CN113604581A - Multiple PCR detection method for zoonosis parasites - Google Patents
Multiple PCR detection method for zoonosis parasites Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 206010048282 zoonosis Diseases 0.000 title claims abstract description 43
- 244000045947 parasite Species 0.000 title claims abstract description 40
- 238000007403 mPCR Methods 0.000 claims abstract description 37
- 230000003321 amplification Effects 0.000 claims abstract description 29
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 29
- 239000008280 blood Substances 0.000 claims abstract description 28
- 210000004369 blood Anatomy 0.000 claims abstract description 28
- 238000000246 agarose gel electrophoresis Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 210000003608 fece Anatomy 0.000 claims abstract description 8
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 238000000137 annealing Methods 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000003292 glue Substances 0.000 claims description 9
- 238000007400 DNA extraction Methods 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 238000012257 pre-denaturation Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 239000011543 agarose gel Substances 0.000 claims description 4
- 238000013461 design Methods 0.000 claims description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 4
- 229960005542 ethidium bromide Drugs 0.000 claims description 4
- 239000000499 gel Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004513 sizing Methods 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 241000283707 Capra Species 0.000 abstract description 6
- 208000030852 Parasitic disease Diseases 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 41
- 238000002474 experimental method Methods 0.000 description 15
- 238000010586 diagram Methods 0.000 description 14
- 239000012634 fragment Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 6
- 241000223936 Cryptosporidium parvum Species 0.000 description 3
- 241000224467 Giardia intestinalis Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 241000223777 Theileria Species 0.000 description 3
- 241000223095 Trypanosoma evansi Species 0.000 description 3
- 241001147660 Neospora Species 0.000 description 2
- 241000223997 Toxoplasma gondii Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 244000000053 intestinal parasite Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 230000035945 sensitivity Effects 0.000 description 2
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- 241001147662 Neospora caninum Species 0.000 description 1
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Abstract
The application discloses a multiple PCR detection method for zoonosis parasites, which comprises the steps of respectively obtaining DNA from animal blood and feces; respectively preparing the obtained blood DNA and the obtained feces DNA into DNA solutions with the concentration of 25 ng/mu L, mixing the DNA solutions of the blood DNA and the feces DNA, and diluting the mixture into a plurality of concentration gradients according to a 10-fold ratio after mixing; performing multiplex PCR amplification on the obtained DNA solution with a plurality of concentration gradients; and (3) carrying out agarose gel electrophoresis observation, taking the former concentration without obvious bands as the lowest detection concentration, and carrying out DNA structure and function analysis on the amplification solution with the lowest detection concentration and above concentration so as to judge the types of the parasites. The scheme has higher practicability, convenience and rapidness, so that the time for clinically detecting the zoonosis parasitic diseases is greatly shortened, the detection efficiency is effectively guaranteed, and a technical support is provided for detecting the zoonosis parasitic diseases susceptible to goats.
Description
Technical Field
The application relates to the technical field of zoonosis epidemic diseases, in particular to a zoonosis parasite multiple PCR detection method.
Background
Bred animals such as goats and the like are susceptible to various zoonosis, on one hand, the health of the animals is affected, the breeding economic benefit is reduced, and on the other hand, the health of breeding practitioners, consumers and scientific and technical staff is seriously harmed. At present, most of detection technologies for the epidemic diseases adopt ELISA or PCR detection of single pathogen, so that the efficiency is low, and a high-efficiency detection technology for simultaneously aiming at a plurality of pathogens is to be developed.
Disclosure of Invention
The main objective of the present application is to provide a multiple PCR detection method for zoonosis parasites, so as to improve the problem of low detection efficiency in the related art.
