CN111500790A - A primer-probe set for detection of African swine fever by fluorescence quantitative PCR and its application - Google Patents

Abstract

The invention discloses a primer probe set for detecting African swine fever by fluorescence quantitative PCR (polymerase chain reaction) and application thereof, wherein the primers are MGF360-F and MGF360-R, the probe is MGF360-Taq Man, the nucleotide sequence of MGF360-F is shown as SEQ ID No.1, the nucleotide sequence of MGF360-R is shown as SEQ ID No.2, and the nucleotide sequence of MGF360-Taq Man is shown as SEQ ID No.3, and a method and a kit for detecting African swine fever by using the primer probe set are provided. The primer probe group has high sensitivity and specificity, makes up the defects of the prior art, and lays a solid technical reserve and theoretical foundation for the revival of the domestic pig industry.

Description

A primer-probe set for detection of African swine fever by fluorescence quantitative PCR and its application

technical field

The invention belongs to the technical field of biology, and in particular relates to a primer probe set for detecting African swine fever by fluorescence quantitative PCR and its application.

Background technique

African swine fever is an acute, severe and highly contagious infectious disease of domestic pigs and wild boars. The virulent strain can kill domestic pigs within about 5-14 days of infection, and the mortality rate is close to 100%. There is no effective preventive vaccine. There is no effective treatment drug. On August 3, 2018, the first case of African swine fever was reported in Shenbei New District, Shenyang City, Liaoning Province, my country. The occurrence of African swine fever has brought great harm to my country's pig breeding industry. In order to prevent the rapid spread of African swine fever, early diagnosis and early treatment are the main measures to prevent and control African swine fever at this stage.

The PCR detection method is the detection method officially recommended by OIE. This method has low requirements on the purity of pathogenic nucleic acid, simple and rapid operation, high sensitivity and specificity, and is especially suitable for identifying pathogens that cannot be isolated in vitro.

Real-time quantitative PCR (qPCR) is a new quantitative test technology introduced by Applied Biosystems in the United States in 1996. It uses fluorescent dyes or fluorescently labeled specific probes to label and track PCR products and monitor the reaction online in real time. In the process, the product can be analyzed in combination with the corresponding software, and the initial concentration of the sample template to be tested can be calculated. On January 4, 2019, the Ministry of Agriculture and Rural Affairs of China announced the first batch of ASFV on-site rapid detection reagents, of which more than 60% use real-time qPCR detection methods. This method can detect the reaction process in real time, determine the absolute amount of the template, and has strong specificity, which makes qPCR more valuable in the actual investigation of ASFV.

TaqMan probe method is a highly specific quantitative qPCR technology. Its working principle is that there are a pair of PCR primers and a probe in the PCR reaction system. The 5' end of the probe is labeled with a reporter group, and the 3' end is labeled with a fluorescent quenching group. The probe only binds specifically to the template. , whose binding site is between the two primers. When the probe is intact, the fluorescence energy of the reporter group is absorbed by the quencher group, so the instrument cannot collect the signal. As the reaction progresses, the Taq enzyme encounters the probe and uses the 3′→5′ exonuclease The activity of the probe cuts off the probe, so that the fluorescence energy of the reporter group cannot be absorbed by the quencher group, resulting in a fluorescent signal, so the intensity of the signal represents the copy number of the template DNA.

In this application, TaqMan probe method is used for detection, and primers designed according to the conserved gene fragments of ASFV can specifically identify ASFV. Compared with the fluorescence quantitative PCR recommended by OIE, OIE is mainly based on the gene design of B646L (p72), an early diagnosis target of ASFV, and this application mainly detects the MFG360 gene of African swine fever virus.

SUMMARY OF THE INVENTION

The invention provides an African swine fever fluorescent quantitative PCR detection primer, a probe and a method thereof, which can detect the African swine fever virus MGF360 gene by real-time fluorescent quantitative PCR. Make up for the shortcomings of the existing technology and lay a solid technical reserve and theoretical foundation for the revival of my country's pig industry.

The present invention is specifically realized through the following technical solutions;

A primer probe set for detecting African swine fever by fluorescence quantitative PCR, the primers are MGF360-F and MGF360-R, the probe is MGF360-Taq Man, and the nucleotide sequence of the MGF360-F As shown in SEQ ID NO. 1, the nucleotide sequence of MGF360-R is shown in SEQ ID NO. 2, and the nucleotide sequence of MGF360-Taq Man is shown in SEQ ID NO. 3.

The 5' end of the probe is labeled with a reporter group, and the 3' end is labeled with a fluorescence quenching group, the reporter group is FAM, and the fluorescence quenching group is BHQ1.

In another aspect of the present invention, there is provided the application of primers shown in SEQ ID NO. 1 to 2 or probes shown in SEQ ID NO. 3 in the detection of African swine fever.

In another aspect of the present invention, there is provided a kit for detecting African swine fever by fluorescence quantitative PCR, the kit includes primer probe sets with nucleotide sequences such as SEQ ID NO. 1-3.

