CN112680546A - Specific amplification primer pair and fluorescent quantitative PCR kit - Google Patents
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Abstract
本发明公开了特异性扩增疙瘩皮肤病病毒的GPCR基因的上游引物和下游引物。还公开了含有上述特异性扩增引物对的荧光定量PCR试剂盒。其采用的是荧光染料法，其特异性好，对GTPV疫苗株无扩增信号，且可检测到7.11×102copies/μL的LSDV DNA。该方法经济实惠，可以做溶解曲线，分析全部PCR产物的Tm值，不易受外界因素影响。The invention discloses an upstream primer and a downstream primer for specifically amplifying the GPCR gene of pimple skin disease virus. Also disclosed is a fluorescent quantitative PCR kit containing the above-mentioned specific amplification primer pair. The fluorescent dye method is used, which has good specificity, no amplification signal for GTPV vaccine strains, and can detect 7.11×10 2 copies/μL of LSDV DNA. This method is economical and can be used to do a dissolution curve and analyze the Tm value of all PCR products, and is not easily affected by external factors.
Description
Technical Field
The disclosure belongs to the technical field of gene editing, and particularly relates to a specific amplification primer pair and a fluorescent quantitative PCR kit containing the primer.
Background
Bovine sarcoidosis (LSD) is a bovine viral disease caused by LSDV, a double-stranded DNA virus of the Poxviridae (Poxviridae), myelopoxvirusidae (Chordopoxvirinae), Capripoxvirus (capropxvirus) genera. The LSDV genome is approximately 150kb in length, and like other poxviruses, the viral genome consists of a central core region and inverted repeats at both ends. The clinical manifestations of the affected cattle are fever, hard nodules or ulcers on the skin surface, and salivation, lacrimation and rhinorrhea in the course of disease; listlessness, weight loss and secondary bacterial infections; the milk yield is reduced; and in severe cases, death. LSD is listed as epidemic disease which needs to be notified by the world animal health Organization (OIE) and is listed as animal epidemic disease by the entry epidemic disease name of China.
In 1929, the disease was first discovered in Zanzia, introduced into boretvania in 1943, and then into south Africa, with significant economic losses. And continues to spread in the continental africa. The spread in the middle east of 2013 to 2017 continues to occur to many countries and is spreading to european countries such as russia, greece, etc., gradually becoming a global spread trend. In view of the prevalence of LSD, corresponding detection techniques are urgently needed. Therefore, establishing an accurate and rapid LSDV diagnosis method is of great significance.
Serological diagnosis of LSD employs agar gel immunodiffusion and indirect enzyme-linked immunosorbent assays (ELISA) which do not distinguish between infected and vaccinated animals, nor do they distinguish between antibodies produced by LSDV infection and antibodies produced by other capripoxvirus infections. Western blotting has good sensitivity and specificity for detecting poxvirus, but has certain limitation in practical application due to time and labor waste and difficult operation. The detection of the antibody level is difficult to realize in the early stage of the animal disease, so that the detection result is delayed, and the prevention and control of the LSD are not facilitated.
There are several existing methods for detecting LSD. An LSD detection method based on a loop-mediated isothermal amplification technology designs two specific inner primers and two specific outer primers according to six specific regions of a conserved region of bovine sarcoidosis 16S rRNA, can specifically distinguish bovine sarcoidosis virus from other pathogenic viruses, and ensures high specificity of loop-mediated isothermal amplification and reliability of a detection result. The disadvantages are as follows: the kit is easily influenced by external factors in the using process, and the probability of false positive is increased.
Disclosure of Invention
In one aspect, the present invention discloses a pair of specific amplification primers to solve one or more of the problems of the prior art and to provide at least one useful choice or condition. The specific amplification primer pair comprises an upstream primer and a downstream primer which are used for specifically amplifying GPCR genes of the bovine sarcoidosis virus. The G-protein-coupled chemokine receptor (GPCR) gene encodes a protein related to a G protein-coupled chemokine receptor subfamily, is one of variable genes in the capripoxvirus, and is a suitable target for distinguishing the capripoxvirus genes. Therefore, GPCR genes are used as the basis of the invention, and a real-time fluorescence quantitative PCR method is established to quickly detect the diseases caused by LSDV, so that early infected animals can be conveniently found.
Further, the sequence of the upstream primer qGPCR-F is 5'-AGTCGAATATAAAGTAATCAGTC-3' (SEQ ID NO.1), and the sequence of the downstream primer qGPCR-R is 5'-CCGCATATAATACAACTTATTATAG-3' (SEQ ID NO. 2). The length of the GPCR gene fragment specifically amplified is 125 bp.
On the other hand, the invention also discloses a fluorescent quantitative PCR kit containing the specific amplification primer pair. The fluorescent quantitative PCR kit also comprises TB Green Mix mixed solution and a positive standard substance.
