CN109781981B - Molecular probe for detecting African swine fever virus and Realtime-PCR detection method - Google Patents
Molecular probe for detecting African swine fever virus and Realtime-PCR detection method Download PDFInfo
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Abstract
The invention discloses a molecular probe for detecting African swine fever virus and a Realtime-PCR detection method, and aims to solve the technical problem that the existing detection technology is poor in reliability and practicability. The nucleotide sequence of the molecular probe for detecting the African swine fever virus is shown as SEQ ID NO. 2. The invention prepares an RT-PCR reaction solution for detecting African swine fever virus, prepares an RT-PCR reaction solution freeze-dried powder for detecting the African swine fever virus, an African swine fever virus detection kit and provides qualitative and quantitative detection methods of the African swine fever virus. The molecular probe and the method for establishing the RT-PCR detection have the characteristics of high sensitivity, good repeatability and strong specificity, improve the reliability and usability of the PCR technology for detecting the ASFV, realize pollution-free and high-flux detection, and can be used for detecting the ASFV nucleic acid in susceptible animals and related animal products.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a molecular probe for detecting African swine fever virus and a Realtime-PCR detection method.
Background
African Swine Fever Virus (ASFV) infects livestock or wild boars causing African Swine Fever (ASF), which can lead to high morbidity and mortality, and cause significant economic losses to the swine industry. ASFV was first discovered in Kenya in 1921, and then the presence of ASFV was found in many countries in the south and east of Africa. ASF spreads rapidly and rapidly, and is spread from Africa to all over the world for as short as several decades, almost wrapping around the world. In 2018, 8, 2, 17 pm, the five-five community of Shenbei street (New City) in Shenbei New zone of Shenyang city has suspected African swine fever epidemic situation, and the diagnosis is confirmed at 8, 3, 11 am. By 8 months, 3 days and 15 days in 2018, 913 head pigs in the epidemic spot are completely killed and harmlessly treated. Then the provinces such as Heilongjiang, Jiangsu, Anhui and the like appear ASF epidemic situation in succession. Therefore, it is urgent to enhance the quarantine and control of ASF.
Some features of ASFV resemble iridovirus and poxvirus, but it is the only member of the african swine fever virus family (Asfarviridae) of the genus african swine fever virus (Asfivirus). The ASFV genome is a double-stranded DNA having a size of 170 to 190 kb. ASFV is the only known DNA arbovirus that infects swine, such as domestic and wild pigs, bush pigs, warts and giant forest pigs, and ticks. Tick transmission is considered to be the major infection pathway of ASFV in african regions. However, the outbreaks of ASF in southern europe and latin america are closely related to the importation of meat products infected with ASFV and the subsequent infection of local ticks by the virus.
The clinical features of swine infection with ASFV are high fever, diarrhea, bleeding and redness of the skin. Some clinical symptoms and autopsy results, such as splenomegaly, lymph node and kidney bleeding, were indistinguishable from Classical Swine Fever Virus (CSFV) infection symptoms in pigs. Therefore, the rapid and reliable detection of ASFV is crucial-the rapid detection of ASFV is not only necessary for the prevention and control of ASF, but also necessary for the differential diagnosis of swine diseases similar to the clinical symptoms thereof.
At present, the foreign diagnosis methods for African swine fever include 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 conventional method for laboratory diagnosis of ASFV is Virus Isolation (VI) in Porcine Bone Marrow (PBM) cell culture, which, although reliable and sensitive, takes too long (6 days) for this assay. 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 PCR technology enables the ASFV diagnosis to be completed within hours after the sample collection, and shortens the diagnosis time. The PCR method for detecting African swine fever virus has the characteristics of specificity, sensitivity and rapidness, but has the characteristics of time consumption, easy pollution, electrophoretic detection after amplification, small number of samples for each detection and the like.
Therefore, there is a need to develop a new and efficient method for detecting African swine fever virus, which can be used for detecting ASFV nucleic acid in susceptible animals and related animal products.
Disclosure of Invention
The invention aims to solve the technical problem of providing a molecular probe for detecting African swine fever virus and a Realtime-PCR detection method so as to solve the technical problem of poor reliability and practicability of the existing detection technology.
