CN111172304B - Triple PCR kit for diagnosing FHT/MP/HPS and detection method thereof - Google Patents
Triple PCR kit for diagnosing FHT/MP/HPS and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the field of animal epidemic disease molecular biology diagnosis in animal medicine and discloses a triple PCR kit for diagnosing FHT/MP/HPS and a detection method thereof, wherein the kit comprises 3 pairs of specific primers, wherein FHT-F/FHT-R is a swine eperythrozoon specific amplification primer set, MP-F/MP-R is a swine mycoplasma pneumoniae specific amplification primer set, and HPS-F/HPS-R is a haemophilus parasuis specific amplification primer set; the kit can amplify corresponding specific genome fragments from the disease materials of the diseased pigs, can diagnose single infection or mixed infection of three pathogens of eperythrozoon suis, mycoplasma pneumonia of pigs and haemophilus parasuis by one-time detection, has high detection sensitivity, good specificity, high stability and visual result, and the detection method is easy to operate, convenient and quick, can greatly shorten the detection time and provides technical support for clinical control of infection.
Description
Technical Field
The invention relates to the field of animal medical animal epidemic disease molecular biological diagnosis, in particular to a triple PCR kit for diagnosing FHT/MP/HPS and a detection method thereof, which are used for detecting erythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis.
Background
The eperythrozoon of pigs can cause fever, anemia and jaundice of pig groups, and the disease is commonly infected in pig farms in recent years, which causes great economic loss for pig industry. Vertical transmission and blood-borne transmission are considered to be the primary pathways of infection of eperythrozoons. Eperythrozoon cannot be cultured in vitro, so that diagnosis of swine eperythrozoonosis is difficult. The existing diagnosis error diagnosis rate by microscopic examination is high, the accuracy and the specificity of diagnosis are improved, and a molecular biological technology is needed.
Mycoplasma hyopneumoniae is also known as swine endemic pneumonia, commonly known as swine enzootic pneumonia, and is a chronic respiratory infectious disease of pigs caused by mycoplasma hyopneumoniae. The main symptoms are cough and asthma, and the lesions are characterized by the meaty or shrimp-like metaplasia of the tip, heart, middle and diaphragm anterior edges of the lung. The pathogen is mycoplasma hyopneumoniae, and belongs to mycoplasma family and mycoplasma genus members. Since there is no cell wall, it is a polymorphic microorganism. The mycoplasma hyopneumoniae has high nutrition requirement, high pathogen separation and culture difficulty and long time consumption, and is not suitable for clinical rapid diagnosis.
Haemophilus parasuis is a common opportunistic pathogen in the upper respiratory tract of pigs, can invade the body under specific conditions, causes systemic epidemic diseases mainly characterized by cellulose polyarthritis, meningitis and serositis, and is one of the most serious pathogens affecting the pig industry at present. The haemophilus parasuis disease has high mortality rate, is not easy to cure after the disease is transmitted, and has the mortality rate of up to 50 percent. The disease has strong infectivity and seriously harms the health of piglets and young pigs. The disease has a popular trend in China, and brings great economic loss to the pig industry. Therefore, the establishment of the early rapid detection method has very important significance for preventing and controlling haemophilus parasuis infection. The traditional bacterial separation and serological detection method has the defects of long time consumption, high technical requirements on detection personnel, complex operation, poor sensitivity and the like.
Along with the maturation and popularization of molecular diagnosis technology, PCR technology is widely applied to animal epidemic disease diagnosis with the advantages of rapidness, sensitivity, easy operation and the like. The three epidemic disease pathogens belong to pathogenic microorganisms which are difficult to culture or can not be cultured, and researchers also establish a PCR diagnosis method of the related pathogens, but no development report of a triple PCR detection technology is seen, so that the diagnosis kit for rapidly identifying FHT/MP/HPS infection has important practical significance for rapidly diagnosing corresponding diseases in clinic.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a triple PCR kit for diagnosing FHT/MP/HPS and a detection method thereof, the kit refers to the gene sequences of swine eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis published in GenBank, 3 pairs of specific primers are designed, corresponding specific genome fragments can be amplified from diseased swine, single infection or mixed infection of three pathogens of swine eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis can be confirmed by one-time detection, and the kit has high detection sensitivity, good specificity, high stability and visual result, and the detection method is easy to operate, convenient and quick, can greatly shorten detection time, and provides technical support for clinical control of infection.
In order to achieve the above purpose, the present invention is realized by the following technical scheme.
