CN116103420A - Goat slurry-free separation and identification method - Google Patents

Goat slurry-free separation and identification method Download PDF

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CN116103420A
CN116103420A CN202310218024.3A CN202310218024A CN116103420A CN 116103420 A CN116103420 A CN 116103420A CN 202310218024 A CN202310218024 A CN 202310218024A CN 116103420 A CN116103420 A CN 116103420A
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goat
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彭永帅
张曼
张利卫
李月勤
王军
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Henan University of Animal Husbandry and Economy
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Abstract

The application provides a goat slurry-free separation and identification method, and relates to the technical field of pathogen analysis. A method for separating and identifying goat slurry-free, comprising the following steps: a1, sample treatment; a2, separating red blood cells; a3, erythrocyte lysis; a4, pathogen separation and purification: loading magnetic particles into the pathogenic suspension, centrifuging at 4deg.C and 7000-10000g centrifugal force for 20-50min, separating supernatant, loading magnetic particles into supernatant, centrifuging at 30000g centrifugal force for 60-90min, and separating precipitate; re-suspending the precipitate with 1 XPBS phosphate buffer solution, loading into magnetic particles, centrifuging at 30000g centrifugal force for 60-90min, and repeatedly washing for 3 times to obtain final precipitate; a5, extracting pathogenic DNA; a6, pathogen identification; a7, pathogen freezing and resuscitating; a8, pathogen detection. The method has good separation effect, high pathogen content after separation, strong vitality and simple and convenient operation.

Description

Goat slurry-free separation and identification method
Technical Field
The application relates to the technical field of pathogen analysis, in particular to a goat slurry-free separation and identification method.
Background
The anabrosis classification belongs to the order of rickettsia, anabrosis family, and comprises 6 species and 1 tentative species, namely phagocytophilic anabrosis (A.phagosotophilium), bovine anabrosis (A.bovis), sheep anabrosis (A.ovis), edge anabrosis (A.mariginale), central anabrosis (A.centralis) and flat anabrosis (A.platys), and goat anabrosis (A.capra) is tentative species. In 2001, scholars re-divided the order of rickettsiae based on germ line analysis of 16SrRNA and groEL genes, updated the genus erigeron of anaplasmatidae and the genus erigeron including only marginal erigeron and central erigeron, when bovine erigeron was classified into erigeron, niu Aili grams of erigeron and platyceros were reclassified into bovine erigeron and platyceros, phagocytophil, marigomeroclox and human granulocyte erigeron were unified into phagocytophil due to the similarity of genetic genes. The goat ungluring body is a unglured body species newly discovered in recent years, the first report of China is that the ungluring body species is detected in an asymptomatic goat body, later Li (2015) is detected in a patient with a hard tick bite history in a peony and river forest center hospital in Heilongjiang province of China, the ungluring body-based 16S rRNA, msp4, gltA and groEL gene sequence analysis is considered to be a ungluring body novel species, and is named as a 'goat ungluring body', and later researchers determine that the ungluring body is a zoonotic pathogen capable of being transmitted by hard ticks.
Slurry-free propagation media are diverse and mainly comprise 3 modes: arthropod transmission, egg or placenta transmission, and blood transfusion or blood contaminant transmission. When cultured in vitro, the goat plasma-free body can successfully infect human HL-60 cells and THP-1 cell lines in vitro, which shows that the goat plasma-free body has the infection capacity on human cells. The pathogenicity of the non-slurry is related to the percentage of infected cells, to the stage of growth of the pathogen, the susceptibility of the host and the invasiveness of the strain. Infected hosts may develop thrombocytopenia, neutropenia, decreased neutrophil function, etc., resulting in an immunosuppressive state, which in turn promotes the occurrence of opportunistic infections. Nonspecific symptoms of infection of goats with no plasma appearance include: fever, headache, general debilitation, rash and chills, etc. Since goat slurry is considered as a zoonotic pathogen, pathogenicity is important to the attitude of people to treat the pathogen, so research on the pathogenicity of the pathogen should be more focused, but an effective separation and identification method for the goat slurry is lacked in the prior art.
Disclosure of Invention
The method for separating and identifying the goat slurry-free body has the advantage of effectively separating and identifying the goat slurry-free body.
The technical problem of the application is solved by adopting the following technical scheme.
