS 269-2019), for the culture of Brucella, a full-automatic blood culture apparatus, a biphase culture bottle and a selective medium can be selected for culture, but for the suspected colonies obtained by culture, the suspected colonies are taken out from a biosafety laboratory of a corresponding grade for subsequent traditional biochemical identification and molecular biological identification, which is long in time consumption, high in cost and requires higher professional skills.
Disclosure of Invention
The invention aims to provide a method for separating and culturing brucella ovis.
The invention is characterized in that: the inventor discovers that the Brucella melitensis has resistance to part of small molecular medicines by screening more than 2000 kinds of small molecular medicines of bovine species, ovine species, porcine species and canine species, and confirms that each biotype of the Brucella melitensis has resistance to part of small molecular medicines through retests of 8 subtypes (types 1, 2, 3, 4, 5, 6, 7 and 9) of bovine species, 3 subtypes (types 1, 2 and 3) of ovine species, 5 subtypes (types 1, 2, 3, 4 and 5) of porcine species and 1 subtype of canine species, and confirms that each biotype of the Brucella melitensis has resistance to part of small molecular medicines through a broth dilution method. According to the results, the selective biphase culture bottle and the selective aerobic blood culture bottle of the brucella melitensis are manufactured, and by combining the results of common culture, whether the brucella melitensis is infected or not can be judged.
In order to achieve the aim of the invention, in a first aspect, the invention provides the application of a composition in preparing a culture medium for separating and culturing brucella ovis, wherein the composition consists of erythromycin and ampicillin sodium, and the contents of the components in the culture medium are as follows: 6-15mg/L erythromycin and 2-7mg/L ampicillin sodium.
In a second aspect, the present invention provides a selective biphasic medium, comprising a solid medium and a liquid medium;
wherein the solid culture medium comprises 15g/L of casein pancreatin digest, 5g/L of soybean papain hydrolysate, 5g/L of sodium chloride, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium and 15g/L of agar;
the liquid culture medium comprises 15g/L of casein pancreatin digest, 3g/L of soybean papain hydrolysate, 5g/L of sodium chloride, 2.5g/L of glucose, 2.5g/L of dipotassium hydrogen phosphate, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium, 35mg/L of Sodium Polyanisole Sulfonate (SPS) and 5mg/L of compound vitamin.
In a third aspect, the invention provides a method for preparing the selective biphasic medium, comprising the steps of:
A. preparation of solid medium: weighing 15g of casein pancreatin digest, 5g of soybean papain hydrolysate, 5g of sodium chloride and 15g of agar, heating and dissolving with 1L of deionized water, packaging 20-30mL, and placing in a biphasic culture bottle, and sterilizing at 121deg.C for 15-20min to obtain culture medium; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into non-coagulated culture medium cooled to 50deg.C, wherein the final concentration is 2-7mg/L; weighing erythromycin, dissolving with absolute ethanol, filtering, sterilizing, and adding into a non-coagulated culture medium cooled to 50 ℃ to obtain a final concentration of 6-15mg/L; evenly mixing and horizontally placing until cooling and solidifying;
B. preparation of liquid medium: weighing 15g of casein pancreatin digest, 3g of soybean papain hydrolysate, 5g of sodium chloride and 2.5g of dipotassium hydrogen phosphate, dissolving with 1L of deionized water, and sterilizing at 121 ℃ for 15-20min under high pressure to obtain a solution I; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into the solution I, wherein the final concentration is 2-7mg/L; weighing erythromycin, dissolving with absolute ethyl alcohol, filtering, sterilizing, and adding into the solution I to obtain final concentration of 6-15mg/L; weighing glucose, sodium polyanisole sulfonate and compound vitamin, dissolving with deionized water to make the concentrations of the glucose, the sodium polyanisole sulfonate and the compound vitamin be 250g/L, 35g/L and 5g/L respectively, filtering, sterilizing, adding the solution I to make the final concentrations of the glucose, the sodium polyanisole sulfonate and the compound vitamin be 2.5g/L respectively, and subpackaging 25-35mL in each double-phase culture bottle.
In a fourth aspect, the invention provides the use of said selective biphasic medium for the isolation and cultivation of Brucella melitensis (including for non-disease diagnostic purposes).