(one) a triple PCR kit for diagnosing FHT/MP/HPS, comprising lysate, amplification reaction mixture, negative control and positive control; wherein the components of the lysate are DNAiso Reagent; the amplification reaction mixture comprises sterilized triple distilled water, PCR Buffer, dNTP, FHT-F upstream primer, FHT-R downstream primer, MP-F upstream primer, MP-R downstream primer, HPS-F upstream primer, HPS-R downstream primer and rTaq DNA polymerase; the negative control is sterilized triple distilled water; the positive control is a positive plasmid mixture of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis.
Preferably, the FHT-F upstream primer sequence is: 5'-TAACTTACTTATCTGAGGAA-3'
The sequence of the FHT-R downstream primer is as follows: 5'-TTTAACGCGTTAGCTACA-3'
The MP-F upstream primer sequence is as follows: 5'-ATTATTTAAAAATTTTAGA-3'
The MP-R downstream primer sequence is as follows: 5'-AATTCGTAATTTTATGTT-3'
The sequence of the HPS-F upstream primer is as follows: 5'-GACTTAAGTGAGATGTGAA-3'
The HPS-R downstream primer sequence is as follows: 5'-AGTATTTTCCCAAGGGGCT-3'.
Preferably, the components of the lysate are DNAiso Reagent, and 20 reactions are added to 20mL, and the obtained mixture is filled into 1 bottle; the amplification reaction mixture contains sterilized triple distilled water, 10 XPCR Buffer and 2.5 mmol.L -1 dNTP、25μmol·L -1 FHT-F upstream primer, 25. Mu. Mol.L -1 FHT-R downstream primer, 25. Mu. Mol.L -1 MP-F upstream primer, 25. Mu. Mol.L -1 MP-R downstream primer, 25. Mu. Mol.L -1 HPS-F upstream primer, 25. Mu. Mol.L -1 HPS-R downstream primer and 5U/. Mu.L rTaq DNA polymerase, wherein the volume ratio of each reaction is 15.0:2.5:2:0.5:0.5:0.5:0.5:0.5:0.5, the total amount of the two primers is 23 mu.L, the total amount of the two primers is 460 mu.L, and the two primers are packaged into 1 tube; the negative control is sterilized three distilled water, and the total of 20 reactions is 2.0mL, and the negative control is filled into 1 bottle; the positive control is a mixture of pig eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis positive plasmid, and the total of 20 reactions is 40.0 mu L, and the mixture is packaged into 1 tube.
(II) a detection method for diagnosing FHT/MP/HPS triple PCR kit, comprising the following detection steps:
Substep 1.1, extracting DNA of a sample to be detected: sucking the sample to be detected, placing the sample into a sterile centrifuge tube, adding a lysate, reversing and uniformly mixing, standing and cracking, adding absolute ethyl alcohol, reversing and uniformly mixing, standing and precipitating, centrifuging, discarding supernatant, washing, discarding washing liquid, inverting the sterile centrifuge tube, naturally drying in air, and dissolving with NaOH to obtain a sample DNA extract to be detected;
substep 1.2, negative control sample extraction: absorbing sterilized three distilled water, placing into a sterile centrifuge tube, adding a lysate, reversing and mixing uniformly, standing and cracking, adding absolute ethyl alcohol, reversing and mixing uniformly, standing and precipitating, centrifuging, discarding supernatant, washing, discarding washing liquid, inverting the sterile centrifuge tube, naturally drying in air, and dissolving with NaOH to obtain a negative control sample extract;
Substep 2.1, PCR amplification reaction of the sample to be detected: adding the amplification reaction mixed solution into the sample DNA extracting solution to be detected, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a sample PCR amplification product to be detected;
substep 2.2, negative control sample PCR amplification reaction: adding the amplification reaction mixed solution into the negative control sample extracting solution, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a negative control sample PCR amplification product;
substep 2.3, positive control sample PCR amplification reaction: adding a mixture of a pig eperythrozoon and a mycoplasma hyopneumoniae positive plasmid into the amplification reaction mixture, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a positive control sample PCR amplification product;
the amplification reaction mixture comprises sterilized triple distilled water, PCR Buffer, dNTP, FHT-F upstream primer, FHT-R downstream primer, MP-F upstream primer, MP-R downstream primer, HPS-F upstream primer, HPS-R downstream primer and rTaq DNA polymerase;
And respectively carrying out electrophoresis detection on the PCR amplification products of the sample to be detected, the PCR amplification products of the negative control sample and the PCR amplification products of the positive control sample by agarose gel electrophoresis, and judging.
Preferably, in the substep 1.1, the sample to be detected is a tissue disease sample, a serum sample or a whole blood sample.
Further preferably, the tissue sample is lung, liver or spleen.
