CN106755585B - Real-time fluorescent quantitative PCR primer for distinguishing classic and Korean duck hepatitis viruses - Google Patents
Real-time fluorescent quantitative PCR primer for distinguishing classic and Korean duck hepatitis viruses Download PDFInfo
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Abstract
The invention provides a group of real-time fluorescent quantitative PCR primers for distinguishing classic duck hepatitis viruses and Korean duck hepatitis viruses, which are designed by utilizing the difference of GC content of characteristic nucleotide sequences of the classic duck hepatitis viruses and the Korean duck hepatitis viruses. The primer set can be used for effectively amplifying both classic and Korean duck hepatitis virus real-time fluorescent quantitative PCR reactions, but the melting temperature (Tm) difference exists in the melting curve generated after the real-time fluorescent quantitative PCR reactions of classic and Korean duck hepatitis viruses, so that the classic and Korean duck hepatitis viruses can be effectively distinguished. By matching with a computer connected with a real-time fluorescent quantitative PCR instrument and utilizing the analysis software carried by the instrument, the specific differential diagnosis of the typical and Korean duck hepatitis virus infection conditions can be realized only by one group of primers. The identification method is simple, and has high efficiency and accuracy.
Description
Technical Field
The invention belongs to the field of animal infectious disease science, and particularly relates to a group of real-time fluorescent quantitative PCR primers for distinguishing classic duck hepatitis viruses and Korean duck hepatitis viruses.
Background
Duck hepatitis is a highly lethal infectious disease which is caused by duck hepatitis virus and attacks ducklings, is clinically characterized by acute hepatitis, mainly attacks ducklings within 3 weeks of age, particularly ducklings within 1 week of age, and has a fatality rate of 100%. At present, with the increase of duck feeding density, the non-standard treatment of sick ducks and ducks died of diseases, the increasing frequency of various trade activities, the occurrence of immunosuppressive diseases and the like in China, the duck hepatitis is more and more serious, and becomes one of the most serious epidemic diseases in the duck breeding industry.
In China, duck hepatitis viruses prevalent in duck groups are mainly typical duck hepatitis viruses, but reports on Korean duck hepatitis virus infection are increasing. However, both classic duck hepatitis virus and Korean duck hepatitis virus mainly attack ducklings within 3 weeks of age, dead ducks mostly present angular bow-shaped reversal, liver swelling and bleeding can be seen in a autopsy, and effective differential diagnosis of the ducklings and the ducks cannot be carried out from clinical autopsy.
The real-time fluorescent quantitative PCR primer group for distinguishing the classic and Korean duck hepatitis viruses only needs 1 primer pair (2 primers) to effectively distinguish the classic and Korean duck hepatitis viruses. The primer is designed according to the characteristic nucleotide sequence difference existing between the typical duck hepatitis virus and the Korean duck hepatitis virus, the Tm value of a melting curve generated after real-time fluorescent quantitative PCR reaction is positively correlated with the GC content of the melting curve, and the Tm value difference of the melting curve generated after the real-time fluorescent quantitative PCR amplification reaction of the typical duck hepatitis virus and the Korean duck hepatitis virus by the group of primers is observed, so that the infection conditions of the typical duck hepatitis virus and the Korean duck hepatitis virus can be accurately identified and diagnosed, the relevant research is not reported in domestic and foreign documents, and the invention can fill in the blank of the relevant fields.
Disclosure of Invention
The invention aims to provide a group of real-time fluorescent quantitative PCR primers for distinguishing classic duck hepatitis virus and Korean duck hepatitis virus and application thereof, wherein the primers can effectively distinguish classic duck hepatitis virus infection (or coinfection) and Korean duck hepatitis virus infection, and provide technical support for scientific prevention and control of duck hepatitis virus infection.
The invention designs a group of real-time fluorescent quantitative PCR primers according to the characteristic nucleotide sequence difference of the classic and Korean duck hepatitis viruses. The primer can obtain specific strips by carrying out PCR amplification on the classic and Korean duck hepatitis viruses, utilizes the nucleotide GC content difference of the amplification region between the classic and Korean duck hepatitis viruses, carries out real-time fluorescent quantitative PCR amplification reaction on the classic and Korean duck hepatitis viruses by establishing a real-time fluorescent quantitative PCR method based on Eva Green, obtains melting curves with different melting temperature (Tm) differences, and can directly carry out differential diagnosis on the classic and Korean duck hepatitis virus infection conditions according to the Tm value difference of the melting curves.