In a fifth aspect, the present invention provides a method for isolating and culturing Brucella melitensis (including the purpose of non-disease diagnosis), comprising the steps of:
(1) Dividing a liquid sample to be measured into two partsEqual parts, one part is injected into a selective biphasic culture flask, the other part is injected into a common biphasic culture flask, and the biphasic culture flask is placed into 5-10% CO at 37 DEG C 2 Culturing in an incubator, wherein the liquid phase fully infiltrates the solid phase for 4-12 hours, and then performing inclined culture;
wherein the selective biphasic culture flask is filled with the selective biphasic culture medium;
the common biphasic culture bottle is provided with a common biphasic culture medium, and the common biphasic culture medium does not contain the composition of the invention;
(2) After 1-4 weeks of culture (preferably 1-2 weeks), the culture results are observed:
a. the liquid phase part is turbid, and the solid phase part is single colony growth;
b. if the single colony is round, the diameter is 1-2mm, the edge is smooth, and the colony is shiny and semitransparent pale yellow, which is suspected Brucella;
c. if the single colony grows in the selective double-phase culture bottle and the common double-phase culture bottle, the Brucella melitensis is the Brucella melitensis; if the selective biphasic culture bottle does not grow the single colony, and the common biphasic culture bottle grows the single colony, the selective biphasic culture bottle is sheep, pig or canine Brucella; if the selective double-phase culture bottle has the single colony growth, and the common double-phase culture bottle does not have the single colony growth, the test fails and the verification needs to be repeated; if the liquid phase of the selective biphasic medium and the common medium are partially clear and the solid phase of the selective biphasic medium does not grow single colonies/lawn, no bacterial infection exists in the sample.
In a sixth aspect, the present invention provides a selective aerobic blood culture that is a liquid culture comprising 17g/L of casein pancreatin digest, 3g/L of soy papain hydrolysate, 35mg/L of sodium polyanisole sulfonate, 1mg/L of pyridoxine hydrochloride, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium, 5mg/L of amino acid complex, and 5mg/L of multivitamin.
In a seventh aspect, the present invention provides a method of preparing the selective aerobic blood culture, the method comprising: weighing 17g of casein pancreatin digest and 3g of soybean papain hydrolysate, dissolving with 1L of deionized water, packaging into blood culture bottles, and sterilizing at 121deg.C under high pressure for 15-20min; weighing amino acid compound, compound vitamin, sodium polyanisole sulfonate and pyridoxine hydrochloride, dissolving with deionized water, filtering, sterilizing, adding into a blood culture bottle to make the final concentration of the mixture be 5mg/L, 35mg/L and 1mg/L respectively, weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into the blood culture bottle to make the final concentration of the mixture be 2-7mg/L; weighing erythromycin, dissolving with absolute ethanol, filtering, sterilizing, and adding into a blood culture bottle with final concentration of 6-15mg/L.
In an eighth aspect, the invention provides the use of said selective aerobic blood culture in the isolated culture of Brucella melitensis (including for non-disease diagnostic purposes).
In a ninth aspect, the present invention provides a method for isolating and culturing Brucella melitensis (including the purpose of non-disease diagnosis), comprising the steps of:
1) Serological detection of brucellosis positive samples brucellosis was isolated in aerobic blood flasks: dividing a liquid sample to be detected into two equal parts, wherein one part is injected into a selective aerobic blood culture bottle, the other part is injected into a common aerobic blood culture bottle, and the blood culture bottle is placed into a full-automatic blood culture instrument for culture;
wherein the selective aerobic blood culture flask contains the selective aerobic blood culture medium;
the normal aerobic blood culture bottle is provided with a normal aerobic blood culture medium, and the normal aerobic blood culture medium does not contain the composition;
2) After 1-4 weeks (preferably 1-2 weeks) of culture, the presence or absence of Brucella melitensis in the sample is determined by observing the growth curve: if the selective blood culture bottle and the common blood culture bottle have strain growth curves, the strain is brucella ovis; if the selective blood culture bottle has no growth curve and the common blood culture bottle has a growth curve, the selective blood culture bottle is bovine species, porcine species or canine species brucella; if the selective blood culture bottle and the common blood culture bottle have no growth curve, bacteria are cultured in the sample; if the selective blood culture flask has a growth curve and the common blood culture flask does not have a growth curve, the test fails and repeated verification is needed. Aerobic flasks with growth curves were recommended to transfer 5-10mL of liquid to biphasic flasks for single colony acquisition.
In the invention, the liquid sample to be detected is selected from any one of liquid samples such as blood, serum, milk sample, tissue homogenate, aerosol liquid and the like, and is a serological detection brucellosis positive sample.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
according to the invention, the selective biphase culture bottle and the selective aerobic blood culture bottle are manufactured, so that the selective culture of the brucella ovis suspected of being the brucella colony is simple to operate, high expertise is not needed, whether the brucella ovis is infected or not can be judged by observing the control culture and the existence of the selectively cultured colony, and the method can be applied to researches, diagnoses and the like related to the preliminary identification of the brucella ovis type, so that technical support is provided for prevention and control of brucellosis in China.
