CN111607656A - Triple PCR detection primer, method and kit for tapeworm and sporozoon in canine feces - Google Patents
Triple PCR detection primer, method and kit for tapeworm and sporozoon in canine feces Download PDFInfo
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Abstract
A triple PCR detection primer, a triple PCR detection method and a triple PCR detection kit for tapeworm and sporozoite in dog excrement are used for detecting two tapeworms of echinococcus granulosus and echinococcus multilocularis and two sporozoites of cryptosporidium parvum and cryptosporidium canis. The triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention can amplify specific gene segments of four parasites simultaneously in one reaction system, and makes up the defects of low detectable rate, poor detection accuracy, time and labor waste in morphology by adopting electron microscope observation. The detection method established by the invention has the characteristics of high specificity and high sensitivity, can realize the high-flux detection of multiple pathogens in a single sample, not only saves the detection cost of the single sample, but also improves the detection rate, and simultaneously improves the detection accuracy to a great extent. The three groups of primers contained in the invention meet the requirement of multiplex PCR detection, and have profound economic value and social benefit.
Description
Technical Field
The invention relates to the technical field of microbiological determination, and particularly relates to a taenia and sporozoon triple PCR detection primer in canine excrement, a taenia and sporozoon triple PCR detection method in canine excrement and a taenia and sporozoon triple PCR detection kit in canine excrement.
Background
The invasion of the host by the parasite can produce a variety of deleterious effects in the host, such as deprivation of nutrients, resulting in host malnutrition, or mechanical damage within the host, or toxic effects on the host caused by the production of toxins by the parasite, all of which can severely affect host development.
After the animal is infected with the parasite, many eggs, oocysts or larvae of the parasite can be excreted with the feces of the host, and by examining the feces of the animal, it can be confirmed whether the animal is infected with the parasite and the kind of infection.
Regarding whether animals are infected with parasites or not, the animal feces are usually observed by an electron microscope, but the electron microscope observation method is easy to cause a problem of false detection due to the closeness of eggs or oocysts of many parasites.
Disclosure of Invention
In view of the above, how to improve the accuracy of animal parasite testing becomes a problem to be solved by those skilled in the art.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a triple PCR detection primer for tapeworm and sporozoon in dog feces, wherein the tapeworm is Echinococcus granulosus and Echinococcus multilocularis, and the sporozoon is Cryptosporidium parvum and Cryptosporidium caninum;
the primer is as follows:
echinococcus granulosus primer 1: 5'-CGTTGTCCTCGTCGTATT-3', respectively;
echinococcus granulosus primer 2: 5'-AACAGCCGTCTTCACATC-3', respectively;
the length of the target fragment is 515 bp;
echinococcus multilocularis primer 1: 5'-TTGATTATTGGAGGCTATGC-3', respectively;
echinococcus multilocularis primer 2: 5'-CAACGACAACATAACATCAC-3', respectively;
the length of the target fragment is 353 bp;
cryptosporidium parvum and Cryptosporidium caninum common primer 1: 5'-CTCAACTCAAGCCACCAT-3', respectively;
cryptosporidium parvum and Cryptosporidium caninum common primer 2: 5'-CCGACCAAGACAACATCA-3', respectively;
the target fragment is 186bp in length.
The invention provides a triple PCR detection kit for tapeworm and sporozoon in dog feces, wherein the tapeworm is Echinococcus granulosus and Echinococcus multilocularis, and the sporozoon is Cryptosporidium parvum and Cryptosporidium canis; the kit comprising the primers as set forth in claim 1 in their entirety.
Preferably, in the triple PCR detection kit for detecting taenia and sporozoon in canine feces provided by the invention, the reaction system for performing the triple PCR detection in the kit is as follows: the method comprises the following steps of: 20-25 parts of PCR buffer solution, 4-6 parts of all primers (equal amount), 2-3 parts of deoxyribose-containing deoxynucleoside triphosphate and 50-60 parts of DECP water.
The invention provides a triple PCR detection method for tapeworm and sporozoon in dog feces, which does not aim at diagnosis and treatment of diseases.
The triple PCR detection method for tapeworm and sporozoon in canine feces comprises the following steps: preparing a primer, extracting DNA, carrying out PCR (polymerase chain reaction) and carrying out electrophoresis detection; wherein, the PCR step uses the triple PCR detection kit for the tapeworm and the sporozoon in the canine feces.
