CN113755610A - Gene for detecting sensitivity of haemonchus contortus to ivermectin and application - Google Patents
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- CN113755610A CN113755610A CN202111171379.9A CN202111171379A CN113755610A CN 113755610 A CN113755610 A CN 113755610A CN 202111171379 A CN202111171379 A CN 202111171379A CN 113755610 A CN113755610 A CN 113755610A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6858—Allele-specific amplification
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- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a gene for detecting sensitivity of haemonchus contortus to ivermectin and application thereof, belonging to the technical field of parasite molecule detection. The gene is used for preparing a detection kit for detecting the sensitivity of the ivermectin of the haemonchus contortus, has strong specificity and convenient use, and can provide guidance for veterinary clinical medication.
Description
Technical Field
The invention belongs to the technical field of parasite molecule detection, and particularly relates to a gene for detecting sensitivity of haemonchus contortus to ivermectin and application thereof.
Background
Haemonchus contortus is one of the major parasites in sheep; the infectious larvae start to suck blood after entering the sheep body for 3 days, bleeding and inflammatory reaction of parasitic parts are caused, and anemia can occur in the host 10-12 days after infection; for adult haemonchus contortus, each worm can cause 30-80 mu L of blood loss of sheep per day, and the degree of anemia is largely related to the blood compensatory formation capability of sheep. The risk of haemonchus contortus infection is closely related to the number of ingested infective larvae, the host's resistance to it and the ability to produce blood.
The haemonchus contortus has extremely strong genetic diversity and has extremely strong potential for different environments when facing complex environmental changes. Thus, in all areas where sheep are active, haemonchus contortus is almost present and outbreaks of haemonchus contortus disease are likely to occur at any time.
The drug prevention and control technology for haemonchus contortus mainly comprises preventive anthelmintic and therapeutic anthelmintic. In the aspect of drug selection, the ivermectin has the characteristics of broad spectrum, high efficiency, easy operation and the like, is well received by farmers and herders and breeding owners, and is widely used for preventing and controlling the haemonchus contortus disease of sheep. However, frequent and blind administration for a long time can cause the haemonchus contortus to have extensive drug resistance to ivermectin, so that the haemonchus contortus can hardly achieve the ideal anthelmintic effect. To date, no accepted molecular biological method has been found that is suitable for detecting sensitivity of Haemonchus contortus to ivermectin.
Therefore, how to detect the sensitivity of haemonchus contortus to ivermectin by using a molecular method becomes a problem to be solved in the field.
Disclosure of Invention
The invention discloses a gene for detecting sensitivity of Haemonchus contortus to ivermectin, and the gene can be used for carrying out specificity detection on the sensitivity of Haemonchus contortus to ivermectin.
In order to achieve the purpose, the invention adopts the following technical scheme:
the gene for detecting the sensitivity of haemonchus contortus to ivermectin has a nucleotide sequence shown in SEQ ID NO: 1:
CTAGTTCAATGCAAGATTTCAATCGTTCATTCGATTCRCAAAGTGCACTAGCATACGGTGGACCTCAAGCAACTCCGATTGGAGTTGGTGTAGCGCCTCAACAAGTTCCGATCACAATGGATCCYAGACCTGTMCCACTCTACCGAGGAAATCAG。
the gene for detecting the sensitivity of haemonchus contortus to ivermectin has a nucleotide sequence shown in SEQ ID NO. 2:
CATGATGGGCGTCTCGGAGTTGGAGATAGAATTTTAGCTGTGAGACTTTTTTTTCATAAAATCCAGATTTCTCTTTTAAATGGCTAGGATGTACTCTTAAAAAAAGACTTTCAGGTTGATGACATCGGACTTGAGAATGTTACACATGAGTATGCCGTCGATGTATTGAAGCGAACCGGCACGAAAGTCACATTGTTGGTGCTTAAAGCAGACCCAAATGTGAATGTCAGCTCAATTGAAGAAACTGCAAGCCGTCAGTATGTTGCATCCACTCCACAATATCCTCAGACAACGCCAATCAGATGTGAGCTTCCTCCATTTTTTATGTGTTCTCGTGTGGTGCTCATGAGACTTGGAATCCTGAAGTGTATAGAATACCAATGGGCCGTTTTAGCTAGTTCAATGCAAGATTTCAATCGTTCATTCGATTCRCAAAGTGCACTAGCATACGGTGGACCTCAAGCAACTCCGATTGGAGTTGGTGTAGCGCCTCAACAAGTTCCGATCACAATGGATCCYAGACCTGTMCCACTCTACCGAGGAAATCAGGCACGGTTATTCCTTTATTATTGTAGTGACGGATTATTTCATGCTTGCCTTTGTGTTTTTTCATTAAGTTTTCGTTCTTCCAAAAGGGTTACAGTGGGTAAGACTCTTGGGCACAGCATGAGATGAGGATGCGGCATGATCGA；
wherein 395-549bp is a CDS region shown as SEQ ID NO. 1.
