CN107988404A - Chicken ring RNAChr26:2670958 | 2679178 detection primer, method and application - Google Patents

Chicken ring RNAChr26:2670958 | 2679178 detection primer, method and application Download PDF

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Publication number
CN107988404A
CN107988404A CN201810020111.7A CN201810020111A CN107988404A CN 107988404 A CN107988404 A CN 107988404A CN 201810020111 A CN201810020111 A CN 201810020111A CN 107988404 A CN107988404 A CN 107988404A
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rna
ring
chr26
chicken
gene
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Inventor
李显耀
郑林娜
刘丽英
王园美
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Shandong Agricultural University
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Shandong Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a breeder ring RNA Chr26:2670958 | 2679178 detection primer, method and application.The primer includes chicken ring RNA Chr26:2670958 | 2679178 ring-type sense primers and chicken ring RNA Chr26:2670958 | 2679178 ring-type anti-sense primers；Its nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.Using method provided by the present invention, chicken ring RNA Chr26 after chicken infection Bacterium enteritidis can be accurately detected:2670958 | 2679178 relative expression's situations, in this, as molecular markers for identification salmonella infection Resistant Individuals, the genetic breeding for the anti-Bacterium enteritidis infection of chicken is provided fundamental basis and scientific basis.

Description

Chicken ring RNA Chr26:2670958 | 2679178 detection primer, method and application
Technical field
The invention belongs to gene engineering technology field, specifically, is related to a breeder ring RNAChr26:2670958| 2679178 detection primer, method and application.
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, cause to poison by food, be one of important pathogen for influencing people's physical and mental health. Related report is pointed out, during 2004, has 19000 people to be in hospital because of infection salmonella in every 1 million people in the U.S., up to There is death because of salmonella infection in 300 people；It there are about food 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 pass through eggshell and enter 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 strengthen.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 new non-coding RNA.Ring RNA is in closed circular structure, is influenced from RNA excision enzymes, expression It is more stable, it is not degradable.5 ' -3 ' polarity are not contained.But ring RNA tables abundant extensively with tissue specificity in animal Reach.Functionally, research in recent years shows, loop RNA molecule is rich in microRNA (miRNA) binding site, is played in cell The effect of miRNA sponges (miRNA sponge), and then inhibitory action of the miRNA to its target gene is released, raise 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.
The content of the invention
In view of this, the present invention provides a breeder ring RNA Chr26:2670958 | 2679178 detection primer, method And application.
In order to solve the above-mentioned technical problem, the invention discloses a breeder ring RNA Chr26:2670958 | 2679178 Detection primer, including chicken ring RNA Chr26:2670958 | 2679178 ring-type sense primers and chicken ring RNA Chr26:2670958 | 2679178 ring-type anti-sense primers, its nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
The invention also discloses a breeder ring RNA Chr26:2670958 | 2679178 detection method, 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 expansions Increase production thing；
Step 4, using 2-△△CTMethod calculates gene relative expression quantity, and specific formula for calculation is as follows：
△ CT=CTTarget gene-CTGAPDH, △ △ CT=△ 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, according to pcr amplification product and gene relative expression quantity judge whether sample to be tested contains ring RNA 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 extraction of the sample to be tested cDNA in the step 2 is specifically implemented according to following steps：Select enteritis Salmonella negative individuals, Bacterium enteritidis, inoculum concentration 10 are inoculated with by oral method7-109Cfu/, the 7th after inoculation My god, cecal content is taken, bacterial content is measured using colony counting method.Caecal tissue sample is taken, extracts RNA, and use RNaseR 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 Chr26:2670958 | 2679178 ring-type sense primer 0.5 μ L, 10 μM of chicken ring RNA Chr26: 2670958 | 2679178 ring-type anti-sense primer 0.5 μ L, sample to be tested cDNA 2.0 μ L, dH27 μ L of O, above cumulative volume are 20 μ L。
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 circulation；95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s, 1 circulations.
The invention also discloses a kind of above-mentioned chicken ring RNA Chr26:2670958 | 2679178 detection primer is detecting Application in the anti-Bacterium enteritidis infection of chicken.
The invention also discloses a kind of above-mentioned chicken ring RNA Chr26:2670958 | 2679178 detection method is detecting Application in the anti-Bacterium enteritidis infection of chicken.
Compared with prior art, the present invention can be obtained including following technique effect：
Using method provided by the present invention, chicken ring RNA Chr26 after chicken infection Bacterium enteritidis can be accurately detected: 2670958 | 2679178 relative expression's situations, are the anti-intestines of chicken in this, as molecular markers for identification salmonella infection Resistant Individuals The genetic breeding of scorching salmonella infection provides theoretical foundation.
Certainly, implement any of the products of the present invention and it is not absolutely required to reach all the above technique effect at the same time.
Brief description of the drawings
Attached drawing described herein is used for providing a further understanding of the present invention, forms the part of the present invention, this hair Bright schematic description and description is used to explain the present invention, does not form inappropriate limitation of 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 Thing；2,3：CDNA primers are Loop primer；4,5：CDNA primers are linear primer；6 represent genomic DNA primer draws as ring-type Thing, 7,8 represent cDNA primers as Loop primer, and 9,10 represent cDNA primers as linear primer；
Fig. 2 is chicken ring RNA Chr26 of the present invention:2670958 | 2679178 relative quantification expression.
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 solves technical problem and reaches the process of realizing of technical effect to fully understand and according to this implement.
Embodiment 1RNA Chr26:2670958 | the foundation of 2679178 detection method
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, digests linear rna, carries out reverse transcription generation cDNA.
According to chicken ring RNA Chr26:2670958 | 2679178 gene orders, design specific ring-type and linear primer (table 1 and table 2), expanded.Artificial sequence (Artificial sequence)
Table 1：Chicken ring RNA Chr26:2670958 | 2679178 Loop primer sequences
Table 2：Chicken ring RNA Chr26:2670958 | 2679178 linear primer sequences
Quantitative pcr amplification, its pcr amplification reaction system such as table 3 below 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 circulations (95 DEG C, 5s, 59 DEG C, 30s)；1 circulation (95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s).
Regular-PCR amplification, its pcr amplification reaction system such as table 4 below are carried out using above-mentioned linear primer.
Table 4：Pcr amplification reaction system
Regular-PCR amplification condition is：30 circulations (98 DEG C, 10s, 55 DEG C, 30s, 72 DEG C, 1min).
Utilize above-mentioned chicken ring RNA Chr26:2670958 | 2679178 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 Chr26:2670958 | 2679178 opposite tables Up to amount.Utilize above-mentioned chicken ring RNA Chr26:2670958 | 2679178 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 Chr26:2670958 | 2679178 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 Do not come out with the amplification of same 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 uses 2-△△CTMethod 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 circulation 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 Chr26:2670958 | the results are shown in Figure 2 for 2679178 quantitative expression.
7th day chicken ring RNAChr26 after the results show infection Bacterium enteritidis:2670958 | 2679178 relative expression Amount up-regulation infected group is 27.60 times of control group and difference extremely significantly (P<0.01).
Therefore, chicken ring RNA Chr26:2670958 | 2679178 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.
Some preferred embodiments of invention have shown and described in described above, 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 technology or knowledge of above-mentioned teaching or association area Change., then all should be in power appended by invention and changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention In the protection domain that profit requires.
Sequence table
<110>Shandong Agricultural University
<120>Chicken ring RNA Chr26:2670958 | 2679178 detection primer, method and application
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Claims (8)

