CN113817842B - SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance and application thereof - Google Patents
SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, and particularly discloses an SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance and application thereof. The SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance contains a nucleotide sequence with the 166 th polymorphism A/C shown as SEQ ID NO. 1. The polymorphic site of the SNP molecular marker is A, and corresponds to the sensitivity of the diclazuril; the polymorphic site is C, corresponding to diclazuril resistance. Through verification of different clinical drug-resistant strains of Eimeria tenella, the SNP locus can be used for identifying the Eimeria tenella of the Dekkali drug-resistant chicken and the sensitive Eimeria tenella of the chicken, and has higher detection accuracy of the Dekkali drug resistance.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance and application thereof.
Background
Eimeria (Eimeria) coccidium belongs to Dimensa and is widely deposited in the intestinal tracts or liver, kidney and other parts of animals such as chickens, rabbits, ducks, geese and the like. Eimeria species which were colonized by the chicken intestinal tract were 7 species, eimeria tenella (E.tenella), eimeria acervulina (E.acervulina), eimeria necatrix (E.necatrix), eimeria maxima (E.maxima), eimeria mitis (E.mitis), eimeria brunetti (E.brunetti) and Eimeria praecox (E.praecox), respectively. Chicken coccidiosis is extremely harmful to the poultry industry. High costs of medicine are required annually for anticoccidial use.
Since the 40s of the 20 th century, effective drugs have been used in countries to control chicken coccidiosis. However, as early as 1945, insect strains resistant to sulfonamides have been isolated from the field, and in 1954, insect strains resistant to sulfonamides have been isolated from the chicken farm. By the 60 s, different drug resistant strains of different drugs appeared in succession. To date, almost all anticoccidial drugs have emerged as drug-resistant strains, and drug-resistant strains exhibit a degree of cross-resistance to other drugs of the same class or mechanism of action as the drug.
Dekkalii (diclazuril) was an anticoccidial developed by Belgium Yansen in 1986 and its chemical name was cloxazine benzonitrile. The diclazuril has good control effect on coccidiosis of various livestock and poultry such as chickens, turkeys, ducks, geese, peacocks, quails, rabbits and the like, belongs to a non-ionic carrier type anticoccidial drug, and is the anticoccidial drug with the least dosage, wide anticoccidial spectrum and high efficiency at present. Dekkalide was effective against 6 important Eimeria species of infected chickens. However, after the drug has been put into use for a period of time, resistant coccidia against diclazuril are constantly emerging. At present, the detection method of the drug resistance of the diclazuril still stays in a cage feeding experiment, and the detection result of the method is reliable, but the process is complex and long in time consumption, and a large number of fresh coccidian oocysts and experimental animals are needed. Therefore, development of a method capable of rapidly detecting chicken coccidiosis resistance so as to guide clinical coccidiosis medication and reduce the loss of the breeding industry is urgent.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to chicken eimeria tenella diclazuril resistance and a method for detecting chicken eimeria diclazuril resistance by using the SNP molecular marker.
In a first aspect, the invention provides a SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance, which comprises a nucleotide sequence with the 166 th polymorphism A/C of a sequence shown as SEQ ID NO. 1.
The polymorphic site of the SNP molecular marker is A, and corresponds to the sensitivity of the diclazuril; the polymorphic site is C, corresponding to diclazuril resistance.
According to the invention, through extracting the genome of the diclazuril-resistant chicken coccidium and the sensitive chicken coccidium, 100 multiplied by the resequencing is carried out, and the result after resequencing is analyzed, the SNP locus at 166 th locus of EVM0004287 gene is found, the SNP locus of the sensitive insect strain is A, and the SNP locus of the drug-resistant insect strain is C. A large number of chicken coccidium samples are obtained through separation, and verification of clinical drug-resistant strains is carried out, so that the SNP locus can be used for screening and identifying the chicken eimeria tenella with the diclazuril drug resistance and the sensitive chicken eimeria tenella, and the identification result is consistent with that of a cage feeding experiment.
In a second aspect, the invention provides a primer pair for amplifying the SNP molecular marker, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3.
In a third aspect, the present invention provides a kit comprising the primer pair described above.