The genes code L27-1, PDZ, Src homology structure and guanylate kinase domain protein, and the sensitivity of Haemonchus contortus to ivermectin can be analyzed through three SNP sites in the genes.
Further, the application of the gene shown in SEQ ID NO. 1 or SEQ ID NO. 2 in the detection of the sensitivity of Haemonchus contortus to ivermectin: sensitivity of Haemonchus contortus to ivermectin is analyzed according to polymorphism of single nucleotides at positions 38, 125 and 134 of the gene shown in SEQ ID NO. 1.
Furthermore, the 38 th site of the gene shown in SEQ ID NO. 1 shows G/A polymorphism, the 125 th site shows T/C polymorphism, and the 134 th site shows C/A polymorphism;
the haemonchus contortus gene to be detected is sensitive to ivermectin if at least one of 38G, 125T and 134C is present.
The gene shown in SEQ ID NO. 1 or SEQ ID NO. 2 is applied to the preparation of a kit for detecting the sensitivity of Haemonchus contortus to ivermectin.
A kit for detecting sensitivity of Haemonchus contortus to ivermectin comprises an upstream primer and a downstream primer for amplifying a gene shown in SEQ ID NO. 1 or SEQ ID NO. 2.
Preferably, the upstream primer is HCIS-F: 5'-CATGATGGGCGTCTCGGAGT-3', SEQ ID NO: 3;
the downstream primer is HCIS-R: 5'-TCGATCATGCCGCATCCTCA-3', SEQ ID NO: 4.
Preferably, the kit for detecting the sensitivity of the haemonchus contortus to ivermectin further comprises a standard reference substance;
the standard reference substance is a recombinant plasmid carrying the gene shown in SEQ ID NO. 1 or SEQ ID NO. 2.
A method of detecting sensitivity of haemonchus contortus to ivermectin, comprising the steps of:
(1) extracting genomic DNA of haemonchus contortus to be detected;
(2) amplifying a gene shown as SEQ ID NO 1 or SEQ ID NO 2 by using the DNA obtained in the step (1) as a template;
(3) carrying out agarose gel electrophoresis on the amplification product, and sequencing the amplification product with the target band;
(4) and analyzing the sequence obtained by sequencing to determine whether the population to which the haemonchus contortus is to be detected is sensitive to ivermectin.
Preferably, the above-described kit is used for detection;
the PCR amplification reaction conditions are as follows:
pre-denaturation at 95 ℃ for 5 min;
denaturation at 95 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles;
extending for 5min at 72 ℃;
the mixture was stored at 4 ℃.
In conclusion, the gene for detecting the sensitivity of the haemonchus contortus to the ivermectin is provided, the gene is used for preparing the detection kit for detecting the sensitivity of the haemonchus contortus to the ivermectin, the specificity is strong, the use is convenient, and the guidance is provided for veterinary clinical medication.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of the PCR product;
the figure shows partial sample results, and the rest is the same and omitted;
among them, lanes 1 to 5 are samples to be detected, and lane 6 is a negative control (sterilized distilled water).
Figure 2 shows the sequencing results for the CDS region of each sample.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Fixing the newly collected haemonchus contortus with 75% alcohol, taking haemonchus contortus adults (including samples resistant to ivermectin (Hc-R) and sensitive to ivermectin (Hc-S)) stored in the 75% alcohol when starting to detect, and rinsing the haemonchus contortus adults with deionized water for 3-5 times for DNA extraction. All the process of extracting the genome DNA of the polypide is carried out according to the instruction of a DNA extraction kit. After completion of the DNA extraction, all DNA concentration measurements were performed using a nucleic acid protein detector.