Preferably, in step 2, the FHT-F upstream primer sequence is: 5'-TAACTTACTTATCTGAGGAA-3';
the sequence of the FHT-R downstream primer is as follows: 5'-TTTAACGCGTTAGCTACA-3'
The MP-F upstream primer sequence is as follows: 5'-ATTATTTAAAAATTTTAGA-3'
The MP-R downstream primer sequence is as follows: 5'-AATTCGTAATTTTATGTT-3'
The sequence of the HPS-F upstream primer is as follows: 5'-GACTTAAGTGAGATGTGAA-3'
The HPS-R downstream primer sequence is as follows: 5'-AGTATTTTCCCAAGGGGCT-3'.
Preferably, in step 2, the plurality of cycles is 35 cycles.
Preferably, in the step 3, no band is formed in the negative control, and three bands of 758bp, 353bp and 162bp are formed in the positive control, which indicates that the control is established; 758bp single band appears in the PCR amplified product of the sample to be detected, namely single eperythrozoon suis infection; the PCR amplified product of the sample to be detected has 353bp single band, namely single mycoplasma hyopneumoniae infection; the PCR amplified product of the sample to be detected has 162bp single band, namely single haemophilus parasuis infection; two or three bands of 758bp, 353bp and 162bp appear in the PCR amplified product of the sample to be detected, namely the double or triple mixed infection of the corresponding pathogen; the PCR amplified product of the sample to be detected is negative without a strip.
Compared with the prior art, the invention has the beneficial effects that:
1) The triple PCR kit for diagnosing the FHT/MP/HPS provided by the invention can detect three pathogens of the eperythrozoon suis, the mycoplasma hyopneumoniae and the haemophilus parasuis at one time, solves the diagnosis problem that the three pathogens are difficult to culture or can not be cultured, controls the total detection time to be about 2 hours, is easy to operate, is convenient and quick, can achieve the purpose of quick detection, and is suitable for quick detection and epidemiological investigation.
2) The invention provides three pairs of specific primers aiming at swine eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis, wherein FHT-F/FHT-R is a swine eperythrozoon specific amplification primer group, MP-F/MP-R is a swine mycoplasma hyopneumoniae specific amplification primer group, and HPS-F/HPS-R is a haemophilus parasuis specific amplification primer group; the triple kit prepared by the kit can rapidly and specifically detect single pathogen, mixed pathogen and mixed pathogen of the single pathogen and the mixed pathogen in haemophilus parasuis in swine eperythrozoon and mycoplasma hyopneumoniae. Wherein, the eperythrozoon suis presents 758bp one band, the mycoplasma hyopneumoniae presents 353bp one band, and the haemophilus parasuis presents 162bp one band.
3) The detection limit of the triple PCR kit for diagnosing FHT/MP/HPS is 20 copies mu L -1 The diagnosis FHT/MP/HPS triple PCR kit has high sensitivity in detecting swine eperythrozoon and swine mycoplasma pneumoniae, and can shorten diagnosis time, save diagnosis reagent and reduce use cost.
4) The detection result of the triple PCR kit for diagnosing FHT/MP/HPS provided by the invention is consistent within 6 months, which shows that the triple PCR kit for diagnosing FHT/MP/HPS has high stability. The field test proves that the diagnosis FHT/MP/HPS triple PCR kit has good practicability and applicability.
Drawings
The invention will now be described in further detail with reference to the drawings and to specific examples.