In a fourth aspect, the invention provides application of the SNP molecular marker or primer pair or kit in detecting chicken eimeria tenella diclazuril drug resistance, screening chicken eimeria tenella diclazuril drug resistant strains and guiding chicken eimeria tenella drug application.
In a fifth aspect, the invention provides a method for detecting the resistance of chicken eimeria tenella to the dekzuril, which uses DNA or RNA of the chicken eimeria tenella to be detected as a template, adopts a primer pair shown as SEQ ID NO.2-3 to carry out PCR amplification, and judges whether the chicken eimeria tenella to be detected has the dekzuril Li Nai resistance according to the sequence of a PCR amplification product.
The reaction procedure for PCR amplification includes: 98 ℃ for 30-60s;98 ℃ for 15-30s;65 ℃ for 20-30s; cycling at 72 deg.C for 25-35s and 30-40 times; 72 ℃ for 5-10min.
Preferably, the reaction procedure for PCR amplification is: 98 ℃ for 30s;98 ℃ for 15s;65 ℃ for 30s;72 ℃,30s,35 cycles; 72℃for 10min.
The chicken coccidiosis DNA/RNA can be obtained by conventional extraction methods. The sequence of the PCR amplification product may be analyzed by methods conventional in the art, such as sequencing.
And if the nucleotide sequence corresponding to the 166 th position of the sequence shown in SEQ ID NO.1 in the PCR amplification product is C, the Eimeria tenella to be detected is a diclazuril drug-resistant strain.
The application method of the kit for detecting the resistance of the diclazuril of the eimeria tenella comprises the steps of extracting DNA/RNA templates of a sample of a strain to be detected by a conventional method, taking the extracted DNA/RNA as templates, carrying out PCR amplification by using the kit, and verifying the resistance of the diclazuril of the strain according to a PCR amplification result.
The invention has the advantages that:
The SNP molecular marker can be used for distinguishing and identifying the sensitive and drug-resistant chicken eimeria tenella, and has higher detection accuracy of the drug resistance of the decbulide. The identification result of the SNP molecular marker detection primer is consistent with the identification result of a cage feeding experiment, and the SNP molecular marker detection primer can be used as the basis for screening and detecting the diclazuril drug-resistant strain. Meanwhile, the method does not need special equipment, a large number of experimental animals and a large number of fresh coccidian oocysts are not needed, the whole experiment is not limited by factors such as sites, and compared with the traditional cage feeding experiment, the method has the advantages of short time consumption, strong specificity, simplicity in operation, low cost and the like, and can be widely used for detecting the drug resistance of the diclazuril.
Drawings
FIG. 1 shows the polymorphism of 16 isolated local worm strains at the 166 locus of EVM0004287 gene in example 2 of the present invention.
FIG. 2 shows the polymorphism of 15 laboratory-induced insect strains at the 166 locus of EVM0004287 gene in examples 2 and 4 of the present invention.
FIG. 3 shows the result of gel electrophoresis of PCR amplification product in example 3 of the present invention, wherein lane 1 is DNA MARKER, AL5000; lane 2 is the amplification product using the sequences in Table 1 as primers and Eimeria tenella genomic cDNA as template.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The equipment and reagents used in each example were conventionally commercially available unless otherwise specified.
In the following embodiments of the invention, the apparatus and reagents involved include:
pEASY-Blunt Simple Cloning Vector and competent cells Trans 5. Alpha. Were purchased from Beijing all gold biology Co., ltd;
the DNA molecular weight Marker was purchased from Beijing Aidelai Biotechnology Co., ltd;
q5 high fidelity DNA polymerase was purchased from NEB company;
TRIZOL is available from Siemens;
Glass beads were purchased from Sigma;
Reverse transcription kit was purchased from Beijing full gold Co., ltd;
the PCR instrument, the constant temperature shaking table at 37 ℃, the incubator at 37 ℃, the gel imager and the gel electrophoresis apparatus are conventional equipment.
