CN113444839B - Kit for preparing reagent for detecting EP pathogen and ER pathogen and application thereof - Google Patents
Kit for preparing reagent for detecting EP pathogen and ER pathogen and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
According to the kit for preparing and detecting the EP pathogen and the ER pathogen reagent and the application thereof, provided by the invention, by adopting the specific primer group and adding the gold nano-particles, the adsorption ssDNA and the protease into a reaction system, the nonspecific reaction in the heating process can be effectively avoided; the LAMP technology and the microfluidic chip technology after improvement are combined, so that the pathogen of equine Jiao Chong and equine rhinopneumonia can be rapidly and accurately detected and distinguished, meanwhile, the reaction reagent is pre-buried on the microfluidic chip to realize the same double-index detection, the operation of a user is simple and convenient, the detection efficiency is obviously improved, and the LAMP detection kit has great potential development value for preventing Ma Jiaochong diseases and equine rhinopneumonia.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a detection method of horse-associated virus and application thereof, and more particularly relates to a kit for detecting horse Jiao Chong disease pathogen and horse nose pneumonia pathogen and application thereof.
Background
Ma Jiaochong disease (Equireiroplasmosis), also known as Marbabesiasis (Equine babesiosis), is a protozoal disease of horses, mules, donkeys and zebras transmitted by ticks. The pathogens are two blood parasites called the Mareuglena (Theileria equi) and the Mareuglena Bei Sichong (Babesia caballi), the Mareuglena old named Mareuglena (Babesieequi). Infected animals can carry these parasites for long periods of time, becoming a transmission vehicle-the source of tick infection. These parasites are present in erythrocytes of infected animals. Animals carrying the parasite can lead to endemic epidemics of the disease after being introduced into the region where ticks are prevalent, the epidemic high occurrence is in summer and autumn, and the damage to the breeding Ma Ye is great. At present, blood sampling smear inspection is mostly adopted, so that a tester is required to have certain experience, otherwise, the tester is easy to judge errors.
Ma Bi pneumonia (Equine rhinopneumonitis, ER), also known as equine viral abortion, is an infectious disease of equine animals caused by equine herpesvirus. Is an acute febrile infectious disease of horses, and the pathogen is equine herpesvirus I. Clinical manifestations are catarrhal inflammation of the head and upper respiratory mucosa and leukopenia. Pregnant mares are susceptible to abortion when they are infected with this disease. The International veterinary Commission (OIE) ranks equine pneumonitis as a group B disease, and is listed in the legal report disease catalog by International animal health Act (1999). The disease occurs widely around the world, and brings great harm to world culture Ma Ye. Current detection methods are gross dissection, tissue section, antibody detection and diagnosis using PCR. The existing PCR technology is adopted, the reaction is circulated in 2 different temperature areas, the requirement on instruments is high, the cost is relatively high, the PCR technology is only 1 pair of amplified primers, the PCR technology is easy to be interfered, the specificity is relatively insufficient, the result output time is longer, the requirement on operation profession is high, and the steps of micro addition are more. The LAMP technology has 4 different specific primers, so that the accuracy of a detection result is higher, but the detection result is influenced by non-specific amplification easily generated in the temperature rising stage of equipment due to the fact that the LAMP technology is a constant temperature reaction and lacks a hot start enzyme similar to PCR.
The two diseases are taken as pathogens causing main virulent diseases of equine animals, no detection technical means with good detection effect aiming at multiple pathogens of the two diseases exist in the prior detection technology, and the development of prevention causes of the equine Jiao Chong diseases and equine rhinopneumonia is seriously hindered.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a kit for preparing and detecting reagents for EP pathogen and ER pathogen and application thereof, and by adding gold nanoparticles into a reaction system, ssDNA and protease are adsorbed, so that non-specific reaction in the heating process is effectively avoided; the improved LAMP technology and the improved microfluidic chip technology are combined, so that the horse Jiao Chong disease and the horse nose pneumonia can be rapidly and accurately detected and distinguished, meanwhile, the reaction reagent is pre-buried on the microfluidic chip to realize the same double-index detection, the user operation is simple and convenient, the detection efficiency is obviously improved, and the method has great potential development value for preventing Ma Jiaochong disease and horse nose pneumonia.