FIG. 1 is a diagram of electrophoresis of a specific assay of the present invention for diagnosing FHT/MP/HPS triplex PCR kit and a method for detecting the same;
in the figure, M is DL2000DNA molecular mass standard, 1 is a swine eperythrozoon positive plasmid, 2 is a swine mycoplasma pneumoniae positive plasmid, 3 is a haemophilus parasuis positive plasmid, 4 is a swine eperythrozoon, swine mycoplasma pneumoniae positive plasmid mixture, 5 is a swine pseudorabies virus, 6 is a swine parvovirus, 7 is a swine circovirus type 2, 8 is a swine circovirus type 3, 9 is a swine fever virus, 10 is a blue ear virus, 11 is a swine epidemic diarrhea virus, and 12 is a swine transmissible gastroenteritis virus;
FIG. 2 is a sensitivity test electrophoresis chart of the triple PCR kit for diagnosing FHT/MP/HPS and the detection method thereof;
in the figure, M is DL2000DNA molecular mass standard, 1-8 is pig eperythrozoon, pig mycoplasma pneumoniae and haemophilus parasuis positive mixed plasmid, and its concentration range is 2X 10 6 Copy/. Mu.L -1 ～2×10 -1 Copy/. Mu.L -1 ；
FIG. 3 shows the result of electrophoresis detection of a part of clinical samples by using the triple PCR kit for diagnosing FHT/MP/HPS of the present invention; 1-20 are the results of electrophoresis detection of a portion of tissue disease and serum.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
The triple PCR kit for diagnosing FHT/MP/HPS comprises lysate, amplification reaction mixture, negative control and positive control, and is concretely as follows:
1) Lysate: the components are DNAiso Reagent,20 reactions are added to 20mL, and the mixture is filled into 1 bottle;
2) Amplification reaction mixture: from sterilized triple distilled water, 10 XPCR Buffer, 2.5 mmol.L -1 dNTP、25μmol·L -1 FHT-F upstream primer, 25. Mu. Mol.L -1 FHT-R downstream primer, 25. Mu. Mol.L -1 MP-F upstream primer, 25. Mu. Mol.L -1 MP-R downstream primer, 25. Mu. Mol.L -1 HPS-F upstream primer, 25. Mu. Mol.L -1 HPS-R downstream primer and 5U/. Mu.L rTaq DNA polymerase, wherein the volume ratio of each reaction is 15.0:2.5:2:0.5:0.5:0.5:0.5:0.5:0.5, the total amount is 23 mu.L, the total amount of 20 reactions is 460 mu.L, and the mixture is filled into 1 tube; wherein the design and preparation of the primer are as follows:
referring to genome sequences of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis published on GenBank, 6 primers are designed aiming at the three pathogenic genes through comprehensive analysis and comparison, and the sequences of the primers are as follows:
the sequence of the FHT-F upstream primer is as follows: 5'-TAACTTACTTATCTGAGGAA-3'
The sequence of the FHT-R downstream primer is as follows: 5'-TTTAACGCGTTAGCTACA-3'
The MP-F upstream primer sequence is as follows: 5'-ATTATTTAAAAATTTTAGA-3'
The MP-R downstream primer sequence is as follows: 5'-AATTCGTAATTTTATGTT-3'
The sequence of the HPS-F upstream primer is as follows: 5'-GACTTAAGTGAGATGTGAA-3'
The HPS-R downstream primer sequence is as follows: 5'-AGTATTTTCCCAAGGGGCT-3';
the primer is synthesized by biological engineering (Shanghai) limited company;
3) Negative control: for sterilizing three distilled water, the total of 20 reactions is 2.0mL, and the three distilled water is filled into 1 bottle;
4) Positive control: the mixture of positive plasmids of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis was prepared by filling 1 tube with a total of 40.0. Mu.L of 20 reactions.
The kit for diagnosing FHT/MP/HPS triple PCR is prepared through normal temperature preservation of the lysis solution, and preservation of the amplification reaction mixture, negative control and positive control at-20 ℃.
The detection method for diagnosing the FHT/MP/HPS triple PCR kit comprises the following detection steps:
Substep 1.1, extracting DNA of a sample to be detected: sucking 100 μL of sample to be detected, placing into a 1.5mL sterile EP tube, adding 800 μL of lysate, mixing, standing for cracking for 10min, adding 600 μL of ice-cold absolute ethanol, mixing, standing for precipitation for 10min, centrifuging for 10min 12000r/min, discarding supernatant, washing with 75% ethanol for 1 time, discarding ethanol, inverting the EP tube, naturally drying in air, and collecting the supernatant with 40 μL of 8mmol.L -1 Dissolving with NaOH to obtain DNA extract of the sample to be detected;
substep 1.2, negative control sample extraction: absorbing 100 μl of sterilized triple distilled water, placing into a sterile centrifuge tube, adding 800 μl of lysate, mixing, standing for cracking for 10min, adding 600 μl of ice-cold absolute ethanol, mixing, standing for precipitating for 10min, centrifuging for 10min 12000r/min, discarding supernatant, washing with 75% ethanol for 1 time, discarding ethanol, inverting EP tube, naturally drying in air, and collecting 40 μl of 8 mmol.L -1 Dissolving NaOH to obtain a negative control sample extracting solution;
Substep 2.1, PCR amplification reaction of the sample to be detected: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of sample DNA extracting solution to be detected, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction at 72 ℃ for 10min to obtain a sample PCR amplification product to be detected;
substep 2.2, negative control sample PCR amplification reaction: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of negative control sample extracting solution, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction at 72 ℃ for 10min to obtain a negative control sample PCR amplification product;
substep 2.3, positive control sample PCR amplification reaction: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of mixture of eperythrozoon suis and mycoplasma hyopneumoniae positive plasmid, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction for 10min at 72 ℃ to obtain a positive control sample PCR amplification product;
By passing 10 g.L -1 Performing agarose gel electrophoresis on the PCR amplification products of the sample to be detected, the PCR amplification products of the negative control sample and the PCR amplification products of the positive control sample respectively, and judging; the judgment standard is as follows: the PCR amplified product of the negative control sample does not generate bands, and the PCR amplified product of the positive control sample generates three bands of 758bp, 353bp and 162bp, which indicates that the control is established; 758bp single band appears in the PCR amplified product of the sample to be detected, namely single eperythrozoon suis infection; the PCR amplified product of the sample to be detected has 353bp single band, namely single mycoplasma hyopneumoniae infection; the PCR amplified product of the sample to be detected has 162bp single band, namely single haemophilus parasuis infection; two or three bands of 758bp, 353bp and 162bp appear in the PCR amplified product of the sample to be detected, namely the double or triple mixed infection of the corresponding pathogen; and if no strip exists, the result is negative.