The embodiment of the application provides a goat slurry-free separation and identification method, which comprises the following steps:
a1, sample treatment: collecting a goat slurry-free positive goat neck vein blood sample, performing anticoagulation treatment, and then preserving at a low temperature of 4 ℃ to obtain a sample;
a2, red blood cell separation: adding the erythrocyte lysate into a sample, lightly blowing and uniformly mixing, centrifuging for 10min under the condition of 2000g centrifugal force after 10min, removing sediment, centrifuging for 30min under the condition of 2000g centrifugal force, and collecting sediment to obtain erythrocyte suspension;
a3, erythrocyte lysis: adding 3 times of erythrocyte lysate into erythrocyte suspension, and standing at 4deg.C for 3-8min to obtain pathogenic suspension;
a4, pathogen separation and purification: loading magnetic particles into the pathogenic suspension, centrifuging at 4deg.C and 7000-10000g centrifugal force for 20-50min, separating supernatant, loading magnetic particles into supernatant, centrifuging at 30000g centrifugal force for 60-90min, and separating precipitate; re-suspending the precipitate with 1 XPBS phosphate buffer solution, loading into magnetic particles, centrifuging at 30000g centrifugal force for 60-90min, and repeatedly washing for 3 times to obtain final precipitate;
a5, extracting pathogenic DNA: extracting pathogenic DNA from the final precipitate with blood DNA extraction kit, eluting DNA with eluent to obtain DNA sample;
pathogen identification:
1) Construction of plasmids and standard curves: cloning the amplification product of the goat slurry-free groEL gene primer into a pMD-18T vector, amplifying in escherichia coli DH5 alpha competent cells, constructing a plasmid containing the goat slurry-free groEL gene, purifying plasmid DNA of the transformed cells by using a SanPrep column plasmid Mini-Prep Kit, quantifying by using an ultra-micro spectrophotometer, and determining according to the formula:
Figure BDA0004115638770000031
converting the unit into copies/. Mu.L;
2) Pathogen identification and quantitative detection: carrying out DNA quantitative detection on the DNA sample by using a fluorescent quantitative PCR instrument;
a6, pathogen freezing and resuscitating: re-suspending the separated DNA sample with a culture medium containing 10% fetal bovine serum, adding 10% dimethyl sulfoxide as a protective agent, sequentially placing at 4 ℃ for 30min, 20 ℃ for 30min, 80 ℃ for 12h, and then transferring to liquid nitrogen for long-term storage, wherein 1 part of the DNA sample is stored at different freezing temperatures for pathogen morphology detection; when pathogen is recovered, the freezing tube is taken out from the liquid nitrogen tank, and is put into a water bath kettle with the temperature of 37 ℃ for quick thawing, and the freezing tube is shaken for a plurality of times during the period, so that the thawing is completed within 1-2 min;
a7, pathogen detection:
1) And (3) detecting pathogenic morphology: taking pathogenic suspension in freezing tube at 4deg.C, -20deg.C, -80deg.C and liquid nitrogen freezing respectively, centrifuging at 10000g centrifugal force for 30min, fixing with glutaraldehyde electron microscope fixing solution for 2 hr, fixing at 4deg.C for 12 hr, centrifuging to collect cells, and collecting cells with 1 XPBS phosphateBuffer solution was washed 3 times, 10min each, 2% O s O 4 After fixing, washing with water, dehydration with 30%, 50%, 70%, 80%, 90%, 100% and 100% ethanol series, dehydration with acetone for 15min, dehydration with 1:1 and 812 epoxy embedding agent, and then 1:2, mixing acetone and 812 embedding agent, then penetrating for 12 hours, then treating for 5-8 hours by using pure 812 embedding agent, pouring the pure 812 embedding agent into an embedding plate, inserting a sample into the embedding plate, then carrying out polymerization treatment at 37 ℃ for 12 hours and 60 ℃ for 48 hours to obtain a solidified resin block, trimming and slicing the solidified resin block, collecting the solidified resin block on a grid with the specification of 200, then dyeing by using 2% uranyl acetate water and lead citrate, and finally observing and photographing under a transmission electron microscope;
2) Detecting pathogen activity: inoculating resuscitated pathogens into a culture medium containing 10% fetal bovine serum, evenly distributing the culture medium in 24 pore plates for in vitro pure culture, collecting samples after 0d, 1d, 2d, 3d, 4d and 5d of culture, taking 3 repeated pores at each time point, extracting DNA from the collected samples by using a blood DNA extraction kit, and quantitatively detecting the pathogens by using a fluorescent quantitative PCR instrument;
taking 1 goat unglured DNA sample, equally dividing into 7 parts, wherein 1 part is used as an initial concentration control, the other 6 parts are added with culture medium with the same quantity as that of pathogen culture, the culture medium is cultured in a 24-well plate and is harvested along with the pathogen sampling time point, 7 parts of DNA sample is used for extracting DNA according to a pathogen DNA extraction method, and the DNA sample is used as a control group of DNA degradation speed;
3) Pathogen infectivity detection: manually inoculating resuscitated pathogens to in-vitro cultured goat erythrocytes, setting 3 times of repeated inoculation in the test, taking unvaccinated goat erythrocytes as negative control, sampling and checking pathogen infection after normal culture for 3d, washing 3 times with 1 XPBS phosphate buffer solution after sampling, and controlling the centrifugal condition at 500g centrifugal force for 5min during washing in order to prevent the influence of pathogen inoculation of the samples; and (3) preparing a cell smear from the washed cell sample, extracting sample DNA, and respectively performing giemsa staining observation and PCR detection.