The invention is realized by the following technical scheme:
the invention provides a kit for preparing a reagent for detecting EP pathogen and ER pathogen, which comprises a double reaction liquid for detecting the EP pathogen and the ER pathogen, wherein the double reaction liquid comprises primer groups for detecting the EP pathogen, the nucleotide sequences of which are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; comprises a primer group for detecting ER pathogen, and the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, the concentration of each primer pair in the double reaction solution is set to be 90-180 mu M respectively.
More preferably, the EP pathogenic primer concentration is: the primer concentration of SEQ ID NO.1 and SEQ ID NO.2 is 90 mu M, and the primer concentration of SEQ ID NO.3 and SEQ ID NO.4 is 180 mu M; the EP pathogenic primer is used in an amount of 0.5 mu L of SEQ ID NO.1 and 2, and 2 mu L of SEQ ID NO.3 and 4.
More preferably, the ER pathogen primer concentration is: the primer concentration of SEQ ID NO.5 and SEQ ID NO.6 is 90 mu M, and the primer concentration of SEQ ID NO.7 and SEQ ID NO.8 is 180 mu M; ER pathogenic primer is used in an amount of 0.5 mu L of SEQ ID NO.5 and 0.5 mu L of SEQ ID NO.6, and 2 mu L of SEQ ID NO.7 and 8.
Wherein, the kit for preparing the reagent for detecting the EP pathogen and the ER pathogen further comprises: 20mM Tris-HCl 0.4. Mu.L, 40mM KCl 6.8. Mu.L, 100mM (NH 4) 2SO4 1.8. Mu.L, 80mM MgSO4 1.8. Mu.L, 1% Tween-20.8. Mu.L, 28mM dNTPs 0.9. Mu.L, 8000U/mL Bst enzyme 1.8. Mu.L, 1mM SYBRGREEN fluorochrome 0.9. Mu.L, 4.0X10. Mu.L -6 mol/L gold nanoparticle 1.8. Mu.L.
Further, the kit for preparing the reagent for detecting the EP pathogen and the ER pathogen has the following reaction conditions: the temperature was set at 63.5℃and the reaction time was set at 30min; running a program: the low-speed centrifugal speed is 1600r/min, the low-speed centrifugal time is 10sec, the high-speed centrifugal speed is 4600r/min, and the high-speed centrifugal time is 30sec.
Meanwhile, the invention also provides application of the kit for preparing the reagent for detecting the EP pathogen and the ER pathogen in preparing the reagent for simultaneously detecting the EP pathogen and the ER pathogen.
Furthermore, the invention also provides a primer group for efficiently and bigeminally detecting the EP pathogen and the ER pathogen, which comprises a primer group for detecting the EP pathogen and the ER pathogen, wherein the nucleotide sequences of the primer group for detecting the EP pathogen are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer group for detecting ER pathogen are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Furthermore, the invention also provides application of the primer group for efficiently and bigly detecting the EP pathogen and the ER pathogen in preparation of reagents for simultaneously detecting the T.martaylori, the babesia caballi, the I type equine herpesvirus and the IV type equine herpesvirus.
The kit for preparing and detecting the EP pathogen and ER pathogen also comprises common reagents for LAMP reaction and microfluidic chip reaction and other auxiliary reagents, and the characteristics of the reagents and the preparation methods thereof are well known to those skilled in the art; in a specific embodiment of the present invention, a method of reacting the LAMP reaction with the microfluidic chip is used.
The invention provides a kit for preparing a reagent for detecting EP pathogen and ER pathogen. By implementing the specific invention content, the following beneficial effects can be achieved:
according to the kit for preparing and detecting the reagents for detecting the EP pathogen and the ER pathogen and the application thereof, provided by the invention, through adopting a specific primer group and combining the gold nano-particles, the adsorption ssDNA and the protease in a reaction system, the nonspecific reaction in the heating process can be effectively avoided; the improved LAMP technology and the micro-fluidic chip technology are combined, so that the horse Jiao Chong and the horse nose pneumonia can be rapidly and accurately detected and distinguished, and meanwhile, the reaction reagent is pre-buried on the micro-fluidic chip, so that the same double-index detection is realized, the user operation is simple and convenient, and the detection efficiency is remarkably improved. The detection result has high specificity and obviously improves the detection accuracy, and the lowest detection limit of the EP pathogen detection primer is 1 multiplied by 10 1 The lowest limit of detection of the ER pathogen detection primer is 1X 10 2 COPies/. Mu.L. Has great potential development value for preventing Ma Jiaochong diseases and equine rhinopneumonia.
Drawings
Fig. 1 is a 8 sample chip test well bitmap.