The specificity, stability and sensitivity of the triple PCR kit for diagnosing FHT/MP/HPS and the detection method thereof obtained in the example 1 are tested, and the practicability and applicability of the triple PCR kit for diagnosing FHT/MP/HPS obtained in the invention are verified by field tests, specifically as follows:
(1) Specificity test
Extracting positive plasmids of gene fragments of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis by adopting the detection method for diagnosing the FHT/MP/HPS triple PCR kit obtained in the embodiment 1, and mixing the three plasmids to be used as 1 sample template; extracting DNA of DNA viruses such as porcine pseudorabies virus, porcine parvovirus, porcine circovirus type 2 and porcine circovirus type 3 as templates; extracting RNA of swine fever virus, blue ear virus, porcine epidemic diarrhea virus and transmissible gastroenteritis virus, performing reverse transcription to obtain cDNA as a template, performing triple PCR with amplification mixed solution of the kit, and performing electrophoresis observation after amplification. The test results are shown in FIG. 1.
As can be seen from FIG. 1, there was 758bp band in the eperythrozoon suis positive plasmid, 353bp band in the mycoplasma hyopneumoniae, 162bp band in the haemophilus parasuis, 758bp, 353bp and 162bp bands in the mixed sample of the eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis gene fragment positive plasmid, and no band in the porcine pseudorabies virus, porcine parvovirus, porcine circovirus type 2, porcine circovirus type 3, swine fever virus, blue ear virus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus.
And (3) recovering the strips of each positive sample, connecting the strips with a pMD18-T vector after purification, converting DH5 alpha competent cells, taking PCR (polymerase chain reaction) identification as positive bacterial liquid, extracting plasmids for sequencing, and comparing the detected sequences with the gene sequences of the used strains to find that the sequences are completely consistent. The results prove that the kit and the detection method have high specificity, can accurately amplify the gene fragments of the swine eperythrozoon, the mycoplasma hyopneumoniae and the haemophilus parasuis, and can intuitively identify and diagnose the infection of the swine eperythrozoon, the mycoplasma hyopneumoniae and the haemophilus parasuis.
(2) Sensitivity test
Use of one of the products obtained in example 1 forThe detection method of the diagnosis FHT/MP/HPS triple PCR kit extracts positive plasmids of the gene fragments of the swine eperythrozoon, the swine mycoplasma pneumoniae and the haemophilus parasuis, calculates copy numbers after measuring the respective concentrations by an ultraviolet spectrophotometer, and dilutes and adjusts the positive plasmid concentrations to be 2 multiplied by 10 respectively 6 Copy/. Mu.L -1 Three plasmids were mixed as templates and diluted to 10 according to a 10-fold concentration gradient -8 Triple PCR was performed using the amplification mixture provided by the kit, and after amplification, the results of the electrophoresis were as shown in FIG. 2.
As can be seen from FIG. 2, the detection limit of the triple PCR kit for diagnosing FHT/MP/HPS of the present invention is 20 copies/. Mu.L -1 The detection method for diagnosing the FHT/MP/HPS triple PCR kit has high sensitivity.
(3) Stability test
The triple PCR kit for diagnosis FHT/MP/HPS obtained in example 1 was stored at-20℃and tested 1 time per month with positive plasmids of the gene fragments of swine eperythrozoon, mycoplasma hyopneumoniae and Haemophilus parasuis, and the test was continued for 6 months to determine the storage stability of the kit.
The results show that the detection results of the triple PCR kit for diagnosing FHT/MP/HPS within 6 months are completely consistent, and the triple PCR kit for diagnosing FHT/MP/HPS has good stability.