Example 1 acquisition of SNP molecular markers related to resistance to chicken coccidiosis
31 Eimeria tenella Decoct drug-resistant strains were obtained by field isolation and laboratory induction.
And (3) separating drug-resistant strains in the field: 64 chicken coccidium samples from different regions are collected from all over the country, and subjected to propagation by selecting drug resistance evaluation indexes relative to oocyst yield (ROP), optimal anticoccidial activity Percentage (POAA), lesion score reduction Rate (RLS) and anticoccidial index (ACI) (refer to ：Lan L,Sun B,Zuo B,Chen X,Du A.Prevalence and drug resistance of avian Eimeria species in broiler chicken farms of Zhejiang province.China Poult Sci.2017,96:2104–2109. Bi Feifei, han Zhenyan, hao Zhenkai, yong lan, songgao, liu Xianyong. Development of chicken coccidium resistance detection method [ J ]. J. Chinese veterinary journal 2019,55 (06): 69-71.), 16 full-scale drug-resistant chicken strains are obtained. However, considering that the insect strains belong to mixed insect strains, in order to ensure the accuracy of SNP (single nucleotide polymorphism) of subsequent re-sequencing analysis and reduce random errors generated by second-generation sequencing, the drug-resistant eimeria tenella in 16 decubitus drug-resistant strains is separated by a technology of separating intestinal contents and separating multi-sporangia, and then chicken is propagated only, so that 16 decubitus insect strains with drug-resistant properties for decubitus are finally obtained.
The drug resistance evaluation index was selected in this example as relative oocyst yield (ROP), percentage of Optimal Anticoccidial Activity (POAA), lesion score reduction Rate (RLS), and anticoccidial index (ACI).
Relative to oocyst yield (Relative oocysts production, ROP) = (average oocyst yield of infected dosing group/average oocyst yield of infected control group) ×100%, ROP <15%, drug resistance detection result is negative (-), ROP > 15%, drug resistance detection result is positive (+).
Percentage of optimal anticoccidial activity (Percent of optimum anticoccidial activity, POAA) = (infected-non-infected group GSR)/(non-infected-non-infected group GSR) ×100%, where GSR (Growth and survival ratio) = group end average weight/group initial average weight. POAA >50%, the drug resistance detection result is negative (-), POAA is less than or equal to 50%, and the drug resistance detection result is positive (+).
Lesion score reduction rate (Reduction of lesion scores, RLS) = (average lesion score of infected non-dosed group-average lesion score of infected dosed group)/average lesion score of infected non-dosed group x 100%, RLS > 50%, drug resistance test result negative (-), RLS <50%, drug resistance test result positive (+).
Anticoccidial index (Anticoccidia indexes, ACI) = (survival rate + relative weight gain rate) - (lesion value + oocyst value), ACI criterion ∈180, complete sensitivity; less than or equal to 150 percent and complete drug resistance.
When evaluating the drug resistance of chicken coccidiosis, the comprehensive evaluation is carried out by combining the 4 indexes: when the 4 indexes are negative in drug resistance, judging that the medicine is sensitive to the medicine; when 1 index is positive, judging that the medicine is slightly resistant; when the 2 indexes are positive, judging that the medicine is moderately resistant; and when 3 or 4 indexes are positive, judging that the medicine is completely resistant.
Laboratory induced drug resistant strains: in the embodiment, a method of increasing the drug concentration is adopted, and the initial induction concentration is 0.02ppm, and 15 strains of Eimeria tenella resistant to the diclazuril are finally obtained after 20 generations.
Eimeria tenella Haoton strain (E.tenella Houghton (H) stand) with the same genetic background and sensitivity to the diclazuril is selected as a parent strain (Chapman HD,Shirley MW.The Houghton strain of Eimeria tenella:a review of the type strain selected for genome sequencing.Avian Pathol.2003,32(2):115-27.),, oocysts of the strain are divided into 20 groups, AA broiler chicken passaged coccidian without coccidian infection at 7-14 days are selected, and each coccidian chicken in each group is infected with 1X 10 4 sporulated oocysts. Desmoride was added to the drinking water 48 hours prior to inoculation and oocysts were removed from the feces 5-10 days after inoculation. Gradually increasing the drug concentration according to the relative oocyst yield until the diclazuril drug-resistant insect strain is induced. When drug concentration increases, if oocysts are not collected enough for passage, then these oocysts are passed once without drug selection pressure. In the embodiment, the concentration of the passage medicine is 0.02mg/kg drinking water, 0.04mg/kg drinking water, 0.06mg/kg drinking water, 0.08mg/kg drinking water, 0.12mg/kg drinking water, 0.24mg/kg drinking water, 0.3mg/kg drinking water, 0.5mg/kg drinking water, 0.8mg/kg drinking water and 1mg/kg drinking water in sequence, and finally 15 strains of eimeria tenella resistant to the diclazuril are obtained.
