CN112301167A - Primer probe combination for detecting African swine fever virus and detection method based on microfluidic chip Digital PCR - Google Patents
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Abstract
The invention belongs to the technical field of inspection and quarantine, and particularly relates to a primer-probe combination for detecting African swine fever virus and a detection method based on microfluidic chip Digital PCR. The nucleic acid sequence of the primer pair in the primer probe combination is shown as SEQ ID NO. 1-2, and the nucleic acid sequence of the probe in the primer probe combination is shown as SEQ ID NO. 3. The primer probe combination is used for amplifying a conserved sequence of a B646L gene of African swine fever virus, and the conserved sequence is specifically shown as SEQ ID NO. 4. When the primer probe combination is used for carrying out the microfluidic chip-based Digital PCR, the minimum detection limit of the African swine fever virus can be as low as 30.1995copies per microliter of template, and the Digital PCR has good repeatability, and the average CV value of the Digital PCR is 9.56%.
Description
Technical Field
The invention belongs to the technical field of inspection and quarantine, and particularly relates to a primer-probe combination for detecting African swine fever virus and a detection method based on microfluidic chip Digital PCR.
Background
African Swine Fever (ASF) is an acute, hemorrhagic and highly contact infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs or wild pigs, and is characterized by short course of disease, high fever and hemorrhagic lesions, the death rate of acute infection reaches 100 percent, and the global pig industry is seriously threatened but no effective vaccine and treatment method is developed at present. ASFV is the only member of African swine fever virus family, is a large double-stranded DNA virus, mainly replicates in macrophage cytoplasm, and has a genome of about 170-193kb, containing 150-167 open reading frames, and encoding 150-200 proteins. At present, about 50 virus-encoded proteins with known functions are available, most of the virus-encoded proteins are virus structural proteins, and more than half of ASFV-encoded proteins have unknown functions.
At present, the diagnosis method of African swine fever comprises red blood cell adsorption test, direct immunofluorescence test, animal inoculation test, ELISA, polymerase chain reaction and the like. Among them, the sensitivity and specificity of erythrocyte adsorption test and direct immunofluorescence test are not high, and the animal inoculation test is unsafe. The routine laboratory diagnostic method for ASFV is Virus Isolation (VI), which, although reliable and sensitive, takes up to 6 days. Polymerase Chain Reaction (PCR) is an alternative to VI for rapid detection of ASFV, and is particularly useful for screening samples with unrecoverable virus that are of poor quality or degraded. The traditional PCR method for detecting African swine fever virus has the characteristics of specificity, sensitivity and rapidness, but has the defects of time consumption, easy pollution, need of electrophoresis detection after amplification, small number of samples for each detection and the like. The second generation PCR technology, namely fluorescence quantitative PCR (qPCR) detection technology, releases fluorescence through a dye or a probe, monitors the fluorescence change of each PCR cycle in real time, finally generates an amplification curve, and can finally generate a melting curve if the method is a dye method, and analyzes the specificity of a product. However, the detection sensitivity is not high, and absolute quantification cannot be performed. The third generation PCR technology, namely, the digital PCR (digital PCR) technology, divides a sample into tens to tens of thousands of samples, distributes the samples to different reaction units, each unit contains zero or one (or a plurality of copies) of target molecules, performs PCR amplification on the target molecules in each reaction unit, and performs statistical analysis on the fluorescence signals of each reaction unit after the amplification is finished. Compared with the traditional qPCR technology, the Digital PCR has extremely high sensitivity, specificity and accuracy. Therefore, the invention aims to establish a technology for detecting African swine fever viruses based on a microfluidic chip Digital PCR platform so as to provide effective technical support for prevention and control of African swine fever viruses.
Disclosure of Invention
The invention aims to provide a primer probe combination for detecting African swine fever virus and a detection method based on a microfluidic chip Digital PCR (polymerase chain reaction), so that the copy number of the African swine fever virus in serum to be detected can be accurately quantified.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a primer probe combination for detecting African swine fever viruses is disclosed, wherein nucleic acid sequences of primer pairs in the primer probe combination are shown as SEQ ID NO. 1-2, and nucleic acid sequences of probes in the primer probe combination are shown as SEQ ID NO. 3.
Preferably, the primer probe combination is used for amplifying a conserved sequence of the B646L gene of the African swine fever virus, and the conserved sequence is specifically shown as SEQ ID NO. 4.
Preferably, the 5 'end of the probe is modified with a luminescent fluorescent group, and the 3' end of the probe is modified with a fluorescence quenching group.
Preferably, the luminescent fluorophore is FAM; the fluorescence quenching group is MGB.