In order to solve the technical problems, the invention adopts the following technical scheme:
a molecular probe for detecting African swine fever virus is designed, and the nucleotide sequence of the molecular probe is as follows:
5'-X-CCAACCCAGTGGTCATATTAACGTATCC-3' -Y, wherein X is a fluorescence reporter group, and Y is a fluorescence quenching group.
Preferably, the fluorescent reporter group is any one of FAM, HEX and TET.
Preferably, the fluorescence quenching group is TAM-RA or BHQ.
Preparing RT-PCR reaction liquid For detecting African swine fever virus, wherein each 22 mu L of RT-PCR reaction liquid consists of 5pmol of 1 mu L of molecular probe, 50pmol of primer ASFV-For 1 mu L, 50pmol of primer ASFV-Back 1 mu L, 12.5 mu L of TaqMan reaction mix and 6.5 mu L of clean-free water;
the nucleotide sequence of the primer ASFV-For is shown in SEQ ID NO. 3; the nucleotide sequence of the primer ASFV-Back is shown in SEQ ID NO. 4.
The preparation method of the RT-PCR reaction liquid freeze-dried powder for detecting the African swine fever virus comprises the following steps:
(1) preparing a Reaction Mixer;
every 11.5mL Reaction Mixer consisted of 5pmol of the molecular probe 500. mu.L, TaqMan interaction mix 6250. mu.L, 50pmol of the primer ASFV-For 500. mu.L, 50pmol of the primer ASFV-Back 500. mu.L and Nuclear-free water 3750. mu.L;
the nucleotide sequence of the primer ASFV-For is shown in SEQ ID NO. 3; the nucleotide sequence of the primer ASFV-Back is shown in SEQ ID NO. 4;
(2) subpackaging the prepared Reaction Mixer into 10 mL-capacity ampoule according to the amount of 1mL in each ampoule, and freeze-drying in a dark place to obtain powder;
(3) the obtained powder is stored at-40 to-80 ℃.
Preparing an African swine fever virus detection kit which contains the molecular probe, the RT-PCR reaction solution or the RT-PCR reaction solution freeze-dried powder.
The qualitative detection method for the African swine fever virus is provided, and comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) taking the RT-PCR reaction solution or diluting the RT-PCR reaction solution freeze-dried powder according to the amount of 1mL of nucleic-free water per bottle to obtain diluted reaction solution;
(3) adding 3 mu L of the sample DNA into 22 mu L of the reaction solution or the diluted reaction solution to perform RT-PCR reaction;
(4) carrying out agarose gel electrophoresis on the RT-PCR reaction product;
(5) if the Ct value detected by RT-PCR is greater than 40 or the electrophoresis has no amplification curve, the sample to be detected is negative; if the Ct value detected by RT-PCR is less than 40 and the electrophoresis has an amplification curve of 258bp, the sample to be detected is positive.
Preferably, the reaction procedure of the RT-PCR reaction is as follows: 1 cycle at 50 ℃ for 2 min, 1 cycle at 95 ℃ for 10 min, 40 cycles at 95 ℃ for 15 second, and 58 ℃ for 1 min.
Also provides a quantitative detection method of African swine fever virus, which comprises the following steps:
(1) quantitatively diluting by a standard substance in a multiple ratio;
(2) extracting DNA of a sample to be detected;
(3) taking the RT-PCR reaction solution or diluting the RT-PCR reaction solution freeze-dried powder according to the amount of 1mL of nucleic-free water per bottle to obtain diluted reaction solution;
(4) adding 3 mu L of the diluted standard substance and the DNA of the sample to be detected into 22 mu L of the reaction solution or the diluted reaction solution for RT-PCR reaction;
(5) calculating the copy number of the nucleic acid corresponding to each dilution according to the known concentration of the standard substance;
(6) taking the logarithm value of the nucleic acid copy number of the quantitative standard substance as a Y axis, and taking the Ct value detected by RT-PCR as an X axis to make a standard curve;
(7) and substituting the Ct value of the sample to be detected into a linear regression equation of the standard curve to calculate the nucleic acid copy number of the sample to be detected, so as to obtain the nucleic acid concentration of the sample to be detected.
