CN110607404B - Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for porcine enterovirus G and kit thereof - Google Patents

Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for porcine enterovirus G and kit thereof Download PDF

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CN110607404B
CN110607404B CN201911046379.9A CN201911046379A CN110607404B CN 110607404 B CN110607404 B CN 110607404B CN 201911046379 A CN201911046379 A CN 201911046379A CN 110607404 B CN110607404 B CN 110607404B
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欧阳康
杨春杰
刘雪婷
程振孔
米雪
陈樱
韦祖樟
黄伟坚
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Abstract

The invention discloses a real-time fluorescent quantitative PCR detection primer for porcine enterovirus G type, which comprises an upstream primer qEV-G-F and a downstream primer qEV-G-R, which respectively have base sequences of SEQ ID No.1 and SEQ ID No.2 in a sequence table. Accordingly, the inventor establishes a corresponding EV-G real-time fluorescent quantitative PCR detection method. Researches show that the invention has better specificity, sensitivity and repeatability, can complete detection within 3 hours, is quick and time-saving, has low cost, can realize quick detection of EV-G in clinical samples, and has important significance for monitoring and detecting the disease.

Description

Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for porcine enterovirus G type and kit thereof
Technical Field
The invention belongs to the technical field of virus nucleic acid detection, and particularly relates to a real-time fluorescent quantitative PCR detection primer of porcine enterovirus G type and a kit thereof.
Background
In recent years, along with the rapid development of the domestic pig breeding industry, the scale degree is continuously improved, new diarrhea viruses are continuously discovered in the domestic swinery, and the harm caused by the diarrhea viruses is increasingly serious. Porcine Enterovirus G (Porcine Enterovirus G, EV-G) is a ubiquitous Enterovirus in a pig group, pigs are the only natural hosts of EV-G, pigs of different ages of days are susceptible to the virus, and the infected pigs have various clinical manifestations, such as diarrhea, cerebrospinal poliomyelitis, reproductive disorders and the like. Yang et al, 3 months to 5 months in 2008, perform RT-PCR detection on 447 fecal samples collected from 14 pig farms in China, and the results show that EV-G infection commonly exists in the detected swinery, the positive rate of 10-15 week-old pigs reaches 10.1%, and the virus poses great potential threat to the health of the swinery in China.
EV-G belongs to picornaviridae, enterovirus, is a single-stranded positive-strand RNA virus, has no envelope, the diameter of virion is 22-30nm, and is spherical, the genome total length of EV-G is about 7.3kb, the whole genome has only one open reading frame, 3 'end has polyA tail, 5' end has a small protein VPg connected by covalent bond. In clinical manifestations, EV-G is similar to symptoms of Porcine Epidemic Diarrhea Virus (PEDV) and porcine reproductive disorder and respiratory syndrome virus (PRRSV), and is clinically difficult to distinguish, and there are often several enterovirus serotypes in a herd, and pigs of any age are often re-infected with other serotype strains after infection with a serotype strain. At present, a common RT-PCR method is generally adopted for detecting the disease in a laboratory, but the common PCR has lower sensitivity, fussy operation and longer time consumption.
Disclosure of Invention
The invention aims to solve the technical problem of providing the porcine enterovirus G-type real-time fluorescent quantitative PCR detection primer and the kit thereof, which have the advantages of strong specificity, high sensitivity, good repeatability and convenient operation.
In order to solve the technical problems, the invention adopts the following technical scheme:
the real-time fluorescent quantitative PCR detection primer of the porcine enterovirus G type comprises an upstream primer qEV-G-F and a downstream primer qEV-G-R, which respectively have the following base sequences:
the upstream primer qEV-G-F:5'-CTTGTGCATCCGGTTATGCC-3';
downstream primer qEV-G-R:5'-GCAAGCAGGCACAGAGAACGC-3'.
The PCR detection primer is applied to amplification of the ORF gene of the porcine enterovirus G type.
The amplification reaction condition is pre-denaturation at 95 ℃ for 120s; amplification was then performed in 40 cycles with denaturation at 95 ℃ for 15s and extension at 64 ℃ for 60s per cycle.
The real-time fluorescent quantitative PCR detection kit for the porcine enterovirus G type comprises an upstream primer qEV-G-F and a downstream primer qEV-G-R, which respectively have the following base sequences:
the upstream primer qEV-G-F:5'-CTTGTGCATCCGGTTATGCC-3';
downstream primer qEV-G-R:5'-GCAAGCAGGCACAGAGAACGC-3'.
The kit comprises the following reagents: a positive control standard substance, a PCR reaction solution and a negative control; the positive control standard substance is a recombinant plasmid containing a porcine enterovirus G-type gene sequence, and the PCR reaction solution comprises an upstream primer qEV-G-F, a downstream primer qEV-G-R and (2 x) TB Green TM Premix Ex Taq TM II, negative control is double distilled water.
The recombinant plasmid takes pMD18-T as a vector.
The mol ratio of the upstream primer qEV-G-F to the downstream primer qEV-G-R is 1:1.
the kit is used for measuring the content of the porcine enterovirus G type by fluorescent quantitative PCR.
The inventor designs real-time fluorescent quantitative PCR detection primers of porcine enterovirus G type according to ORF genes on the basis of comparing a plurality of EV-G sequences on NCBI, wherein the primers comprise an upstream primer qEV-G-F and a downstream primer qEV-G-R, and the primers respectively have base sequences of SEQ ID No.1 and SEQ ID No.2 in a sequence table. Accordingly, the inventor establishes a corresponding EV-G real-time fluorescent quantitative PCR detection method by adopting a fluorescent dye method through screening of primers and optimization of reaction conditions. The method obtains positive recombinant plasmids through PCR amplification and cloning, then carries out fluorescent quantitative reaction after taking target genes with known concentration as template gradient dilution, draws a standard curve graph and a standard curve, and is used for calculating the virus content in a sample to be detected. Comparing the sample detection result with the standard curve to determine the copy number of the target gene in the sample, and simultaneously, the method can also be used as a method for detecting the virus replication and proliferation curve in a laboratory. The research shows that the detection sensitivity of the method is 2.92 × 10 copies/mu L, the Cq value and the logarithm of the plasmid copy number have good linear relation, and the correlation coefficient R 2 =0.998, with good repeatability; the detection sensitivity is 100 times higher than that of the conventional PCR. Different viruses (including Porcine Epidemic Diarrhea Virus (PEDV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine astrovirus (Pastv), A-type Seneca Virus (SVA) and Classical Swine Fever Virus (CSFV) are used as templates, negative and positive controls are simultaneously arranged for fluorescent quantitative detection, and the result shows that only the positive control shows an amplification curve and no amplification curve exists in other controls, which shows that the specificity of the method is strong.