(4) Field test
The detection and diagnosis of 160 parts of tissue disease materials and serum are carried out by adopting the triple PCR kit for diagnosing FHT/MP/HPS and the detection method thereof, which are obtained in the embodiment 1, and the specific steps are as follows:
example 2
1. Treatment of tissue specimens
Taking 3-5 g of suspected case tissue disease materials such as lung, liver or spleen, cutting into paste, adding 5 times of sterile PBS buffer solution, fully grinding by a tissue grinder, collecting suspension, centrifuging at 4 ℃ for 10min at 12000r/min, and sucking supernatant for later use;
2. DNA extraction
2.1 DNA extraction of tissue disease samples: 100. Mu.L of the resulting supernatant was aspirated and placed in a 1.5mL sterile EP tubeAdding 800 μL of lysate, mixing, standing for cracking for 10min, adding 600 μL of ice-cold absolute ethanol, mixing, standing for precipitation for 10min, centrifuging for 10min 12000r/min, discarding supernatant, washing with 75% ethanol for 1 time, discarding ethanol, inverting EP tube, naturally drying in air, and concentrating with 40 μL of 8mmol.L -1 Dissolving NaOH to obtain tissue disease material DNA extract;
2.2 negative control sample extraction: absorbing 100 μl of sterilized triple distilled water, placing into a sterile centrifuge tube, adding 800 μl of lysate, mixing, standing for cracking for 10min, adding 600 μl of ice-cold absolute ethanol, mixing, standing for precipitating for 10min, centrifuging for 10min 12000r/min, discarding supernatant, washing with 75% ethanol for 1 time, discarding ethanol, inverting EP tube, naturally drying in air, and collecting 40 μl of 8 mmol.L -1 Dissolving NaOH to obtain a negative control sample extracting solution;
3. PCR amplification reaction
3.1 PCR amplification reaction of tissue disease samples: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of tissue disease material DNA extracting solution, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction at 72 ℃ for 10min to obtain a tissue disease material sample PCR amplification product;
3.2 negative control sample PCR amplification reaction: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of negative control sample extracting solution, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction at 72 ℃ for 10min to obtain a negative control sample PCR amplification product;
3.3 positive control sample PCR amplification reaction: taking 23 mu L of amplification reaction mixed solution, adding 2 mu L of mixture of eperythrozoon suis and mycoplasma hyopneumoniae positive plasmid, uniformly mixing, carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, carrying out 35 cycles in total, and finally, carrying out reaction for 10min at 72 ℃ to obtain a positive control sample PCR amplification product;
4. electrophoresis detection
The obtained PCR amplified product of the tissue disease sample, the PCR amplified product of the negative control sample and the PCR amplified product of the positive control sample pass through 10 g.L -1 The agarose gel electrophoresis is used for examination and diagnosis, and the method is concretely as follows:
4.1 weighing 1.0g agarose, adding into 100mL 1 xTAE buffer solution, heating in a microwave oven to melt, adding 5 mu L (100 mg/mL) ethidium bromide, uniformly mixing, pouring into a horizontally placed gel plate with the thickness of 5mm, pulling out a comb after cooling and solidifying, placing gel into an electrophoresis tank, and adding 1 xTAE buffer solution to submerge the gel surface;
4.2, respectively taking 5 mu L of tissue disease sample PCR amplification product, negative control sample PCR amplification product, positive control sample PCR amplification product and loading buffer solution, uniformly mixing, adding into a loading hole, and simultaneously adding 5 mu LDL2000DNA molecular weight standard;
4.3, the voltage is 80V-100V, or the current is 40 mA-50 mA, and electrophoresis is carried out for 30min;
4.4, taking out the gel, placing the gel into an ultraviolet analyzer for observation, or placing the gel into a gel imaging system for observation and photographing;
4.5, taking DL2000DNA molecular mass standard as reference, the PCR amplified product of the negative control sample does not produce bands, and the PCR amplified product of the positive control sample produces three bands of 758bp, 353bp and 162bp, which indicates that the control is established; 758bp single band appears in the PCR amplified product of the sample to be detected, namely single eperythrozoon suis infection; the PCR amplified product of the sample to be detected has 353bp single band, namely single mycoplasma hyopneumoniae infection; the PCR amplified product of the sample to be detected has 162bp single band, namely single haemophilus parasuis infection; two or three bands of 758bp, 353bp and 162bp appear in the PCR amplified product of the sample to be detected, namely the double or triple mixed infection of the corresponding pathogen; and if no strip exists, the result is negative.
Example 3
1. Treatment of serum samples
Collecting blood from the anterior vena cava of the pigs in suspected cases, naturally coagulating, and separating out serum after the serum is separated out for later use;
2. DNA extraction, PCR amplification reaction and electrophoresis detection
The specific detection method is the same as that of example 2, except that the tissue disease sample in example 2 is replaced with a serum sample.