Preferably, the reaction procedure of the RT-PCR reaction is as follows: 1 cycle at 50 ℃ for 2 min, 1 cycle at 95 ℃ for 10 min, 40 cycles at 95 ℃ for 15 second, and 58 ℃ for 1 min.
Compared with the prior art, the invention has the beneficial technical effects that:
1. compared with the traditional PCR method based on agarose gel electrophoresis, the method has the characteristics of high sensitivity, good repeatability, strong specificity, high detection speed, objective result, quantifiability, high throughput and the like, and effectively overcomes the defects that the traditional PCR and nested PCR amplification products have to be subjected to electrophoretic detection, long time consumption, low sensitivity and incapability of quantification.
2. The invention can directly detect PCR amplification products by using the fluorescent type of the TaqMan molecular probe, does not need to open the cover, avoids pollution, is quicker and realizes pollution-free and high-flux detection.
3. The method can make a quantitative and objective estimation, can accurately judge the detection sample, and definitely defines that the Ct value is 40.00 and is a critical value of a positive result and a negative result.
4. The freeze-dried powder of the RT-PCR reaction mixed solution can be stored for a long time and is convenient to transport and carry.
5. The method has high sensitivity, and can accurately detect the samples of low-content ASFV virus and recessive infection or continuously toxic host samples.
6. The method of the invention effectively improves the reliability and the usability of the PCR technology for detecting ASFV, can be used for detecting a large batch of samples for separating viruses, and is beneficial to application and popularization.
Drawings
FIG. 1 is a Realtime-PCR standard graph;
FIG. 2 is a photograph of a Realtime-PCR product agarose gel electrophoresis;
in the figure, 1 is marker 2000, 2-12 are ASFV VP72 amplifications with different copy numbers, and 13 is a negative control;
FIG. 3 is a diagram showing Realtime-PCR sensitivity evaluation;
FIG. 4 is an agarose gel electrophoresis of a viral gene such as CSFV;
in the figure, 1 is marker 2000, 2 is CSFV, 3 is PCV2, 4 is PRRSV (Mac145), 5 is PRRSV (pam), 6 is PEDV, and 7 is PPV;
FIG. 5 is a diagram showing the evaluation of the specificity of Realtime-PCR;
FIG. 6 is a chart of the lyophilized reaction solution Realtime-PCR standard;
FIG. 7 is a Realtime-PCR standard curve diagram of the reaction solution prepared in situ;
FIG. 8 is a standard graph of Realtime-PCR of the commercial kit;
FIG. 9 is a diagram showing Realtime-PCR sensitivity evaluation of the reaction mixture prepared in situ;
FIG. 10 is a diagram showing the evaluation of Realtime-PCR sensitivity of the reaction solution of lyophilized powder;
FIG. 11 is a diagram showing the sensitivity evaluation of Realtime-PCR in a reaction solution of a commercial kit.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.
Example 1: design of molecular probe for detecting ASFV
According to 54 African swine fever virus VP72 genes on GeneBank, a conserved sequence with the nucleotide sequence size of 258bp at the 3' end of the VP72 gene is screened by comparison, and is shown as SEQ ID NO. 1:
CGTATCCGATCACATTACCTATTATTAAAAACATTTCCGTAACTGCTCATGGTATCAATCTTATCGATAAATTTCCATCAAAGTTCTGCAGCTCTTACATACCCTTCCACTACGGAGGCAATGCGATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGGTCATATTAACGTATCCAGAGCAAGAGAATTTTATATTAGTTGGGACACGGATTACG。
taking a conserved sequence as a target gene, comprehensively considering various factors and combining years of practical experience, and designing a molecular probe, which is shown as SEQ ID NO. 2:
5'-[6-carboxy-fluorescein(FAM)]-CCAACCCAGTGGTCATATTAACGTATCC-3'-[6-carboxy-tetramethyl-rhodamine(TAM-RA)]。
the probe is characterized in that the fluorescence reporter group marked at the 5 'end is 6-carboxyl-Fluorescein (FAM), and the fluorescence quencher group marked at the 3' end of the probe is 6-carboxyl-tetramethyl-rhodomine (TAM-RA).