In some embodiments of the present application, the anticoagulation treatment described above is specifically performed with heparin lithium.
In some embodiments of the present application, the mass ratio of the erythrocyte lysate to the sample is 1:8.
in some embodiments of the present application, the aforementioned pathogen identification step 2) the fluorescent quantitative PCR apparatus for pathogen identification and quantitative detection performs DNA quantitative detection of DNA samples, specifically in a 20. Mu.L system, comprising 12.5. Mu.L TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions were 95℃for 30s of pre-denaturation, 95℃for 5s of denaturation, 55℃for 30s of annealing and extension, 35 cycles.
In some embodiments of the present application, the PCR reaction of the fluorescent quantitative PCR apparatus for pathogen viability detection of the A7 step 2) above is performed in a 20. Mu.L system comprising 12.5. Mu.L TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions were 95℃for 30s of pre-denaturation, 95℃for 5s of denaturation, 55℃for 30s of annealing and extension, 35 cycles.
In some embodiments of the present application, the giemsa staining method described above is specifically as follows: and respectively preparing goat plasma-free negative and positive red blood cell smears, fixing the smears with methanol for 10min, drying, dyeing the smears with giemsa staining solution for 10min, and observing pathogen inclusion bodies in cells under an optical microscope.
In some embodiments of the present application, the PCR detection method specifically includes: the quadruple PCR system comprises: 10 XLaBuffer 2.5. Mu. L, dNTPs (2.5 mM) 4. Mu.L, la Taq DNA polymerase 0.25. Mu.L, 0.5. Mu.L for each primer (20. Mu. Mol/L) and 2. Mu.L of DNA template, sterilized pure water was made up to 25. Mu.L; PCR reactions based on different gene loci were all performed in 25. Mu.L reaction system, comprising: 10 xLa Buffer 2.5. Mu.L, dNTPs 4. Mu.L, upstream and downstream primers (20. Mu. Mol/L) each 0.5. Mu.L, la Taq enzyme 0.25. Mu.L, 2. Mu.L DNA template, sterilized pure water to 25. Mu.L; the PCR reactions are all carried out in a PCR amplification instrument, and the reaction conditions are as follows: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for 1min, and extension at 72℃for a further 10min for 40 cycles.
Compared with the prior art, the embodiment of the application has at least the following advantages or beneficial effects:
according to the method, the goat ungluring body in the goat body is subjected to external culture, and then the external cultured goat ungluring body is detected by combining multiple detection methods, so that the detection speed is high, the sensitivity is good, the repeatability is good and the reliability is high.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered limiting the scope, and that other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of pathogen identification and quantitative detection in examples 1-4 of the present application;
FIG. 2 is a diagram of the detection of pathogenic morphology according to example 1 of the present application;
FIG. 3 is a diagram of the pathogen viability assay of example 1 of the present application;
FIG. 4 is a graph showing the results of the giemsa staining of example 1 of the present application;
FIG. 5 is a graph showing the results of PCR detection in example 1 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present application will be described in detail with reference to specific examples.
The embodiment of the application provides a goat slurry-free separation and identification method, which comprises the following steps:
a1, sample treatment: collecting a goat slurry-free positive goat neck vein blood sample, performing anticoagulation treatment, and then preserving at a low temperature of 4 ℃ to obtain a sample;
a2, red blood cell separation: adding the erythrocyte lysate into a sample, lightly blowing and uniformly mixing, centrifuging for 10min under the condition of 2000g centrifugal force after 10min, removing sediment, centrifuging for 30min under the condition of 2000g centrifugal force, and collecting sediment to obtain erythrocyte suspension;
a3, erythrocyte lysis: adding 3 times of erythrocyte lysate into erythrocyte suspension, and standing at 4deg.C for 3-8min to obtain pathogenic suspension;
a4, pathogen separation and purification: loading magnetic particles into the pathogenic suspension, centrifuging at 4deg.C and 7000-10000g centrifugal force for 20-50min, separating supernatant, loading magnetic particles into supernatant, centrifuging at 30000g centrifugal force for 60-90min, and separating precipitate; re-suspending the precipitate with 1 XPBS phosphate buffer solution, loading into magnetic particles, centrifuging at 30000g centrifugal force for 60-90min, and repeatedly washing for 3 times to obtain final precipitate;
a5, extracting pathogenic DNA: extracting pathogenic DNA from the final precipitate with blood DNA extraction kit, eluting DNA with eluent to obtain DNA sample;
pathogen identification:
1) Construction of plasmids and standard curves: cloning the amplification product of the goat slurry-free groEL gene primer into a pMD-18T vector, amplifying in escherichia coli DH5 alpha competent cells, constructing a plasmid containing the goat slurry-free groEL gene, purifying plasmid DNA of the transformed cells by using a SanPrep column plasmid Mini-Prep Kit, quantifying by using an ultra-micro spectrophotometer, and determining according to the formula:
Figure BDA0004115638770000081
converting the unit into copies/. Mu.L;
2) Pathogen identification and quantitative detection: carrying out DNA quantitative detection on the DNA sample by using a fluorescent quantitative PCR instrument;
a6, pathogen freezing and resuscitating: re-suspending the separated DNA sample with a culture medium containing 10% fetal bovine serum, adding 10% dimethyl sulfoxide as a protective agent, sequentially placing at 4 ℃ for 30min, 20 ℃ for 30min, 80 ℃ for 12h, and then transferring to liquid nitrogen for long-term storage, wherein 1 part of the DNA sample is stored at different freezing temperatures for pathogen morphology detection; when pathogen is recovered, the freezing tube is taken out from the liquid nitrogen tank, and is put into a water bath kettle with the temperature of 37 ℃ for quick thawing, and the freezing tube is shaken for a plurality of times during the period, so that the thawing is completed within 1-2 min;
a7, pathogen detection:
1) And (3) detecting pathogenic morphology: taking pathogenic suspension in freezing tube frozen at 4deg.C, -20deg.C, -80deg.C and liquid nitrogen respectively, centrifuging at 10000g centrifugal force for 30min, fixing with glutaraldehyde electron microscope fixing solution for 2 hr, fixing at 4deg.C for 12 hr, centrifuging to collect cells, washing with 1×PBS phosphate buffer solution for 3 times, 10min each time, 2% O s O 4 After fixing, washing with water, dehydration with 30%, 50%, 70%, 80%, 90%, 100% and 100% ethanol series, dehydration with acetone for 15min, dehydration with 1:1 and 812 epoxy embedding agent, and then 1:2, mixing acetone and 812 embedding agent, then penetrating for 12 hours, then treating for 5-8 hours by using pure 812 embedding agent, pouring the pure 812 embedding agent into an embedding plate, inserting a sample into the embedding plate, then carrying out polymerization treatment at 37 ℃ for 12 hours and 60 ℃ for 48 hours to obtain a solidified resin block, trimming and slicing the solidified resin block, collecting the solidified resin block on a grid with the specification of 200, then dyeing by using 2% uranyl acetate water and lead citrate, and finally observing and photographing under a transmission electron microscope;
2) Detecting pathogen activity: inoculating resuscitated pathogens into a culture medium containing 10% fetal bovine serum, evenly distributing the culture medium in 24 pore plates for in vitro pure culture, collecting samples after 0d, 1d, 2d, 3d, 4d and 5d of culture, taking 3 repeated pores at each time point, extracting DNA from the collected samples by using a blood DNA extraction kit, and quantitatively detecting the pathogens by using a fluorescent quantitative PCR instrument;
taking 1 goat unglured DNA sample, equally dividing into 7 parts, wherein 1 part is used as an initial concentration control, the other 6 parts are added with culture medium with the same quantity as that of pathogen culture, the culture medium is cultured in a 24-well plate and is harvested along with the pathogen sampling time point, 7 parts of DNA sample is used for extracting DNA according to a pathogen DNA extraction method, and the DNA sample is used as a control group of DNA degradation speed;
3) Pathogen infectivity detection: manually inoculating resuscitated pathogens to in-vitro cultured goat erythrocytes, setting 3 times of repeated inoculation in the test, taking unvaccinated goat erythrocytes as negative control, sampling and checking pathogen infection after normal culture for 3d, washing 3 times with 1 XPBS phosphate buffer solution after sampling, and controlling the centrifugal condition at 500g centrifugal force for 5min during washing in order to prevent the influence of pathogen inoculation of the samples; and (3) preparing a cell smear from the washed cell sample, extracting sample DNA, and respectively performing giemsa staining observation and PCR detection.
In some embodiments of the present application, the anticoagulation treatment described above is specifically performed with heparin lithium.
In some embodiments of the present application, the mass ratio of the erythrocyte lysate to the sample is 1:8.
in some embodiments of the present application, the aforementioned pathogen identification step 2) the fluorescent quantitative PCR apparatus for pathogen identification and quantitative detection performs DNA quantitative detection of DNA samples, specifically in a 20. Mu.L system, comprising 12.5. Mu.L TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions were 95℃for 30s of pre-denaturation, 95℃for 5s of denaturation, 55℃for 30s of annealing and extension, 35 cycles.
In some embodiments of the present application, the PCR reaction of the fluorescent quantitative PCR apparatus for pathogen viability detection of the A7 step 2) above is performed in a 20. Mu.L system comprising 12.5. Mu.L TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions were 95℃for 30s of pre-denaturation, 95℃for 5s of denaturation, 55℃for 30s of annealing and extension, 35 cycles.
