CN108179197A - Chicken ring RNA Chr9:10814512 | 10838667 genetic test primers, method and application - Google Patents

Chicken ring RNA Chr9:10814512 | 10838667 genetic test primers, method and application Download PDF

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CN108179197A
CN108179197A CN201810019252.7A CN201810019252A CN108179197A CN 108179197 A CN108179197 A CN 108179197A CN 201810019252 A CN201810019252 A CN 201810019252A CN 108179197 A CN108179197 A CN 108179197A
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rna
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chicken
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target gene
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CN108179197B (en
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李显耀
郑林娜
刘丽英
王园美
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Shandong Agricultural University
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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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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract

The invention discloses a breeder ring RNA Chr9:10814512 | detection primer, method and the application of 10838667 genes.The detection primer includes chicken ring RNA Chr9:10814512 | 10838667 cyclic annular sense primers and chicken ring RNA Chr9:10814512 | 10838667 cyclic annular downstream primers, nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.Using above-mentioned primer and method, Bacterium enteritidis infection resistance correlative link rna expression situation can be accurately detected, is that the anti-Bacterium enteritidis of chicken infects the screening of Resistant Individuals and breeding for disease resistance is provided fundamental basis and scientific basis.

Description

Chicken ring RNA Chr9:10814512 | 10838667 genetic test primers, method and Using
Technical field
The invention belongs to gene engineering technology field, specifically, being related to a breeder ring RNA Chr9:10814512| Detection primer, method and the application of 10838667 genes.
Background technology
Bacterium enteritidis (Salmonella enteritidis, SE) is common infecting both domestic animals and human pathogen, because of its nothing Host specificity, therefore it is lethal that the diseases such as poultry acute gastroenteritis can not only be caused even to fall ill, and is caused to aviculture huge Economic loss, and human health is seriously endangered, it causes to poison by food, is one of important pathogen for influencing people's physical and mental health. Related report is pointed out, during 2004, has 19000 people to be hospitalized when infecting salmonella in every 1 million people in the U.S., up to There is death because of salmonella infection in 300 people；There are about foods caused by 99020 cause salmonella infections between European Union, 2010 Source property illness outbreak.Poultry and its related converted products are the major source of infection of Bacterium enteritidis, and caecum is its main host, Bacterium enteritidis can enter through eggshell in scrambled egg product, and breeding can be deposited in genital tract.Control Salmonella at present The main method of bacterium infection is to use vaccine and antibiotic, but this cannot tackle the problem at its root, and antibiotic is a large amount of Using the drug resistance of food-safety problem and bacterium can be caused, and drug tolerance of strain is caused to enhance.With the hair of molecular genetics Exhibition improves genetic resistance of the livestock and poultry to disease and cause of disease, and the infection of salmonella in order to control provides new possibility.
Ring RNA is a kind of novel non-coding RNA.Ring RNA is in closed circular structure, is not influenced by RNA excision enzymes, is expressed It is more stable, it is not degradable.5 ' -3 ' polarity are not contained.But ring RNA has the extensive abundant table of tissue specificity in animal It reaches.Functionally, in recent years research shows that, loop RNA molecule be rich in microRNA (miRNA) binding site, played in cell The effect of miRNA sponges (miRNA sponge), and then inhibiting effect of the miRNA to its target gene is released, increase derived genes Expression；This mechanism of action is referred to as competitive endogenous RNA (ceRNA) mechanism.Pass through the miRNA phases with disease association Interaction, ring RNA play important regulating and controlling effect in disease.
Invention content
In view of this, the present invention provides a breeder ring RNA Chr9:10814512 | the detection of 10838667 genes is drawn Object, method and application.