Preferably, in the triple PCR detection method for tapeworm and sporozoon in canine feces provided by the invention, a primer design step is included before the primer preparation step;
in the primer design step, according to the comparison analysis result of the mitochondrial genome sequence blast of the echinococcus granulosus, echinococcus multilocularis and cryptosporidium parvum, or according to the comparison analysis result of the mitochondrial genome sequence blast of the echinococcus granulosus, echinococcus multilocularis and cryptosporidium canis, the primer is designed based on the principle that the primer must have specificity and the size of the DNA fragment to be amplified by the primer must have obvious difference;
the primers obtained by the primer design procedure were as follows:
echinococcus granulosus primer 1: 5'-CGTTGTCCTCGTCGTATT-3', respectively;
echinococcus granulosus primer 2: 5'-AACAGCCGTCTTCACATC-3', respectively;
the length of the target fragment is 515 bp;
echinococcus multilocularis primer 1: 5'-TTGATTATTGGAGGCTATGC-3', respectively;
echinococcus multilocularis primer 2: 5'-CAACGACAACATAACATCAC-3', respectively;
the length of the target fragment is 353 bp;
cryptosporidium parvum and Cryptosporidium caninum common primer 1: 5'-CTCAACTCAAGCCACCAT-3', respectively;
cryptosporidium parvum and Cryptosporidium caninum common primer 2: 5'-CCGACCAAGACAACATCA-3', respectively;
the target fragment is 186bp in length.
Preferably, in the triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention, the DNA extraction steps are specifically as follows:
dissolving excrement in water, performing first centrifugal separation to obtain an upper layer liquid, mixing the captured upper layer liquid with a Trizol solution, adding chloroform, performing second centrifugal separation, adding an isopropanol solution into the mixed solution after the second centrifugal separation for performing third centrifugal separation, and adding ethanol into the liquid after the third centrifugal separation for performing fourth centrifugal separation to obtain an RNA precipitate;
preferably, in the first centrifugal separation, the rotating speed of the centrifugal separation is 3000-3500r/min, the time of the centrifugal separation is 2-2.5min, and the upper liquid of 1/3-1/4 is taken for standby;
preferably, in the second centrifugal separation, the rotating speed of the centrifugal separation is 15000r/min, and the time of the centrifugal separation is 15 min;
preferentially, in the third centrifugal separation, the rotating speed of the centrifugal separation is 15000r/min, and the time of the centrifugal separation is 10 min;
preferably, in the fourth centrifugation, the rotation speed of the centrifugation is 7500r/min, and the time of the centrifugation is 5 min.
Preferably, in the triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention, the PCR steps are specifically operated as follows:
mixing 20-25 parts of PCR buffer solution, 4-6 parts of all primers (equal amount), 2-3 parts of deoxyribose-containing deoxynucleoside triphosphate, 50-60 parts of DECP water and 20-25 parts of DNA template according to volume parts;
heating the mixed solution at 68-72 deg.C for 3 min;
then, heating for 3min under the condition of dry bath at 95 ℃;
adding 5-7 parts of TAG enzyme by volume, and centrifuging for 1min at 12000r/min under airtight condition;
and carrying out 38-42 cycles of 95-65 s, 56-50 s and 72-95 s of the solution according to the following cycle, and then annealing at the constant temperature of 72 ℃ for 10-12 min.
Preferably, in the triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention, the electrophoresis detection steps are specifically operated as follows:
preparing 0.5mol/L EDTA solution with pH of 8.0: adding 160ml of distilled water into 37.2g of tetrasodium ethylene diamine tetraacetate, stirring, adjusting the pH value to 8.0 by using sodium hydroxide, fixing the volume to 200ml by using distilled water, and then carrying out high-pressure sterilization under the airtight condition;
preparing 50 TAE solution: dissolving 121g of trihydroxymethyl aminomethane in 400ml of distilled water, adding 28.55ml of glacial acetic acid and 50ml of ethylene diamine tetraacetic acid solution, and adding distilled water to reach the constant volume of 500 ml;
preparing 10mg/ml ethidium bromide: adding 0.2g of ethidium bromide into 20ml of deionized water, and stirring for 3-5 h;
and (3) taking 10ml of the 50 TAE solution, diluting to 500ml, taking 50ml of the diluted solution, dissolving 0.5g of agarose in the diluted solution, heating, cooling the solution to 60 ℃, adding 5 microliters of 10mg/ml ethidium bromide, introducing the ethidium bromide into an electrophoresis plate, standing at room temperature for 45-60min, performing electrophoresis for 1h, and detecting by adopting an ultraviolet lamp for irradiation.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a triple PCR detection primer, a triple PCR detection method and a triple PCR detection kit for tapeworm and sporozoon in dog feces, wherein the triple PCR detection method is used for detecting two kinds of tapeworms, namely echinococcus granulosus and echinococcus multilocularis, and detecting two kinds of sporozoons, namely cryptosporidium parvum and cryptosporidium caninum.