Drawings
FIG. 1 is a growth curve of Brucella melitensis selectively cultured using an aerobic flask according to a preferred embodiment of the present invention. A is a growth curve of Brucella melitensis in a common aerobic blood culture bottle, the instrument reports positive when the Brucella melitensis is cultured for 5 days, and the bottle is taken out; b is a selective aerobic blood culture bottle sheep Brucella growth curve; c is the growth curve of the bovine brucella in a common aerobic blood culture bottle, the instrument reports positive when the culture is carried out for 5 days, and the bottle is taken out; d is a selective aerobic blood culture flask bovine brucella growth curve; e is a growth curve of Brucella suis in a common aerobic blood culture bottle, the instrument reports positive when the Brucella suis is cultured for 5 days, and the bottle is taken out; f is a selective aerobic blood culture flask Brucella suis growth curve, G is a common aerobic blood culture flask Brucella suis growth curve, and H is a selective aerobic blood culture flask Brucella suis growth curve.
FIG. 2 is a single colony fluorescent quantitative amplification curve of a selective biphasic flask mixed culture of multiple Brucella bacteria according to an embodiment of the present invention.
Detailed Description
The invention aims to provide a rapid, simple and effective method for identifying the brucella ovis infection in a brucella infection sample, which is used for solving the problem of rapid species identification in the clinical, prevention and control processes of the brucellosis. The invention relates to a method for determining whether the Brucella infection is sheep Brucella infection or not by visual inspection without complex identification test, which is common in instruments and can be applied to identification of clinical infection samples.
The invention adopts the following technical scheme:
the invention relates to a selective biphasic culture flask and a selective aerobic blood culture flask, which are respectively as follows:
the selective biphasic culture flask consists of a solid culture medium and a liquid culture medium, wherein each flask contains 20mL of solid culture medium and 25mL of liquid culture medium, and the solid culture medium contains 15g/L of casein pancreatin digest, 5g/L of soybean papain hydrolysate, 5g/L of sodium chloride, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium and 15g/L of agar; the liquid phase culture medium comprises 15g/L of casein pancreatin digest, 3g/L of soybean papain hydrolysate, 5g/L of sodium chloride, 2.5g/L of glucose, 2.5g/L of dipotassium hydrogen phosphate, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium, 35mg/L of Sodium Polyanisole Sulfonate (SPS) and 5mg/L of compound vitamin.
Each of the selective aerobic blood culture flasks contained 30mL of liquid medium containing 17g/L of casein pancreatin digest, 3g/L, SPS mg/L of soybean papain hydrolysate, 1mg/L of pyridoxine hydrochloride, 6-15mg/L of erythromycin, 2-7mg/L of ampicillin sodium, 5mg/L of other complex amino acids (i.e., amino acid complex), and 5mg/L of complex vitamins.
The common biphase culture flask and the common aerobic blood culture flask for control are respectively: the common biphasic culture bottle consists of a solid culture medium and a liquid culture medium, wherein each bottle contains 20mL of solid culture medium and 25mL of liquid culture medium, and the solid culture medium contains 15g/L of casein pancreatin digest, 5g/L of soybean papain hydrolysate, 5g/L of sodium chloride and 15g/L of agar; the liquid phase culture medium comprises 15g/L of casein pancreatin digest, 3g/L of soybean papain hydrolysate, 5g/L of sodium chloride, 2.5g/L of glucose, 2.5g/L of dipotassium hydrogen phosphate, 35mg/L of Sodium Polyanisole Sulfonate (SPS) and 5mg/L of compound vitamin.
A common aerobic blood culture bottle contains 30mL of liquid culture medium, and contains 17g/L of casein pancreatin digest, 3g/L, SPS mg/L of soybean papain hydrolysate, 1mg/L of pyridoxine hydrochloride, 5mg/L of other compound amino acids (namely amino acid compound) and 5mg/L of compound vitamin.
The invention provides a method for separating and culturing brucella ovis (related to a selective biphase culture bottle), which comprises the following steps:
(1) 10mL of whole blood is aseptically taken and is ready to be checked and liquid is injected into a selective biphasic culture flask, meanwhile, 10mL of whole blood is aseptically taken and is injected into a common biphasic culture flask, and the biphasic culture flask is put into a carbon dioxide incubator at 37 ℃ for culture (CO 2 The content is 5-10 percent), and the liquid phase is fully infiltrated into the solid phase for 4 hours and then is obliquely cultured.
(2) The growth condition of the culture flask is observed every other day, and the culture flask is gently shaken at the same time, so that the liquid phase is uniformly paved on the surface of the solid phase.