The 31-strain diclazuril resistant strain is subjected to genome extraction, then subjected to second-generation high-throughput sequencing, the sequencing depth is 100×, the re-sequenced data are compared (BWA) with a reference genome (GCF_000499545.2_ETH001), and strategies such as GATK CALL SNP and SnpEff annotation genes are combined to annotate mutant genes. The mutant gene was anchored at the EVM0004287 gene using both parameters of population solidification index (Fst) Heterozygosity (HP). In order to avoid errors caused by high-throughput sequencing, the sequencing of the EVM0004287 gene amplification product was further verified by using a high-fidelity DNA polymerase amplification method, and the SNP site at 166 th position of the EVM0004287 gene of coccidian was found, wherein the SNP site A corresponds to the sensitivity of diclazuril, and the SNP site C corresponds to the resistance of diclazuril.
Example 2 detection of Dekkalide resistance of samples for resequencing based on SNP molecular markers
The EVM0004287 gene of 16 isolated endemic strains described in example 1 for re-sequencing was introduced into APE software, and it was found by sequence alignment that these 16 isolated endemic strains were C at 166 locus and reference sensitive strain (WT) was a, i.e. 16 isolated endemic strains were all drug resistant strains in which the polymorphism at 166 locus was mutated from a to C. See in particular fig. 1. The identification result is consistent with that of the cage feeding experiment.
The EVM0004287 gene of 15 laboratory-induced insect strains described in example 1 for re-sequencing was introduced into APE software, and sequence alignment revealed that these 15 laboratory-induced insect strains were C at 166 locus and reference sensitive strain (WT) was a, i.e., the 15 laboratory-induced insect strains were all drug-resistant strains by mutating the polymorphism at 166 locus from a to C. See in particular fig. 2. The identification result is consistent with that of the cage feeding experiment.
Example 3 design of primers for detection of SNP molecular markers associated with Decoccyx chicken resistance
In the primer design and screening process, since two intronic sequences exist in the Eimeria tenella EVM0004287 gene, the template should be a reverse transcribed cDNA sample in order to obtain a DNA fragment capable of efficiently and specifically amplifying the SNP site. For such sequences, the primer design should also be directed to cDNA. Primers were designed upstream and downstream of the EVM0004287 gene, and primers capable of efficiently and specifically amplifying DNA fragments containing the SNP sites were obtained by screening, and the primers were synthesized by the company of Biotechnology, inc. of the family Beijing at Boxing, and the primer sequences (SEQ ID NO.2 and 3) are specifically shown in Table 1 below.
TABLE 1
PCR amplification was performed using Eimeria tenella genomic cDNA as a template and the primers shown in Table 1 above.
The specific PCR reaction system and the procedure are as follows:
PCR reaction system: 1 mu L of each of the upstream primer and the downstream primer, 1 mu L of the DNA template, 1 mu L of dNTP solution, 10 mu L of 5 times Reaction buffer solution, 0.5 mu L of Q5 enzyme and the balance of 50 mu L with deionized water;
PCR reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 15s; annealing temperature 65 ℃ for 30s; the extension temperature is 72 ℃ for 30s; cycling for 35 times; finally, the extension is carried out for 10min at 72 ℃.
The target fragments of the lengths shown in Table 1 were obtained by PCR amplification. The results are shown in FIG. 3. Example 4SNP molecular markers for detecting the differences between Dicreril drug-resistant insect strains and parent insect strains thereof produced by an experimental evolution system
The SNP molecular markers obtained in example 1 and amplification primers (SEQ ID NO.2 and 3) thereof were used to detect the resistance of 15 Eimeria tenella decubitus drug-resistant strains produced under the experimental evolution conditions in example 1, and the specific method is as follows:
1. Preparation of DNA templates
After purification of the final concentration of induced oocysts, each chicken (13 day old AA broiler) was inoculated with 2×10 6 oocysts, 3 chickens total, the cecum was taken at 110-120 hours, and the second generation merozoites were extracted for DNA extraction.