Fig. 2 is a diagram of a microfluidic chip detector.
FIG. 3 is a graph showing the results of amplification of primer sensitivity of BAR Bei Sichong (EP) and equine herpesvirus type I (EV).
Wherein, panel A is the EP sensitivity amplification result, the amplification curves are 1X 10 from left to right, respectively 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 COPIES/. Mu.L and 1X 10 1 Plasmid fragments of copies/. Mu.L; panel B shows EV-sensitive amplification results, the amplification curves being 1X 10, respectively, from left to right 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 COPIES/. Mu.L and 1X 10 2 Plasmid fragment of copies/. Mu.L.
FIG. 4 shows the repetitive amplification patterns of babaci Bei Sichong (EP) and equine herpesvirus type I (EV) primers.
Wherein, FIG. A shows the result of EP repetitive amplification, and FIG. B shows the result of EV repetitive amplification.
FIG. 5 shows amplification patterns specific for BAR Bei Sichong (EP) and equine herpesvirus type I (EV) primers.
Detailed Description
The present invention will be described with reference to the following examples, but the present invention is not limited to the examples.
The materials and the reagents in the invention are as follows: nucleic acids of Vab Bei Sichong (EP) and Martensitic virus type I (EV) were both maintained by the Urufigo customs technology center, and plasmid extraction kits were purchased from TaKaRa, tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Tween-20, dNTPs, bst enzyme, SYBRGREEN fluorescent dye, and gold nanoparticles were purchased from Siemens.
The instruments used in the present invention are: CO 2 Incubator was purchased from tenmei company; the NanoDrop ND-1000 trace nucleic acid protein detector, the fluorescence detector, the microflow hole chip, the sealing film, the pipette, the gun head and the centrifuge tube are purchased from the Simer femto company. The reagents and materials can be purchased through public channels, and the equipment and instruments adopted in the process are all common equipment in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but are not limiting of the practice of the invention, and other reagents and equipment known in the art may be suitable for use in the practice of the following embodiments of the invention.
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Embodiment one: kit for preparing reagent for detecting EP pathogen and ER pathogen
The invention provides a kit for preparing a reagent for detecting EP pathogen and ER pathogen, which comprises a double reaction liquid for detecting the EP pathogen and the ER pathogen, wherein the double reaction liquid comprises primer groups for detecting the EP pathogen, the nucleotide sequences of which are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; comprises a primer group for detecting ER pathogen, and the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Preferably, the concentration of each primer pair in the double reaction solution is set to be 90-180 mu M respectively.
More preferably, the EP pathogenic primer concentration is: the primer concentration of SEQ ID NO.1 and SEQ ID NO.2 is 90 mu M, and the primer concentration of SEQ ID NO.3 and SEQ ID NO.4 is 180 mu M; the EP pathogenic primer is used in an amount of 0.5 mu L of SEQ ID NO.1 and 2, and 2 mu L of SEQ ID NO.3 and 4.
More preferably, the ER pathogen primer concentration is: the primer concentration of SEQ ID NO.5 and SEQ ID NO.6 is 90 mu M, and the primer concentration of SEQ ID NO.7 and SEQ ID NO.8 is 180 mu M; ER pathogenic primer is used in an amount of 0.5 mu L of SEQ ID NO.5 and 0.5 mu L of SEQ ID NO.6, and 2 mu L of SEQ ID NO.7 and 8.
Wherein, the kit for preparing the reagent for detecting the EP pathogen and the ER pathogen further comprises: 20mM Tris-HCl 0.4. Mu.L, 40mM KCl 6.8. Mu.L, 100mM (NH 4) 2SO4 1.8. Mu.L, 80mM MgSO4 1.8. Mu.L, 1% Tween-20.8. Mu.L, 28mM dNTPs 0.9. Mu.L, 8000U/mL Bst enzyme 1.8. Mu.L, 1mM SYBRGREEN fluorochrome 0.9. Mu.L, 4.0X10. Mu.L -6 mol/L gold nanoparticle 1.8. Mu.L.
Further, the reaction conditions for preparing the kit for detecting the EP pathogen and the ER pathogen are as follows: the temperature was set at 63.5℃and the reaction time was set at 30min; running a program: the low-speed centrifugal speed is 1600r/min, the low-speed centrifugal time is 10sec, the high-speed centrifugal speed is 4600r/min, and the high-speed centrifugal time is 30sec.