(3) And (3) result judgment:
in the case of bacterial infection:
in the double-phase culture flask, the liquid phase part is seen to be turbid and the solid phase part is seen to be single colony growing within 1-2 weeks of culture. If the single colony is round, the diameter is 1-2mm, the edge is smooth, and the colony is shiny and semitransparent pale yellow, which is suspected Brucella. If the single colony grows in the selective double-phase culture bottle and the common double-phase culture bottle, the Brucella melitensis is the Brucella melitensis; if the selective biphasic culture bottle does not grow the single colony, and the common biphasic culture bottle grows the single colony, the selective biphasic culture bottle is brucella such as bovine species, porcine species or canine species; if the selective dual-phase culture flask has the single colony growth, and the common dual-phase culture flask does not have the single colony growth, the test fails and the verification needs to be repeated.
No bacterial infection:
if the liquid phase of the selective biphasic medium and the common medium are partially clear and the solid phase of the selective biphasic medium does not grow single colonies/lawn, no bacterial infection exists in the sample.
The invention provides a method for separating and culturing brucella ovis (relating to a selective aerobic blood culture bottle), which comprises the following steps:
(1) 10mL of whole blood waiting to be detected is aseptically taken and injected into a selective aerobic blood culture bottle, and meanwhile 10mL of whole blood waiting to be detected is aseptically taken and injected into a common aerobic blood culture bottle, and the aerobic blood culture bottle is placed into a full-automatic blood culture instrument for culture.
(2) And observing a growth curve of the corresponding position of the full-automatic blood culture instrument. Typically, the culture is carried out for 1-2 weeks, and the maximum observation is 4 weeks.
(3) And (3) result judgment: in the blood culture bottle, judging whether the Brucella melitensis exists in the sample by observing a growth curve. If the selective blood culture bottle and the common blood culture bottle have strain growth curves, the strain is brucella ovis; if the selective blood culture bottle has no growth curve and the common blood culture bottle has a growth curve, the selective blood culture bottle is brucella such as bovine species, porcine species or canine species; if the selective blood culture bottle and the common blood culture bottle have no growth curve, bacteria are not cultured in the sample; if the selective blood culture flask has a growth curve and the common blood culture flask does not have a growth curve, the test fails and repeated verification is needed.
The method for separating and culturing the brucella ovis provided by the invention directly aims at identifying the brucella ovis infection, and does not acquire disease diagnosis results.
The invention provides a method for rapidly, effectively and intuitively judging whether the strain is Brucella melitensis infection, which can be applied to researches, diagnosis and the like related to Brucella melitensis type identification. The invention relates to a common instrument, can judge whether the strain is Brucella infection of sheep species by observing the existence of a colony without higher professional skills, can be applied to researches, diagnoses and the like related to Brucella type identification, can be fully popularized and applied, and is beneficial to promoting the diagnosis, prevention and control of Brucella.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The test materials and test instruments used in the following examples were as follows:
1. test materials
The sheep type 1 standard strain 16M, the cattle type 1 standard strain 2308, the pig type 1 standard strain 1330 and the canine type standard strain RM6/66 are all stored by a Beijing livestock veterinary research institute animal biosafety and public health prevention and control team of China academy of agricultural sciences, and recovery, passage and inoculation are completed in a Beijing city animal epidemic prevention and control center biosafety third-level laboratory (BSL-3).
Casein pancreatin digests, soy papain hydrolysates, and agar from BD company were used to prepare the culture medium; sodium chloride, glucose, sodium Polyanisole Sulfonate (SPS), dipotassium hydrogen phosphate, pyridoxine hydrochloride were purchased from chinese medicine company; the compound vitamins, erythromycin, ampicillin sodium and compound amino acids are purchased from Soy Bao company; the flask was customized as required by the instrument, and autoclaved with the double distilled water as required. Other biochemical reagents are imported and split-packed or domestic analytically pure; the Brucella bovine species, ovine species and porcine species nucleic acid detection kit (probe method) is purchased from Tokyo corporation in Qingdao.
2. Test instrument
CO 2 Incubator (Thermo Fisher Co.), full automatic blood incubator (Mei Liai Co.), bacterial turbidimeter (Mei Liai Co.), fluorescent quantitative PCR instrument (Thermo Fisher Co.).
Example 1 preparation of biphase culture flask
(1) Preparation of common biphase culture flask
a. Autoclaving the solid phase medium. Weighing 15g of casein pancreatin digest, 5g of soybean papain hydrolysate, 5g of sodium chloride and 15g of agar, heating and dissolving with 1L of deionized water, subpackaging 20mL, placing into a double-phase culture flask, sterilizing at 121 ℃ for 15-20min, and horizontally placing until cooling and solidifying.
b. Autoclaving the liquid phase medium. Weighing 15g of casein pancreatin digest, 3g of soybean papain hydrolysate, 5g of sodium chloride and 2.5g of dipotassium hydrogen phosphate, dissolving with 1L of deionized water, and sterilizing at 121 ℃ for 15-20min under high pressure to obtain a liquid culture medium; weighing glucose, sodium Polyanisole Sulfonate (SPS) and compound vitamin, dissolving with deionized water to make the concentrations of the glucose, the Sodium Polyanisole Sulfonate (SPS) and the compound vitamin be 250g/L, 35g/L and 5g/L respectively, filtering, sterilizing, adding the mixture into a liquid culture medium to make the final concentrations of the glucose, the Sodium Polyanisole Sulfonate (SPS) and the compound vitamin be 2.5g/L, SPS mg/L respectively, and subpackaging the mixture into 25mL in each double-phase culture bottle.