The detection method specifically operates as follows: extracting total DNA of the dog excrement sample, using the DNA as a template, utilizing a primer combination to carry out PCR amplification, and judging whether the sample contains cryptosporidium caninum and cryptosporidium parvum infection or not and whether the sample contains echinococcus granulosus or echinococcus multilocularis infection or not through electrophoretic analysis of reaction products. In the present invention, the primer combination consists of six primer nucleotides. The detection method provided by the invention has the characteristics of rapidness, accuracy, strong specificity and the like, provides technical support for diagnosis and detection of the canine dung echinococcus granulosus, echinococcus multilocularis, cryptosporidium parvum and cryptosporidium caninum, and has very important significance for prevention and control of important human and canine co-morbidities.
The triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention can amplify specific gene segments of four parasites simultaneously in one reaction system, and makes up the defects of low detectable rate, poor detection accuracy, time and labor waste in morphology by adopting electron microscope observation. The detection method established by the invention has the characteristics of high specificity and high sensitivity, can realize the high-flux detection of multiple pathogens in a single sample, not only saves the detection cost of the single sample, but also improves the detection rate, and simultaneously improves the detection accuracy to a great extent. The three groups of primers contained in the invention meet the requirement of multiplex PCR detection, and have profound economic value and social benefit.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.
The invention provides a triple PCR detection primer, a triple PCR detection method and a triple PCR detection kit for tapeworm and sporozoon in dog feces, wherein the triple PCR detection method is used for detecting two kinds of tapeworms, namely echinococcus granulosus and echinococcus multilocularis, and detecting two kinds of sporozoons, namely cryptosporidium parvum and cryptosporidium caninum.
The detection method specifically operates as follows: extracting total DNA of the dog excrement sample, using the DNA as a template, utilizing a primer combination to carry out PCR amplification, and judging whether the sample contains cryptosporidium caninum and cryptosporidium parvum infection or not and whether the sample contains echinococcus granulosus or echinococcus multilocularis infection or not through electrophoretic analysis of reaction products. In the present invention, the primer combination consists of six primer nucleotides. The detection method provided by the invention has the characteristics of rapidness, accuracy, strong specificity and the like, provides technical support for diagnosis and detection of the canine dung echinococcus granulosus, echinococcus multilocularis, cryptosporidium parvum and cryptosporidium caninum, and has very important significance for prevention and control of important human and canine co-morbidities.
The triple PCR detection method for tapeworm and sporozoon in dog feces provided by the invention can amplify specific gene segments of four parasites simultaneously in one reaction system, and makes up the defects of low detectable rate, poor detection accuracy, time and labor waste in morphology by adopting electron microscope observation. The detection method established by the invention has the characteristics of high specificity and high sensitivity, can realize the high-flux detection of multiple pathogens in a single sample, not only saves the detection cost of the single sample, but also improves the detection rate, and simultaneously improves the detection accuracy to a great extent. The three groups of primers contained in the invention meet the requirement of multiplex PCR detection, and have profound economic value and social benefit.
The method for detecting the tapeworm and the sporozoon in the dog excrement by triple PCR comprises the following specific steps:
step one, designing a primer
Based on the results of alignment analysis of the mitochondrial genomic sequence blast of Echinococcus granulosus, Echinococcus multilocularis, Cryptosporidium parvum (or Cryptosporidium canis), according to: in principle one, the primer must have specificity; principle two, the sizes of the DNA fragments to be amplified by the primers must have obvious differences, and the following three groups of primers are designed:
echinococcus granulosus primer 1: 5'-CGTTGTCCTCGTCGTATT-3', respectively;
echinococcus granulosus primer 2: 5'-AACAGCCGTCTTCACATC-3', respectively;
the length of the target fragment is 515 bp;
echinococcus multilocularis primer 1: 5'-TTGATTATTGGAGGCTATGC-3', respectively;
echinococcus multilocularis primer 2: 5'-CAACGACAACATAACATCAC-3', respectively;
the length of the target fragment is 353 bp;
cryptosporidium parvum and Cryptosporidium caninum common primer 1: 5'-CTCAACTCAAGCCACCAT-3', respectively;
cryptosporidium parvum and Cryptosporidium caninum common primer 2: 5'-CCGACCAAGACAACATCA-3', respectively;
the length of the target fragment is 186 bp;
wherein, the primer 1 is an upstream primer, and the primer 2 is a downstream primer.
