CN108179197B - Chicken ring RNA Chr9:10814512|10838667 gene detection primer, method and application - Google Patents

Chicken ring RNA Chr9:10814512|10838667 gene detection primer, method and application Download PDF

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CN108179197B
CN108179197B CN201810019252.7A CN201810019252A CN108179197B CN 108179197 B CN108179197 B CN 108179197B CN 201810019252 A CN201810019252 A CN 201810019252A CN 108179197 B CN108179197 B CN 108179197B
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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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Abstract

The invention discloses a detection primer, a method and application of chicken circular RNA Chr9:10814512|10838667 gene. The detection primers comprise a chicken circular RNA Chr9:10814512|10838667 circular upstream primer and a chicken circular RNA Chr9:10814512|10838667 circular downstream primer, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. By using the primers and the method, the expression condition of the loop RNA related to the infection resistance of the salmonella enteritidis can be accurately detected, and a theoretical basis and a scientific basis are provided for the screening and disease-resistant breeding of the chicken salmonella enteritidis infection resistance individuals.

Description

Chicken ring RNA Chr9:10814512|10838667 gene detection primer, method and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a detection primer, a method and application of chicken circular RNA Chr9:10814512|10838667 gene.
Background
Salmonella Enteritidis (SE) is common pathogenic bacteria of human and livestock, and has no host specificity, so that the Salmonella Enteritidis (SE) can cause diseases such as acute gastroenteritis of poultry and even death, causes huge economic loss to poultry industry, seriously harms human health, causes food poisoning, and is one of important pathogenic bacteria affecting human body and heart health. Reports indicate that 19000 people were hospitalized with salmonella infection and up to 300 people died with salmonella infection in every 1 million people in the united states during 2004; in the european union, there was an outbreak of food-borne illness caused by infection with salmonella of about 99020 origin in 2010. Poultry and their related processed products are the main sources of infection by salmonella enteritidis, the caecum is the main host, and salmonella enteritidis can enter egg products through egg shells and can be deposited and propagated in the reproductive tract. At present, the main method for controlling salmonella infection is to use vaccines and antibiotics, but the problems cannot be solved fundamentally, and the large-scale use of the antibiotics can cause food safety problems and drug resistance of bacteria, and cause the drug resistance of strains to be enhanced. With the development of molecular genetics, the genetic resistance of livestock and poultry to diseases and pathogens is improved, and a new possibility is provided for controlling the infection of salmonella.
Circular RNA is a novel non-coding RNA. The ring RNA is in a closed ring structure, is not influenced by RNA exonuclease, is more stable in expression and is not easy to degrade. Does not contain 5 '-3' polarity. However, circular RNA has a tissue-specific, widely abundant expression in animals. Functionally, recent researches show that the ring RNA molecule is rich in microRNA (miRNA) binding sites and plays a role of miRNA sponge (miRNA sponge) in cells, so that the inhibition of miRNA on target genes is relieved, and the expression level of source genes is increased; this mechanism of action is known as the competitive endogenous rna (cerna) mechanism. Through the interaction with disease-associated mirnas, loop RNAs play important regulatory roles in disease.
Disclosure of Invention
In view of the above, the invention provides a detection primer, a method and an application of chicken circular RNA Chr9:10814512|10838667 gene.
In order to solve the technical problem, the invention discloses a detection primer of chicken circular RNA Chr9:10814512|10838667 gene, which comprises a chicken circular RNA Chr9:10814512|10838667 circular upstream primer and a chicken circular RNA Chr9:10814512|10838667 circular downstream primer, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention also discloses a detection method of the chicken circular RNA Chr9:10814512|10838667 gene, which comprises the following steps:
step 1, extracting RNA of a sample to be detected;
step 2, carrying out reverse transcription to obtain cDNA;
step 3, carrying out quantitative PCR amplification on the cDNA of a sample to be detected by using the primer in claim 1 to obtain a PCR amplification product;
step 4, adopt
Figure BDA0001542995980000021
The method calculates the relative expression quantity of the gene, and the specific calculation formula is as follows:
ΔCT＝CTtarget gene-CTGAPDH，ΔΔCT＝ΔCTTarget gene-△CTReference gene；
Wherein, the delta CT represents the CT value of the gene to be verified minus the CT value of the reference gene;
CTtarget geneCT values representing the genes to be verified;
CTGAPDHrepresents the CT value of the reference gene;
delta CT represents the delta CT value of the target gene minus the average value of the delta CT of the target gene of the control group;
△CTtarget geneRepresents the Δ CT value of the target gene;
△CTreference geneRepresents the average value of the target gene delta CT of the control group;
and 5, judging whether the sample to be detected contains chicken circular RNA Chr9:10814512|10838667 gene and the expression condition thereof according to the PCR amplification product and the relative gene expression quantity.
Further, the extraction of the RNA of the sample to be detected in step 1 specifically comprises: the total RNA of the sample is extracted by a Trizol method, and the extracted total RNA is subjected to quality inspection by an Agilent bioanalyzer 2100 and a Qubit 2.0.
Further, the reverse transcription performed in the step 2 to obtain the cDNA is specifically performed according to the following steps: selecting Salmonella enteritidis negative individuals, and inoculating Salmonella enteritidis by oral administration in an amount of 107-109cfu/piece, 7 days after inoculation, the cecum contents were taken, the bacterial content was determined by plate counting, cecum tissue samples were taken, RNA was extracted, the extracted RNA was treated with RNase R, linear RNA was digested, and reverse transcription was performed to generate cDNA.
Further, the quantitative PCR reaction system in step 3 is as follows: 2x SYBR Premix Ex TaqTM 10 uL, 10 uM chicken circular RNA Chr9:10814512|10838667 circular upstream primer 0.5 uL, 10 uM chicken circular RNA Chr9:10814512|10838667 circular downstream primer 0.5 uL, sample cDNA 2.0 uL to be detected, dH2O7. mu.L, the total volume above is 20. mu.L.