(2) Preparation of selective biphase culture flask
a. Autoclaving the solid phase medium. Weighing 15g of casein pancreatin digest, 5g of soybean papain hydrolysate, 5g of sodium chloride and 15g of agar, heating and dissolving with 1L of deionized water, packaging 20mL, and placing in a biphasic culture flask, and sterilizing at 121 ℃ for 20min to obtain a culture medium; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into a non-coagulated culture medium cooled to about 50deg.C, wherein the final concentration is 3mg/L; weighing erythromycin, dissolving with absolute ethyl alcohol, filtering, sterilizing, adding into a non-coagulated culture medium cooled to about 50 ℃, and obtaining a final concentration of 6mg/L; and (5) evenly mixing, and horizontally placing until cooling and solidifying.
b. Autoclaving the liquid phase medium. Weighing 15g of casein pancreatin digest, 3g of soybean papain hydrolysate, 5g of sodium chloride and 2.5g of dipotassium hydrogen phosphate, dissolving with 1L of deionized water, and sterilizing at 121 ℃ for 15-20min under high pressure to obtain a liquid culture medium; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into liquid culture medium with final concentration of 3mg/L; weighing erythromycin, dissolving with absolute ethyl alcohol, filtering, sterilizing, and adding into a liquid culture medium, wherein the final concentration is 6mg/L; weighing glucose, SPS and compound vitamin, dissolving with deionized water to make the concentrations of the glucose, SPS and compound vitamin be 250g/L, 35g/L and 5g/L respectively, filtering, sterilizing, adding the mixture into a liquid culture medium to make the final concentrations of the glucose, SPS and compound vitamin be 2.5g/L, SPS mg/L and 5mg/L respectively, and subpackaging 25mL in each double-phase culture bottle.
Example 2 preparation of aerobic blood culture flask
(1) Preparation of common aerobic blood culture bottle
Weighing 17g of casein pancreatin digest and 3g of soybean papain hydrolysate, dissolving with 1L of deionized water, packaging 30mL of the mixture per bottle, and sterilizing at 121 ℃ under high pressure for 15-20min; weighing the amino acid compound, the compound vitamin, the SPS and the pyridoxine hydrochloride, dissolving the amino acid compound, the compound vitamin, the SPS and the pyridoxine hydrochloride in deionized water, filtering, sterilizing and adding the mixture into a blood culture flask to ensure that the final concentration is 5mg/L, 35mg/L and 1mg/L respectively.
(2) Selective aerobic blood culture flask preparation
Weighing 17g of casein pancreatin digest and 3g of soybean papain hydrolysate, dissolving with 1L of deionized water, packaging 30mL of the mixture per bottle, and sterilizing at 121 ℃ under high pressure for 15-20min; weighing amino acid compound, vitamin complex, SPS and pyridoxine hydrochloride, dissolving with deionized water, filtering, sterilizing, adding into a blood culture bottle to obtain culture solution with final concentration of 5mg/L, 35mg/L and 1mg/L respectively, weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into culture solution with final concentration of 3mg/L; and (3) weighing erythromycin, dissolving the erythromycin with absolute ethyl alcohol, filtering, sterilizing, and adding the erythromycin into a culture solution, wherein the final concentration is 6mg/L.
Example 3 Selective cultivation of Brucella melitensis (involving a Selective biphase culture flask)
(1) Preparation of bacterial liquid
After streaking different Brucella strains for 96 hours, the specific turbidity of the strain was adjusted to 0.5McF (about 1X 10) 8 CFU/mL)。
(2) Standard strain culture of brucella
The prepared bacterial liquid is diluted by 100 times, and 100 mu L of bacterial liquid is taken and added into a common and selective biphase culture bottle. The diphase flask was placed in a 37℃carbon dioxide incubator for cultivation (CO 2 The content is 5-10 percent), and the liquid phase fully infiltrates the solid phase for 4-12 hours and then is obliquely cultured. The growth condition of the culture flask is observed every other day, and the culture flask is gently shaken at the same time, so that the liquid phase is uniformly paved on the surface of the solid phase. Typically, the culture is carried out for 1-2 weeks, and the maximum observation is 4 weeks.