The specific method comprises the following steps:
To the collected second merozoite suspension was added 500. Mu.L of CTAB buffer, 40. Mu.L of proteinase K, and digested in a metal bath at 55℃for 2-3 hours, during which time the mixture was inverted and homogenized several times. Add 40. Mu.L RNase and digest for 30min at 37℃in a metal bath with inversion and mixing several times. An equal volume of CTAB solution was added, vigorously shaken several times, centrifuged at 1X 10 4 rpm, and the mixture was centrifuged for 10min. Transferring the supernatant to a new 1.5mL centrifuge tube, adding equal volume of isopropanol into each tube, mixing, and standing at-20deg.C for more than 30 min.
Centrifuging at 4deg.C at 1×10 4 rpm for 15min, discarding supernatant, retaining precipitate, adding 1mL75% ethanol, centrifuging at 4deg.C at 1×10 4 rpm for 5min, discarding supernatant, adding 100 μL ddH 2 O for dissolving after ethanol is completely volatilized, and standing at-20deg.C.
2. RNA template preparation and reverse transcription
1ML TRIZOL was added to the second merozoites, and the mixture was repeatedly blown with a pipette until the merozoites were completely lysed, and allowed to act at room temperature for 5min. Adding 200 μl chloroform, shaking vigorously for 15s, standing for 3min, centrifuging at 12000rpm at 4deg.C for 5min, transferring the upper water phase to precooled 500 μl isopropanol, precipitating RNA in the water phase, and standing for 10min; centrifuging at 12000rpm for 15min at 4deg.C, discarding supernatant, washing the precipitate with 75% ethanol, centrifuging at 12000rpm for 15min at 4deg.C, and discarding supernatant. Volatilizing clean ethanol, adding enzyme-free sterile double distilled water according to the precipitation amount, measuring RNA concentration by using a spectrophotometer, and preserving the RNA sample in a refrigerator at-80 ℃.
RNA samples were reverse transcribed according to the requirements of the full gold kit, the reverse transcription system is as follows in Table 2.
TABLE 2 reverse transcription system
| Reagent(s) | Volume (mu L) |
| F-Primer | 1 |
| 2×R-Primer | 10 |
| DNA Remover | 1 |
| Random Primer | 1 |
| Oligo | 1 |
| RNase free water | Depending on the sample concentration |
| RNA | Make up to 20 mu L |
3. PCR amplification
Using the cDNA obtained in step 2 as a template, and using the primers shown in table 1 for PCR amplification, the PCR reaction system and the procedure were as follows:
PCR reaction system: 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the DNA template, 1. Mu.L of dNTP solution, 10. Mu.L of 5 Xreaction buffer solution, 0.5. Mu.L of Q5 enzyme, and 50. Mu.L of the mixture were made up with deionized water.
PCR reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 15s; annealing temperature 65 ℃ for 30s; the extension temperature is 72 ℃ for 30s; cycling for 35 times; finally, the extension is carried out for 10min at 72 ℃.
4. PCR product detection and sequencing after ligation transformation
And (3) detecting gel electrophoresis of a PCR product: 1.5% gel block was prepared, 130V voltage, electrophoresis was performed for 10min, and gel imaging was performed. The size of the PCR product was 585bp.
After cutting the strips, they were recovered and ligated into pEASY-Blunt Simple Cloning Vector, then transformed into competent cells Trans 5. Alpha. And spread evenly onto plates containing 20. Mu.g/ml ampicillin and incubated overnight in an incubator at 37 ℃. After the monoclonal was picked, PCR was identified and positive clones were selected and sequenced by the biotechnology company of the family Boxing, beijing.
5. Analysis of detection results
The sequence of PCR products of the parent susceptible insect strain and the induced drug-resistant insect strain are aligned, and the result is shown in figure 2. The results show that the 166 th position of the EVM0004287 gene of the sensitive insect strain is A, and the drug-resistant insect strain is C. The sequenced base sequence is translated into an amino acid sequence through software, and the result shows that the mutation of A at 166 th site of EVM0004287 gene into C causes the mutation of 56 th site of amino acid, the 56 th site of amino acid of a sensitive insect strain is serine, and the drug-resistant insect strain is arginine.