In order to achieve the purpose, the invention adopts the following technical scheme:
a group of real-time fluorescent quantitative PCR primers for distinguishing classic and Korean duck hepatitis viruses has the nucleotide sequence as follows:
the upstream primer F1: 5'-AAACGGATTACCGGTAGTAGCATC-3',
the downstream primer R1: 5'-GACCAGCCGCGACCCTAT-3'.
The real-time fluorescent quantitative PCR method based on Eva Green is established through the primers, and the melting curves generated after the amplification reaction of the real-time fluorescent quantitative PCR reaction have different melting temperatures (Tm values) according to the nucleotide characteristic difference, namely the GC content difference, of the typical and Korean duck hepatitis viruses amplified by the primers, so that the infection of the typical and Korean duck hepatitis viruses can be directly identified.
The method specifically comprises the following steps:
1. designing and synthesizing the specific primer pair F1/R1;
2. and (3) extracting virus RNA: respectively extracting typical and Korean duck hepatitis virus RNA from the detection sample;
3. and (3) RT-PCR amplification: the extracted nucleic acid RNA is reversely transcribed by DHVF/DHVR of duck hepatitis specific primers, then PCR amplification is carried out, the target fragment amplified by PCR is subjected to gel recovery and purification, and then cloned on a T vector to obtain a positive recombinant plasmid containing typical and Korean duck hepatitis viruses, and after OD value is measured to calculate copy number, the positive recombinant plasmid is continuously diluted in multiple proportions and used as a standard substance of real-time fluorescent quantitative PCR reaction.
The nucleotide sequence of DHVF/DHVR of the duck hepatitis specific primer pair is as follows:
DHVF: 5’- GTTGTGAAACGGATTACCGGTAGTA -3’；
DHVR: 5’- ACTCGACCAGCCGCGACC -3’。
4. real-time fluorescent quantitative PCR: and carrying out Eva Green real-time fluorescent quantitative PCR amplification on the positive recombinant plasmid containing the classic and Korean duck hepatitis viruses by using the real-time fluorescent quantitative primers F1 and R1, obtaining a corresponding amplification curve after the reaction is finished, and generating a melting curve of the Eva Green real-time fluorescent quantitative PCR amplification.
5. And (3) judging the virus infection condition: after the real-time fluorescent quantitative PCR reaction is finished, whether the classic and Korean duck hepatitis virus infection exists is judged by observing an amplification curve; the classic and korean duck hepatitis viruses can be identified by observing the difference in Tm values of melting curves.
The designed real-time fluorescent quantitative PCR primer needs to meet the following requirements:
(1) the specificity is strong: the real-time fluorescent quantitative PCR product needs to select a region design conserved after the nucleotide sequences of the typical and Korean duck hepatitis viruses are analyzed, so that only one group of primers can amplify the typical and Korean duck hepatitis viruses, namely, the amplification curve after the real-time fluorescent quantitative PCR reaction is observed to judge the infection of the typical and Korean duck hepatitis viruses.
(2) The amplified regions of the two viruses differ in GC content: the primer set has GC content difference through the nucleotide sequence of the amplified region of the typical and Korean duck hepatitis virus. Based on the difference of Tm peak values of melting curves generated by the real-time fluorescent quantitative PCR reaction (the difference is in positive correlation with the GC content difference of a nucleotide sequence), the typical and Korean duck hepatitis virus infection can be judged by observing the melting curves after the real-time fluorescent quantitative PCR reaction (the typical duck hepatitis virus or the Korean duck hepatitis virus can be effectively distinguished).
Wherein, the virus infection condition judgment method in the step (5) is as follows:
if a single specific Tm peak appears at Tm = (87.2 +/-0.20) DEG C, the virus is judged to be positive by the classic duck hepatitis virus;
if a single specific Tm peak appears at Tm = (84.8 +/-0.24) ° C, the Korean duck hepatitis virus is judged to be positive;
if double peaks appear at Tm = (87.2 +/-0.20) ° C and Tm = (84.8 +/-0.24) ° C, the duck hepatitis virus is judged to be doubly infected by the typical and Korean duck hepatitis viruses;
otherwise, the test was negative.