Meanwhile, the invention also provides application of the kit for preparing the reagent for detecting the EP pathogen and the ER pathogen in preparing the reagent for simultaneously detecting the horse Jiao Chong disease and the equine rhinopneumonia.
Furthermore, the invention also provides a primer group for preparing the reagent for detecting the EP pathogen and the ER pathogen, which comprises a primer group for detecting the EP pathogen and the ER pathogen, wherein the nucleotide sequences of the primer pair for detecting the EP pathogen are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer group for detecting ER pathogen are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Furthermore, the invention also provides application of the primer group for preparing the reagent for detecting the EP pathogen and the ER pathogen in preparing the reagent for simultaneously detecting the horse Jiao Chong disease and the equine rhinopneumonia pathogen.
Embodiment two: kit for preparing reagent for detecting EP pathogen and ER pathogen and application thereof
And driving the sample to enter the reaction hole of the microfluidic chip by using centrifugal force, and performing isothermal amplification. If the sample contains target fragments to obtain isothermal amplification, effectively combining the amplification products with fluorescent substances, capturing fluorescent signals in real time by a fluorescent detector, intuitively reacting the generation of the amplification products, and judging whether the sample contains equine Jiao Chong and equine rhinopneumovirus according to the occurrence time, the intensity and the position of the real-time fluorescent signals.
(1) Primer design
The target sequence is first searched through NCBI GenBank, and primers are designed for the target sequence.
(2) Template extraction and pretreatment
And respectively fixing the nucleic acid templates at corresponding positions of the microfluidic chip, packaging the microfluidic chip, adding the nucleic acid templates extracted from horse blood, nasal/oral secretions, excretions, environmental samples and cell cultures into the packaged microfluidic chip, and then placing the packaged microfluidic chip into a microfluidic chip detector with a centrifugal function, a constant temperature function and real-time fluorescence detection, as shown in figure 2.
The specific operation steps are as follows:
1. the composition of 18 μl of the reaction solution in the microfluidic chip detection system is shown in table 1 below:
table 1: composition table of reaction solution
| Concentration of | Reagent(s) | Volume of |
| 20mM | Tris-HCl | 0.4μL |
| 40mM | KCl | 6.8μL |
| 100mM | (NH 4 ) 2 SO 4 | 1.8μL |
| 80mM | MgSO 4 | 1.8μL |
| 1％ | Tween-20 | 1.8μL |
| 28mM | dNTPs | 0.9μL |
| 8000U/mL | Bst enzyme | 1.8μL |
| 1mM | SYBRGREEN fluorescent dyes | 0.9μL |
| 4.0×10 -6 mol/L | Gold nanoparticles | 1.8μL |
In the reaction system, the concentration and the volume of the Ma Jiao insect primer are as follows: 90. Mu.M of EP-F3 primer 0.5. Mu.L, 90. Mu.M of EP-B3 primer 0.5. Mu.L, 180. Mu.M of EP-FIP primer 2. Mu.L, 180. Mu.M of EP-BIP primer 2. Mu.L. The concentration and volume of the equine rhinopneumonia primer are as follows: 90. Mu.M of ER-F3 primer 0.5. Mu.L, 90. Mu.M of ER-B3 primer 0.5. Mu.L, 180. Mu.M of ER-FIP primer 2. Mu.L, 180. Mu.M of ER-BIP primer 2. Mu.L. The kit selects 8 sample chips, namely, 1 sample adding hole corresponds to 4 detection holes, 1 and 2 detection holes are used for embedding and amplifying primers of an EP sequence, 2 and 3 detection holes are used for embedding and amplifying primers of an EV sequence, as shown in figure 1, and freeze-drying is carried out after the treatment is completed.
Selecting 16 mu L of EP gene fragment plasmid; taking 16 mu L of EV gene fragment plasmids, fully and uniformly mixing the two gene fragment plasmids, then mixing 18 mu L of reaction solution with 32 mu L of EP and EV gene fragment plasmids, adding the mixture into a sample adding hole of a chip, sealing the sample adding hole by using a sealing film, and loading the mixture into a machine;
the temperature was set at 63.5℃and the reaction time was set at 30min.
(3) Performing on-machine amplification on a microfluidic chip:
because the method adopts isothermal amplification, the whole reaction process is completed under the condition of constant temperature without the temperature change processes of denaturation, annealing, extension and the like of PCR amplification, and the amplification process is as follows: the temperature was set at 63.5℃and the reaction time was set at 30min. Running a program: the low-speed centrifugal speed is 1600r/min, the low-speed centrifugal time is 10sec, the high-speed centrifugal speed is 4600r/min, and the high-speed centrifugal time is 30sec.