Example 4
1. Treatment of whole blood samples
Adding heparin sodium anticoagulant into the injector, collecting blood from the anterior vena cava of the pigs in suspected cases, and uniformly mixing to obtain anticoagulation for later use;
2. DNA extraction, PCR amplification reaction and electrophoresis detection
The specific detection method is the same as that of example 2, except that the tissue disease sample in example 2 is replaced with a whole blood sample.
The detection results of examples 2-4 show that the single eperythrozoon suis in 160 clinical samples is positive for 13 parts, and the positive rate is 8.1%; 22 parts of single mycoplasma hyopneumoniae are positive, and the positive rate is 13.8%; 6 parts of single haemophilus parasuis are positive, and the positive rate is 3.8%; 24 parts of pig eperythrozoon and mycoplasma hyopneumoniae are double-positive, and the positive rate is 15%; 17 parts of mycoplasma hyopneumoniae and haemophilus parasuis double positive, and the positive rate is 10.6%; pig eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis are 6 positive parts, and the positive rate is 3.8%.
Wherein FIG. 3 shows the results of partial tissue disease and serum detection. As can be seen from FIG. 3, 1/8/14 is positive for mycoplasma hyopneumoniae alone; 4/5/10/12/17/18 is a positive result of haemophilus parasuis alone; 11 is a single pig eperythrozoon positive result; 6 is the double positive result of the eperythrozoon suis and haemophilus parasuis; 20 is the double positive result of eperythrozoon suis and mycoplasma hyopneumoniae; 3/16 is a triple positive result of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis; the remaining non-banded persons were negative. The field test proves that the diagnosis FHT/MP/HPS triple PCR kit has good practicability and applicability.
While the invention has been described in detail in this specification with reference to the general description and the specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. The triple PCR kit for diagnosing FHT/MP/HPS is characterized by comprising a lysate, an amplification reaction mixture, a negative control and a positive control; wherein the components of the lysate are DNAiso Reagent; the amplification reaction mixture comprises sterilized triple distilled water, PCR Buffer, dNTP, FHT-F upstream primer, FHT-R downstream primer, MP-F upstream primer, MP-R downstream primer, HPS-F upstream primer, HPS-R downstream primer and rTaq DNA polymerase; the negative control is sterilized triple distilled water; the positive control is a positive plasmid mixture of eperythrozoon suis, mycoplasma hyopneumoniae and haemophilus parasuis;
wherein FHT is eperythrozoon suis, MP is mycoplasma hyopneumoniae, HPS is haemophilus parasuis;
the sequence of the FHT-F upstream primer is as follows: 5'-TAACTTACTTATCTGAGGAA-3'
The sequence of the FHT-R downstream primer is as follows: 5'-TTTAACGCGTTAGCTACA-3'
The MP-F upstream primer sequence is as follows: 5'-ATTATTTAAAAATTTTAGA-3'
The MP-R downstream primer sequence is as follows: 5'-AATTCGTAATTTTATGTT-3'
The sequence of the HPS-F upstream primer is as follows: 5'-GACTTAAGTGAGATGTGAA-3'
The HPS-R downstream primer sequence is as follows: 5'-AGTATTTTCCCAAGGGGCT-3'.
2. The triple PCR kit for diagnosing FHT/MP/HPS according to claim 1, wherein the components of the lysate are DNAiso Reagent,20 reactions total 20mL, 1 bottle; the amplification reaction mixture comprises sterilized triple distilled water, 10 x PCR Buffer, 2.5 mmol.L-1 dNTP, 25 mu mol.L-1 FHT-F upstream primer, 25 mu mol.L-1 FHT-R downstream primer, 25 mu mol.L-1 MP-F upstream primer, 25 mu mol.L-1 MP-R downstream primer, 25 mu mol.L-1 HPS-F upstream primer, 25 mu mol.L-1 HPS-R downstream primer and 5U/. Mu.L rTaq DNA polymerase, wherein each reaction volume ratio is 15.0:2.5:2.5:0.5:0.5:0.5:0.5:0.5, and the total of 23 mu L and 20 reactions is 460 mu L and is filled into a tube; the negative control is sterilized three distilled water, and the total reaction amount of 20 reactions is 2.0mL, and the negative control is 1 bottle; the positive control is a mixture of pig eperythrozoon, mycoplasma hyopneumoniae and haemophilus parasuis positive plasmid, and the total of 20 reactions is 40.0 mu L, and the mixture is packaged into 1 tube.