Example 5 detection of Dekkalide resistance of Mixed chicken coccidium samples collected in the field based on SNP molecular markers
The SNP molecular markers obtained in example 1 and their amplification primers (SEQ ID NO.2 and 3) against Eimeria tenella were used to test 12 Eimeria mixed samples collected from different regions (for experimental and isolation methods see ：Geng T,Ye C,Lei Z,Shen B,Fang R,Hu M,Zhao J,Zhou Y.Prevalence of Eimeria parasites in the Hubei and Henan provinces of China.Parasitol Res.2021,120:655–663.) for Desmoril resistance of Eimeria tenella. Morphological and PCR tests confirm that 12 mixed samples all contained Eimeria tenella oocysts. After that, further genomic preparation and PCR resistance tests were performed as follows:
1. Preparation of DNA templates
The sporulated 12 field mixed chicken coccidiosis samples were inoculated into coccidiosis-free chickens of appropriate ages (7-14 days old) at a dose of 5X 10 3-2×104 oocysts. Oocysts were collected from the faeces 5-10 days after inoculation.
After 48 hours of oocyst sporulation, purification was performed. Pouring potassium dichromate containing oocysts into a clean centrifuge tube, centrifuging at 3600rpm for 5min, pouring out the upper liquid, re-suspending and precipitating with PBS, and repeating for 2-3 times; resuspension the pellet with saturated saline, centrifuging at 3600rpm for 5min, introducing the upper layer liquid containing oocysts into another clean centrifuge tube, adding 5 times volume of PBS, centrifuging at 3600rpm for 5min; pouring out the supernatant, then re-suspending the sediment by sodium hypochlorite solution, placing on ice for preferably not more than 5min, centrifuging at 3600rpm for 5min, sucking the liquid containing oocysts on the upper layer into another clean centrifuge tube, adding 5 times of volume of PBS, centrifuging, repeating for 3-5 times until no odor of sodium hypochlorite remains, and obtaining the sediment which is the purified oocysts.
2Mm glass beads were rinsed 2-3 times with PBS (total volume of about 3 ml) and poured into the oocysts and allowed to act on the vortex for 5-10min; sucking the upper liquid and transferring into a clean EP tube, and centrifuging at 12000rpm at 4deg.C for 5-10min; the supernatant was discarded, 500ml of CTAB solution and 40. Mu.L of proteinase K were added and allowed to act at 55℃for 2 hours; after the temperature was reduced to 37 ℃, 20. Mu.L of RNase was added and the mixture was allowed to act at 37℃for 30min; after the temperature is reduced to room temperature, adding DNA extracting solution (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), shaking vigorously for 30s, and centrifuging at 1000rpm for 5min; discarding the supernatant, adding equal volume of isopropanol, standing at-20deg.C for 30min, and centrifuging at 12000rpm at 4deg.C for 15min; the supernatant was discarded, 1ml of a 75% ethanol solution was added, and after complete evaporation of ethanol, 200. Mu.L of ddH 2 O was added for dissolution and placed at-20℃for further use.
2. RNA template preparation and reverse transcription
The specific procedure is as in step 2 of example 4.
3. PCR amplification
The reaction conditions and the reaction system were the same as in example 4.
4. PCR product detection and sequencing after ligation transformation
The PCR product detection and sequencing method after ligation transformation were the same as in example 4.
5. Analysis of detection results
And (3) carrying out sequence alignment on the PCR products of the mixed insect strains obtained in the step (4). The results showed that there were 166A to C mutations in the EVM0004287 gene of 4 strains of the 12 field isolation mixed insect strains. The results are shown in Table 3 below.
Cage feeding experiments were performed on the 4 mutant mixed insect strains and 8 wild mixed insect strains identified above, and the drug resistance phenotype verification indexes (refer to ：Lan L,Sun B,Zuo B,Chen X,Du A.Prevalence and drug resistance of avian Eimeria species in broiler chicken farms of Zhejiang province.China Poult Sci.2017,96:2104–2109. Bi Feifei, han Zhenyan, hao Zhenkai, on Yong lan, solanum, liu Xianyong. Chicken coccidian drug resistance detection method research progress [ J ]. Chinese veterinary journal 2019,55 (06): 69-71) and the results are shown in Table 4. The results show that the mutant mixed insect strain has the resistance to the diclazuril and the wild mixed insect strain has no resistance, and the detection result of the SNP molecular marker is consistent with the cage feeding experimental result. Therefore, the PCR detection method based on SNP molecular markers can be used for detecting the resistance of the diclazuril of pure Eimeria tenella and can be well applied to the detection of clinical samples (mixed insect strains).