The kit also includes reagents: 2×Pro TaqHS Probe Premix, MGF360-F, MGF360-R, MGF360-Probe, DNase/RNase-Free Deionized Water.

In another aspect of the present invention, a method for detecting African swine fever by fluorescence quantitative PCR is provided, comprising the following steps: extracting viral genomic DNA using a kit, and using the above primers and probes to amplify according to the reaction procedure.

The reaction system is 25 μL: 12.5 μL of 2×Pro TaqHS Probe Premix, 1 μL of upstream and downstream primers (50 pmol/μL), 1 μL of probe (50 pmol/μL), 2 μL of template DNA, and DNase/RNase-Free Deionized Water is added to 25 μL .

The reaction procedure described was: 50°C for 2 min; pre-denaturation at 95°C for 10 min; 95°C for 15 s, 55°C for 1 min, 40 cycles.

The beneficial effects of the present invention are:

The real-time fluorescent PCR method designed for the detection of African swine fever MGF360 gene can specifically detect the MGF360 gene, and can detect the reaction process in real time and determine the absolute amount of the template. This method has the advantages of high sensitivity, high specificity and high precision.

Description of drawings

Fig. 1 is the PCR identification result of the recombinant plasmid standard product of the present invention; wherein, M: DL2000 DNA Marker; 1: MGF360 gene fragment; 2: negative control;

Fig. 2 is the MGF360 fluorescence quantitative PCR standard curve of the present invention;

Figure 3 is the result of the sensitivity test of the present invention.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Main test materials:

A plasmid containing the MFG360 gene sequence (GenBank: MK333180.1, gene length 7559bp, located at the 27942-35500 nucleotide sequence position) was synthesized by the company.

Porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, parvovirus, Japanese Japanese encephalitis, porcine epidemic diarrhea virus, swine fever virus vaccine and foot-and-mouth disease virus vaccine are kept in this laboratory.

DH5α competent cells, viral genomic DNA extraction kit, common agarose gel DNA recovery kit, and common plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.; pMD19-T vector, 2×Taq PCR Master Mix, DL2000 DNA Marker and others were purchased from Bao Bioengineering (Dalian) Co., Ltd.

Example 1 Construction and identification of recombinant plasmid standard

According to the nucleic acid sequence of ASFV (MK333180.1), specific primers and fluorescent probes for the MGF360 gene were designed (Table 1). The primers and probes were synthesized by Chengdu Youkang Biotechnology Co., Ltd. and Shanghai Sangon Bioengineering Technology Co., Ltd., respectively.

Table 1 Primer and TaqMan probe sequence information

According to the MFG360 gene sequence of ASFV on GenBank, the 143bp gene sequence of the target fragment (the 27942-35500 nucleotide sequence of the accession number MK333180.1) was artificially synthesized as a positive template for subsequent experiments. Expand the culture on the template. The specific primers MGF360-F/MGF360-R were used to amplify the target gene. After detection by 1% agarose gel electrophoresis, the target fragment was recovered and cloned into the pMD19-T vector. The positive cloned product was provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Company sequencing identification. Proliferation-positive strains were cultured, and the recombinant plasmid was extracted with a plasmid extraction kit and named pMD19-ASFV-MGF360.

The concentration of recombinant plasmid standard was determined by NanoDrop 2000 nucleic acid and protein analyzer, and its copy number was calculated. According to the formula: copy number of recombinant plasmid (copy/μL)=(plasmid concentration×10 ⁻⁹ ×6.02×10 ²³ )/(660 Daltons/base×base number), the copy number was calculated.

The results showed that the strains were amplified to obtain target bands of about 143 bp (Fig. 1). After the PCR products were recovered and purified, they were cloned into the pMD19-T vector to construct the recombinant plasmid standard pMD19-ASFV-MGF360. After sequencing, compared with the reference sequence (accession number: MK333180.1), no MGF360 gene mutation was found, and the results were in line with expectations. It indicated that the plasmid standard pMD19-ASFV-MGF360 was constructed correctly. After calculation, the plasmid standard was 1.8×10 ⁸ copies/μL.

Example 2 Fluorescence quantitative PCR standard curve

The constructed pMD19-T recombinant plasmid was used as the standard for pMD19-ASFV-MGF360 quantification, and 10-fold gradient dilution was performed on the standard according to the determined plasmid concentration, and a total of 7 gradients (10 ¹ to 10 ⁷ ) were diluted. Fluorescence quantitative PCR was performed with different concentrations of recombinant plasmids as templates, the Ct values of each gradient standard were recorded, and the quantitative standard curve of the relationship between plasmid copy number and its corresponding relationship was established.

Using a 25 μL reaction system, using the same concentration of 2 μL positive plasmid as a template, selecting primers and probes of different concentrations, using the matrix method to optimize the optimal concentrations of primers and probes, the lowest Ct value and the highest fluorescence intensity increase value of the reaction were obtained. (ΔRn), to improve the amplification efficiency and sensitivity of the reaction, optimize the annealing temperature (55°C to 62°C), and set a blank control at the same time.

