Primer and probe for detecting FMO3 gene A1034T mutation of chicken

A technology of A1034T and FMO3-, which is applied in the field of TaqMan MGB probes, can solve the problems that the detection results are easily affected by experimental conditions, cumbersome steps, and low sensitivity, and achieve reliable detection results, simple workflow, and high sensitivity.

Inactive Publication Date: 2013-03-27
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to overcome the shortcomings of the existing PCR-RFLP method to detect the existence of the mutation site, which is cumbersome, time-consuming, low sensitivity, and detection results are easily affected by experimental conditions, and to provide a method that can accurately and efficiently detect the FMO3 gene. Ap

Method used

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  • Primer and probe for detecting FMO3 gene A1034T mutation of chicken
  • Primer and probe for detecting FMO3 gene A1034T mutation of chicken
  • Primer and probe for detecting FMO3 gene A1034T mutation of chicken

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 detects the design of specific primers and probes of chicken FMO3 gene A1034T mutation site

[0047] The invention obtains a pair of fluorescent quantitative PCR primers for detecting the A1034T mutation site of the chicken FMO3 gene and a TaqMan probe used in conjunction with the primers through repeated screening and verification.

[0048] Upstream primer FMO3 F: 5'-GTGCAGGACGACCTCGAT-3' (SEQ ID NO.1)

[0049] Downstream primer FMO3 R: 5'-CTCCATGAAAGGAAAGGAGTGAGA-3' (SEQ ID NO.2).

[0050] The upstream primer consists of 18 bases, corresponding to bases 1001-1018 of the chicken FMO3 gene cDNA sequence in GenBank (accession number: AJ431390); the downstream primer consists of 24 bases, corresponding to the 1043rd base of the chicken FMO3 gene cDNA sequence ~1066 base sequences. The length of the amplified fragment of the primer pair is 66bp.

[0051] The nucleotide sequence of the TaqMan fluorescent probe used in conjunction with the above specific prim...

Embodiment 2

[0055] Example 2 Chicken Anticoagulant Genomic DNA Extraction

[0056] This example contains a total of 50 Beijing oil chickens, of which 6 young chickens (8 weeks old) are used for the establishment of the TaqMan probe real-time fluorescent quantitative PCR detection method, and the other 44 hens are 40-week-old hens waiting for pure breeding. Choose to keep the hen. They were raised in the Yufa Breeding Chicken Farm of Beijing Academy of Agriculture and Forestry Sciences, blood was collected from the subwing vein, and anticoagulated with ACD (citric acid 0.48g, sodium citrate 1.32g, glucose 1.47g, added distilled water to 100ml).

[0057] Chicken Anticoagulant Genomic DNA Extraction Kit DP318 (Tiangen Biochemical Technology Co., Ltd., Beijing) was used to extract genomic DNA. The specific steps are as follows:

[0058] 1) Take out the sample, after melting, pipette 20μL into a 2mL centrifuge tube;

[0059] 2) Add 180 μL buffer GS, 20 μL proteinase K, and mix well;

[0060...

Embodiment 3

[0071] Example 3 Establishment of TaqMan probe real-time fluorescent quantitative PCR detection method for chicken FMO3 gene A1034T mutation

[0072] 1. Determination of sample DNA genotype

[0073] The DNA concentration of the 6 chickens obtained in Example 2 was detected again using NanoDrop2000 chromatography, and the DNA concentrations of chickens A, B, C, D, E, and F were respectively: 14.5ng / μL and 14.5ng / μL , 13.3ng / μL, 32.2ng / μL, 23.2ng / μL, 29.9ng / μL.

[0074]Using the genomic DNA of the above 6 chickens as a template, PCR amplification was performed using the following primer pair (Zhang Longchao, 2006). The upstream primer FMO_F1: 5'-CAGGGTGGTATCAAGCCTGT-3', the downstream primer FMO_R1: 5'-GATCCAAAGGACTGGACCAA-3' . The amplification reaction system is a 25 μL system, including: 2 μL (50 ng) of the genomic DNA template of 6 chickens obtained in Example 2, 0.5 μL of Taq enzyme (Takara), 2.5 μL of 10×buffer, 2.0 μL of dNTP, upstream and downstream primers (10 pmol / ...

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Abstract

The invention provides a primer and a probe for detecting FMO3 gene A1034T mutation of a chicken. A nucleotide sequence is shows as EQ ID NO.1-4. The primer and the probe can be utilized to quickly and sensitively detect FMO3 gene A1034T mutation sites of the chicken by adopting the real-time fluorescence quantification polymerase chain reaction (PCR) technology, namely gene type of fishlike smell sensitive genes, so that whether the chicken carries fishlike smell sensitive gene sites or not can be accurately judged. Compared with the prior art, the primer and probe have the advantages of being simple in operation, high in detection speed, capable of judging types by only one PCR, high in automation degree, high in throughout and high in sensitivity, saving time and improving detection efficiency. The whole reaction process is conducted in a closed tube, and cross pollution is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers and specific TaqMan MGB probes for detecting chicken FMO3 gene A1034T mutation, a detection method and a kit. Background technique [0002] In 2005, Honkatukia et al. found that a missense mutation in the flavine containing monooxygenase 3 (FMO3) gene located on chicken chromosome 8 was the cause of fishy-smelling eggs. The mutation is located on exon 7 of the FMO3 gene, and the 1034th base adenine (A) of the mutant individual cDNA sequence (GenBank accession number: AJ431390) is replaced by thymine (T), which makes the 329th base of the encoded protein The amino acid at the position is changed from threonine (T) to serine (S), so this mutation is also named T329S mutation. The mutation from threonine to serine at position 329 changes a highly conserved region (FATGY) of flavin monooxygenase. It is speculated that this region may be the binding region for substrate recogniti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 初芹刘华贵朱珊珊张剑王海宏耿爱莲
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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