In order to achieve the above object, the present application provides a multiplex PCR detection method for zoonosis parasites, which specifically comprises the following steps:
s1, obtaining DNA, and respectively obtaining DNA from animal blood and feces;
s2, mixing, namely preparing the obtained blood DNA and the obtained excrement DNA into DNA solutions with the concentration of 25 ng/mu L respectively, mixing the DNA solutions of the blood DNA and the excrement DNA, and diluting the DNA solutions into a plurality of concentration gradients according to a 10-fold ratio after mixing;
s3, performing multiplex PCR amplification on the obtained DNA solution with a plurality of concentration gradients, wherein the parameters of the multiplex PCR amplification are as follows: mu.L of mixed substrate, 0.5. mu.L of each of upstream and downstream primers, 2 XHiFi Amplification Mix 10. mu.L, ddH2Supplementing 20 mu L of O, performing pre-denaturation at 94 ℃ for 2min, performing denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, performing extension at 68 ℃ for 15s, and repeating 35 cycles to finish the amplification process;
and S4, obtaining a result, observing by agarose gel electrophoresis, taking the former concentration without obvious bands as the lowest detection concentration, and analyzing the DNA structure and function of the amplification solution with the lowest detection concentration and the concentration above, thereby judging the type of the parasite.
In an embodiment of the present application, in S1, the DNA is obtained by first obtaining a blood sample and a stool sample, and extracting DNA according to the operation requirements of the blood DNA extraction kit and the stool DNA extraction kit, respectively, to obtain the blood DNA and the stool DNA.
In one embodiment of the present application, in S2, there are 9 concentration gradients, and each concentration gradient is 2.5 × 101ng/μL、2.5×100ng/μL、2.5×10-1ng/μL、2.5×10-2ng/μL、2.5×10-3ng/μL、2.5×10-4ng/μL、2.5×10-5ng/μL、2.5×10-6ng/. mu.L and 2.5X 10-7ng/μL。
In an embodiment of the present application, in S3, the determination manner of the number of cycles is: the number of design cycles was 20, 25, 30, 35 and 40 cycles, 2.5 × 10 respectively-51. mu.L of ng/. mu.L DNA mixture, 0.5. mu.L of each of the upstream and downstream primers, 2. mu.L of the HiFi Amplification Mix, 10. mu.L of ddH2O was added to 20. mu.L, pre-denatured at 94 ℃ for 2min, denatured at 98 ℃ for 10s, annealed at 60 ℃ for 30s, and extended at 68 ℃ for 15s, and observed by agarose gel electrophoresis, with the cycle number at which a clear band appeared being the optimum cycle number, and the optimum cycle number being 35.
In an embodiment of the present application, the agarose gel electrophoresis observation is specifically performed by: electrophoresis is carried out for 35min by 2 percent agarose gel under the voltage of 120V, observation is carried out under an EB ethidium bromide staining ultraviolet instrument, and a gel preparation system is as follows:
small glue: 0.4g of agar powder, 20ml of TAE and 2 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
and (3) medium sizing: 0.8g of agar powder, 40ml of TAE and 4 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
large glue: 1.6g of agar powder, 80ml of TAE and 8 microliter of EB, heating for 3-4 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
In an embodiment of the present application, in S3, the annealing temperature is determined by: setting annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃, and observing the brightness and the definition of the amplified band to select the optimal annealing temperature, wherein the optimal annealing temperature is 60 ℃.
Compared with the prior art, the beneficial effects of this application are: by the designed multiple PCR detection method for the zoonosis parasites, multiple parasites can be detected at the same time, and the method is efficient and convenient, not only saves time, but also saves reagents, and has great clinical significance; the kit has higher practicability, convenience and rapidness, greatly shortens the time for clinically detecting the zoonosis parasitic diseases, effectively ensures the detection efficiency, and provides technical support for detecting the zoonosis parasitic diseases susceptible to goats.