Drawings
FIG. 1 is a schematic diagram of the fluorescent quantitative PCR amplification kinetic curve of the present invention.
FIG. 2 is a schematic diagram of a standard curve of the fluorescent quantitative PCR of the present invention.
FIG. 3 is a schematic diagram of a melting curve of the fluorescent quantitative PCR of the present invention.
FIG. 4 is an electrophoretogram of a common PCR assay for EV-G positive samples, in which the lanes are, from left to right: m: DL2000DNA Marker;1 negative control; 2, positive.
FIG. 5 is a general PCR detection sensitivity electrophoresis diagram, wherein the lanes from left to right are: m: DL2000DNA Marker;1:2.92*10 6 copies/μL；2：2.92*10 5 copies/μL；3：2.92*10 4 copies/μL；4：2.92*10 3 copies/μL；5：2.92*10 2 copies/. Mu.L; 6:2.92 × 10copies/μ L;7: a positive control; 8: and (5) negative control.
FIG. 6 is a diagram showing the results of the specific detection of the fluorescent quantitative PCR of the present invention, in which: 1: negative control; 2: PEDV;3: poRV;4: PRV;5: PRRSV;6: (iv) Pastv;7: SVA;8: CSFV;9: EV-G.
Detailed Description
Example 1 establishment of porcine Enterovirus G-type fluorescent quantitative PCR method
(1) Design and Synthesis of primers
According to ORF gene sequences of porcine enterovirus strains registered by GenBank, 1 pair of specific primers is designed by applying Primer5.0 software, the size of an amplified target fragment is 104bp (SEQ. ID. NO. 3), the primers are synthesized by Jie Li Shengwu (Shanghai) technology Limited company, and the specific sequences are as follows:
the upstream primer qEV-G-F:5'-CTTGTGCATCCGGTTATGCC-3' (seq. Id. No. 1);
downstream primer qEV-G-R:5'-GCAAGCAGGCACAGAGAACGC-3' (seq. Id. No. 2).
(2) Cloning of ORF Gene
According to EV-G positive samples stored by the applicant, total RNA is extracted and reversely transcribed according to an AxyPrep humoral virus DNA/RNA small-quantity kit, the total cDNA samples are stored to-80 ℃, a 25-mu-L reaction system is adopted, the reaction condition is 94 ℃ for pre-denaturation for 3min, then amplification is carried out in 35 cycles, each cycle is 94 ℃ for denaturation 25s,58 ℃ for extension for 25s, and finally 72 ℃ for extension for 10min. After amplification was complete, 10. Mu.L of the product was identified by electrophoresis on a 1.0% agarose gel. And (3) purifying and recovering the PCR product identified as positive by using an Omega Gel extraction kit (20) Gel recovery kit, cloning to a pMD18-T vector and transforming to DH5 alpha competent cells, selecting the positive clone, and sequencing and identifying.
(3) Establishment of a Standard Curve
Extracting recombinant plasmid with plasmid extraction kit, measuring OD260nm with ultraviolet spectrophotometer, converting standard plasmid concentration into copies/μ L, and diluting to final concentration of 2.92 × 10 8 -2.92*10 2 copies/μL。
The total volume of the fluorescent quantitative reaction system is 20 mu L and comprises (2 x) TB Green TM Premix Ex Taq TM II 10. Mu.L of each of the upstream and downstream primers 1. Mu. L, DNA template 2. Mu.L and sterile water 6. Mu.L.
The reaction conditions were as follows: pre-denaturation at 95 ℃ for 120s; amplification was performed in 40 additional cycles, with denaturation at 95 ℃ for 15s and extension at 64 ℃ for 60s per cycle. And collecting fluorescent signals for real-time detection when the extension temperature of each cycle is finished, and finally analyzing the specificity of the PCR amplification product by using a melting curve.
As shown in fig. 2, in the diluted linear concentration range, the template amount is linearly related to the corresponding Cq value with a correlation coefficient of 0.998. The slope of the standard curve is-4.023, the intercept is 43.51, and the equation for the straight line: y = -4.023x +43.51.
Where y represents the Cq value and x represents Log (number of viruses) in copy number/. Mu.L.
(4) Sensitivity test
As shown in FIG. 5, the lowest detectable level in conventional PCR was 2.92 x 10 3 copies/. Mu.L, whereas the lowest detectable amount for the fluorescent quantitative PCR of the present invention was 2.92 × 10 copies/. Mu.L. Therefore, the fluorescence quantitative PCR established by the experiment has higher sensitivity which is 100 times higher than the sensitivity of the conventional PCR.
(5) Experiment of specificity
Performing conventional RT-PCR detection on EV-G, PEDV, poRV, PRV, PRRSV, pastv, SVA and CSFV, and applying the fluorescent quantitative PCR amplification of the invention after ensuring that the positive sample is obtained. As shown in FIG. 6, only EV-G amplification is positive, cq value is 20.08, and other samples have no specific amplification, indicating that the method has strong specificity.
(6) Repeatability test
Selecting 1X 10 -2 -1×10 -5 The three positive standard plasmids with different dilutions are respectively subjected to the repeatability tests in groups and among groups, the amplification results are shown in table 1, and the variation coefficient is between 0.26% and 1.57%, so that the fluorescent quantitative PCR detection method has good repeatability.
TABLE 1 repeatability test results of fluorescent quantitative PCR
Figure BDA0002254243400000041
Example 2
The results of the real-time fluorescent quantitative PCR detection of 27 clinical disease samples collected in Guangxi area by applying the invention are shown in Table 2.
TABLE 2 results of clinical sample testing using the present invention
Figure BDA0002254243400000042
And (4) conclusion: the real-time fluorescent quantitative PCR method established by the invention is used for detecting 27 clinical positive samples, wherein 5 samples are positive, and the positive rate is 18.52%.
Sequence listing
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<120> real-time fluorescent quantitative PCR detection primer of porcine enterovirus G type and kit thereof
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cttgtgcatc cggttatgcc 20
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cttgtgcatc cggttatgcc aatgaaggag attcacgaat ctatcaggtg gaccagggat 60
gcgcgcaaca cacaagacca cgtgcgttct ctgtgcctgc ttgc 104