The real-time fluorescent quantitative PCR primer pair F1/R1 designed by the invention can be used for preparing a kit for distinguishing classic and Korean duck hepatitis virus infection.
The invention has the beneficial effects that: the identification method is simple, and the efficiency and the accuracy are high. The real-time fluorescent quantitative PCR detection of 5 strains of pre-isolated classic duck hepatitis virus and 3 strains of Korean duck hepatitis virus by using a set of real-time fluorescent quantitative PCR primers provided by the research is consistent with the expectation. And only 1 primer pair (2 primers) is needed to effectively distinguish the classic and Korean duck hepatitis viruses.
Drawings
FIG. 1 shows the design region of the quantitative PCR primer for duck hepatitis virus;
FIG. 2 shows nucleotide differences in the amplified region of the duck hepatitis virus gene;
FIG. 3 shows the melting curve of the specific primer pair F1/R1 in real-time fluorescent quantitative PCR reaction with typical duck hepatitis virus;
FIG. 4 is a melting curve of real-time fluorescent quantitative PCR reaction of the specific primer pair F1/R1 on Korean duck hepatitis virus;
FIG. 5 melting curves of the specific primer pair F1/R1 for real-time fluorescent quantitative PCR reaction of typical and Korean duck hepatitis viruses.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
1. Strain:
both classic duck hepatitis virus (JX 1132 strain) and Korean duck hepatitis virus (FJ 1521 strain) were maintained by the animal husbandry and veterinary institute of agricultural and scientific institute of Fujian province.
2. Primer design and Synthesis
Primers F1 and R1 of the real-time fluorescent quantitative PCR reaction are designed according to the nucleotide characteristic difference region of the typical and Korean duck hepatitis viruses, and the primer sequences are as follows:
the upstream primer F1: 5'-AAACGGATTACCGGTAGTAGCATC-3' the flow of the air in the air conditioner,
the downstream primer R1: 5'-GACCAGCCGCGACCCTAT-3'.
3. Viral RNA extraction and RT-PCR amplification:
viral RNA of classic duck hepatitis virus (JX 1132 strain) and Korean duck hepatitis virus (FJ 1521 strain) was extracted by a conventional method. Performing PCR amplification on the extracted nucleic acid RNA by using a duck hepatitis specific primer pair DHVF/DHVR, performing gel recovery and purification on a target fragment amplified by the PCR, cloning the target fragment to a T vector to obtain a positive recombinant plasmid containing typical and Korean duck hepatitis viruses, measuring the OD value of the positive recombinant plasmid to calculate the copy number, and performing continuous dilution by multiple times to obtain a standard substance of real-time fluorescent quantitative PCR reaction. The nucleotide sequence of DHVF/DHVR of the duck hepatitis specific primer pair is as follows:
DHVF: 5’- GTTGTGAAACGGATTACCGGTAGTA -3’；
DHVR: 5’- ACTCGACCAGCCGCGACC -3’。
4. real-time fluorescent quantitative PCR reaction:
the optimized 20 muL optimal reaction system is a system: eva Green 10. mu.L, F1 with the concentration of 10. mu. mol/L, R1 each 0.2. mu.L, template 2. mu.L, water make up to 20. mu.L. The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; and (3) carrying out 40 cycles of 95 ℃ for 10 s and 59 ℃ for 15 s, and after the cycles are finished, making corresponding melting curves according to conditions.
5. And (3) judging the virus infection condition:
and after the real-time fluorescent quantitative PCR reaction is finished, observing an amplification curve, and judging whether the established method has specific amplification. The melting curve made after the real-time fluorescent quantitative PCR reaction is finished is directly analyzed (can be directly observed by naked eyes) on a computer connected with a fluorescent quantitative PCR instrument by using software corresponding to the real-time fluorescent quantitative PCR, and the typical and Korean duck hepatitis virus infection conditions are judged by the judging method:
if a single specific Tm peak appears at Tm = (87.2 +/-0.20) DEG C, the virus is judged to be positive by the classic duck hepatitis virus;
if a single specific Tm peak appears at Tm = (84.8 +/-0.24) ° C, the Korean duck hepatitis virus is judged to be positive;
if double peaks appear at Tm = (87.2 +/-0.20) ° C and Tm = (84.8 +/-0.24) ° C, the duck hepatitis virus is judged to be doubly infected by the typical and Korean duck hepatitis viruses;
otherwise, the test was negative.