In order to solve the above-mentioned technical problem, the invention discloses a breeder ring RNA Chr9:10814512 | 10838667 bases The detection primer of cause, including chicken ring RNA Chr9:10814512 | 10838667 cyclic annular sense primers and chicken ring RNA Chr9: 10814512 | 10838667 cyclic annular downstream primers, nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
The invention also discloses a breeder ring RNA Chr9:10814512 | the detection method of 10838667 genes, including with Lower step：
The extraction of step 1, sample to be tested RNA；
Step 2, progress reverse transcription obtain cDNA；
Step 3, with the primer described in claim 1, quantitative pcr amplification is carried out to sample to be tested cDNA, obtains PCR expansions Increase production object；
Step 4 usesMethod calculates gene relative expression quantity, and specific formula for calculation is as follows：
Δ CT=CTTarget gene-CTGAPDH, Δ Δ CT=Δs CTTarget gene-△CTReference gene；
Wherein, △ CT represent the CT values that the required CT values for verifying gene subtract reference gene；
CTTarget geneThe CT values of verification gene needed for representing；
CTGAPDHRepresent the CT values of reference gene；
The △ CT values that △ △ CT represent target gene subtract the average value of control group target gene △ CT；
△CTTarget geneRepresent the △ CT values of target gene；
△CTReference geneRepresent the average value of control group target gene △ CT；
Step 5 judges whether sample to be tested contains chicken ring RNA according to pcr amplification product and gene relative expression quantity Chr9:10814512 | 10838667 genes and its expression.
Further, the extraction of the sample to be tested RNA in the step 1 is specially：It is total using Trizol methods extraction sample RNA, extracting gained total serum IgE carry out quality inspection through agilent bio-analyser 2100 and Qubit 2.0.
Further, the carry out reverse transcription in the step 2 obtains cDNA and is specifically implemented according to the following steps：Select enteritis Salmonella negative individuals are inoculated with Bacterium enteritidis, inoculum concentration 10 by oral method7-109Cfu/, the 7th after inoculation My god, cecal content is taken, bacterial content is measured using colony counting method, takes caecal tissue sample, extracts RNA, and with RNase R RNA processing to extraction digests linear rna, carries out reverse transcription generation cDNA.
Further, the quantitative PCR reaction system in the step 3 is as follows：2x SYBR Premix Ex TaqTM 10μ L, 10 μM of chicken ring RNA Chr9:10814512 | 10838667 cyclic annular sense primer 0.5 μ L, 10 μM of chicken ring RNA Chr9: 10814512 | 10838667 cyclic annular downstream primer 0.5 μ L, sample to be tested cDNA 2.0 μ L, dH27 μ L of O, more than total volume are 20 μL。
Further, the quantitative pcr amplification condition in the step 3 is：95 DEG C, 30s；95 DEG C, 5s, 59 DEG C, 30s, 40 A cycle；95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s, 1 cycles.
The invention also discloses a kind of above-mentioned chicken ring RNA Chr9:10814512 | the detection primer of 10838667 genes Application in the anti-Bacterium enteritidis infection of detection chicken.
The invention also discloses a kind of above-mentioned chicken ring RNA Chr9:10814512 | the detection method of 10838667 genes Application in the anti-Bacterium enteritidis infection of detection chicken.
Compared with prior art, the present invention can be obtained including following technique effect：
The present invention is according to chicken ring RNA Chr9:10814512 | it is cyclic annular and linear to design specificity for 10838667 gene orders Primer, using the expression of fluorescence quantitative PCR detection ring RNA, analysis ring rna expression is related to cecal content bacterial content Property.This method provides chicken ring RNA Chr9:10814512 | PCR conditions, utilization are above-mentioned used in 10838667 quantitative expressions Primer and method can accurately detect Bacterium enteritidis infection resistance correlative link RNA expressions, be the anti-enteritis sramana of chicken Salmonella infects the screening of Resistant Individuals and breeding for disease resistance is provided fundamental basis and scientific basis.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaches all the above technique effect.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and forms the part of the present invention, this hair Bright illustrative embodiments and their description do not constitute improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is PCR product electrophoretogram of the present invention；Wherein, M:DL2000marker；1:Genomic DNA primer draws for ring-type Object；2,3：CDNA primers are Loop primer；4,5：CDNA primers are linear primer；6 represent genomic DNA primer draws as ring-type Object, 7,8 represent cDNA primers as Loop primer, and 9,10 represent cDNA primers represents genomic DNA primer as linear primer, 11 For Loop primer, 12,13cDNA primers are Loop primer, and 14,15 represent cDNA primers as linear primer；