Step two, DNA extraction
When collecting feces, the feces should be kept fresh and uncontaminated. Under convenient condition, can follow the rectum and directly gather, when carrying out excrement and urine collection to large-scale dog class, can adopt the excrement and urine's of rectum collection mode, to small-size dog class, then adopt the mode of on-the-spot collection. When collecting the feces on the spot, the collecting bag should be kept clean to avoid the condition that the feces are polluted to influence the detection result.
The invention mainly adopts a cleaning precipitation method for collecting the worm eggs. The method comprises the following specific steps:
5-10g of the feces are taken and placed in a clean container, such as a beaker. Then a small amount of deionized or distilled water is added, taking care not to use tap water or water from natural sources to avoid external contamination. The ratio of water to feces is 20-40:1 in parts by weight. Scattering the excrement in water, uniformly stirring, filtering the water by adopting a gauze, and carrying out centrifugal separation on the filtrate, wherein the rotating speed of the centrifugal separation is 3000-3500r/min, and the time of the centrifugal separation is 2-2.5 min. Taking 1/3-1/4 upper layer liquid for standby.
And (3) introducing the extracted upper layer liquid into an EP (EP) tube, dropwise adding a Trizol solution, vibrating for dispersion, and standing for 7-9min at room temperature for later use. Wherein the volume ratio of the obtained supernatant liquid to the Trizol solution is 0.4-0.8: 1. Then adding chloroform, wherein the volume ratio of the chloroform solution to the mixed solution is 0.15-0.2: 1. Shaking for 30s under airtight condition, standing in ice water bath for 7-9min, and centrifuging at 15000r/min for 15 min.
And taking the upper aqueous phase solution of the centrifuged mixed solution and putting the upper aqueous phase solution into a clean EP tube for later use. Then adding isopropanol, centrifuging, and controlling the volume ratio of the mixed solution to the isopropanol solution to be 0.4-0.5: 1. Then, the mixture was left standing in a constant temperature environment of-25 ℃ for 1.5 hours under an airtight condition, and centrifuged at 15000r/min in the environment for 10 minutes.
And (3) extracting the supernatant of the solution after the second centrifugation, reserving a precipitate, adding 75% ethanol, washing the precipitate (precipitated RNA) under the oscillation condition, and centrifuging for 5min at 7500r/min after washing. Wherein, ethanol is prepared on site and precooled.
And (4) extracting the supernatant of the solution after the third centrifugation, reserving the precipitate, then placing the precipitate in a clean vessel, and performing air cooling blow-drying on a superclean bench, wherein the precipitate cannot be completely dried according to vision. Then, DEPC water is added into the vessel, the temperature is gradually increased, the temperature rising speed is 0.2-0.3 ℃/min, and the temperature is increased by adopting a water bath. And (3) performing cosolvent at 60-62 ℃ for 15 min.
Step three, PCR step
According to the volume parts, 20-25 parts of PCR buffer solution, 4-6 parts of all primers (equal amount), 2-3 parts of deoxynucleoside triphosphate containing deoxyribose, 50-60 parts of DECP water and 20-25 parts of DNA template are mixed and then slightly shaken to be uniformly dispersed. Then heating the mixed solution at 68-72 deg.C for 3min, preferably dry bath, and keeping the temperature below 0.2 deg.C. After heating, the mixture was cooled to room temperature in an ice water bath. Immediately thereafter, heating was carried out for 3min under the condition of a dry bath at 95 ℃. Then, 5-7 parts by volume of TAG enzyme was added and centrifuged at 12000r/min for 1min under airtight conditions.
The solution is circulated for 38-42 cycles of 95-65 s, 56-50 s and 72-95 s. And after the circulation is completed, annealing is carried out in a constant temperature environment of 72 ℃ for 10-12 min.
Step four, electrophoresis detection
Preparing 0.5mol/L EDTA solution with pH of 8.0. The method comprises the following specific steps: to 37.2g of tetrasodium ethylenediaminetetraacetate, 160ml of distilled water was added and stirred (vigorously). Sodium hydroxide was used to adjust the pH to 8.0. Distilled water was used to bring the volume to 200 ml. Then autoclaved under airtight conditions.