In some embodiments of the present application, the giemsa staining method described above is specifically as follows: and respectively preparing goat plasma-free negative and positive red blood cell smears, fixing the smears with methanol for 10min, drying, dyeing the smears with giemsa staining solution for 10min, and observing pathogen inclusion bodies in cells under an optical microscope.
In some embodiments of the present application, the PCR detection method specifically includes: the quadruple PCR system comprises: 10 XLaBuffer 2.5. Mu. L, dNTPs (2.5 mM) 4. Mu.L, la Taq DNA polymerase 0.25. Mu.L, 0.5. Mu.L for each primer (20. Mu. Mol/L) and 2. Mu.L of DNA template, sterilized pure water was made up to 25. Mu.L; PCR reactions based on different gene loci were all performed in 25. Mu.L reaction system, comprising: 10 xLa Buffer 2.5. Mu.L, dNTPs 4. Mu.L, upstream and downstream primers (20. Mu. Mol/L) each 0.5. Mu.L, la Taq enzyme 0.25. Mu.L, 2. Mu.L DNA template, sterilized pure water to 25. Mu.L; the PCR reactions are all carried out in a PCR amplification instrument, and the reaction conditions are as follows: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for 1min, and extension at 72℃for a further 10min for 40 cycles.
In some embodiments of the present application, the four-fold PCR detection primers, the multiplex locus based goat slurry-free PCR identification primer fragment lengths, and the annealing temperatures at PCR are shown in Table 1.
TABLE 1
Figure BDA0004115638770000101
Figure BDA0004115638770000111
The features and capabilities of the present application are described in further detail below in connection with the examples.
Example 1
A method for separating and identifying goat slurry-free, comprising the following steps:
the main instrument is as follows: ultra-high speed refrigerated centrifuges (Beckman Avanti JXN-30, U.S.), fluorescent quantitative PCR instruments (qTOWER 3G, yena, germany), ultra-micro spectrophotometers (Nanodrop OneC, thermo Fisher Scientific, U.S.), high speed centrifuges (Hunan) H1650, hunan), transmission electron microscopy (Hitachi HT7700, japan), liquid gas tanks, -20℃freezer, microscopes (lycra, japan), eppendorf pipettes (1000 mL, 100mL, 10mL, 2.5 mL), 5% CO2 cell incubators (Thermo, 3111, U.S.), 24 well culture plates, shakers, water baths, and the like.
Main consumable: EDTA-K2 anticoagulation blood collection tube, blood collection needle, 50 mu L PCR tube, 1000mL pipette tip, 100mL pipette tip, 10mL pipette tip, 1.5mL centrifuge tube, latex glove, PE glove, etc.
Chemical reagent: blood DNA extraction Kit (OMEGA, USA), erythrocyte separation Kit (Hebei, N.P.), la Taq DNA polymerase (Takara, dalian), sanPrep column plasmid Mini-Prep Kit purification Kit (Takara, japan), E.coli DH 5. Alpha. Competent cells (Shanghai Biotechnology Co., ltd.), pMD-18T (Takara, dalian), TB Green TM Premix Ex Taq (Takara, japan), fetal bovine serum (Gibco, usa), RPMI 1640 medium (500 mL, hyclone, usa), 10% dimethyl sulfoxide (Sigma, germany), glutaraldehyde electron microscope fixative (Solarbio, beijing), giemsa stain (Solarbio, beijing), 1 x PBS phosphate buffer, red blood cell lysate (Solarbio, beijing), absolute ethanol, agarose powder (agarsose, 500 g), DL-2000Marker (daybia, 1 mL), and the like.