Drawings
FIG. 1 is a schematic diagram showing the sizes of single PCR-amplified fragments of six zoonosis parasites in an experiment according to the multiplex PCR detection method for zoonosis parasites provided by the embodiment of the application;
FIG. 2 is a schematic diagram showing the sizes of amplified fragments obtained by simultaneously amplifying single PCR and multiple PCR of four zoonosis parasites in an experiment according to the method for detecting zoonosis parasites provided in the embodiment of the present application;
FIG. 3 is a schematic diagram showing the sizes of amplified fragments obtained by simultaneously performing single PCR and multiplex PCR on two zoonosis parasites in an experiment according to the method for detecting zoonosis parasites provided in the embodiment of the present application;
FIG. 4 is a schematic diagram showing the annealing temperature optimization and amplified fragment size of quadruple PCR in an experiment of the multiple PCR detection method for zoonosis parasites according to the embodiment of the application;
FIG. 5 is a schematic diagram showing the annealing temperature optimization and amplified fragment size of the double PCR in the experiment of the multiple PCR detection method for zoonosis parasites according to the embodiment of the present application;
FIG. 6 is a diagram showing the sensitivity of detecting a multiplex PCR reaction system and the size of amplified fragments in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples of the present application;
FIG. 7 is a diagram showing the DNA mixing of four hematoparasites, selecting appropriate amplification cycles, and the sizes of the amplified fragments in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the example of the present application;
FIG. 8 is a schematic diagram showing the DNA mixing of two hematoparasites, selecting appropriate amplification cycles, and the sizes of the amplified fragments in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the example of the present application;
FIG. 9 is a first schematic diagram of a multiplex PCR system for amplifying 68 clinical blood samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples of the present application;
FIG. 10 is a second schematic diagram of a multiplex PCR system for amplifying 68 clinical blood samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples of the present application;
FIG. 11 is a third schematic diagram of a multiplex PCR system for amplifying 68 clinical blood samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples of the present application;
FIG. 12 is a first schematic diagram of a multiplex PCR system for amplifying clinical 40 stool samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples of the present application;
FIG. 13 is a second schematic diagram of a multiplex PCR system for amplifying clinical 40 stool samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the examples herein;
FIG. 14 is a third schematic diagram of a multiplex PCR system for amplifying clinical 40 stool samples in an experiment according to the multiplex PCR detection method for zoonosis parasites provided in the example of the application.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to a particular orientation or to be constructed and operated in a particular orientation.
Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meaning of these terms in this application will be understood by those of ordinary skill in the art as appropriate.
In addition, the term "plurality" shall mean two as well as more than two.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1
Referring to fig. 1-14, the present application provides a multiplex PCR method for detecting zoonosis parasites, which comprises the following steps:
s1, obtaining DNA, and respectively obtaining DNA from animal blood and feces;
s2, mixing, namely preparing the obtained blood DNA and the obtained excrement DNA into DNA solutions with the concentration of 25 ng/mu L respectively, mixing the DNA solutions of the blood DNA and the excrement DNA, and diluting the DNA solutions into a plurality of concentration gradients according to a 10-fold ratio after mixing;
s3, performing multiplex PCR amplification on the obtained DNA solution with a plurality of concentration gradients, wherein the parameters of the multiplex PCR amplification are as follows: mu.L of mixed substrate, 0.5. mu.L of each of upstream and downstream primers, 2 XHiFi Amplification Mix 10. mu.L, ddH2Supplementing 20 mu L of O, performing pre-denaturation at 94 ℃ for 2min, performing denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, performing extension at 68 ℃ for 15s, and repeating 35 cycles to finish the amplification process;
and S4, obtaining a result, observing by agarose gel electrophoresis, taking the former concentration without obvious bands as the lowest detection concentration, and analyzing the DNA structure and function of the amplification solution with the lowest detection concentration and the concentration above, thereby judging the type of the parasite.
In an embodiment of the present application, in S1, the DNA is obtained by first obtaining a blood sample and a stool sample, and extracting DNA according to the operation requirements of the blood DNA extraction kit and the stool DNA extraction kit, respectively, to obtain the blood DNA and the stool DNA.
In one embodiment of the present application, in S2, there are 9 concentration gradients, and each concentration gradient is 2.5 × 101ng/μL、2.5×100ng/μL、2.5×10-1ng/μL、2.5×10-2ng/μL、2.5×10-3ng/μL、2.5×10-4ng/μL、2.5×10-5ng/μL、2.5×10-6ng/. mu.L and2.5×10-7ng/μL。
in an embodiment of the present application, in S3, the determination manner of the number of cycles is: the number of design cycles was 20, 25, 30, 35 and 40 cycles, 2.5 × 10 respectively-51. mu.L of ng/. mu.L DNA mixture, 0.5. mu.L of each of the upstream and downstream primers, 2. mu.L of the HiFi Amplification Mix, 10. mu.L of ddH2O was added to 20. mu.L, pre-denatured at 94 ℃ for 2min, denatured at 98 ℃ for 10s, annealed at 60 ℃ for 30s, and extended at 68 ℃ for 15s, and observed by agarose gel electrophoresis, with the cycle number at which a clear band appeared being the optimum cycle number, and the optimum cycle number being 35.