(4) And judging the result of the micro-fluidic chip:
1. microfluidic chip detector threshold line setting
The threshold line is generally set to 800 (which can be adjusted according to practical conditions, the setting principle is that the threshold line just exceeds the highest point of an atypical S-shaped amplification curve, and the Ct value is displayed as 30), and the automatic analysis result is carried out by the matched software of the instrument.
2. Result determination
2.1 conditions for establishment of experiments
Positive control: the reaction wells of the positive control had obvious typical S-type amplification curves with Ct < 30.
Negative control: the reaction wells of the negative control had no typical S-type amplification curve with Ct < 30.
2.2 criterion for determination
Positive: within 30 minutes of reaction time, obvious amplification curves appear on the detection hole sites of the items, and the Ct value is less than 30, so that the detection items corresponding to the holes are judged to be positive.
Negative: within 30 minutes of reaction time, no obvious amplification curve appears on the detection hole site of the item, and the detection item corresponding to the hole is judged to be negative.
Performing isothermal amplification of the microfluidic chip on a microfluidic chip detector, performing real-time fluorescence detection by the detector, judging according to an effective amplification curve of the fluorescence detection, and judging positive if a standard S-shaped amplification curve exists in any detection hole, namely, if the sample contains virus nucleic acid corresponding to the detection hole; the detection well without amplification curve is judged as negative, i.e. the sample does not contain viral nucleic acid corresponding to the detection well.
Embodiment III: verification of sensitivity and detection limits
The kit for preparing the reagent for detecting the EP pathogen and the ER pathogen provided by the invention is used for detecting the nucleic acid of equine Jiao Chong diseases and equine rhinopneumonia pathogens of equine animals, and verifying the specificity.
(1) Experimental materials
Reagent: a reaction solution; 1X 10 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 The copies/. Mu.L of plasmid with EP and EV gene fragments; a negative control; positive control.
Instrument: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(2) Detection system
Reference is made to the inspection in the first and second embodimentsThe test system is subjected to experimental operation, and then the loaded chip is put into a isothermal amplification instrument for experimental detection, the amplification result can be shown as figure 3, and the lowest detection limit of the EP (3A) primer is 1 multiplied by 10 as shown in the detection limit 1 The lowest limit of detection of the plasmids, the EV (3B) primers, of the copies/. Mu.L was 1X 10 2 The Ct values of the plasmids with the copies/mu L are all smaller than 30min, which shows that the kit provided by the invention and the test kit have higher sensitivity.
Embodiment four: verification of repeatability
(1) Experimental materials
Reagent: a reaction solution; EP and EV primers 1X 10 4 Plasmids of the copies/. Mu.L gene fragment; a negative control; positive control. Instrument: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(2) Detection system
The experimental operation was performed with reference to the detection system in the first and second embodiments, and then the chip was put into a isothermal amplification apparatus for experimental detection.
(3) Amplification results
The amplification results of the reproducibility assay are shown in FIG. 4. The results of the measurement of the coefficient of variation (CV,%) of Ct values of EP and EV are shown in Table 2.
Table 2: results of measurement of coefficient of variation in Ct values of EP and EV
As shown in Table 2, the coefficient of variation (CV,%) of Ct value of the primer EP is calculated to be 2.0%, less than 5%, and the repeatability is good, so that the primer EP meets the requirements; the coefficient of variation (CV,%) of Ct value of primer EV is 2.7%, less than 5%, and has good repeatability and stability, and meets the requirements.
Fifth embodiment: specificity verification
(1) Experimental materials
Reagent: reagent: a reaction solution; babesia caballi; equine herpesvirus type i nucleic acid; negative control. Instrument: a constant temperature amplification instrument; a palm centrifuge; a pipette.
(2) Detection system
The experimental operation was performed with reference to the detection systems in the first and second embodiments, and then the chip was put into a isothermal amplification apparatus for experimental detection. The comparison of the amplification results shows that the amplification results of the EP and EV specific experiments can be referred to as shown in FIG. 5, and the amplification results of the EP prove that the amplification results of the sample nucleic acid except Ma Jiaochong are positive, and the amplification curves of the Ma Bi pneumonia vaccine nucleic acid and the negative control are not available, so that the EP primer can only specifically amplify and detect the equine burnt worm sample nucleic acid, has good specificity and generally does not generate cross reaction with equine rhinopneumovirus. As can be seen from the EV amplification results, the amplification results of the vaccine nucleic acid except Ma Bi pneumonia are positive, and the Ma Jiaochong sample nucleic acid and the negative control have no amplification curve, so that the EV primer can only specifically amplify and detect the vaccine nucleic acid of equine rhinopneumonia, has good specificity and generally does not generate cross reaction with horse Jiao Chong.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.