A1, sample treatment: collecting a goat slurry-free positive goat neck vein blood sample, performing anticoagulation treatment by using heparin lithium, and then preserving at a low temperature of 4 ℃ to obtain a sample;
a2, red blood cell separation: the mass ratio of the sample to the sample is 1:8, taking the erythrocyte lysate according to the proportion, adding the erythrocyte lysate into a sample, gently blowing and uniformly mixing, centrifuging for 10min under the condition of 2000g centrifugal force after 10min, removing sediment, centrifuging for 30min under the condition of 2000g centrifugal force, and collecting sediment to obtain erythrocyte suspension;
a3, erythrocyte lysis: adding 3 times of erythrocyte lysate into erythrocyte suspension, and standing at 4deg.C for 3-8min to obtain pathogenic suspension;
a4, pathogen separation and purification: loading magnetic particles into the pathogenic suspension, centrifuging at 4deg.C and 7000g centrifugal force for 30min, separating supernatant, centrifuging at 30000g centrifugal force for 80min, and separating precipitate; suspending the precipitate with 1×PBS phosphate buffer solution, loading into magnetic particles, centrifuging at 30000g centrifugal force for 80min, and repeatedly washing for 3 times to obtain final precipitate;
a5, extracting pathogenic DNA: extracting pathogenic DNA from the final precipitate with blood DNA extraction kit, eluting DNA with eluent to obtain DNA sample;
pathogen identification:
1) Construction of plasmids and standard curves: cloning the amplification product of the goat slurry-free groEL gene primer into a pMD-18T vector, amplifying in escherichia coli DH5 alpha competent cells, constructing a plasmid containing the goat slurry-free groEL gene, purifying plasmid DNA of the transformed cells by using a SanPrep column plasmid Mini-Prep Kit, quantifying by using an ultra-micro spectrophotometer, and determining according to the formula:
Figure BDA0004115638770000121
converting the unit into copies/. Mu.L;
2) Pathogen identification and quantitative detection: DNA quantitative detection of DNA samples using a fluorescent quantitative PCR apparatus was performed in a 20. Mu.L system containing 12.5. Mu.L TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions are 95 ℃, 30s of pre-denaturation, 95 ℃, 5s of denaturation, 55 ℃ of annealing and extension for 30s,35 cycles;
a6, pathogen freezing and resuscitating: re-suspending the separated DNA sample with a culture medium containing 10% fetal bovine serum, adding 10% dimethyl sulfoxide as a protective agent, sequentially placing at 4 ℃ for 30min, 20 ℃ for 30min, 80 ℃ for 12h, and then transferring to liquid nitrogen for long-term storage, wherein 1 part of the DNA sample is stored at different freezing temperatures for pathogen morphology detection; when pathogen is recovered, the freezing tube is taken out from the liquid nitrogen tank, and is put into a water bath kettle with the temperature of 37 ℃ for quick thawing, and the freezing tube is shaken for a plurality of times during the period, so that the thawing is completed within 1-2 min;
a7, pathogen detection:
1) And (3) detecting pathogenic morphology: taking pathogenic suspension in freezing tube frozen at 4deg.C, -20deg.C, -80deg.C and liquid nitrogen respectively, centrifuging at 10000g centrifugal force for 30min, fixing with glutaraldehyde electron microscope fixing solution for 2 hr, fixing at 4deg.C for 12 hr, centrifuging to collect cells, washing with 1×PBS phosphate buffer solution for 3 times, 10min each time, 2% O s O 4 After fixing, washing with water, dehydration with 30%, 50%, 70%, 80%, 90%, 100% and 100% ethanol series, dehydration with acetone for 15min, dehydration with 1:1 and 812 epoxy embedding agent, and then 1:2, mixing acetone and 812 embedding agent, then penetrating for 12 hours, then treating for 5-8 hours by using pure 812 embedding agent, pouring the pure 812 embedding agent into an embedding plate, inserting a sample into the embedding plate, then polymerizing for 48 hours at 37 ℃ to obtain a solidified resin block, trimming and slicing the solidified resin block, collecting the solidified resin block on a grid with the specification of 200, then dyeing by using 2% uranyl acetate water and lead citrate, finally observing and photographing under a transmission electron microscope, as shown in fig. 2;
2) Detecting pathogen activity: inoculating resuscitated pathogen into culture medium containing 10% fetal bovine serum, evenly distributing in 24-well plate for in vitro pure culture, collecting samples after 0d, 1d, 2d, 3d, 4d and 5d, respectively, sampling 3 repeated wells at each time point, extracting DNA from the collected samples with blood DNA extraction kit, quantitatively detecting pathogen DNA with fluorescent quantitative PCR instrument, and performing PCR reaction in 20 μl system containing 12.5 μl TB Green TM Premix Ex Taq, 0.5. Mu.L of each of the upstream and downstream primers, 2.5. Mu.L of sterile deionized H 2 O, 4. Mu.L of sample DNA; the PCR reaction conditions were 95℃for 30s of pre-denaturation, 95℃for 5s of denaturation, 55℃for 30s of annealing and extension, and the results are shown in FIG. 3;
taking 1 goat unglured DNA sample, equally dividing into 7 parts, wherein 1 part is used as an initial concentration control, the other 6 parts are added with culture medium with the same quantity as that of pathogen culture, the culture medium is cultured in a 24-well plate and is harvested along with the pathogen sampling time point, 7 parts of DNA sample is used for extracting DNA according to a pathogen DNA extraction method, and the DNA sample is used as a control group of DNA degradation speed;
3) Pathogen infectivity detection: manually inoculating resuscitated pathogens to in-vitro cultured goat erythrocytes, setting 3 times of repeated inoculation in the test, taking unvaccinated goat erythrocytes as negative control, sampling and checking pathogen infection after normal culture for 3d, washing 3 times with 1 XPBS phosphate buffer solution after sampling, and controlling the centrifugal condition at 500g centrifugal force for 5min during washing in order to prevent the influence of pathogen inoculation of the samples; making a cell smear from the washed cell sample, extracting sample DNA, and respectively performing giemsa staining observation and PCR detection;
the giemsa staining method is specifically as follows: respectively preparing goat plasma-free negative and positive red blood cell smears, fixing with methanol for 10min, drying, staining with giemsa staining solution for 10min, and observing pathogenic inclusion bodies in cells under an optical microscope, wherein the result is shown in figure 4;
the PCR detection method specifically comprises the following steps: the quadruple PCR system comprises: 10 XLaBuffer 2.5. Mu. L, dNTPs (2.5 mM) 4. Mu.L, la Taq DNA polymerase 0.25. Mu.L, 0.5. Mu.L for each primer (20. Mu. Mol/L) and 2. Mu.L of DNA template, sterilized pure water was made up to 25. Mu.L; PCR reactions based on different gene loci were all performed in 25. Mu.L reaction system, comprising: 10 xLa Buffer 2.5. Mu.L, dNTPs 4. Mu.L, upstream and downstream primers (20. Mu. Mol/L) each 0.5. Mu.L, la Taq enzyme 0.25. Mu.L, 2. Mu.L DNA template, sterilized pure water to 25. Mu.L; the PCR reactions are all carried out in a PCR amplification instrument, and the reaction conditions are as follows: the results of the pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for 1min and extension at 72℃for another 10min for 40 cycles are shown in FIG. 5.