In an embodiment of the present application, the agarose gel electrophoresis observation is specifically performed by: electrophoresis is carried out for 35min by 2 percent agarose gel under the voltage of 120V, observation is carried out under an EB ethidium bromide staining ultraviolet instrument, and a gel preparation system is as follows:
small glue: 0.4g of agar powder, 20ml of TAE and 2 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
and (3) medium sizing: 0.8g of agar powder, 40ml of TAE and 4 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
large glue: 1.6g of agar powder, 80ml of TAE and 8 microliter of EB, heating for 3-4 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
In an embodiment of the present application, in S3, the annealing temperature is determined by: setting annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃, and observing the brightness and the definition of the amplified band to select the optimal annealing temperature, wherein the optimal annealing temperature is 60 ℃.
Through test verification, the specific test steps are as follows:
68 parts of goat blood sample and 40 parts of goat feces (both from Chongming herbivore test station of agricultural science institute of Shanghai city) stored at-80 ℃ are taken out, unfrozen at 4 ℃, DNA is extracted according to the operation requirements of a blood DNA extraction kit and a feces DNA extraction kit respectively, and the four-fold PCR and two-fold PCR systems established by the research are used for detecting the two extracted DNAs.
As shown in fig. 1: 6 zoonosis parasites are respectively subjected to single PCR amplification, and the samples added from the first hole to the sixth hole are as follows: cryptosporidium parvum, Theileria taenii, neospora, Giardia duodenalis, Trypanosoma evansi, Toxoplasma gondii. The sizes of the amplified fragments were in order: 700bp, 492bp, 221bp, 447bp, 556bp and 124 bp. The results for each well were consistent with the expected length of the desired product.
As shown in FIG. 2, the single PCR and the multiplex PCR of four zoonosis parasites were amplified simultaneously and used as a control to check whether the products of the multiplex PCR amplification are correct, and the samples added from the first well to the fourth well were: theileria taylonica, neospora, trypanosoma evansi, toxoplasma. The sizes of the amplified fragments were in order: 492bp, 221bp, 556bp and 124 bp. All products were fit to the expected target fragment size.
As shown in fig. 3: simultaneously amplifying single PCR and multiple PCR of two zoonosis parasites, and checking whether the multiple PCR amplification product is correct by using the PCR amplification product as a control, wherein the first hole is an amplification sequence of cryptosporidium parvum, and the size of the first hole is 700 bp; the second hole is a giardia duodenalis amplification sequence with the size of 447 bp; the third well is a multiplex PCR amplification sequence for two intestinal parasites. All products were fit to the expected target fragment size.
As shown in fig. 4: the annealing temperature of the quadruple PCR was optimized. Setting different annealing temperatures, and selecting the optimal annealing temperature by amplifying the brightness and the definition of the strip, wherein the annealing temperatures set in the first hole to the sixth hole are respectively as follows: 50 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C. The 4 bands amplified from each channel are matched with the size, and the optimal temperature is selected to be 60 ℃.
As shown in FIG. 5, the annealing temperature of the duplex PCR was optimized. Setting different annealing temperatures, and selecting the optimal annealing temperature by amplifying the brightness and the definition of the strip, wherein the annealing temperatures set in the first hole to the sixth hole are respectively as follows: 50 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C. Two bands amplified from each pore channel are matched with the size, and the optimal band is selected at 60 ℃.