Using qualified sample DNA as a template to amplify HCOI01200700 gene (shown in SEQ ID NO: 2):
CATGATGGGCGTCTCGGAGTTGGAGATAGAATTTTAGCTGTGAGACTTTTTTTTCATAAAATCCAGATTTCTCTTTTAAATGGCTAGGATGTACTCTTAAAAAAAGACTTTCAGGTTGATGACATCGGACTTGAGAATGTTACACATGAGTATGCCGTCGATGTATTGAAGCGAACCGGCACGAAAGTCACATTGTTGGTGCTTAAAGCAGACCCAAATGTGAATGTCAGCTCAATTGAAGAAACTGCAAGCCGTCAGTATGTTGCATCCACTCCACAATATCCTCAGACAACGCCAATCAGATGTGAGCTTCCTCCATTTTTTATGTGTTCTCGTGTGGTGCTCATGAGACTTGGAATCCTGAAGTGTATAGAATACCAATGGGCCGTTTTAGCTAGTTCAATGCAAGATTTCAATCGTTCATTCGATTCRCAAAGTGCACTAGCATACGGTGGACCTCAAGCAACTCCGATTGGAGTTGGTGTAGCGCCTCAACAAGTTCCGATCACAATGGATCCYAGACCTGTMCCACTCTACCGAGGAAATCAGGCACGGTTATTCCTTTATTATTGTAGTGACGGATTATTTCATGCTTGCCTTTGTGTTTTTTCATTAAGTTTTCGTTCTTCCAAAAGGGTTACAGTGGGTAAGACTCTTGGGCACAGCATGAGATGAGGATGCGGCATGATCGA；
wherein 395-549bp is a CDS region shown as SEQ ID NO. 1.
The amplification primers were designed as follows:
the upstream primer HCIS-F: 5'-CATGATGGGCGTCTCGGAGT-3', SEQ ID NO: 3;
the downstream primer HCIS-R: 5'-TCGATCATGCCGCATCCTCA-3', SEQ ID NO: 4.
And (3) PCR reaction system: mu.L of each HCIS-F, HCIS-R (20mmol/L), 12.5. mu.L of 2 XPCR Mix, 1. mu.L of DNA template, and 10.5. mu.L of sterile, enzyme-free water were added to a 25. mu.L system.
PCR amplification conditions: 95 ℃ for 5 min; 95 ℃, 30s, 56 ℃, 30s, 72 ℃, 1min, 35 cycles; 72 ℃ for 5 min; and preserving at 4 ℃.
The result of agarose gel electrophoresis detection is shown in figure 1, all detection samples have target bands of about 690bp on the agarose gel, which shows that the gene amplification is successful, and the products can be sequenced and used for SNP analysis of specific sites.
The product was sent directly to the company for sequencing (two-way).
The polymorphism of single nucleotide at position 38, 125 and 134 in the CDS region sequence of the successfully sequenced haemonchus contortus HCOI01200700 gene is analyzed, and the result is shown in figure 1 and table 1.
TABLE 1
In all the 38 Hc-S samples with successful sequencing, the percentage of 3 SNPs at positions 38(G), 125(T) and 134(C) was 52.6%, 57.9% and 57.9%, respectively, and in the 13 Hc-R samples, the percentage of 38(G), 125(T) and 134(C) was 0%. It was shown that 38(G), 125(T) and 134(C)3 SNPs were present only in Haemonchus contortus which was sensitive to ivermectin. Of the 13 Hc-R samples, 38(A), 125(C) and 134(A) accounted for 100%.
Further, the sensitivity of Haemonchus contortus to ivermectin was evaluated by using different combinations of the 38(G), 125(T) and 134(C) sites as evaluation indices, respectively, as shown in Table 2.
TABLE 2
The specificity in the table refers to the proportion of samples sensitive to ivermectin in haemonchus contortus that meet the corresponding evaluation criteria; sensitivity refers to the proportion of sensitive haemonchus contortus in the total amount of sensitive haemonchus contortus that meets the corresponding evaluation criteria. The specificity of each group was 100%, and the sensitivity was 52.6% to 63.2%. As can be seen, the detection method of the present invention has strong specificity, and the presence of at least one of 38(G), 125(T) and 134(C) in the gene of Haemonchus contortus to be detected is sensitive to ivermectin.
Furthermore, genes shown in SEQ ID NO. 2 corresponding to 38(G), 125(T) and 134(C) can be inserted into the vector to construct a recombinant plasmid as a sensitive standard control.
Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention shall not be limited to the examples shown herein, nor to the selected gene fragments, but any gene fragment containing the above 3 SNP sites obtained from the entire HCOI01200700 gene.