Prepare 50% TAE solution. The method comprises the following specific steps: 121g of tris (hydroxymethyl) aminomethane was dissolved in 400ml of distilled water, and then 28.55ml of glacial acetic acid and 50ml of the above-mentioned ethylenediaminetetraacetic acid solution were added thereto, and distilled water was added thereto to make the volume of 500 ml.
10mg/ml of ethidium bromide is prepared. The method comprises the following specific steps: 0.2g of ethidium bromide is added to 20ml of deionized water, magnetically stirred for 3-5h and then transferred to a dark flask (dark brown).
Diluting the 50 × TAE solution 10ml to 500ml, dissolving 0.5g agarose in 50ml diluent, heating to boil, cooling to 60 deg.C after agarose is completely dissolved, adding 10mg/ml ethidium bromide 5 μ l, and mixing. The warm gel mixture was introduced into an electrophoresis plate and allowed to stand at room temperature for 45-60 min. And then carrying out electrophoresis for 1h, and detecting by using an ultraviolet lamp.
Test examples
The kit prepared in the embodiment is used for testing actual sample detection, 156 fecal samples of different dogs (Bingmu, Tady and Hatschy) in a pet shop in Hebei are collected for detection, 156 fecal genomic DNAs are extracted, triple PCR amplification is carried out according to the system operation and optimized reaction conditions, the operation is repeated for 3 times, 5 parts (3.2%) of echinococcus granulosus positive fecal samples, 7 parts (4.5%) of cryptosporidium parvum and cryptosporidium caninum positive fecal samples and 13 parts (8.3%) of echinococcus multilocularis positive fecal samples are detected by the triple PCR.
And (4) conclusion: the detection examples prove that the kit disclosed by the invention has accuracy and stability. Can carry out triple PCR rapid detection on the Echinococcus granulosus, Echinococcus multilocularis, cryptosporidium parvum and cryptosporidium caninum, and lays a foundation for future chemical investigation on canine cestodiasis or sporozoosis. Meanwhile, technical support is provided for the quality standard of experimental cattle.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A triple PCR detection primer for tapeworm and sporozoon in dog feces is characterized in that,
the tapeworm is echinococcus granulosus and echinococcus multilocularis, and the sporozoon is cryptosporidium parvum and cryptosporidium canis;
the primer is as follows:
echinococcus granulosus primer 1: 5'-CGTTGTCCTCGTCGTATT-3', respectively;
echinococcus granulosus primer 2: 5'-AACAGCCGTCTTCACATC-3', respectively;
the length of the target fragment is 515 bp;
echinococcus multilocularis primer 1: 5'-TTGATTATTGGAGGCTATGC-3', respectively;
echinococcus multilocularis primer 2: 5'-CAACGACAACATAACATCAC-3', respectively;
the length of the target fragment is 353 bp;
cryptosporidium parvum and Cryptosporidium caninum common primer 1: 5'-CTCAACTCAAGCCACCAT-3', respectively;
cryptosporidium parvum and Cryptosporidium caninum common primer 2: 5'-CCGACCAAGACAACATCA-3', respectively;
the target fragment is 186bp in length.
2. A triple PCR detection kit for tapeworm and sporozoon in dog feces is characterized in that,
the tapeworm is echinococcus granulosus and echinococcus multilocularis, and the sporozoon is cryptosporidium parvum and cryptosporidium canis;
the kit comprising the primers as set forth in claim 1 in their entirety.
3. The triple PCR detection kit for tapeworm and sporozoite in canine feces according to claim 2,
the reaction system for carrying out the triple PCR detection in the kit is as follows:
the method comprises the following steps of: 20-25 parts of PCR buffer solution, 4-6 parts of all primers (equal amount), 2-3 parts of deoxyribose-containing deoxynucleoside triphosphate and 50-60 parts of DECP water.
4. A triple PCR detection method for tapeworm and sporozoon in dog feces, which does not aim at the diagnosis and treatment of diseases,
it is characterized in that the preparation method is characterized in that,
the method comprises the following steps: preparing a primer, extracting DNA, carrying out PCR (polymerase chain reaction) and carrying out electrophoresis detection;
wherein the PCR step uses the triple PCR detection kit for detecting the tapeworm and the sporozoite in the canine feces as described in claim 2 or 3.
