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Method using markers of defensin genes to improve resistance ability of chicken salmonella

A technology for chicken salmonellosis and salmonella, which is applied in the field of genetic engineering, can solve the problems of lack of an effective specific vaccine for the prevention of chicken salmonellosis, inability to prevent and spread salmonellosis, and difficulty in the prevention and control of salmonella, achieving low cost, high accuracy, time saving effect

Pending Publication Date: 2019-08-02
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] In summary, the problems in the prior art are: chicken salmonellosis is mainly caused by Salmonella typhimurium (Salmonella Typhimurium, ST) and Salmonella enterica (Salmonella Enterica, SE) infection, and there are multiple serotypes in the two types of bacteria , there is no effective specific vaccine for the prevention of chicken salmonellosis
The treatment measures are mainly antibiotics, including drugs such as tetracycline and oxytetracycline. These antibiotics can only inhibit or kill Salmonella in the body, but cannot prevent the spread of bacteria in chicken flocks
Because chicken salmonellosis has a vertical transmission mode from adult carrier chickens to breeding eggs and then to chicks, the current isolation and drug treatment measures adopted by chicken farms cannot prevent and spread salmonellosis in chicken farms
[0011] The difficulty and significance of solving the above technical problems: the unique parallel and vertical transmission methods of Salmonella chicken bring great difficulties to the prevention and control of Salmonella. Control, and improve poultry's own immunity, breeding chickens with high resistance to Salmonella may be one of the most effective ways to solve this problem

Method used

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  • Method using markers of defensin genes to improve resistance ability of chicken salmonella
  • Method using markers of defensin genes to improve resistance ability of chicken salmonella
  • Method using markers of defensin genes to improve resistance ability of chicken salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Obtaining of 3 Polymorphic Sites of Chicken Defensin Genes

[0047] 1 Chicken blood sample collection

[0048] In the sample collection, the sex, age, feather color, tail traits and pedigree information of each chicken were recorded in detail, and 2-3ml of whole blood was collected from the wing vein using a heparin sodium anticoagulated vacuum blood collection tube, transported to the laboratory at 4°C, and then Store at -20°C.

[0049] 2 Genomic DNA Extraction

[0050] Chicken wing vein blood collection, all blood samples were 1mL / bird, anticoagulated with EDTA, and the blood samples were frozen at -20°C. Genomic DNA was extracted from the above-mentioned chicken blood by the conventional phenol-chloroform method, and detected by 1.0% agarose electrophoresis. It can be seen that the DNA bands are clear, bright, and free of impurities, which fully meets the requirements of the subsequent PCR test.

[0051] 3 Primer design and synthesis

[0052] According ...

Embodiment 2

[0075] Example 2 Genetic markers of chicken three defensin genes bidirectional allele-specific PCR (Bi-PASA)

[0076] 1 AvBD4 Bi-PASA molecular marker

[0077] According to the sequencing results, the C / T mutation at position 613 of the AvBD4 gene caused a variation from histidine to tyrosine, and a pair of forward and reverse primers were designed at the mutation site, called AvBD4-C (5'-ggggcggggACCCCTGGGAAATGCCCTC-3') and

[0078] AvBD4-Y (5'-ggggcggggCCCAGGTAAGCATTCCCATA-3').

[0079] Chicken AvBD4 gene Bi-PASA amplified 3 fragments in total, one is 845bp fragment, which is the product amplified by primers AvBD4-FI and AvBD4-R1, which can be used as a positive control for PCR amplification; the other is 616bp fragment, which is the primer The amplified product of AvBD4-F1 and AvBD4-T is TT type; the other is a 229bp fragment amplified by primers AvBD4-C and AvBD4-R1, which is CC type. Chicken AvBD4 gene Bi-PASA electrophoresis detection results see Figure 7 .

[0080...

Embodiment 3

[0089] Example 3 Association Analysis of 3 Chicken Defensin Genes and Salmonella Susceptibility

[0090] 1. Association analysis of the susceptibility of Salmonella with a single SNP of 3 defensin genes

[0091] 1(1) Association analysis of single SNPs of AvBD4 gene and the susceptibility to Salmonella

[0092] Using the logistic regression method, the correlation between the single SNPs of the AvBD4 gene and the susceptibility to Salmonella was analyzed under the codominant inheritance mode, and no correlation was found between the SNP site and the susceptibility to Salmonella (P>0.05). The results are shown in Table 15 .

[0093] Table 15 Correlation analysis between AvBD4 gene polymorphism and susceptibility to Salmonella chicken

[0094]

[0095]

[0096] Note: "NA" means absent or absent; OR: odds ratio; CI: confidence interval; AIC: Akaike information criterion; BIC::Bayesian information criterion.

[0097] (2) Association analysis of single SNPs of AvBD5 gene a...

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Abstract

The invention belongs to the technical field of mutation or genetic engineering, discloses a method using Bi-PASA markers of three defensin genes to improve resistance ability of chicken salmonella, aprimer and a kit, and provides bidirectional allele specific PCR (polymerase chain reaction) (Bi-PASA) genetic markers of three defensin genes. The method is characterized in that the Bi-PASA technique is adopted for the first time; on the basis of analysis of susceptibility of chicken salmonellosis correlated with the variance of the three defensin genes, after the SNP (single nucleotide polymorphism) site and gene haplotype genetic variance are analyzed, the three defensin genes as function genes for resisting the chicken salmonellosis are disclosed, the simple, time-shortening, low-cost, quick and effective Bi-PASA detection marker for the three defensin genes is established, and the positive guidance meaning is realized on the gene purification and disease-resistant breeding. The three defensin genes are used for the gene detection of resistance ability of chicken salmonellosis and the molecular marker-assisted breeding, and have the characteristics of early screening, quick identification, time shortening, low cost, high accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method, a primer and a kit for improving chicken Salmonella resistance by using three defensin gene Bi-PASA markers. Background technique [0002] At present, the commonly used prior art in the industry is as follows: Salmonellosis is a common zoonotic infectious disease, which not only causes huge economic losses to animal husbandry production, but also seriously threatens human health. Salmonellosis in chickens manifests as pullorum in young chickens and typhoid fever in adult chickens. Chicken Salmonella spreads vertically and horizontally. On the one hand, sick chickens and infected chickens can infect poultry raised in the same group through secretions of the digestive tract and respiratory tract. Become a congenital carrier. Currently, there are no effective control measures for Salmonella infection. Therefore, cultivating Salmonella-resistant c...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156Y02A50/30
Inventor 史宪伟李杨陈多珍杨向东罗袁山
Owner YUNNAN AGRICULTURAL UNIVERSITY
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