The total volume of the optimized fluorescence quantitative PCR reaction system is 25 μL, including 12.5 μL of 2×Pro TaqHS ProbePremix, 1 μL of upstream and downstream primers (50 pmol/μL), 1 μL of probe (50 pmol/ μL), 2 μL of template DNA, DNase/RNase- Free Deionized Water was added to 25 μL. Bio-Rad CFX96 fluorescence quantitative PCR instrument was used for amplification according to the following procedures: 50 °C for 2 min; 95 °C for pre-denaturation for 10 min; 95 °C for 15 s; Fluorescence channel selection FAM.

^Seven dilutions (1.8×10 ¹ to 1.8× ^{10 7} ) of quantitative standards were detected using the optimized fluorescence quantitative PCR reaction system. A good linear relationship, with a correlation coefficient of 0.999, yielded a linear equation of standard copy number and Ct value (Figure 2).

Example 3 Fluorescence quantitative PCR sensitivity detection

The standard was diluted 10 times serially with ddH ₂ O into 7 gradients (1.8×10 ¹ -1.8×10 ⁷ copies/μL) as templates, respectively, and the optimal reaction conditions were used for detection, and the established fluorescence quantitative PCR detection method was determined. The minimum detection amount used to evaluate the sensitivity of the method.

As shown in Figure 3, it was determined that the minimum detection amount of the established fluorescence quantitative PCR detection method was 1.8×100 ^copies /μL, which indicated that the method had high sensitivity.

Example 4 Specificity test

Extract genomic DNA of porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, parvovirus, Japanese Japanese encephalitis, porcine epidemic diarrhea virus, swine fever virus vaccine and foot-and-mouth disease virus vaccine preserved in this laboratory and use it as a template , while using recombinant plasmid standard as positive control and ddH ₂ O as negative control, the fluorescence quantitative PCR method established in this study was used for amplification to evaluate the specificity of the method.

Specificity tests were carried out with corresponding DNA samples such as extracted porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, and recombinant plasmid samples, and a negative control was set up to amplify according to the optimized fluorescence quantitative PCR system and conditions. The results showed that only the recombinant plasmid samples were positive, other viruses and negative control samples did not show any obvious amplification curves, and the detection results were all negative, indicating that the method has good specificity.

Example 5 Repeatability test

In order to evaluate the repeatability of the detection method, 3 concentrations of recombinant plasmids at 1.8×10 ² to 1.8×10 ⁴ copies/μL were tested, and each concentration was repeated 3 times, and the obtained Ct values were counted to calculate the average, Standard deviation and coefficient of variation.

Table 2 Fluorescence quantitative PCR repeatability detection results

It can be seen from Table 2 that the established method for the detection of African swine fever MGF360 genotype by real-time quantitative PCR has good repeatability, and can perform stable and reliable detection of African swine fever MGF360 genotype samples.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principle and spirit of the invention Variations, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

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Claims (10)

1. A primer-probe group for the detection of African swine fever by fluorescence quantitative PCR, characterized in that the nucleotide sequence of the primer is shown in SEQ ID NO.1～2, and the nucleotide sequence of the probe The sequence is shown in SEQ ID NO.3. 2. a kind of primer probe set for the detection of African swine fever according to claim 1, it is characterized in that, the 5' end of described probe is marked with a reporter group, and the 3' end is marked with fluorescence quenching group. 3 . The primer-probe set for detecting African swine fever according to claim 2 , wherein the reporter group is FAM, and the fluorescence quenching group is BHQ1. 4 . 4. the application of the primer probe set described in claim 1 in detecting African swine fever. 5. A kit for detecting African swine fever by fluorescence quantitative PCR, characterized in that the kit comprises a primer probe set with nucleotide sequences such as SEQ ID NO. 1-3. 6 . The kit according to claim 6 , wherein the kit comprises reagents: 2×Pro TaqHSProbe Premix, MGF360-F, MGF360-R, MGF360-Taq Man, DNase/RNase-Free Deionized Water. 7 . 7. A method for detecting African swine fever by fluorescence quantitative PCR, characterized in that, a kit is used to extract viral genomic DNA, and the primers and probes of claim 1 are used to carry out PCR amplification according to the reaction program. 8. The method for detecting African swine fever according to claim 7, wherein the reaction system is 25 μL: 12.5 μL of 2×ProTaqHS Probe Premix, 1 μL of upstream and downstream primers, 1 μL of probe, 2 μL of template DNA, DNase/RNase - Free Deionized Water was added to 25 μL. 9 . The method for detecting African swine fever according to claim 8 , wherein the concentration of the PCR upstream and downstream primers and probes is 50 pmol/μL. 10 . 10 . The method for detecting African swine fever according to claim 7 , wherein the PCR reaction program is: 50° C. for 2 min; 95° C. for 10 min of pre-denaturation; 95° C. for 15 s, 55° C. for 1 min, 40 cycles. 11 .

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