Example 2: design of specific primer for detecting ASFV
Taking a conserved sequence shown in SEQ ID NO.1 as a target gene, comprehensively considering various factors and combining years of practical experience, and designing a pair of specific primers as shown in SEQ ID NO.3 and SEQ ID NO. 4:
ASFV-For：5’-CGTATCCGATCACATTACC-3’
ASFV-Back：5’-CGTAATCCGTGTCCCAAC-3’
example 3: extraction of ASFV Total DNA
(1) Preparing a reagent:
separating buffer solution: 10mmoL/L Tris-Cl pH 7.4, 10mmoL/L NaCl, 25mmoL/L EDTA;
other reagents: 10% SDS, protein kinase K (20 mg/mL or powder), diethyl ether, phenol: chloroform: isoamyl alcohol (25: 24: 1), absolute ethyl alcohol and 70% ethyl alcohol, 5mol/L NaCl, 3mol/L NaAc, TE;
(2) cutting off about 5g of tissue, removing connective tissue, sucking dry blood with absorbent paper, cutting into pieces (the more pieces are better), and placing into a mortar;
(3) pouring liquid nitrogen, grinding into powder, and adding 10mL of separation buffer solution;
(4) add 10mL 10% SDS and mix until the sample becomes very viscous;
(5) adding 50 mu L or 1mg of protein kinase K, and keeping the temperature at 37 ℃ for 1-2 h to guide the tissue to be completely dissociated;
(6) adding 1mL of 5mol/L NaCl, uniformly mixing, and centrifuging at 5000rpm for several seconds;
(7) taking the supernatant into a new centrifuge tube, and adding equal volume of phenol: chloroform: extracting isoamyl alcohol (25: 24: 1), and centrifuging at 3000rpm for 5 min after layering;
(8) taking the upper layer of water phase to a clean centrifuge tube, adding 2 times of volume of ether for extraction (operation under ventilation condition);
(9) removing the ether at the upper layer, and reserving the water phase at the lower layer;
(10) adding 1/10 volumes of 3mol/L NaAc and 2 volumes of absolute ethyl alcohol to reversely mix the DNA; standing for 10-20 min at room temperature, and precipitating DNA to form white floccules;
(11) hooking the DNA precipitate with a glass rod, rinsing in 70% ethanol, blotting on absorbent paper, dissolving in 1mL TE, and storing at-20 deg.C;
(12) if insoluble particles exist in the DNA solution, the DNA solution can be instantaneously centrifuged at 5000rpm, and a supernatant is taken; to remove RNA, 5. mu.L of RnaseA (10. mu.g/. mu.L) was added, incubated at 37 ℃ for 30 min, extracted with phenol, and then DNA was reprecipitated according to the procedures (10) (11).
Example 4: RT-PCR reaction solution for preparing and detecting ASFV
Each reaction system is 25 mu L, and the reaction liquid comprises the following components in percentage by weight:
1. mu.L of the molecular probe of example 1 (5 pmol); ASFV-For of example 2 (50 pmol) 1. mu.L; ASFV-Back (50 pmol) of example 2 by 1. mu.L; 12.5 mu L of TaqMan interaction mix; mu.L of nucleic-free water was prepared as a reaction solution. In use, 22. mu.L of the mixture was added to each well of the quantification plate, and 3. mu.L of template was added to each well.
Example 5: freeze-dried powder for preparing RT-PCR reaction solution for detecting ASFV
The preparation method of the freeze-dried powder of the detection reaction mixed liquid comprises the following steps:
prepare 11.5mL Reaction Mixer, calculate according to 500 Reaction systems:
TaqMan Probe of example 1, 5pmol 500. mu.L (1X 500);
TaqMan reaction mix 6250μL(12.5×500)；
ASFV-For of example 2, 50pmol 500. mu.L (1X 500);
ASFV-Back of example 2, 50pmol 500. mu.L (1X 500);
Nuclease-free water 3750μL(7.5×500)。
the mixture was dispensed into 10mL ampoules, 1mL Reaction Mixer per vial, and lyophilized to a powder using the Freezone storage train Dryers System of photogen, Inc., and the powder was stored in a freezer at-80 deg.C, protected from light.