Further, the construction of the positive standard comprises the following steps: extracting, amplifying, purifying and recovering GPCR genes of the pimple skin disease virus, cloning a PCR product into a pMD19-T vector, and constructing a recombinant plasmid pMD19T-LSDV which is the positive standard.
Further, the reaction system of the fluorescent quantitative PCR kit is 20 muL, and comprises 10 muL of the TB Green Mix mixed solution, 0.8 muL of the upstream primer, 0.8 muL of the downstream primer, 1 muL of the positive standard, and ddH for the rest2And (4) complementing O. The reaction conditions are as follows: maintaining at 95 deg.C for 30s for 1 cycle; maintaining the temperature at 95 ℃ for 10s and the temperature at 60 ℃ for 30s for 40 cycles, and measuring a standard curve of-3.1852 x +37.764 and a correlation coefficient R20.9993, the amplification Efficiency, 106.04%.
The detection method established by the invention adopts a fluorescent dye method, has good specificity, has no amplification signal to the GTPV vaccine strain, and can detect 7.11 multiplied by 102copies/. mu.L of LSDV DNA. The method is economical and practical, can be used for preparing a dissolution curve and analyzing the Tm values of all PCR products, and is not easily influenced by external factors.
Drawings
FIG. 1 is a gel electrophoresis image of example 1 for amplifying GPCR genes;
FIG. 2 is a standard curve diagram of the fluorescent quantitative PCR detection method of example 3;
FIG. 3 is a diagram of the fluorescent quantitative PCR specificity test in example 4;
FIG. 4 is a graph of the sensitivity assay for the fluorescent quantitative PCR detection of pMD19T-LSDV of example 5.
Detailed Description
The following steps are only used for illustrating the technical scheme of the disclosure and are not limited; although the present disclosure has been described in detail with reference to the foregoing steps, those of ordinary skill in the art will understand that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the scope of the respective technical solutions of the steps of the present disclosure.
Example 1 design and Synthesis of specific amplification primer pairs
GPCR genes were amplified and aligned for analysis according to the LSDV GPCR gene sequence published in GenBank (accession number: MN 508357). Designing a pair of specific primers, wherein an upstream primer qGPCR-F: 5'-AGTCGAATATAAAGTAATCAGTC-3', downstream primer qGPCR-R: 5'-CCGCATATAATACAACTTATTATAG-3', the length of the amplified fragment is 125 bp. The primer pair is synthesized by Shanghai Bioengineering Co., Ltd.
Example 2 construction of Positive Standard
LSDV DNA was extracted according to Mini BEST viral RNA/DNA Extraction Kit, and its GPCR gene was amplified using ordinary PCR. The reaction system is 25 μ L: 2 × Premix Tap 12.5 μ L, upstream and downstream primers 1.0 μ L (10 μmol/L), template 1.0 μ L, ddH2O7.5. mu.L. The amplification program is 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃. The PCR amplification product was subjected to 1.0% agarose electrophoresis as shown in FIG. 1. Then, according to the procedure of the DNA Purification Kit instruction, the PCR product is recovered and purified, and is cloned in a pMD19-T vector to construct a recombinant plasmid pMD19T-LSDV, namely a positive standard substance. In FIG. 1, lane number 1 is the recombinant plasmid pMD19T-GPCR, and lane number 2 is the negative control.
The recombinant plasmid pMD19T-LSDV was sent to Shanghai Bioengineering Co., Ltd for sequencing. Determination of OD of recombinant plasmid Using Spectrophotometer260nm/OD280nmRatio and concentration according to the formula: plasmid copy number (copies/. mu.L) ═ plasmid concentration × 10-9X dilution multiple x 6.02 x 1023) (660 daltons/base x number), standard plasmid concentration is converted to copy number.
Example 3 establishment of a Standard Curve for fluorescent quantitative PCR
The recombinant plasmid pMD19T-LSDV is subjected to gradient dilution to obtain 7.11X 102copies/μL～7.11×1098 dilutions of the standard, copies/. mu.L, were used as reaction templates. 3 sample replicates per dilution gradient and a negative control was established. The fluorescent quantitative PCR reaction system is 20 mu L: TB Green (2X) 10. mu.L, upstream and downstream primers 0.8. mu.L each (10. mu. mol/L), template 1. mu.L, ddH2O7.4. mu.L. The reaction conditions are as follows: 30s at 95 ℃ for 1 cycle; 10s at 95 ℃; 60 ℃ for 30s, for 40 cycles. Taking logarithm of standard substance concentration as X axis, Cq value as Y axis, drawing standard curve as shown in FIG. 2, wherein the standard curve is-3.1852X +37.764, and correlation coefficient R20.9993, the amplification Efficiency, 106.04%.