(3) Result determination
In the case of bacterial infection:
in the double-phase culture flask, the liquid phase part is seen to be turbid and the solid phase part is seen to be single colony growing within 1-2 weeks of culture. If the single colony is round, the diameter is 1-2mm, the edge is smooth, and the colony is shiny and semitransparent pale yellow, which is suspected Brucella. If the single colony grows in the selective double-phase culture bottle and the common double-phase culture bottle, the Brucella melitensis is the Brucella melitensis; if the selective biphasic culture bottle does not grow the single colony, and the common biphasic culture bottle grows the single colony, the selective biphasic culture bottle is brucella such as bovine species, porcine species or canine species; if the selective double-phase culture bottle and the common double-phase culture bottle do not have the single colony growth, bacteria are not cultured in the sample; if the selective dual-phase culture flask has the single colony growth, and the common dual-phase culture flask does not have the single colony growth, the test fails and the verification needs to be repeated.
No bacterial infection: if the liquid phase of the selective biphasic medium and the common medium are partially clear and the solid phase of the selective biphasic medium does not grow single colonies/lawn, no bacterial infection exists in the sample.
The growth curve of selective culture of Brucella melitensis using an aerobic flask is shown in FIG. 1.
Example 4 Selective cultivation of Brucella melitensis (involving a Selective aerobic blood culture flask)
(1) Preparation of bacterial liquid
After streaking different Brucella strains for 96 hours, the specific turbidity of the strain was adjusted to 0.5McF (about 1X 10) 8 CFU/mL)。
(2) Standard strain culture of brucella
The prepared bacterial liquid is diluted by 100 times, and 100 mu L of bacterial liquid is taken and added into a common and selective aerobic blood culture flask. The aerobic blood culture flask is placed into a fully automatic blood culture apparatus for culture. For the blood culture flask, a growth curve was observed at the corresponding position of the fully automatic blood culture apparatus. Typically, the culture is carried out for 1-2 weeks, and the maximum observation is 4 weeks.
(3) Result determination
In the blood culture bottle, judging whether the Brucella melitensis exists in the sample by observing a growth curve. If the selective blood culture bottle and the common blood culture bottle have strain growth curves, the strain is brucella ovis; if the selective blood culture bottle has no growth curve and the common blood culture bottle has a growth curve, the selective blood culture bottle is brucella such as bovine species, porcine species or canine species; if the selective blood culture bottle and the common blood culture bottle have no growth curve, bacteria are not cultured in the sample; if the selective blood culture flask has a growth curve and the common blood culture flask does not have a growth curve, the test fails and repeated verification is needed. Aerobic flasks with growth curves were recommended to transfer 5-10mL of liquid to biphasic flasks for single colony acquisition.
EXAMPLE 5 selective two-phase flask culture of Brucella
Since single colonies cannot be observed in the selective blood flask, the mixed culture identification was performed by taking the selective two-phase flask as an example.
(1) Preparation of bacterial liquid
After streaking different Brucella strains for 96 hours, the specific turbidity of the strain was adjusted to 0.5McF (about 1X 10) 8 CFU/mL). Each strain was diluted to 100CFU/mL in multiple ratio.
(2) Brucella culture
100 mu L of each of the different brucella is taken and added into a common and selective biphasic culture flask after being evenly mixed. The diphase flask was placed in a 37℃carbon dioxide incubator for cultivation (CO 2 The content is 5-10 percent), and the liquid phase is fully infiltrated into the solid phase for 4 hours and then is subjected to inclined culture. The growth condition of the culture flask is observed every other day, and the culture flask is gently shaken at the same time, so that the liquid phase is uniformly paved on the surface of the solid phase.
(3) Fluorescent quantitative detection
And (3) adding the single colony grown in the solid phase into 100 mu L of physiological saline to prepare bacterial suspension, detecting 2 mu L of bacterial suspension by using a fluorescent quantitative kit, and preparing a system according to the description of the kit to identify the Brucella.
(4) Result determination
In the biphasic flask, the liquid phase portion was seen as cloudy and the solid phase portion was seen as 8 single colonies growing within 5 days of culture. The single colony is round, the diameter is 1-2mm, the edge is smooth, and the colony is shiny and semitransparent pale yellow.