Fig. 2 is chicken ring RNA Chr9 of the present invention:10814512 | 10838667 relative quantification expression.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand Section can fully understand and implement according to this to solve technical problem and reach the realization process of technical effect.
1 chicken ring RNA Chr9 of embodiment:10814512 | the foundation of the detection method of 10838667 genes
Bacterium enteritidis negative individuals are selected, Bacterium enteritidis, inoculum concentration 10 are inoculated with by oral method7- 109Cfu/ only, the 7th day after inoculation, takes cecal content, and bacterial content is measured using colony counting method.Caecal tissue sample is taken, RNA is extracted, and with RNA processing of the RNase R to extraction, digest linear rna, carry out reverse transcription generation cDNA.
According to chicken ring RNA Chr9:10814512 | 10838667 gene orders design specificity ring-type and linear primer (table 1-2) is expanded.
Table 1：Chicken ring RNA Chr9:10814512 | 10838667 Loop primer sequences
Table 2：Chicken ring RNA Chr9:10814512 | 10838667 linear primer sequences
Quantitative pcr amplification, pcr amplification reaction system such as the following table 3 are carried out using above-mentioned specific quantification Loop primer.
Table 3：Quantitative PCR reaction system (20 μ L)
Quantitative pcr amplification condition is：95 DEG C, 30s；40 cycles (95 DEG C, 5s, 59 DEG C, 30s)；1 cycle (95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s).
Regular-PCR amplification, pcr amplification reaction system such as the following table 4 are carried out using above-mentioned linear primer.
Table 4：Pcr amplification reaction system
Regular-PCR amplification condition is：30 cycles (98 DEG C, 10s, 55 DEG C, 30s, 72 DEG C, 1min).
Utilize above-mentioned chicken ring RNA Chr9:10814512 | 10838667 specific Loop primer, according to above-mentioned 20 μ L bodies System is loaded, and is reacted by PCR amplification condition, so as to analyze chicken ring RNA Chr9:10814512 | 10838667 opposite tables Up to amount.Utilize above-mentioned chicken ring RNA Chr9:10814512 | 10838667 linear primer is added according to above-mentioned 50 μ L systems Sample is reacted by PCR amplification condition, so as to analyze chicken ring RNA Chr9:10814512 | 10838667 amplified productions.
The amplifiable out product only under the conditions of Loop primer and cDNA as can be seen from Figure 1, and in genomic DNA It is not come out with the amplification of similary Loop primer.Product also can not be expanded with the linear primer and cDNA of ring RNA.
Data are arranged using Excel.Gene relative expression quantity usesMethod calculates, and specific formula for calculation is as follows：
△ CT=CTTarget gene-CTGAPDH, △ △ CT=△ CTTarget gene-△CTReference gene。
Wherein, CT values are meant that：Fluorescence signal in each reaction tube reaches the cycle undergone during the thresholding of setting Number；
△ CT verify that the CT values of gene subtract the CT values of reference gene needed for representing；
CTTarget geneThe CT values of verification gene needed for representing；
CTGAPDHRepresent the CT values of reference gene；
The △ CT values that △ △ CT represent target gene subtract the average value of control group target gene △ CT；
△CTTarget geneRepresent the △ CT values of target gene；
△CTReference geneRepresent the average value of control group target gene △ CT.
Chicken ring RNA Chr9:10814512 | the results are shown in Figure 2 for 10838667 quantitative expression.
As a result show infection Bacterium enteritidis after the 7th day chicken ring RNA Chr9:10814512 | 10838667 opposite table It it is 5.95 times and difference extremely significantly (P of control group up to amount up-regulation infected group<0.05).
Therefore, chicken ring RNA Chr9:10814512 | 10838667 expression quantity infect the notable phase of resistance with Bacterium enteritidis Close, can be used as Bacterium enteritidis infection resistance molecular labeling, for Bacterium enteritidis infection Resistant Individuals selection with The resistant heredity breeding of chicken.
Several preferred embodiments of invention have shown and described in above description, but as previously described, it should be understood that invention is not Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification And environment, and can be carried out in the scope of the invention is set forth herein by the above teachings or related fields of technology or knowledge Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in power appended by invention In the protection domain of profit requirement.
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Claims (8)