An application of the primer probe combination in detecting African swine fever virus.
The method for detecting African swine fever virus by using the primer probe combination is characterized in that the primer probe combination is used for carrying out fluorescence quantitative PCR or microfluidic chip Digital PCR, and the addition concentration of the primer pair is 12.5-100 pmol/L; the concentration of the probe added was 1.25 to 10 pmol/L.
Preferably, the concentration of the primer pair is 12.5 pmol/L; the concentration of the probe added was 10 pmol/L.
Preferably, when the primer probe combination is used for performing Digital PCR based on a microfluidic chip, the minimum detection limit is 30-35 copies.
Through earlier researches, primers and probes are designed aiming at a conserved sequence of a B646L gene of African swine fever virus, and the optimal addition concentration of a primer probe combination is explored by utilizing fluorescent quantitative PCR. The result shows that when the primer probe combination is used for carrying out fluorescence quantitative PCR, the lowest detection limit of the African swine fever virus is 1000copies per microliter of the template; when the primer probe combination is used for carrying out the microfluidic chip-based Digital PCR, the minimum detection limit of the African swine fever virus can be as low as 30.1995copies per microliter of template, and the Digital PCR has good repeatability, and the average CV value of the Digital PCR is 9.56%. The method is high in sensitivity, accuracy and repeatability, is provided for detecting the African swine fever virus, is beneficial to quickly and efficiently detecting the African swine fever virus, and lays a foundation for preventing and blocking the spread of the virus.
Drawings
FIG. 1 phylogenetic tree of African swine fever virus;
FIG. 2 shows a fluorescent quantitative PCR amplification curve under different standard concentration conditions;
FIG. 3 is a comparison of the microfluidic chip based Digital PCR and fluorescent quantitative PCR standard curves. (a) A Digital PCR standard curve, (b) a fluorescent quantitative PCR standard curve;
FIG. 4 is a comparison of detection limits based on the microfluidic chip Digital PCR and the fluorescent quantitative PCR; (a) a logistic regression curve of Digital PCR, (b) a logistic regression curve of fluorescent quantitative PCR, (c) a Digital PCR amplification curve, (d) a fluorescent quantitative PCR amplification curve;
FIG. 5 is a comparison of the repeatability of Digital PCR and fluorescent quantitative PCR based on microfluidic chips; (a) the CV value comparison curves of the two detection methods; (b) the average CV values of the two detection methods are compared.
Detailed Description
The invention is discussed in further detail below with reference to specific examples.
Reagent and consumable
Nucleic-free water (Promega), primers and probes (Shanghai Biotech Co., Ltd.),
optical 96-well reaction plate(Applied Biosystems)、
master mix(Applied Biosystems)、
5700Sequence Detection System(Applied Biosystems)、QuantStudioTM 3D Digital PCR Instrument(ThermoFisher)、Dual Flat Block GeneAmpTM PCR System 9700(ThermoFisher)；
Other reagents and consumables used herein are either commonly available or available to those skilled in the art through published work.
EXAMPLE 1 design of primers and probes
According to the previous research results, p72 is an important structural protein expressed by ASFV, is coded by ORF B646L gene, has high conservation among different strains of African swine fever virus, and therefore, the B646L gene of ASFV is selected as a target gene to carry out PCR amplification.
The B646L sequences of 53 different ASFV epidemic strains are aligned and analyzed, a phylogenetic tree of ASFV is constructed by using MEGA 7.0 software and the adjacent method (as shown in figure 1), and phylogenetic analysis is carried out, and the number on the branch indicates bootstrap value >50(1,000 times of repetition). It can be seen from the evolutionary tree that the ASFV strains circulating in china are closely related to the circulating strains in grurgia, germany and polish. Therefore, 53 ASFV gene sequences (GenBank accession numbers are shown in Table 1) which have a close genetic relationship with the epidemic Anhui strain in China are selected and subjected to Blastn comparison to obtain a highly conserved region sequence of the B646L gene of the ASFV main structural protein p72, which is shown in SEQ ID No.4 and specifically comprises the following steps:
ACGTTTCCTCGCAACGGATATGACTGGGACAACCAAACACCCTTAGAGGGCGCCGTTTACACG
PRIMERs and probes are designed aiming at the high-conservative region by using PRIMER EXPRESS version 1.5 software of the company of Applied Biosystems in the United states, and are compared with the whole gene sequence of each strain of ASFV, and the specific comparison result is shown in Table 1;
TABLE 1 alignment of primers and TaqMan-MGB probes with 53 ASFV epidemic strains and 5 other virus strains
Selecting a primer and a probe sequence which are mismatched with the sequences of the strains with the least errors as a primer pair and a probe for subsequent fluorescent quantitative PCR and PCR reaction based on a microfluidic chip according to a comparison result, wherein the nucleic acid sequence of the primer pair is shown as SEQ ID NO. 1-2, the nucleic acid sequence of the probe is shown as SEQ ID NO.3, and the specific sequence is shown in Table 2 (synthesized by Shanghai Biotech, Ltd.);
TABLE 2 TaqMan-MGB Probe sequences and PCR primer sequences
In order to facilitate the observation of reaction results, FAM fluorescent molecules and MGB fluorescent quencher molecules are respectively modified at two ends of the TaqMan-MGB probe. When a sequence to be amplified is in a PCR system, the TaqMan-MGB probe is combined with the sequence to be detected through a complementary pairing principle, and is hydrolyzed by Taq enzyme in the PCR process, so that FAM fluorescein and MGB fluorescence quencher molecules are separated, and fluorescence is emitted.