Further, the quantitative PCR amplification conditions in step 3 are: at 95 ℃ for 30 s; 95 ℃, 5s, 59 ℃, 30s, 40 cycles; at 95 ℃ for 1 min; 61 ℃, 30 s; 95 ℃ for 30s, 1 cycle.
The invention also discloses application of the detection primer of the chicken circular RNA Chr9:10814512|10838667 gene in detection of chicken salmonella enteritidis infection resistance.
The invention also discloses an application of the detection method of the chicken circular RNA Chr9:10814512|10838667 gene in detection of chicken salmonella enteritidis infection resistance.
Compared with the prior art, the invention can obtain the following technical effects:
according to the invention, specific circular and linear primers are designed according to the chicken circular RNA Chr9:10814512|10838667 gene sequence, the expression of the circular RNA is detected by utilizing fluorescent quantitative PCR, and the correlation between the circular RNA expression and the bacterial content of the caecum content is analyzed. The method provides PCR conditions for quantitative expression of chicken circular RNA Chr9:10814512|10838667, and by using the primers and the method, the expression condition of the circular RNA related to the infection resistance of the salmonella enteritidis can be accurately detected, so that a theoretical basis and a scientific basis are provided for screening and disease-resistant breeding of chicken resistant individuals to the infection resistance of the salmonella enteritidis.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is an electrophoretogram of a PCR product of the present invention; wherein, M is DL2000 marker; 1, the genome DNA primer is a circular primer; 2,3: the cDNA primer is a circular primer; 4,5: the cDNA primer is a linear primer; 6 represents that the genomic DNA primer is a circular primer, 7, 8 represents that the cDNA primer is a circular primer, 9, 10 represents that the cDNA primer is a linear primer, 11 represents that the genomic DNA primer is a circular primer, 12, 13cDNA primers are circular primers, 14, 15 represent that the cDNA primer is a linear primer;
FIG. 2 shows the relative quantitative expression of the chicken circular RNA Chr9:10814512| 10838667.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Example 1 establishment of detection method of Chicken circular RNA Chr9:10814512|10838667 Gene
Selecting Salmonella enteritidis negative individuals, and inoculating Salmonella enteritidis by oral administration in an amount of 107-109cfu/mouse, 7 days after inoculation, cecal contents were taken and the bacterial content was determined by plate counting. And extracting RNA from the cecum tissue sample, treating the extracted RNA with RNase R, digesting linear RNA, and performing reverse transcription to generate cDNA.
Specific circular and linear primers (Table 1-2) were designed based on the sequence of the chicken circular RNA Chr9:10814512|10838667 gene for amplification.
Table 1: chicken circular RNA Chr9:10814512|10838667 circular primer sequence
Figure BDA0001542995980000051
Table 2: chicken circular RNA Chr9:10814512|10838667 linear primer sequence
Figure BDA0001542995980000052
The specific quantitative circular primers were used to perform quantitative PCR amplification, and the PCR amplification reaction system is shown in Table 3 below.
Table 3: quantitative PCR reaction system (20. mu.L)
Figure BDA0001542995980000053
The quantitative PCR amplification conditions were: at 95 ℃ for 30 s; 40 cycles (95 ℃, 5s, 59 ℃, 30 s); 1 cycle (95 ℃, 1 min; 61 ℃, 30 s; 95 ℃, 30 s).
The above linear primers were used for general PCR amplification, and the PCR amplification reaction system is shown in Table 4 below.
Table 4: PCR amplification reaction system
Figure BDA0001542995980000054
Figure BDA0001542995980000061
The common PCR amplification conditions were: 30 cycles (98 ℃, 10s, 55 ℃, 30s, 72 ℃, 1 min).
The relative expression level of the chicken circular RNA Chr9:10814512|10838667 is analyzed by using the specific circular primer of the chicken circular RNA Chr9:10814512|10838667, loading the sample according to the 20-microliter system, and carrying out reaction according to the PCR amplification condition. The linear primers of the chicken circular RNA Chr9:10814512|10838667 were used to sample the solution according to the 50. mu.L system, and the PCR amplification conditions were used to analyze the amplification products of chicken circular RNA Chr9:10814512| 10838667.
It can be seen from FIG. 1 that the product was amplified only under the conditions of the circular primer and cDNA, but not in the genomic DNA using the same circular primer. No product was amplified with linear primers for circular RNA and cDNA.
The data was collated using Excel. Relative expression amount of gene
Figure BDA0001542995980000062
The method comprises the following specific calculation formula:
△CT＝CTtarget gene-CTGAPDH，△△CT＝△CTTarget gene-△CTReference gene。
Wherein, the meaning of CT value is: the number of cycles that the fluorescence signal in each reaction tube undergoes when it reaches a set threshold;
the delta CT represents the CT value of the gene to be verified minus the CT value of the reference gene;
CTtarget geneRepresenting the desired validation geneA CT value of (1);
CTGAPDHrepresents the CT value of the reference gene;
delta CT represents the delta CT value of the target gene minus the average value of the delta CT of the target gene of the control group;
△CTtarget geneRepresents the Δ CT value of the target gene;
△CTreference geneRepresents the mean value of the control group target gene Δ CT.
The results of quantitative expression of chicken circular RNA Chr9:10814512|10838667 are shown in FIG. 2.
The results show that the relative expression level of chicken circular RNA Chr9:10814512|10838667 at 7 days after infection of salmonella enteritidis is 5.95 times of that of the control group and the difference is very obvious (P < 0.05).
Therefore, the expression level of the chicken circular RNA Chr9:10814512|10838667 is obviously related to the resistance to the salmonella enteritidis infection, and the chicken circular RNA Chr9:10814512|10838667 can be used as a molecular marker of the resistance to the salmonella enteritidis infection and used for selecting individuals resistant to the salmonella enteritidis infection and carrying out disease-resistant genetic breeding on chickens.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Shandong university of agriculture
<120> chicken circular RNA Chr9:10814512|10838667 gene detection primer, method and application
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Claims (4)

