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A set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes

A genetic defect and kit technology is applied in the field of a set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes, which can solve the problems of easy introduction of errors, misjudgments, poor repeatability, etc. Detecting difficult effects

Active Publication Date: 2020-03-17
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] China authorized invention patent, CN200610150317.9, a method for detecting harmful genes of bovine CVM, the invention provides a method for detecting bovine vertebral deformity syndrome (CVM) carriers, the method first designs synthetic primers for bovine DNA samples Carry out polymerase chain reaction (PCR), then carry out single-strand conformation polymorphism (SSCP) analysis to the PCR product, judge whether the cattle carry CVM harmful gene according to the result of polyacrylamide gel electrophoresis, its shortcoming is that can only detect Single CVM carrying case, and poor repeatability
[0010] China authorized invention patent, CN201310617444.5, a primer composition for the detection of harmful genes of tauridylate synthase deficiency and its kit and application, the invention discloses a detection of harmful genes of tauridin synthase deficiency The primer composition and kit and application thereof, the detection method of the invention is to first extract the whole group of DNA in bovine blood as a template, carry out nested PCR (NestedPCR) amplification, and the obtained PCR products are sequenced, and according to the sequenced results can be Directly understand the base change of the mutation site, which is characterized by two amplifications and sequencing of the product, which is easy to introduce errors and can only detect a single defective gene
[0011] China authorized invention patent, CN200910272954.7, invented a method for detecting tauridylate synthase deficiency by using AS-PCR. The method of enzyme deficiency, prepared a specific PCR primer for the detection method of tauridylate synthase deficiency, introduced mismatched bases in the amplified product, and needed to use electrophoresis for genotyping, because the product The length is short, it is easy to cause misjudgment, and it is not suitable for high-throughput and large-scale herd detection
[0012] The Chinese authorized invention patent CN201110440150.0 discloses a method for screening bovine degenerative axon disease carriers and its kit. The invention uses the genomic DNA of the cattle to be tested as a template, and designs upstream primers and downstream primers to carry it out. PCR amplification reaction, and then recover the PCR amplified fragment, digest it with FastDigest restriction enzyme TspRI, and perform gel detection after digestion in the enzyme digestion reaction system. This screening method is simple, fast, and low in cost, but there are also limitations Can detect the deficiency of a single genetic defect gene
[0013] At present, there is no research on methods for detecting other genetic defect genes and lethal haplotypes at home and abroad, and there is no research on high-throughput detection methods that can simultaneously detect hundreds of known genetic defect genes in cattle.

Method used

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  • A set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes
  • A set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes
  • A set of primer combinations and kits for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Construction of a method for simultaneous detection of 93 bovine genetic defect genes

[0083] 1. PCR amplification reaction primer design and condition optimization

[0084] Organize the SNP sequence information into a standard format, design PCR amplification reaction primers and single-base extension primers for 93 causal mutation sites (SNPs, insertions or deletions of short fragments), and check the conditions that affect the detection reaction system and PCR procedures. Optimization, including reagent concentration, temperature, time, number of reaction cycles, etc., and then a 384-well PCR reaction (93 causal mutation sites were divided into 4 PCR wells for simultaneous detection). The PCR amplification reaction is as follows:

[0085] (1) Use AssayDesigner3.1 software to design primers for each mutation site sequence, analyze the primers after the optimization test, and mix the PCR primers required for detecting mutation sites in each well with deion...

Embodiment 2

[0104] Embodiment 2: The kit for detecting 93 kinds of bovine genetic defect genes

[0105] The composition of the kit includes: 1-4 well amplification primer mix (500nM each), 1-4 well extension primer mix (7μM: 14μM), dNTP (25mM each), MgCl 2 (25mM), 0.100μl HotStar Taq (5U / μl), PCR Bμfferwith 15mM MgCl 2 , Shrimp Alkaline Phosphatase Buffer, Shrimp Alkaline Phosphatase (1U / μl), iPLEX Bμffer Plus, iPLEX Termination mix, iPLEX Enzyme, ddH 2 O, 384Dimple board, 384 sample board, resin.

[0106] Wherein, the sequence of the amplification primer mixture and the extension primer mixture described in the kit is as described in the summary of the invention above. The detection steps of bovine tissue samples using the kit are as follows:

[0107] (1) Use the reagents in the kit to carry out PCR amplification reaction on the DNA in bovine blood, semen or hair follicles qualified for quality control. The causal mutation sites of 93 kinds of bovine genetic defects in each sample are...

Embodiment 3

[0116] Example 3: Sample detection of different genetic defect genes and characteristic milk genes of cattle

[0117] The blood samples of 96 Holstein cows were collected, and the causal mutation sites (SNP, insertion or deletion of short fragments) of the cow samples were detected by using the reagents in the kit of Example 2 of the present invention and related steps.

[0118] It was found that some cattle carried (heterozygous) genetic defect genes and lethal haplotypes ( Figure 3-Figure 10 ) and verified by sequencing. The genotype detection results of the specific samples are shown in Table 5 below:

[0119] Table 5. Detection results of bovine genetic defect genes and lethal haplotypes (phenotype traits)

[0120] Genetic defect (phenotypic trait) Number of samples (genotype) Lethal Haplotype (HH1) 3 heads carry Lethal Haplotype (HH3) 7 heads carry Spinal deformity syndrome (HHC) 4 heads carry Recessive Red Hair 373 2 carry ...

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Abstract

The invention discloses a primer combination and a kit for simultaneous detection of 93 bovine genetic defect genes and lethal haplotypes. Through use of the primer combination and the kit, casual mutation sites (SNP and insertion or deletion of short fragments) of the 93 genetic defect genes (such as PIRM syndrome, crooked tail syndrome, epidermolysis bullosa and the like) and the lethal haplotypes (HH1, HH3, HH4, HH5 and JH1) can be detected in one time, cattle with monogenic inheritance defect gene carriers and lethal haplotype carriers can be screened, and accurate guidance is provided for cattle breeding and genetic evaluation, herd selection and assortative mating, population genetic improvement, cultivation of new varieties and genetic resource protection. The primer combination and the kit have the characteristics of high throughput, low cost, high accuracy, and the like, is suitable for the varieties of beef cattle and dairy cattle, and can be widely used in the breeding and reproduction field of the cattle.

Description

technical field [0001] The invention relates to the technical field of animal husbandry, and relates to a detection method for determining whether cattle carry 93 bovine genetic defect genes and lethal haplotypes, specifically, using DNA extraction, PCR multiple amplification technology, single base Extended technology and flight mass spectrometry technology, genotyping 93 genetic defect genes and lethal haplotypes at the same time, to determine whether the cattle carry recessive genetic defect genes and lethal haplotypes, involving recessive genetic defect genes and lethal haplotypes in cattle Type elimination and individual labeling, selection and matching of cattle herds, screening methods for dairy cows with specific type of β-casein milk, etc. Background technique [0002] So far, there are 506 known genetic defects in cattle, 233 of which belong to Mendelian inheritance, of which 133 are known to cause mutations (http: / / omia.angis.org.au / home / ), most Mutations are har...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2521/525C12Q2533/101C12Q2565/627
Inventor 黄金明王秀革姜强鞠志花张燕李建斌杨春红王长法孙艳戴蕴平仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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