As shown in fig. 6: diluting 6 substrate DNA mixed templates with the concentration of 25 ng/mu L by 10 times, and detecting the multiple PCR reaction systemSensitivity was 25 ng/. mu.L in the first well and 2.5X 10 in the second well0ng/. mu.L, third well 2.5X 10-1ng/. mu.L, fourth well 2.5X 10-2ng/. mu.L, fifth well 2.5X 10-3ng/. mu.L, six wells 2.5X 10-4ng/. mu.L, seventh well 2.5X 10-5ng/. mu.L, eighth well 2.5X 10-6ng/. mu.L, ninth well 2.5X 10-7ng/. mu.L. 4 amplified nucleic acid fragments with 1-7 channels have correct sizes.
As shown in FIG. 7, the concentration of 2.5X 10 was selected-5ng/. mu.L of the four hematoparasite pooled DNA, set for different numbers of cycles, respectively, and the appropriate number of amplification cycles was selected, 20 cycles for the first well, 25 cycles for the second well, 30 cycles for the third well, 35 cycles for the fourth well, and 40 cycles for the fifth well. After sequence comparison, the fragments amplified from 4-5 channels are in accordance with the expected size, and the optimal number of 35 cycles is selected.
As shown in fig. 8: selecting the concentration of 2.5 × 10-5ng/. mu.L of 2 mixed DNA of intestinal parasites, setting different numbers of cycles respectively, and selecting proper amplification cycles, wherein the first hole is 20 cycles, the second hole is 25 cycles, the third hole is 30 cycles, the fourth hole is 35 cycles, and the fifth hole is 40 cycles. After sequence comparison, the sizes of the bands amplified from 4-5 channels are correct, and 35 cycles are selected to be the best.
As shown in fig. 9-11: the multiplex PCR system established above was used to amplify 68 clinical blood samples, and all the results are shown in Table 1;
| name (R) | Toxoplasma gondii | Theileria taenii (Lee) Johnson | Trypanosoma evansi | Neospora caninum |
| Positive detected quantity (quantity) | 0 | 0 | 0 | 0 |
| Positive detection Rate (%) | 0 | 0 | 0 | 0 |
TABLE 168 blood sample test results
As shown in fig. 12-14: amplifying the clinical 40 stool samples by using the multiplex PCR system established above, wherein all detection results are shown in Table 2;
| name (R) | Giardia duodenalis | Cryptosporidium parvum |
| Positive detected quantity (quantity) | 0 | 0 |
| Positive detection Rate (%) | 0 | 0 |
Table 240 stool sample test results
Testing the sensitivity;
after mixing 25 ng/. mu.L of DNA of zoonosis, 10-fold diluted into 9 concentration gradients, which are respectively: 2.5X 101ng/μL、2.5×100ng/μL、2.5×10-1ng/μL、2.5×10-2ng/μL、2.5×10-3ng/μL、2.5×10-4ng/μL、2.5×10-5ng/μL、2.5×10-6ng/μL、2.5×10-7ng/. mu.L, multiplex PCR was performed. The reaction system is as follows: mu.L of mixed substrate, 0.5. mu.L of each of upstream and downstream primers, 2 XHiFi Amplification Mix 10. mu.L, ddH2O make up to 20. mu.L. Pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 15s, and repeating 35 cycles. Agarose gel electrophoresis observation is carried out, and the former concentration without obvious bands is taken as the lowest detection concentration.
Optimizing the number of cycles;
the number of design cycles is 20, 25, 30, 35, 40 cycles, 2.5 × 10-5ng/. mu.L DNA mixture 1. mu.L, upstream and downstream primers 0.5. mu.L each, 2 XHiFi Amplification Mix 10. mu.L, ddH2O to 20. mu.L. Pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, and extension at 68 ℃ for 15 s. Agarose gel electrophoresis was observed, and the number of cycles at which a clear band appeared was used as the optimum number of cycles.