Example 6: setting reaction program for detecting ASFV
The reaction procedure is as follows:
1 cycle at 50 ℃ for 2 min, 1 cycle at 95 ℃ for 10 min, 40 cycles at 95 ℃ for 15 second, and 58 ℃ for 1 min.
Example 7: method for detecting ASFV
(1) In a 1.5mL centrifuge tube, the RT-PCR Reaction solution prepared in the example 4 is taken, or the Reaction Mixer freeze-dried powder prepared in the example 5 is diluted by 1mL of nucleic-free water diluent;
(2) adding 22 mu L of prepared RT-PCR Reaction liquid or diluted Reaction Mixer into the MicroAmp optical Reaction plate holes;
(3) adding 3 mu L of template into the MicroAmp optical extraction plate holes in the step (2);
(4) the reaction plate was centrifuged for 1 min;
(5) the Realtime-PCR program set up in example 6 was followed and PCR amplification was performed;
(6) preparing 1.5% agarose gel to identify PCR amplification products, judging the result according to the Ct value, wherein the Ct value is more than 40 or no amplification curve is negative, and the result indicates that the African swine fever virus exists in the sample; ct value <40 and a typical amplification curve appears, positive, indicating the presence of African swine fever virus in the sample.
(7) The quantitative determination method comprises the following steps:
firstly, preparing a standard curve according to standard substance dilution, carrying out multiple-ratio dilution on a quantitative standard substance, and then carrying out RT-PCR detection on the quantitative standard substance and an unknown sample at the same time; then calculating the nucleic acid copy number corresponding to each dilution according to the known standard concentration; taking the logarithm value of the nucleic acid copy number of the quantitative standard product as the Y axis, taking the Ct value detected by RT-PCR as the X axis as a scatter diagram, and adding a trend line to obtain a standard curve, a linear regression equation and R2(ii) a And substituting the detection Ct value of the sample into a linear regression equation of a standard curve to calculate the nucleic acid copy number of the unknown sample so as to obtain the nucleic acid concentration.
Example 8: drawing of standard curve of ASFV RT-PCR detection method and evaluation of repeatability, sensitivity and specificity
(1) Material
The RT-PCR template is ASFV VP72, CSFV, PCV2, PRRSV (Mac145), PRRSV (PAM), PEDV, PPV, a disease sample, a standard substance and a diluent.
(2) Drawing of standard curve
Will be 1 × 1019Copy/. mu.L plasmid gradient was diluted 10-fold to 1X 100Copy/μ L, do 3 replicates per concentration gradient for the fluorescent quantitative PCR test, automatically generate a standard curve by the ABI 7500 fluorescent quantitative PCR instrument. And subjected to nucleic acid electrophoresis using 1.5% agarose gel to identify amplified fragments. The RT-PCR reaction solution was the reaction solution of example 4, and the reaction procedure was the procedure of example 6.
The results are shown in FIGS. 1 and 2.
By taking the logarithm of the plasmid concentration as the abscissa and the Ct value as the ordinate, the slope of the curve is-1.16, the intercept is 22.43, the correlation coefficient is 0.997, and the standard curve equation is obtained as follows: y = -1.16X + 22.43; after nucleic acid electrophoresis on a 1.5% agarose gel, a specific band of about 258bp in size was observed (FIG. 2), consistent with the expected results.
(3) Evaluation of reproducibility
By 1X 101～1×1018Replicate experiments were performed in 3 replicates per concentration gradient of plasmid per μ L and coefficient of variation CV was calculated from the Ct values obtained.
The results of the repeatability tests are shown in table 1:
TABLE 1 coefficient of variation of Ct value for each concentration gradient
Table 1 shows that the method has good repeatability, and the coefficient of variation Cv of each concentration gradient is less than 0.25%, which indicates that the real-time fluorescence quantitative PCR detection method has good stability.