Sequence listing
<110> inner Mongolia autonomous region academy of agriculture and animal husbandry
<120> gene for detecting sensitivity of haemonchus contortus to ivermectin and application
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ctagttcaat gcaagatttc aatcgttcat tcgattcrca aagtgcacta gcatacggtg 60
gacctcaagc aactccgatt ggagttggtg tagcgcctca acaagttccg atcacaatgg 120
atccyagacc tgtmccactc taccgaggaa atcag 155
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catgatgggc gtctcggagt tggagataga attttagctg tgagactttt ttttcataaa 60
atccagattt ctcttttaaa tggctaggat gtactcttaa aaaaagactt tcaggttgat 120
gacatcggac ttgagaatgt tacacatgag tatgccgtcg atgtattgaa gcgaaccggc 180
acgaaagtca cattgttggt gcttaaagca gacccaaatg tgaatgtcag ctcaattgaa 240
gaaactgcaa gccgtcagta tgttgcatcc actccacaat atcctcagac aacgccaatc 300
agatgtgagc ttcctccatt ttttatgtgt tctcgtgtgg tgctcatgag acttggaatc 360
ctgaagtgta tagaatacca atgggccgtt ttagctagtt caatgcaaga tttcaatcgt 420
tcattcgatt crcaaagtgc actagcatac ggtggacctc aagcaactcc gattggagtt 480
ggtgtagcgc ctcaacaagt tccgatcaca atggatccya gacctgtmcc actctaccga 540
ggaaatcagg cacggttatt cctttattat tgtagtgacg gattatttca tgcttgcctt 600
tgtgtttttt cattaagttt tcgttcttcc aaaagggtta cagtgggtaa gactcttggg 660
cacagcatga gatgaggatg cggcatgatc ga 692
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Claims (10)
1. A gene for detecting sensitivity of Haemonchus contortus to ivermectin,
the nucleotide sequence of the gene is shown as SEQ ID NO: 1:
CTAGTTCAATGCAAGATTTCAATCGTTCATTCGATTCRCAAAGTGCACTAGCATACGGTGGACCTCAAGCAACTCCGATTGGAGTTGGTGTAGCGCCTCAACAAGTTCCGATCACAATGGATCCYAGACCTGTMCCACTCTACCGAGGAAATCAG。
2. a gene for detecting sensitivity of Haemonchus contortus to ivermectin,
the nucleotide sequence of the gene is shown as SEQ ID NO: 2:
CATGATGGGCGTCTCGGAGTTGGAGATAGAATTTTAGCTGTGAGACTTTTTTTTCATAAAATCCAGATTTCTCTTTTAAATGGCTAGGATGTACTCTTAAAAAAAGACTTTCAGGTTGATGACATCGGACTTGAGAATGTTACACATGAGTATGCCGTCGATGTATTGAAGCGAACCGGCACGAAAGTCACATTGTTGGTGCTTAAAGCAGACCCAAATGTGAATGTCAGCTCAATTGAAGAAACTGCAAGCCGTCAGTATGTTGCATCCACTCCACAATATCCTCAGACAACGCCAATCAGATGTGAGCTTCCTCCATTTTTTATGTGTTCTCGTGTGGTGCTCATGAGACTTGGAATCCTGAAGTGTATAGAATACCAATGGGCCGTTTTAGCTAGTTCAATGCAAGATTTCAATCGTTCATTCGATTCRCAAAGTGCACTAGCATACGGTGGACCTCAAGCAACTCCGATTGGAGTTGGTGTAGCGCCTCAACAAGTTCCGATCACAATGGATCCYAGACCTGTMCCACTCTACCGAGGAAATCAGGCACGGTTATTCCTTTATTATTGTAGTGACGGATTATTTCATGCTTGCCTTTGTGTTTTTTCATTAAGTTTTCGTTCTTCCAAAAGGGTTACAGTGGGTAAGACTCTTGGGCACAGCATGAGATGAGGATGCGGCATGATCGA；
wherein 395-549bp is a CDS region shown as SEQ ID NO. 1.
The application of the gene shown in SEQ ID NO. 1 or SEQ ID NO. 2 in the detection of the sensitivity of Haemonchus contortus to ivermectin,
sensitivity of Haemonchus contortus to ivermectin is analyzed according to polymorphism of single nucleotides at positions 38, 125 and 134 of the gene shown in SEQ ID NO. 1.
4. Use according to claim 3,
the gene shown in SEQ ID NO. 1 shows G/A polymorphism at the 38 th site, T/C polymorphism at the 125 th site and C/A polymorphism at the 134 th site;
the haemonchus contortus gene to be detected is sensitive to ivermectin if at least one of 38G, 125T and 134C is present.