Sequence listing
<110> Uruziq customs technical center
<120> kit for preparing reagent for detecting EP pathogen and ER pathogen and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
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<213> Baba Bei Sichong (Babesia caballi)
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accgtcgtag tcctaacyat 20
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<213> Baba Bei Sichong (Babesia caballi)
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cctwcctytg tctggacctg 20
<210> 3
<211> 41
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<213> Baba Bei Sichong (Babesia caballi)
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tccccccaga acccaaagac ttgccgacta grgattggag g 41
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<212> DNA
<213> Baba Bei Sichong (Babesia caballi)
<400> 4
aaggaattga cggaagggca ccgagtttcc ccgtgttgag tc 42
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<213> Martensitic Virus type I (Equid alphaherpesvirus 1)
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<213> Martensitic Virus type I (Equid alphaherpesvirus 1)
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<212> DNA
<213> Martensitic Virus type I (Equid alphaherpesvirus 1)
<400> 8
ctacgcacgt ctttagcggt gagcataggt ctgaatgcaa t 41
Claims (5)
1. Preparing a kit for detecting the EP pathogen and the ER pathogen, wherein the kit comprises a double reaction solution for detecting the EP pathogen and the ER pathogen, and the double reaction solution comprises primer groups for detecting the EP pathogen, the nucleotide sequences of which are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; comprises a primer group for detecting ER pathogen, and the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8;
the concentration of the EP pathogenic primer is as follows: the primer concentration of SEQ ID NO.1 and SEQ ID NO.2 is 90 mu M, and the primer concentration of SEQ ID NO.3 and SEQ ID NO.4 is 180 mu M; the using amount of the EP pathogenic primer is 0.5 mu L of SEQ ID NO.1 and SEQ ID NO.2, and 2 mu L of SEQ ID NO.3 and SEQ ID NO. 4;
the concentration of the ER pathogenic primer is as follows: the primer concentration of SEQ ID NO.5 and SEQ ID NO.6 is 90 mu M, and the primer concentration of SEQ ID NO.7 and SEQ ID NO.8 is 180 mu M; ER pathogenic primer is used in an amount of 0.5 mu L of SEQ ID NO.5 and 0.5 mu L of SEQ ID NO.6, and 2 mu L of SEQ ID NO.7 and 8;
the detection kit also comprises: 20mM Tris-HCl 0.4. Mu.L, 40mM KCl 6.8. Mu.L, 100mM (NH) 4 ) 2 SO 4 1.8μL、80mM MgSO 4 1.8. Mu.L, 1% Tween-20.8. Mu.L, 28mM dNTPs 0.9. Mu.L, 8000U/mL Bst enzyme 1.8. Mu.L, 1mM SYBRGREEN fluorescent dye 0.9. Mu.L, 4.0X10) -6 mol/L gold nanoparticle 1.8. Mu.L.
2. Kit for the preparation of a reagent for the detection of EP pathogen and ER pathogen according to claim 1, wherein the reaction conditions for the detection of EP pathogen and ER pathogen are: the temperature was set at 63.5℃and the reaction time was set at 30min; running a program: the low-speed centrifugal speed is 1600r/min, the low-speed centrifugal time is 10sec, the high-speed centrifugal speed is 4600r/min, and the high-speed centrifugal time is 30sec.
3. Use of a kit for the preparation of a reagent for the detection of EP pathogen and ER pathogen according to any of claims 1-2 for the preparation of a reagent for the simultaneous detection of EP pathogen and ER pathogen.
4. The primer group for detecting the EP pathogen and the ER pathogen in a high-efficiency duplex manner is characterized by comprising a primer group for detecting the EP pathogen and the ER pathogen, wherein the nucleotide sequences of the primer group for detecting the EP pathogen are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer group for detecting ER pathogen are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
5. The use of the primer set according to claim 4 for preparing a reagent for simultaneously detecting T.martaylori, babesia caballi and equine herpesvirus type I.
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