  1. A 1. breeder ring RNA Chr9:10814512 | the detection primer of 10838667 genes, which is characterized in that including chicken ring RNA Chr9:10814512 | 10838667 cyclic annular sense primers and chicken ring RNA Chr9:10814512 | draw in 10838667 cyclic annular downstreams Object, nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
  2. A 2. breeder ring RNA Chr9:10814512 | the detection method of 10838667 genes, which is characterized in that including following step Suddenly：
    The extraction of step 1, sample to be tested RNA；
    Step 2, progress reverse transcription obtain cDNA；
    Step 3, with the primer described in claim 1, quantitative pcr amplification is carried out to sample to be tested cDNA, obtains PCR amplification production Object；
    Step 4, using 2- Δ ΔsCTMethod calculates gene relative expression quantity, and specific formula for calculation is as follows：
    Δ CT=CTTarget gene-CTGAPDH, Δ Δ CT=Δs CTTarget gene-ΔCTReference gene；
    Wherein, Δ CT represents the CT values that the required CT values for verifying gene subtract reference gene；
    CTTarget geneThe CT values of verification gene needed for representing；
    CTGAPDHRepresent the CT values of reference gene；
    The Δ CT values that Δ Δ CT represents target gene subtract the average value of control group target gene Δ CT；
    ΔCTTarget geneRepresent the Δ CT values of target gene；
    ΔCTReference geneRepresent the average value of control group target gene Δ CT；
    Step 5 judges whether sample to be tested contains chicken ring RNA Chr9 according to pcr amplification product and gene relative expression quantity: 10814512 | 10838667 genes and its expression.
  3. 3. detection method according to claim 2, which is characterized in that the extraction tool of the sample to be tested RNA in the step 1 Body is：Sample total serum IgE is extracted using Trizol methods, extracting gained total serum IgE is through agilent bio-analyser 2100 and Qubit 2.0 Carry out quality inspection.
  4. 4. detection method according to claim 2, which is characterized in that the carry out reverse transcription in the step 2 obtains cDNA It is specifically implemented according to the following steps：Bacterium enteritidis negative individuals are selected, Bacterium enteritidis is inoculated with by oral method, is connect Kind amount is 107-109Cfu/ only, the 7th day after inoculation, takes cecal content, measures bacterial content using colony counting method, takes blind Intestinal tissue sample extracts RNA, and with RNA processing of the RNase R to extraction, digests linear rna, carries out reverse transcription generation cDNA。
  5. 5. detection method according to claim 2, which is characterized in that the quantitative PCR reaction system in the step 3 is such as Under：2x SYBR Premix Ex TaqTM 10 μ L, 10 μM of chicken ring RNA Chr9:10814512 | draw 10838667 cyclic annular upstreams Object 0.5 μ L, 10 μM of chicken ring RNA Chr9:10814512 | 10838667 cyclic annular downstream primer 0.5 μ L, 2.0 μ of sample to be tested cDNA L, dH27 μ L of O, more than total volume are 20 μ L.
  6. 6. detection method according to claim 2, which is characterized in that the quantitative pcr amplification condition in the step 3 is： 95 DEG C, 30s；95 DEG C, 5s, 59 DEG C, 30s, 40 cycles；95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s, 1 cycles.
  7. 7. chicken ring RNA Chr9 described in claim 1:10814512 | the detection primer of 10838667 genes is in the detection anti-intestines of chicken Application in scorching salmonella infection.
  8. 8. the chicken ring RNA Chr9 described in claim 2-6:10814512 | the detection method of 10838667 genes resists in detection chicken Application in Bacterium enteritidis infection.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013027146A1 (en) * 2011-08-25 2013-02-28 Proteon Pharmaceuticals S.A. The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013027146A1 (en) * 2011-08-25 2013-02-28 Proteon Pharmaceuticals S.A. The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
INTERNATIONAL CHICKEN GENOME SEQUENCING CONSORTIUM: ""Gallus gallus isolate RJF #256 breed Red Jungle fowl, inbred line UCD001 chromosome 9, Gallus_gallus-5.0, whole", 《GENBANK》 *

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