1. The application of the detection primer of the chicken circular RNA Chr9:10814512|10838667 gene in the preparation of the reagent for detecting the infection of the salmonella enteritidis is characterized in that the primer comprises a chicken circular RNA Chr9:10814512|10838667 circular upstream primer and a chicken circular RNA Chr9:10814512|10838667 circular downstream primer, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The use of claim 1, wherein the chicken circular RNA Chr9:10814512|10838667 gene amplification method comprises the following steps: performing quantitative PCR amplification using the primers of claim 1 to obtain a PCR amplification product.
3. The use of claim 2, wherein the quantitative PCR reaction system is as follows: 10 mu L of 2x SYBR Premix Ex TaqTM, 0.5 mu L of 10 mu M chicken circular RNA Chr9:10814512|10838667 circular upstream primer, 0.5 mu L of 10 mu M chicken circular RNA Chr9:10814512|10838667 circular downstream primer, 2.0 mu L of sample cDNA to be detected and 20 mu L of dH2O 7 mu L of sample cDNA to be detected.
4. The use according to claim 2, wherein the quantitative PCR amplification conditions are: at 95 ℃ for 30 s; 95 ℃, 5s, 59 ℃, 30s, 40 cycles; at 95 ℃ for 1 min; 61 ℃, 30 s; 95 ℃ for 30s, 1 cycle.
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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

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* 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

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* Cited by examiner, † Cited by third party
Title
"Gallus gallus isolate RJF #256 breed Red Jungle fowl, inbred line UCD001 chromosome 9, Gallus_gallus-5.0, whole;International Chicken Genome Sequencing Consortium;《GenBank》;20160104;第1-2页 *

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