Example 2 primer and Probe concentration optimization
In order to obtain the optimal addition concentrations of primers and probes, the use concentrations of primers and probes in example 1 were optimized using conventional fluorescent quantitative PCR.
The fluorescent quantitative PCR reaction system is 10 mu L, and the adding amount of each component is as follows:
5. mu.L of master mix, 0.4. mu.L of primer Fwd, 0.4. mu. L, TaqMan of primer Rev-MGB probe 0.4. mu. L, Nuclease-free water 1.8. mu.L, and 2. mu.L of template. The reaction procedure is as follows: reacting at 95 ℃ for 10min in one cycle; the reaction was carried out at 58 ℃ for 60s for 40 cycles. Each proportion is repeated for three times; the Ct values for different primer and probe concentrations are shown in Table 3.
TABLE 3 Ct value determination results under different primer and probe concentration conditions
As can be seen from the above table, since the Ct value of the amplification result is the lowest at a primer concentration of 12.5pmol/L and a probe concentration of 10pmol/L, the concentration ratio (i.e., the upstream and downstream primer concentrations are both 12.5pmol/L and the probe concentration is 10pmol/L) is selected as the concentration to be used in the subsequent reaction.
Example 3 validation of primers and probes in fluorescent quantitative PCR
The synthesized African swine fever virus B646L gene (GeneBank Accession: MH766894.1) conserved sequence is inserted into a baculovirus vector pFastBacI to construct pFastBacI-P72 recombinant plasmid, the recombinant plasmid is transformed into DH5a competent cells to enable the recombinant plasmid to proliferate in large quantity, pFastBacI-P72 plasmid with correct sequence is obtained through cloning and sequencing, the concentration is determined, and pFastBacI-P72 is used as standard plasmid. Measuring pFastBacI-P72 standard plasmid concentration with micro ultraviolet spectrophotometer, and calculating to obtain 1010Plasmid concentration corresponding to copies, ten fold dilution of standard plasmid to 1010copies、109copies、108copies、107copies……100copies, left at 4 ℃ for use.
Performing fluorescent quantitative PCR amplification by using the standard plasmids with different concentration gradients as templates, wherein the reaction system is as follows:
5. mu.L of master mix, 0.4. mu.L of primer Fwd, 0.4. mu. L, TaqMan of primer Rev-MGB probe 0.4. mu. L, Nuclease-free water 1.8. mu.L, and 2. mu.L of template. The total volume of the reaction system per well was 10. mu.L. The PCR reaction program is: reacting at 95 ℃ for 10min in one cycle; the reaction was carried out at 58 ℃ for 60s for 40 cycles. Three replicates were made for each standard gradient. The fluorescent quantitative PCR amplification curve of the standard plasmid with different concentration gradients is shown in FIG. 2; as can be seen from FIG. 2, when the optimal primer and probe concentrations obtained in example 2 were used for conventional qPCR amplification, the amplification curve was smooth and complete, and the amplification sensitivity could reach 103copies。
Example 4 comparison of amplification effects of Digital PCR and fluorescent quantitative PCR
The negative control method is to detect negative controls several times, and 2 times of the maximum detection value of the negative controls is used as the detection limit. Dilution method i.e. 10-fold gradient dilution of standard plasmid: adding 45 μ L of water into 5 μ L of the above template supernatant, mixing wellTaking 5 mu L to 45 mu L water in the 2 nd tube, and repeating the dilution in sequence to obtain the final dilution concentration of 104～10-1. And (3) taking an equal amount of sample from each gradient, respectively amplifying by using the traditional fluorescent quantitative PCR and the microfluidic chip-based Digital PCR, setting 2 parallel samples for each concentration gradient to perform parallel determination, and evaluating the detection limit of the detection method, namely the detection sensitivity.