Claims (8)

1. The real-time fluorescent quantitative PCR detection primer of the porcine enterovirus G type is characterized by comprising the following components in parts by weight: comprises an upstream primer qEV-G-F and a downstream primer qEV-G-R which are respectively the following base sequences:
the upstream primer qEV-G-F:5'-CTTGTGCATCCGGTTATGCC-3';
downstream primer qEV-G-R:5'-GCAAGCAGGCACAGAGAACGC-3'.
2. The non-diagnostic use of the PCR detection primers of claim 1 for amplifying the ORF gene of porcine enterovirus type G.
3. The non-diagnostic application of claim 2, wherein: the amplification reaction condition is pre-denaturation at 95 ℃ for 120s; amplification was then performed in 40 cycles with denaturation at 95 ℃ for 15s and extension at 64 ℃ for 60s per cycle.
4. A real-time fluorescent quantitative PCR detection kit for porcine enterovirus G type is characterized by comprising an upstream primer qEV-G-F and a downstream primer qEV-G-R which are respectively the following base sequences:
the upstream primer qEV-G-F:5'-CTTGTGCATCCGGTTATGCC-3';
downstream primer qEV-G-R:5'-GCAAGCAGGCACAGAGAACGC-3'.
5. The kit according to claim 4, characterized in that it comprises the following reagents: a positive control standard substance, a PCR reaction solution and a negative control; the positive control standard substance is a recombinant plasmid containing a porcine enterovirus G-type gene sequence, and the PCR reaction solution comprises an upstream primer qEV-G-F, a downstream primer qEV-G-R and 2 xTB Green TM Premix Ex Taq TM II, negative control is double distilled water.
6. The kit of claim 5, wherein: the recombinant plasmid takes pMD18-T as a vector.
7. The kit of claim 5, wherein: the molar ratio of the upstream primer qEV-G-F to the downstream primer qEV-G-R is 1:1.
8. the kit of claim 4 for use in non-diagnostic fluorescent quantitative PCR assay of porcine enterovirus type G.
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