The real-time fluorescent quantitative PCR detection of 5 strains of pre-isolated classic duck hepatitis virus and 3 strains of Korean duck hepatitis virus by using a set of real-time fluorescent quantitative PCR primers provided by the research is consistent with the expectation. And only 1 primer pair (2 primers) is needed to effectively distinguish the classic and Korean duck hepatitis viruses.
Example 2
Detecting 75 suspected samples which are clinically submitted; the typical duck hepatitis virus infection is positive by 6 parts, and the positive rate is 8 percent (6/75); the Korean duck hepatitis virus infection is positive by 2 parts, and the positive rate is 2.67 percent (2/75); 1 sample co-infected with classic duck hepatitis virus and korean duck hepatitis virus.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> real-time fluorescent quantitative PCR primer for differentiating classic and Korean duck hepatitis virus
<130>4
<160>4
<170>PatentIn version 3.3
<210>1
<211>24
<212>DNA
<213>F1
<400>1
aaacggatta ccggtagtag catc 24
<210>2
<211>18
<212>DNA
<213>R1
<400>2
gaccagccgc gaccctat 18
<210>3
<211>25
<212>DNA
<213>DHVF
<400>3
gttgtgaaac ggattaccgg tagta 25
<210>4
<211>18
<212>DNA
<213>DHVR
<400>4
actcgaccag ccgcgacc 18
Claims (2)
1. A group of real-time fluorescent quantitative PCR primers for distinguishing classic and Korean duck hepatitis viruses is characterized in that: the nucleotide sequence of the PCR primer is as follows:
the upstream primer F1: 5'-AAACGGATTACCGGTAGTAGCATC-3',
the downstream primer R1: 5'-GACCAGCCGCGACCCTAT-3'.
2. Use of the set of real-time fluorescent quantitative PCR primers for differentiating classic and Korean duck hepatitis virus according to claim 1 for preparing a kit for differentiating classic and Korean duck hepatitis virus.
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| CN109487015A (en) * | 2019-01-12 | 2019-03-19 | 福建省农业科学院畜牧兽医研究所 | The real-time fluorescence quantitative PCR detection primer and its kit of 2 type hepatitis A virus of duck |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969914A (en) * | 2016-07-26 | 2016-09-28 | 福建省农业科学院畜牧兽医研究所 | Group of real-time fluorescence quantification PCR primers for distinguishing duck circovirus genotypes |
| CN106065419A (en) * | 2016-07-26 | 2016-11-02 | 福建省农业科学院畜牧兽医研究所 | The quick real-time fluorescence quantitative PCR detection primer differentiating duck circovirus genotype and detection method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969914A (en) * | 2016-07-26 | 2016-09-28 | 福建省农业科学院畜牧兽医研究所 | Group of real-time fluorescence quantification PCR primers for distinguishing duck circovirus genotypes |
| CN106065419A (en) * | 2016-07-26 | 2016-11-02 | 福建省农业科学院畜牧兽医研究所 | The quick real-time fluorescence quantitative PCR detection primer differentiating duck circovirus genotype and detection method thereof |
Non-Patent Citations (4)
| Title |
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| Differential diagnosis between type-specific duck hepatitis virus type 1 (DHV-1) and recent Korean DHV-1-like isolates using a multiplex polymerase chain reaction;Min-Chul Kim等;《Avian Pathology》;20080430;171-177 * |
| I型与新I型鸭肝炎病毒分离鉴定及实时荧光定量RT-PCR方法的建立;陈玉环;《中国优秀硕士学位论文全文数据库》;20131015(第10期);D050-174 * |
| 一株新型鸭肝炎病毒的分离与鉴定;陈生雷等;《中国兽药杂志》;20160120;19-23 * |
| 分子生物学技术在鸭肝炎病毒;于新友等;《家禽科学》;20150910;51-54 * |
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