Detecting by agarose gel electrophoresis;
agarose gel electrophoresis: the experiment was carried out on 2% agarose gel at 120V for 35min, and the gel was observed on an EB ethidium bromide staining UV instrument. The glue preparation system comprises the following components:
small glue: 0.4g of agar powder, 20ml of TAE and 2 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
And (3) medium sizing: 0.8g of agar powder, 40ml of TAE and 4 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
Large glue: 1.6g of agar powder, 80ml of TAE and 8 microliter of EB, heating for 3-4 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
In conclusion, the multiple PCR detection method for the zoonosis parasites can be used for detecting multiple parasites simultaneously, is efficient and convenient, saves time and reagents, and has great clinical significance; the kit has higher practicability, convenience and rapidness, greatly shortens the time for clinically detecting the zoonosis parasitic diseases, effectively ensures the detection efficiency, and provides technical support for detecting the zoonosis parasitic diseases susceptible to goats.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. A multiple PCR detection method for zoonosis parasites is characterized by comprising the following steps:
s1, obtaining DNA, and respectively obtaining DNA from animal blood and feces;
s2, mixing, namely preparing the obtained blood DNA and the obtained excrement DNA into DNA solutions with the concentration of 25 ng/mu L respectively, mixing the DNA solutions of the blood DNA and the excrement DNA, and diluting the DNA solutions into a plurality of concentration gradients according to a 10-fold ratio after mixing;
s3, performing multiplex PCR amplification on the obtained DNA solution with a plurality of concentration gradients, wherein the parameters of the multiplex PCR amplification are as follows: mu.L of mixed substrate, 0.5. mu.L of each of upstream and downstream primers, 2 XHiFiamplification Mix 10. mu.L, ddH2Supplementing 20 mu L of O, performing pre-denaturation at 94 ℃ for 2min, performing denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, performing extension at 68 ℃ for 15s, and repeating 35 cycles to finish the amplification process;
and S4, obtaining a result, observing by agarose gel electrophoresis, taking the former concentration without obvious bands as the lowest detection concentration, and analyzing the DNA structure and function of the amplification solution with the lowest detection concentration and the concentration above, thereby judging the type of the parasite.
2. The method for multiplex PCR detection of zoonosis parasites according to claim 1, wherein in S1, DNA is obtained by obtaining a blood sample and a stool sample, and extracting DNA according to the operation requirements of a blood DNA extraction kit and a stool DNA extraction kit respectively, so as to obtain blood DNA and stool DNA.
3. The method of claim 1, wherein 9 concentration gradients are set at S2, and each concentration gradient is 2.5X 101ng/μL、2.5×100ng/μL、2.5×10-1ng/μL、2.5×10-2ng/μL、2.5×10-3ng/μL、2.5×10-4ng/μL、2.5×10-5ng/μL、2.5×10-6ng/. mu.L and 2.5X 10-7ng/μL。
4. The method for multiplex PCR detection of zoonosis parasites according to claim 1, wherein in S3, the number of cycles is determined by: the number of design cycles was 20, 25, 30, 35 and 40 cycles, 2.5 × 10 respectively-51. mu.L of ng/. mu.L DNA mixture, 0.5. mu.L of each of the upstream and downstream primers, 2. mu.L of the HiFi Amplification Mix, 10. mu.L of ddH2O was added to 20. mu.L, pre-denatured at 94 ℃ for 2min, denatured at 98 ℃ for 10s, annealed at 60 ℃ for 30s, and extended at 68 ℃ for 15s, and observed by agarose gel electrophoresis, with the cycle number at which a clear band appeared being the optimum cycle number, and the optimum cycle number being 35.
5. The method for multiplex PCR detection of zoonosis parasites according to claim 1 or 4, wherein the agarose gel electrophoresis observation is carried out in a specific manner: electrophoresis is carried out for 35min by 2 percent agarose gel under the voltage of 120V, observation is carried out under an EB ethidium bromide staining ultraviolet instrument, and a gel preparation system is as follows:
small glue: 0.4g of agar powder, 20ml of TAE and 2 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
and (3) medium sizing: 0.8g of agar powder, 40ml of TAE and 4 microliter of EB, heating for 2-3 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb;
large glue: 1.6g of agar powder, 80ml of TAE and 8 microliter of EB, heating for 3-4 min in a microwave oven until the solution is transparent and clear, pouring into a container, and inserting a comb.
6. The method for multiplex PCR detection of zoonosis parasites according to claim 1, wherein in S3, the annealing temperature is determined by: setting annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃, and observing the brightness and the definition of the amplified band to select the optimal annealing temperature, wherein the optimal annealing temperature is 60 ℃.
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