The composition of the mixed solution of the Digital PCR reaction is as follows: 10.0 muL of Master Mix, 1.8 muL of upstream primer, 1.8 muL of downstream primer, 0.5 muL of probe TaqMan-MGB, 3.9 muL of Water, 2 muL of template and 20.0 muL of total volume (the volumes above all contain 20% of the loss of pipette tip wall hanging of a micropipette). The Digital PCR reaction program is: the Digital PCR amplification program is: the first step is as follows: hot start at 96 ℃ for 10 minutes; the second step is that: annealing at 60 ℃ for 2 minutes; 60 seconds at 98 ℃; amplifying for 39 cycles; the third step: reacting at 60 ℃ for 2 minutes; keeping the temperature at 10 ℃.
Making a standard curve by using curve expert software (figure 3); as can be seen from FIG. 3, R of the standard curve for Digital PCR20.9985, compared with the traditional fluorescent quantitative PCR (R)20.9881) has better linearization.
Example 5 minimum detection Limit based on microfluidic chip Digital PCR
Evaluating the detection range in the detection limit evaluation method, performing 10-fold gradient dilution on the standard plasmid for detection, performing linear regression statistical treatment on the regression equation, performing linear test and a nonlinear logit model according to R2Value, the lowest detection limit of the detection method is evaluated.
The experimental results show that through logistic regression analysis, the nonlinear logistic regression equation obtained is:
wherein R is2＝0.998；
It follows that the minimum detection limit for microfluidic chip-based Digital PCR reaches 30.1995copies per reaction, see fig. 4 (a). The same primers and probes are used for real-time fluorescent quantitative PCR (qPCR) amplification, and a qPCR nonlinear logistic regression equation obtained by logistic regression analysis is as follows:
from this, it can be concluded that the lowest detection limit of fluorescent quantitative PCR is 1000copies per reaction. The result shows that the detection limit of the fluorescent quantitative PCR is 33 times of that of the Digital PCR, and the Digital PCR based microfluidic chip has higher sensitivity.
Example 6 repeatability analysis
The repeatability of the detection method in the detection limit evaluation method is evaluated for the standard substance diluted by 10 times, 3 times of repeated detection is carried out, and the repeatability of the detection method is evaluated by calculating the Relative Standard Deviation (RSD) of the detection result. As shown in FIG. 5, the results of the experiment showed that the Digital PCR had very good reproducibility, the average CV value was 9.56%, which was lower than that of the fluorescent quantitative PCR (12.67%), and the difference between the average CV values was 26.99%.
SEQUENCE LISTING
<110> Zhengzhou university
HENAN ZHONGZE BIOLOGICAL ENGINEERING Co.,Ltd.
<120> primer probe combination for detecting African swine fever virus and detection method based on microfluidic chip Digital PCR
<130> NONE
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<170> PatentIn version 3.5
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Claims (8)
1. A primer probe combination for detecting African swine fever virus is characterized in that: the nucleic acid sequence of the primer pair in the primer probe combination is shown as SEQ ID NO. 1-2, and the nucleic acid sequence of the probe in the primer probe combination is shown as SEQ ID NO. 3.
2. The primer probe combination of claim 1, wherein: the primer probe combination is used for amplifying a conserved sequence of a B646L gene of African swine fever virus, and the conserved sequence is specifically shown as SEQ ID NO. 4.
3. The primer probe combination of claim 1, wherein: the 5 'end of the probe is modified with a luminous fluorescent group, and the 3' end of the probe is modified with a fluorescent quenching group.
4. The primer probe combination of claim 3, wherein: the luminescent fluorophore is FAM; the fluorescence quenching group is MGB.
5. The primer probe combination of claim 1, wherein the primer probe combination is used for detecting African swine fever virus.
6. A method for detecting african swine fever virus using the primer probe combination of claim 1, wherein the method comprises: performing fluorescent quantitative PCR or microfluidic chip-based Digital PCR by using the primer probe combination, wherein the addition concentration of the primer pair is 12.5-100 pmol/L; the concentration of the probe added is 1.25 to 10 pmol/L.
7. The method of claim 6, wherein: the addition concentration of the primer pair is 12.5 pmol/L; the concentration of the probe added was 10 pmol/L.
8. The method of claim 6, wherein: when the primer probe combination is used for carrying out Digital PCR based on a microfluidic chip, the lowest detection limit is 30-35 copies per microliter of template.
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