Example 4 specificity test
Genomic DNA of GTPV (CVCC AV41 accession number MH381810) was extracted using a kit, genomic RNA of PPRV (Clone 9 strain) vaccine strain was extracted using TRIZO method, and cDNA thereof was obtained using a reverse transcription kit. The constructed LSDV fluorescent quantitative PCR is used for detection, the recombinant plasmid pMD19T-LSDV is used as a positive control, and a negative control is set at the same time, so that the specificity of the detection method is verified. The results are shown in FIG. 3, in which reference numeral 1 is the amplification curve of the recombinant plasmid pMD19T-LSDV, and reference numerals 2, 3 and 4 are the amplification curves of GTPV, PPRV and negative control, respectively. Therefore, the fluorescent quantitative PCR kit has good LSDV specificity.
Example 5 sensitivity test
The recombinant plasmid pMD19T-LSDV was diluted 10-fold to obtain 7.11X 102copies/μL～7.11×1098 dilutions of the standard such as copies/. mu.L were used as templates for the sensitive detection of the fluorescent quantitative PCR. Meanwhile, the same template is subjected to conventional PCR amplification, and the sensitivity of the two detection methods is observed. As shown in FIG. 4, the reference numerals 1 to 8 are respectively: 7.11X 109copies/μL、7.11×108copies/μL、7.11×107copies/μL、7.11×106copies/μL、7.11×105copies/μL、7.11×104copies/μL、7.11×103copies/μL、7.11×102copies/. mu.L; reference numeral 9 is a negative control. It can be seen that even the concentration is 7.11X 102Amplification detection can also be successfully achieved with copies/. mu.L samples.
Example 6 repeatability test
Selecting 7.11X 102copies/μL～7.11×108Duplicate experiments in batches were carried out at 7 concentrations copies/. mu.L; at 3 different times, the above 7 dilutions of plasmid standard were subjected to a batch-to-batch reproducibility test, with 3 replicates per concentration. And calculating the variation Coefficient (CV) between batches according to the Cq value of the template, and verifying the reliability and repeatability of the fluorescent quantitative PCR method. The results are shown in Table 1.
TABLE 1 repeatability test of the fluorescent quantitative PCR method (n ═ 3)
Example 7 detection of clinical samples
Samples from cattle farms in the Guangdong region were collected as: 10 parts of each of skin nodules, tear swabs and nasal swabs are diluted by a proper amount of PBS solution, ground by using a tissue grinder, 30cycles/s for 2min, then centrifuged at 12000rpm/min for 1min, tissue supernatant is taken, DNA is extracted according to a TIANAmp Genomic DNA Kit extraction Kit, the sample is detected by using a fluorescence quantitative PCR Kit established in the experiment, the experiment result is compared with the ordinary PCR detection result, and the LSDV positive detection rate is calculated. The results are shown in Table 2.
Table 2 testing of clinical samples with two test methods (n ═ 3)
SEQUENCE LISTING
<110> institute of Buddha science and technology
<120> specific amplification primer pair and fluorescent quantitative PCR kit
<130> 2020
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence
<400> 1
agtcgaatat aaagtaatca gtc 23
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
ccgcatataa tacaacttat tatag 25
Claims (6)
1. A specific amplification primer pair, comprising an upstream primer and a downstream primer for specifically amplifying a GPCR gene of bovine sarcoidosis virus.
2. The specific amplification primer pair of claim 1, wherein the sequence of the upstream primer is shown as SEQ ID No.1, and the sequence of the downstream primer is shown as SEQ ID No. 2.
3. A fluorescent quantitative PCR kit comprising the specific amplification primer set according to claim 1 or 2.
4. The fluorescent quantitative PCR kit of claim 3, further comprising TB Green Mix mixture and a positive standard.
5. The fluorescent quantitative PCR kit of claim 3, wherein the construction of the positive standard comprises the steps of: extracting, amplifying, purifying and recovering GPCR genes of the bovine sarcoidosis virus, cloning a PCR product into a pMD19-T vector, and constructing a recombinant plasmid pMD19T-LSDV, namely the positive standard substance.
6. The fluorescent quantitative PCR kit of claim 3, wherein the reaction system of the fluorescent quantitative PCR kit is 20 μ L, and comprises 10 μ L of the TB Green Mix mixture, 0.8 μ L of the upstream primer, 0.8 μ L of the downstream primer, 1 μ L of the positive standard, and the balance ddH2And (4) complementing O.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114736952A (en) * | 2022-03-29 | 2022-07-12 | 佛山科学技术学院 | Real-time fluorescent quantitative PCR detection primer, method and application of streptococcus equi subsp zooepidemicus |
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