The 8 single-colony fluorescence quantitative detection FAM channels all have amplification curves, and according to the kit, the FAM channels are all of brucella ovis, the positive control is a standard strain colony of the ovine, the FAM channels also have amplification curves, and the other channels all have no amplification curves. The amplification curve is shown in fig. 2, 8 single colonies to be detected and 1 standard strain colony of sheep species are detected in total, and the amplification curve is a brucella curve of sheep species and is consistent with the expected result.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. Preparation of composition for separating and culturing brucella ovisBrucella melitensis) The composition consists of erythromycin and ampicillin sodium, and the contents of the components in the culture medium are respectively as follows: 6-15mg/L erythromycin and 2-7mg/L ampicillin sodium;
the culture medium for separating and culturing the brucella ovis is a selective biphase culture medium or a selective aerobic blood culture medium;
the selective biphase culture medium consists of a solid culture medium and a liquid culture medium;
wherein the solid culture medium comprises casein pancreatin digest 15g/L, soybean papain hydrolysate 5g/L, sodium chloride 5g/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, agar 15g/L;
the liquid culture medium comprises casein pancreatin digest 15g/L, soybean papain hydrolysate 3g/L, sodium chloride 5g/L, glucose 2.5g/L, dipotassium hydrogen phosphate 2.5g/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, sodium polyanisole 35mg/L and vitamin complex 5mg/L;
the selective aerobic blood culture medium is a liquid culture medium comprising casein pancreatin digest 17g/L, soybean papain hydrolysate 3g/L, sodium polyanisole sulfonate 35mg/L, pyridoxine hydrochloride 1mg/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, amino acid complex 5mg/L and multivitamin 5mg/L.
2. The selective biphase culture medium is characterized by comprising a solid culture medium and a liquid culture medium;
wherein the solid culture medium comprises casein pancreatin digest 15g/L, soybean papain hydrolysate 5g/L, sodium chloride 5g/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, agar 15g/L;
the liquid culture medium comprises casein pancreatin digest 15g/L, soybean papain hydrolysate 3g/L, sodium chloride 5g/L, glucose 2.5g/L, dipotassium hydrogen phosphate 2.5g/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, sodium polyanisole 35mg/L and vitamin complex 5mg/L.
3. The method for preparing the selective biphasic medium according to claim 2, comprising the steps of:
A. preparation of solid medium: weighing casein pancreatin digest 15g, soybean papain hydrolysate 5g, sodium chloride 5g, agar 15g, heating with 1L deionized water for dissolving to obtain culture medium, packaging 20-30mL, placing in biphasic culture bottle, and sterilizing at 121deg.C under high pressure for 15-20min; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into non-coagulated culture medium cooled to 50deg.C, wherein the final concentration is 2-7mg/L; dissolving erythromycin in absolute ethanol, filtering, sterilizing, and adding into non-coagulated culture medium cooled to 50deg.C, wherein the final concentration is 6-15mg/L; evenly mixing and horizontally placing until cooling and solidifying;
B. preparation of liquid medium: weighing casein pancreatin digest 15g, soybean papain hydrolysate 3g, sodium chloride 5g and dipotassium hydrogen phosphate 2.5g, dissolving with 1L deionized water, and sterilizing at 121deg.C for 15-20min to obtain solution I; weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into the solution I, wherein the final concentration is 2-7mg/L; weighing erythromycin, dissolving with absolute ethyl alcohol, filtering, sterilizing, and adding into the solution I to obtain final concentrations of 6-15mg/L respectively; weighing glucose, sodium polyanisole sulfonate and compound vitamin, dissolving with deionized water to make the concentrations of the glucose, the sodium polyanisole sulfonate and the compound vitamin be 250g/L, 35g/L and 5g/L respectively, filtering, sterilizing, adding the solution I to make the final concentrations of the glucose, the sodium polyanisole sulfonate 35mg/L and the compound vitamin be 2.5g/L respectively, and sub-packaging the two-phase culture bottles with 25-35mL.
4. Use of the selective biphasic medium of claim 2 for the isolated culture of brucella ovis, said use being for non-disease diagnosis purposes.
5. The method for separating and culturing the brucella ovi is characterized by comprising the following steps of:
(1) Dividing the liquid sample to be tested into two equal parts, one part is injected into a selective biphase culture flask, the other part is injected into a common biphase culture flask, and the biphase culture flask is placed into 37 ℃ and 5-10% CO 2 Culturing in an incubator, wherein the liquid phase fully infiltrates the solid phase 4-12h, and then performing inclined culture;
wherein the selective biphasic culture flask is filled with the selective biphasic culture medium of claim 2;
the common biphasic culture bottle is provided with a common biphasic culture medium;
the preparation method of the common biphase culture bottle comprises the following steps:
a. autoclaving the solid phase medium: weighing casein pancreatin digest 15g, soybean papain hydrolysate 5g, sodium chloride 5g and agar 15g, heating with 1L deionized water for dissolving, packaging 20mL, placing in a biphasic culture flask, sterilizing at 121deg.C under high pressure for 15-20min, and standing until cooling and solidifying;
b. autoclaving the liquid phase medium: weighing casein pancreatin digest 15g, soybean papain hydrolysate 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, dissolving with 1L deionized water, and sterilizing at 121deg.C under high pressure for 15-20min to obtain liquid culture medium; weighing glucose, sodium polyanisole sulfonate and compound vitamin, dissolving with deionized water to make the concentrations of the glucose, the sodium polyanisole sulfonate and the compound vitamin be 250g/L, 35g/L and 5g/L respectively, filtering, sterilizing, adding the mixture into a liquid culture medium to make the final concentrations of the glucose, the sodium polyanisole sulfonate and the compound vitamin be 2.5g/L, 35mg/L and 5mg/L respectively, and subpackaging 25mL in each double-phase culture bottle;
(2) After 1-4 weeks of culture, the culture results were observed:
in the case of bacterial infection:
a. the liquid phase part is turbid, and the solid phase part is single colony growth;
b. if the single colony is round, the diameter is 1-2mm, the edge is smooth, and the colony is shiny and semitransparent pale yellow, which is suspected Brucella;
c. if the single colony grows in the selective double-phase culture bottle and the common double-phase culture bottle, the Brucella melitensis is the Brucella melitensis; if the selective biphasic culture bottle does not grow the single colony, and the common biphasic culture bottle grows the single colony, the selective biphasic culture bottle is bovine species, porcine species or canine species brucella; if the selective double-phase culture bottle has the single colony growth, and the common double-phase culture bottle does not have the single colony growth, the test fails and the verification needs to be repeated;
no bacterial infection:
if the liquid phase part of the selective biphasic culture medium and the common culture medium is clear and the solid phase part does not grow single colony or lawn, no bacterial infection exists in the sample;
the method is for non-disease diagnostic purposes.