Example 2
This embodiment is substantially the same as embodiment 1 except that: a4, pathogen separation and purification steps: the pathogenic suspension is filled with magnetic particles and subjected to centrifugation at a temperature of 4 ℃ and a centrifugal force of 8000g for 30min.
Example 3
This embodiment is substantially the same as embodiment 1 except that: a4, pathogen separation and purification steps: the pathogen suspension is loaded with magnetic particles and centrifuged at 9000g centrifugal force for 30min at 4 ℃.
Example 4
This embodiment is substantially the same as embodiment 1 except that: a4, pathogen separation and purification steps: the pathogenic suspension is filled with magnetic particles and subjected to centrifugal treatment at a temperature of 4 ℃ and a centrifugal force of 10000g for 30min.
Experimental example
The results of the pathogen identification and quantitative detection of the pathogen identification step 2) of examples 1-4 are shown in FIG. 1.
According to FIG. 1, the pathogen was collected by centrifugation at 7000g for 30min, but the recovered amount was small, and the pathogen was recovered most by centrifugation at 1000g for 30min.
FIG. 2 shows the results of the pathogenic morphology detection in A7, pathogenic detection step 1) of example 1.
In the view of fig. 2, the external morphology of the pathogen inclusion body after freezing under different temperature conditions is consistent with the result before freezing, and the internal pathogen amount is not obvious, so that the morphology and structure of the slurry-free goat after freezing under-20 ℃, -80 ℃ and liquid nitrogen conditions are consistent.
A7 of example 1, pathogen detection step 2) the results of pathogen viability detection are shown in FIG. 3.
Observing figure 3, carrying out in-vitro pure culture on the recovered goat slurry-free in a 24-well plate, and carrying out quantitative detection results on pathogens at different time points to find that the total amount of pathogenic DNA of the first 3d in the pathogenic culture process of the goat slurry-free has no growing trend, the pathogenic DNA of the 4d is reduced, and then the total amount of pathogenic DNA shows a slow rising trend; in the control group, the goat plasma-free DNA sample was degraded by more than 50% in culture for 1d, and was degraded substantially completely in culture for 2 d. Thus, the test results show that the recovered goat slurry-free medium has proliferation capacity in the culture solution.
A7 of example 1, pathogen detection step 3) the results of the staining with giemsa for pathogen infectivity detection are shown in FIG. 4.
Looking at fig. 4, the giemsa staining results showed that particulate matter could be observed in positive erythrocytes, presumably without slurry inclusion bodies in goats, while no similar particulate matter was observed inside unvaccinated (negative) goat erythrocytes.
FIG. 5 shows the results of PCR detection of A7 and the pathogen detection step 3) of example 1.
As shown in FIG. 5, the PCR detection result shows that the goat plasma-free gltA gene is amplified in 3 repeated inoculation experiments, the size of the target fragment is 594bp, and the target gene fragment is not detected in the negative control sample.
In summary, the method and the device for detecting the goat ungluring body of the goat, which are used for carrying out external culture on the goat ungluring body of the goat, are combined with various detection methods to detect the goat ungluring body of the goat, and have the advantages of high detection speed, good sensitivity, good repeatability and high reliability.
The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.