  1. A 1. breeder ring RNA Chr26:2670958 | 2679178 detection primer, it is characterised in that including chicken ring RNA Chr26:2670958 | 2679178 ring-type sense primers and chicken ring RNA Chr26:2670958 | 2679178 ring-type anti-sense primers, Its nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
  2. A 2. breeder ring RNA Chr26:2670958 | 2679178 detection method, it is characterised in that comprise the following steps：
    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 Thing；
    Step 4, useMethod calculates gene relative expression quantity, and specific formula for calculation is as follows：
    △ CT=CTTarget gene-CTGAPDH, △ △ CT=△ 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, according to pcr amplification product and gene relative expression quantity judge whether sample to be tested contains ring RNA Chr9: 10814512 | 10838667 genes and its expression.
  3. 3. detection method according to claim 2, it is characterised 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, it is characterised in that the extraction of the sample to be tested cDNA in the step 2 Specifically implement according to 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, and bacterial content is measured using colony counting method.Take 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, it is characterised 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 Chr26:2670958 | 2679178 ring-type sense primers 0.5 μ L, 10 μM of chicken ring RNA Chr26:2670958 | 2679178 ring-type anti-sense primer, 2.0 μ L of 0.5 μ L, sample to be tested cDNA, dH27 μ L of O, above cumulative volume are 20 μ L.
  6. 6. detection method according to claim 2, it is characterised 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 circulations；95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s, 1 circulations.
  7. 7. the chicken ring RNA Chr26 described in claim 1:2670958 | 2679178 detection primer is husky in the anti-enteritis of detection chicken Application in door Salmonella infection.
  8. 8. the chicken ring RNA Chr26 described in claim 2-6:2670958 | 2679178 detection method is in the detection anti-enteritis of chicken Application in salmonella infection.
CN201810020111.7A 2018-01-09 2018-01-09 Chicken ring RNAChr26:2670958 | 2679178 detection primer, method and application Pending CN107988404A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108753776A (en) * 2018-05-31 2018-11-06 广东海洋大学 A kind of circular rna circGHR and detection primer participating in chicken muscle growth and development

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2010084371A1 (en) * 2009-01-26 2010-07-29 Mitoprod Novel circular interfering rna molecules
CN107423579A (en) * 2017-03-30 2017-12-01 范仲凯 Combine the method for screening spinal cord injury chronogeometry with circRNA express spectras using mRNA express spectras

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010084371A1 (en) * 2009-01-26 2010-07-29 Mitoprod Novel circular interfering rna molecules
CN107423579A (en) * 2017-03-30 2017-12-01 范仲凯 Combine the method for screening spinal cord injury chronogeometry with circRNA express spectras using mRNA express spectras

Non-Patent Citations (2)

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Title
LINNA ZHENG等: "Cecal CircRNAs Are Associated With the Response to Salmonella Enterica Serovar Enteritidis Inoculation in the Chicken", 《FRONTIERS IN IMMUNOLOGY》 *
夏天等: "环状RNA: 竞争性内源RNA新成员", 《中国细胞生物学学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108753776A (en) * 2018-05-31 2018-11-06 广东海洋大学 A kind of circular rna circGHR and detection primer participating in chicken muscle growth and development

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