Application of the gene shown in SEQ ID NO. 1 or SEQ ID NO. 2 in preparing a kit for detecting sensitivity of Haemonchus contortus to ivermectin.
6. A kit for detecting sensitivity of Haemonchus contortus to ivermectin is characterized in that,
comprises an upstream primer and a downstream primer for amplifying a gene shown by SEQ ID NO. 1 or SEQ ID NO. 2.
7. The kit for detecting sensitivity of Haemonchus contortus to ivermectin according to claim 6,
the upstream primer is HCIS-F: 5'-CATGATGGGCGTCTCGGAGT-3', SEQ ID NO: 3;
the downstream primer is HCIS-R: 5'-TCGATCATGCCGCATCCTCA-3', SEQ ID NO: 4.
8. The kit for detecting sensitivity of Haemonchus contortus to ivermectin according to claim 6,
also comprises a standard reference substance;
the standard reference substance is a recombinant plasmid carrying a gene shown in SEQ ID NO. 1 or SEQ ID NO. 2.
9. A method of detecting sensitivity of haemonchus contortus to ivermectin, comprising the steps of:
(1) extracting genomic DNA of haemonchus contortus to be detected;
(2) amplifying a gene shown as SEQ ID NO 1 or SEQ ID NO 2 by using the DNA obtained in the step (1) as a template;
(3) carrying out agarose gel electrophoresis on the amplification product, and sequencing the amplification product with the target band;
(4) and analyzing the sequence obtained by sequencing to determine whether the population to which the haemonchus contortus is to be detected is sensitive to ivermectin.
10. The method of claim 9, wherein the sensitivity of Haemonchus contortus to ivermectin is determined by the sensitivity of Haemonchus contortus to ivermectin,
detecting using the kit of claim 7;
the PCR amplification reaction conditions are as follows:
pre-denaturation at 95 ℃ for 5 min;
denaturation at 95 ℃ for 30sec, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles;
extending for 5min at 72 ℃;
the mixture was stored at 4 ℃.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001054507A1 (en) * | 2000-01-28 | 2001-08-02 | Akkadix Corporation | Methods for killing nematodes and nematode eggs using oxadiazole and oxaimidazole compounds |
| CN1401001A (en) * | 2000-02-15 | 2003-03-05 | 联邦科学及工业研究组织 | 5HT3 receptors of nematodes, polynucleotide molecules encoding same, and antagonists thereof |
| CN101848710A (en) * | 2007-11-09 | 2010-09-29 | 英特威国际有限公司 | Anthelmintic combination |
| CN104531892A (en) * | 2015-01-19 | 2015-04-22 | 华中农业大学 | Loop-mediated isothermal amplification detection kit and detection method for haemonchus contortus |
| CN107460243A (en) * | 2017-08-16 | 2017-12-12 | 华中农业大学 | The primer in haemonchus contortus Genes relating to drug resistance mutational site and application |
| CN111537716A (en) * | 2020-06-04 | 2020-08-14 | 南京农业大学 | Haemonchus contortus early infection diagnostic kit |
| CN112794893A (en) * | 2021-01-29 | 2021-05-14 | 华中农业大学 | Construction method and application of Haemonchus contortus Hc-H11-2 recombinant protein |
-
2021
- 2021-10-08 CN CN202111171379.9A patent/CN113755610B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001054507A1 (en) * | 2000-01-28 | 2001-08-02 | Akkadix Corporation | Methods for killing nematodes and nematode eggs using oxadiazole and oxaimidazole compounds |
| CN1401001A (en) * | 2000-02-15 | 2003-03-05 | 联邦科学及工业研究组织 | 5HT3 receptors of nematodes, polynucleotide molecules encoding same, and antagonists thereof |
| CN101848710A (en) * | 2007-11-09 | 2010-09-29 | 英特威国际有限公司 | Anthelmintic combination |
| CN104531892A (en) * | 2015-01-19 | 2015-04-22 | 华中农业大学 | Loop-mediated isothermal amplification detection kit and detection method for haemonchus contortus |
| CN107460243A (en) * | 2017-08-16 | 2017-12-12 | 华中农业大学 | The primer in haemonchus contortus Genes relating to drug resistance mutational site and application |
| CN111537716A (en) * | 2020-06-04 | 2020-08-14 | 南京农业大学 | Haemonchus contortus early infection diagnostic kit |
| CN112794893A (en) * | 2021-01-29 | 2021-05-14 | 华中农业大学 | Construction method and application of Haemonchus contortus Hc-H11-2 recombinant protein |
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