5. The triple PCR detection method for tapeworm and sporozoon in dog feces according to claim 4,
a primer design step is included before the primer preparation step;
in the primer design step, according to the comparison analysis result of the mitochondrial genome sequence blast of the echinococcus granulosus, echinococcus multilocularis and cryptosporidium parvum, or according to the comparison analysis result of the mitochondrial genome sequence blast of the echinococcus granulosus, echinococcus multilocularis and cryptosporidium canis, the primer is designed based on the principle that the primer must have specificity and the size of the DNA fragment to be amplified by the primer must have obvious difference;
the primers obtained by the primer design procedure were as follows:
echinococcus granulosus primer 1: 5'-CGTTGTCCTCGTCGTATT-3', respectively;
echinococcus granulosus primer 2: 5'-AACAGCCGTCTTCACATC-3', respectively;
the length of the target fragment is 515 bp;
echinococcus multilocularis primer 1: 5'-TTGATTATTGGAGGCTATGC-3', respectively;
echinococcus multilocularis primer 2: 5'-CAACGACAACATAACATCAC-3', respectively;
the length of the target fragment is 353 bp;
cryptosporidium parvum and Cryptosporidium caninum common primer 1: 5'-CTCAACTCAAGCCACCAT-3', respectively;
cryptosporidium parvum and Cryptosporidium caninum common primer 2: 5'-CCGACCAAGACAACATCA-3', respectively;
the target fragment is 186bp in length.
6. The triple PCR detection method for tapeworm and sporozoon in dog feces according to claim 4,
the DNA extraction step is specifically operated as follows:
dissolving excrement in water, performing first centrifugal separation to obtain an upper layer liquid, mixing the captured upper layer liquid with a Trizol solution, adding chloroform, performing second centrifugal separation, adding an isopropanol solution into the mixed solution after the second centrifugal separation for performing third centrifugal separation, and adding ethanol into the liquid after the third centrifugal separation for performing fourth centrifugal separation to obtain an RNA precipitate;
preferably, in the first centrifugal separation, the rotating speed of the centrifugal separation is 3000-3500r/min, the time of the centrifugal separation is 2-2.5min, and the upper liquid of 1/3-1/4 is taken for standby;
preferably, in the second centrifugal separation, the rotating speed of the centrifugal separation is 15000r/min, and the time of the centrifugal separation is 15 min;
preferentially, in the third centrifugal separation, the rotating speed of the centrifugal separation is 15000r/min, and the time of the centrifugal separation is 10 min;
preferably, in the fourth centrifugation, the rotation speed of the centrifugation is 7500r/min, and the time of the centrifugation is 5 min.
7. The triple PCR detection method for tapeworm and sporozoon in dog feces according to claim 4,
the PCR steps are specifically operated as follows:
mixing 20-25 parts of PCR buffer solution, 4-6 parts of all primers (equal amount), 2-3 parts of deoxyribose-containing deoxynucleoside triphosphate, 50-60 parts of DECP water and 20-25 parts of DNA template according to volume parts;
heating the mixed solution at 68-72 deg.C for 3 min;
then heating for 3min under the condition of dry bath at 95 ℃;
adding 5-7 parts of TAG enzyme by volume, and centrifuging for 1min at 12000r/min under airtight condition;
and carrying out 38-42 cycles of 95-65 s, 56-50 s and 72-95 s of the solution according to the following cycle, and then annealing at the constant temperature of 72 ℃ for 10-12 min.
8. The triple PCR detection method for tapeworm and sporozoon in dog feces according to claim 4,
the electrophoresis detection step is specifically operated as follows:
preparing 0.5mol/L EDTA solution with pH of 8.0: adding 160ml of distilled water into 37.2g of tetrasodium ethylene diamine tetraacetate, stirring, adjusting the pH value to 8.0 by using sodium hydroxide, fixing the volume to 200ml by using distilled water, and then carrying out high-pressure sterilization under the airtight condition;
preparing 50 TAE solution: dissolving 121g of trihydroxymethyl aminomethane in 400ml of distilled water, adding 28.55ml of glacial acetic acid and 50ml of ethylene diamine tetraacetic acid solution, and adding distilled water to reach the constant volume of 500 ml;
preparing 10mg/ml ethidium bromide: adding 0.2g of ethidium bromide into 20ml of deionized water, and stirring for 3-5 h;
and (3) taking 10ml of the 50 TAE solution, diluting to 500ml, taking 50ml of the diluted solution, dissolving 0.5g of agarose in the diluted solution, heating, cooling the solution to 60 ℃, adding 5 microliters of 10mg/ml ethidium bromide, introducing the ethidium bromide into an electrophoresis plate, standing at room temperature for 45-60min, performing electrophoresis for 1h, and detecting by adopting an ultraviolet lamp for irradiation.
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