TABLE 3 sequence alignment of susceptible and resistant insect strains and mixed populations
Table 44 mutant mixed insect strain and 8 wild mixed insect strain drug resistance phenotype verification index and result
While the invention has been described in detail in the foregoing general description and with reference to 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.
Sequence listing
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Claims (11)
1. SNP molecular marker related to chicken eimeria tenella resistance, characterized in that the SNP molecular marker is a nucleotide sequence with A/C of 166 th polymorphism of the sequence shown as SEQ ID NO. 1.
2. Application of a reagent for detecting SNP molecular markers in detecting chicken eimeria tenella diclazuril drug resistance; the SNP molecular marker is a nucleotide sequence with the 166 th polymorphism A/C of the sequence shown as SEQ ID NO. 1; the use is for non-disease diagnosis or therapeutic purposes.
3. The application of the primer pair in detecting the drug resistance of chicken eimeria tenella diclazuril; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the use is for non-disease diagnosis or therapeutic purposes.
4. The application of the kit in detecting chicken eimeria tenella decubitus drug resistance; the kit comprises a primer pair, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the use is for non-disease diagnosis or therapeutic purposes.
5. Application of a reagent for detecting SNP molecular markers in screening chicken eimeria tenella diclazuril drug-resistant insect strains; the SNP molecular marker is a nucleotide sequence with the 166 th polymorphism A/C of the sequence shown as SEQ ID NO. 1; the use is for non-disease diagnosis or therapeutic purposes.
6. The application of the primer pair in screening chicken eimeria tenella diclazuril drug-resistant insect strains; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the use is for non-disease diagnosis or therapeutic purposes.
7. Application of the kit in screening chicken eimeria tenella decubitus drug-resistant insect strains; the kit comprises a primer pair, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the use is for non-disease diagnosis or therapeutic purposes.
8. Application of a reagent for detecting SNP molecular markers in preparing a product for guiding chicken to resist Eimeria tenella; the SNP molecular marker is a nucleotide sequence with the 166 th polymorphism A/C of the sequence shown as SEQ ID NO. 1; the drug is diclazuril.
9. The application of the primer pair in preparing a kit for guiding the use of chicken anti-eimeria tenella medicines; the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the drug is diclazuril.
10. A method for detecting the resistance of chicken eimeria tenella to the dekzuril for the purpose of non-disease diagnosis or treatment is characterized in that DNA or RNA of the chicken eimeria tenella to be detected is taken as a template, a primer pair shown as SEQ ID NO.2-3 is adopted for PCR amplification, and whether the chicken eimeria tenella to be detected has the resistance to the dekzuril is judged according to the sequence of a PCR amplification product; if the nucleotide sequence corresponding to the 166 th position of the sequence shown in SEQ ID NO.1 in the PCR amplification product is A, the Eimeria tenella to be detected is a diclazuril sensitive strain, and if the nucleotide sequence corresponding to the 166 th position of the sequence shown in SEQ ID NO.1 in the PCR amplification product is C, the Eimeria tenella to be detected is a diclazuril drug resistant strain.
11. The method of claim 10, wherein the reaction procedure for PCR amplification comprises: 98 ℃ for 30-60s;98 ℃ for 15-30s;65 ℃ for 20-30s; cycling at 72 deg.C for 25-35s and 30-40 times; 72 ℃ for 5-10min.
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| CN110709080A (en) * | 2017-03-28 | 2020-01-17 | Irp健康股份有限公司 | Use of berberine alkaloid preparation in preventing and/or treating infectious diseases |
| CN113025738A (en) * | 2021-04-30 | 2021-06-25 | 中国农业大学 | SNP molecular marker related to drug resistance of eimeria coccidioides halofuginone and application thereof |
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| CN110709080A (en) * | 2017-03-28 | 2020-01-17 | Irp健康股份有限公司 | Use of berberine alkaloid preparation in preventing and/or treating infectious diseases |
| CN113025738A (en) * | 2021-04-30 | 2021-06-25 | 中国农业大学 | SNP molecular marker related to drug resistance of eimeria coccidioides halofuginone and application thereof |
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