6. A selective aerobic blood culture, characterized in that the selective aerobic blood culture is a liquid culture comprising casein pancreatin digest 17g/L, soy papain hydrolysate 3g/L, sodium polyanisole sulfonate 35mg/L, pyridoxine hydrochloride 1mg/L, erythromycin 6-15mg/L, ampicillin sodium 2-7mg/L, amino acid complex 5mg/L, and multivitamin 5mg/L.
7. The method of preparing the selective aerobic blood culture of claim 6, wherein the method comprises: weighing casein pancreatin digest 17g and soybean papain hydrolysate 3g, dissolving with 1L deionized water, packaging in blood culture bottle, and sterilizing at 121deg.C under high pressure for 15-20min; weighing amino acid compound, compound vitamin, sodium polyanisole sulfonate and pyridoxine hydrochloride, dissolving with deionized water, filtering, sterilizing, adding into a blood culture bottle to obtain final concentrations of 5mg/L, 5mg/L, 35mg/L and 1mg/L, respectively, weighing ampicillin sodium, dissolving with double distilled water, filtering, sterilizing, and adding into the blood culture bottle to obtain final concentration of 2-7mg/L; weighing erythromycin, dissolving with absolute ethanol, filtering, sterilizing, and adding into blood culture bottle with final concentration of 6-15mg/L.
8. Use of the selective aerobic blood culture of claim 6 for the isolated culture of brucella ovis, said use being for non-disease diagnostic purposes.
9. The method for separating and culturing the brucella ovi is characterized by comprising the following steps of:
1) Dividing a liquid sample to be detected into two equal parts, wherein one part is injected into a selective aerobic blood culture bottle, the other part is injected into a common aerobic blood culture bottle, and the blood culture bottle is placed into a full-automatic blood culture instrument for culture;
wherein the selective aerobic blood culture flask contains the selective aerobic blood culture medium of claim 6;
the common aerobic blood culture bottle is provided with a common aerobic blood culture medium;
the preparation method of the common aerobic blood culture flask comprises the following steps:
weighing casein pancreatin digest 17g and soybean papain hydrolysate 3g, dissolving with 1L deionized water, packaging 30mL bottles each, and sterilizing at 121deg.C under high pressure for 15-20min; weighing the amino acid compound, the compound vitamin, the SPS and the pyridoxine hydrochloride, dissolving the amino acid compound, the compound vitamin, the SPS and the pyridoxine hydrochloride in deionized water, filtering, sterilizing and then adding the mixture into a blood culture flask to ensure that the final concentration is 5mg/L, 5mg/L, 35mg/L and 1mg/L respectively;
2) After 1-4 weeks of culture, judging whether the Brucella melitensis exists in the sample by observing a growth curve: if the selective blood culture bottle and the common blood culture bottle have strain growth curves, the strain is brucella ovis; if the selective blood culture bottle has no growth curve and the common blood culture bottle has the single colony growth, the selective blood culture bottle is bovine species, porcine species or canine species brucella; if the selective blood culture bottle and the common blood culture bottle have no growth curve, bacteria are not cultured in the sample; if the selective blood culture bottle has a growth curve and the common blood culture bottle has no growth curve, the test fails and the verification is needed to be repeated;
the method is for non-disease diagnostic purposes.
10. The method according to claim 5 or 9, wherein the liquid sample to be tested is selected from the group consisting of blood, serum, milk sample, tissue homogenate, aerosol liquid, and is a serological test bruising positive sample.
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