3. Use of the FHT/MP/HPS triple PCR kit according to any one of claims 1 or 2 for the preparation of a FHT/MP/HPS diagnostic product, comprising the following detection steps:
step 1, DNA extraction
Substep 1.1, extracting DNA of a sample to be detected: sucking the sample to be detected, placing the sample into a sterile centrifuge tube, adding a lysate, reversing and uniformly mixing, standing and cracking, adding absolute ethyl alcohol, reversing and uniformly mixing, standing and precipitating, centrifuging, discarding supernatant, washing, discarding washing liquid, inverting the sterile centrifuge tube, naturally drying in air, and dissolving with NaOH to obtain a sample DNA extract to be detected;
substep 1.2, negative control sample extraction: absorbing sterilized three distilled water, placing into a sterile centrifuge tube, adding a lysate, reversing and mixing uniformly, standing and cracking, adding absolute ethyl alcohol, reversing and mixing uniformly, standing and precipitating, centrifuging, discarding supernatant, washing, discarding washing liquid, inverting the sterile centrifuge tube, naturally drying in air, and dissolving with NaOH to obtain a negative control sample extract;
step 2, PCR amplification reaction
Substep 2.1, PCR amplification reaction of the sample to be detected: adding the amplification reaction mixed solution into the sample DNA extracting solution to be detected, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a sample PCR amplification product to be detected;
substep 2.2, negative control sample PCR amplification reaction: adding the amplification reaction mixed solution into the negative control sample extracting solution, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a negative control sample PCR amplification product;
substep 2.3, positive control sample PCR amplification reaction: adding a mixture of a pig eperythrozoon and a mycoplasma hyopneumoniae positive plasmid into the amplification reaction mixture, uniformly mixing, and carrying out PCR amplification reaction, wherein the conditions of the PCR amplification reaction are 95 ℃ pre-denaturation reaction for 5min,94 ℃ reaction for 40s,56 ℃ reaction for 50s and 72 ℃ reaction for 60s in sequence, and carrying out multiple cycles, and finally, carrying out reaction at 72 ℃ for 10min to obtain a positive control sample PCR amplification product;
the amplification reaction mixture comprises sterilized triple distilled water, PCR Buffer, dNTP, FHT-F upstream primer, FHT-R downstream primer, MP-F upstream primer, MP-R downstream primer, HPS-F upstream primer, HPS-R downstream primer and rTaq DNA polymerase;
the sequence of the FHT-F upstream primer is as follows: 5'-TAACTTACTTATCTGAGGAA-3';
the sequence of the FHT-R downstream primer is as follows: 5'-TTTAACGCGTTAGCTACA-3'
The MP-F upstream primer sequence is as follows: 5'-ATTATTTAAAAATTTTAGA-3'
The MP-R downstream primer sequence is as follows: 5'-AATTCGTAATTTTATGTT-3'
The sequence of the HPS-F upstream primer is as follows: 5'-GACTTAAGTGAGATGTGAA-3'
The HPS-R downstream primer sequence is as follows: 5'-AGTATTTTCCCAAGGGGCT-3';
step 3, electrophoresis detection
And respectively carrying out electrophoresis detection on the PCR amplification products of the sample to be detected, the PCR amplification products of the negative control sample and the PCR amplification products of the positive control sample by agarose gel electrophoresis, and judging.
4. Use of the FHT/MP/HPS triple PCR kit according to claim 3 for the preparation of FHT/MP/HPS diagnostic products according to any of claims 1 or 2, wherein in substep 1.1 the sample to be tested is a tissue disease sample, a serum sample or a whole blood sample.
5. Use of the FHT/MP/HPS triple PCR kit according to claim 4 for the preparation of an FHT/MP/HPS diagnostic product, wherein the tissue disease sample is lung, liver or spleen.
6. Use of the FHT/MP/HPS triple PCR kit according to claim 3 for the preparation of FHT/MP/HPS diagnostic products according to any of claims 1 or 2, wherein in step 2 the number of cycles is 35 cycles.
7. Use of the FHT/MP/HPS triple PCR kit according to claim 3 for the preparation of FHT/MP/HPS diagnostic products according to any one of claims 1 or 2, wherein in step 3, the negative control does not strip, the positive control does three strips 758bp, 353bp, 162bp, indicating that the control is true; 758bp single band appears in the PCR amplified product of the sample to be detected, namely single eperythrozoon suis infection; the PCR amplified product of the sample to be detected has 353bp single band, namely single mycoplasma hyopneumoniae infection; the PCR amplified product of the sample to be detected has 162bp single band, namely single haemophilus parasuis infection; two or three bands of 758bp, 353bp and 162bp appear in the PCR amplified product of the sample to be detected, namely the double or triple mixed infection of the corresponding pathogen; the PCR amplified product of the sample to be detected is negative without a strip.
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