Claims (4)

1. A method for separating and identifying a goat slurry-free body, which is characterized by comprising the following steps:
a1, sample treatment: collecting a goat slurry-free positive goat neck vein blood sample, performing anticoagulation treatment, and then preserving at a low temperature of 4 ℃ to obtain a sample;
a2, red blood cell separation: adding the erythrocyte lysate into a sample, lightly blowing and uniformly mixing, centrifuging for 10min under the condition of 2000g centrifugal force after 10min, removing sediment, centrifuging for 30min under the condition of 2000g centrifugal force, and collecting sediment to obtain erythrocyte suspension;
a3, erythrocyte lysis: adding 3 times of erythrocyte lysate into erythrocyte suspension, and standing at 4deg.C for 3-8min to obtain pathogenic suspension;
a4, pathogen separation and purification: loading magnetic particles into the pathogenic suspension, centrifuging at 4deg.C and 7000-10000g centrifugal force for 20-50min, separating supernatant, loading magnetic particles into supernatant, centrifuging at 30000g centrifugal force for 60-90min, and separating precipitate; re-suspending the precipitate with 1 XPBS phosphate buffer solution, loading into magnetic particles, centrifuging at 30000g centrifugal force for 60-90min, and repeatedly washing for 3 times to obtain final precipitate;
a5, extracting pathogenic DNA: extracting pathogenic DNA from the final precipitate with blood DNA extraction kit, eluting DNA with eluent to obtain DNA sample;
a6, pathogen freezing and resuscitating: re-suspending the separated DNA sample with a culture medium containing 10% fetal bovine serum, adding 10% dimethyl sulfoxide as a protective agent, sequentially placing at 4 ℃ for 30min, 20 ℃ for 30min, 80 ℃ for 12h, and then transferring to liquid nitrogen for long-term storage, wherein 1 part of the DNA sample is stored at different freezing temperatures for pathogen morphology detection; when pathogen is recovered, the freezing tube is taken out from the liquid nitrogen tank, and is put into a water bath kettle with the temperature of 37 ℃ for quick thawing, and the freezing tube is shaken for a plurality of times during the period, so that the thawing is completed within 1-2 min;
a7, pathogen detection:
1) And (3) detecting pathogenic morphology: taking pathogenic suspension in freezing tube frozen at 4deg.C, -20deg.C, -80deg.C and liquid nitrogen respectively, centrifuging at 10000g centrifugal force for 30min, fixing with glutaraldehyde electron microscope fixing solution for 2 hr, fixing at 4deg.C for 12 hr, centrifuging to collect cells, washing with 1×PBS phosphate buffer solution for 3 times, 10min each time, 2% O s O 4 After fixing, washing with water, dehydration with 30%, 50%, 70%, 80%, 90%, 100% and 100% ethanol series, dehydration with acetone for 15min, dehydration with 1:1 and 812 epoxy embedding agent, and then 1:2, mixing acetone and 812 embedding agent, then penetrating for 12 hours, then treating for 5-8 hours by using pure 812 embedding agent, pouring the pure 812 embedding agent into an embedding plate, inserting a sample into the embedding plate, then carrying out polymerization treatment at 37 ℃ for 12 hours and 60 ℃ for 48 hours to obtain a solidified resin block, trimming and slicing the solidified resin block, collecting the solidified resin block on a grid with the specification of 200, then dyeing by using 2% uranyl acetate water and lead citrate, and finally observing and photographing under a transmission electron microscope;
2) Detecting pathogen activity: inoculating resuscitated pathogens into a culture medium containing 10% fetal bovine serum, evenly distributing the culture medium in 24 pore plates for in vitro pure culture, collecting samples after 0d, 1d, 2d, 3d, 4d and 5d of culture, taking 3 repeated pores at each time point, extracting DNA from the collected samples by using a blood DNA extraction kit, and quantitatively detecting the pathogens by using a fluorescent quantitative PCR instrument;
taking 1 goat unglured DNA sample, equally dividing into 7 parts, wherein 1 part is used as an initial concentration control, the other 6 parts are added with culture medium with the same quantity as that of pathogen culture, the culture medium is cultured in a 24-well plate and is harvested along with the pathogen sampling time point, 7 parts of DNA sample is used for extracting DNA according to a pathogen DNA extraction method, and the DNA sample is used as a control group of DNA degradation speed;
3) Pathogen infectivity detection: manually inoculating resuscitated pathogens to in-vitro cultured goat erythrocytes, setting 3 times of repeated inoculation in the test, taking unvaccinated goat erythrocytes as negative control, sampling and checking pathogen infection after normal culture for 3d, washing 3 times with 1 XPBS phosphate buffer solution after sampling, and controlling the centrifugal condition at 500g centrifugal force for 5min during washing in order to prevent the influence of pathogen inoculation of the samples; and (3) preparing a cell smear from the washed cell sample, extracting sample DNA, and respectively performing giemsa staining observation and PCR detection.
2. A method for goat slurry-less separation and identification according to claim 1, characterized in that the anticoagulation treatment is specifically with heparin lithium.
3. The method for separating and identifying goat plasma-free body according to claim 1, wherein the mass ratio of the red blood cell lysate to the sample is 1:8.
4. the method for separating and identifying goat slurry-free substance according to claim 1, wherein the giemsa staining method is specifically as follows: and respectively preparing goat plasma-free negative and positive red blood cell smears, fixing the smears with methanol for 10min, drying, dyeing the smears with giemsa staining solution for 10min, and observing pathogen inclusion bodies in cells under an optical microscope.
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