(3) Evaluation of sensitivity
By 1X 100～1×1020Copy/. mu.L plasmid was subjected to a sensitivity test in 3 replicates per concentration gradient to determine the lowest detectable concentration of the method.
The results are shown in FIG. 3.
By 1X 1020To 1X 100Sensitivity test of copy/. mu.L plasmid the method can detect 1X 10 of plasmid at the lowest1When the concentration of the plasmid is more than or equal to 10 copies/. mu.L, an amplification curve can appear, which indicates that the detection method has extremely high sensitivity.
(3) Evaluation of specificity
And carrying out fluorescent quantitative PCR reaction by taking cDNA of Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Epidemic Diarrhea (PEDV), DNA of porcine circovirus (PCV2) and Porcine Parvovirus (PPV) and ASFV pET28a-VP72 as templates to observe whether an amplification curve exists.
The results are shown in FIGS. 4 and 5.
As can be seen from fig. 4: the cDNA of CSFV, PEDV, PRRSV (Mac145) and PSRRV (PAM), and the DNA of PCV2 and PPV are taken as templates, and corresponding primers are respectively used for carrying out traditional PCR to amplify corresponding specific bands, which shows that the extracted templates have good quality and can be used for specific identification.
As can be seen from fig. 5: the cDNA of CSFV, PEDV, PRRSV (Mac145) and PSRRV (PAM), the DNA of PCV2 and PPV and the plasmid pET28a-VP72 of ASFV are taken as templates, and the primers for amplifying ASFV are used for carrying out fluorescence quantitative PCR, so that only the pET28a-VP72 of ASFV has an amplification curve, and the method has good specificity.
Comparative example: kit repeatability and sensitivity contrast for detecting ASFV
(1) Design of experiments
Comparative materials were the Reaction mixer lyophilized powder of example 5, the presently prepared Reaction mixer and the VetMAX African Swine Fever (ASF) qPCR detection kit of Thermo Fisher;
respectively using 1X 100～1×1010Copy/. mu.L, 1X 100～1×109Copy/. mu.L, 1X 100～1×1012Copies/. mu.L of plasmid were made in 3 replicates per concentration gradient and subjected to fluorescent quantitative PCR amplification, the reaction procedure being that of example 6.
And (3) comparing the standard curves, the repeatability and the sensitivity of the fluorescent quantitative PCR of the three contrast media.
(2) Drawing of standard curve
The standard curve of the Reaction mixer lyophilized powder is shown in FIG. 6:
by taking the logarithm value of the plasmid concentration as the abscissa and the Ct value as the ordinate, the slope of the curve is-1.6695, the intercept is 43.18, the correlation coefficient is 0.9934, and the standard curve equation is obtained as follows: y = -1.6695x + 43.18.
The standard curve for the Reaction mixer now fitted is shown in FIG. 7:
by taking the logarithm value of the plasmid concentration as the abscissa and the Ct value as the ordinate, the slope of the curve is-1.5418, the intercept is 40.851, the correlation coefficient is 0.9929, and the standard curve equation is obtained as follows: y = -1.5418x + 40.851.
The standard curve of the commercial fluorescent quantitative detection kit is shown in FIG. 8:
by taking the logarithm value of the plasmid concentration as the abscissa and the Ct value as the ordinate, the slope of the curve is-1.6056, the intercept is 38.782, the correlation coefficient is 0.993, and the standard curve equation is obtained as follows: y = -1.6056x + 38.782.
(3) Evaluation of reproducibility
The reproducibility of the Reaction mixer lyophilized powder is shown in table 2:
TABLE 2 Freeze-drying reaction liquid Realtime-PCR
As can be seen from Table 2: the Reaction mixer freeze-dried powder has good repeatability, and the coefficient of variation Cv of each concentration gradient is less than 1.672%.
The reproducibility of the now-prepared Reaction mixer is shown in Table 3:
TABLE 3 Realtime-PCR in situ
As can be seen from Table 3: the Reaction mixer prepared now has good repeatability, and the coefficient of variation Cv of each concentration gradient is less than 1.46 percent.
The repeatability of the commercial fluorescent quantitative detection kit is shown in table 4:
TABLE 4 commercial fluorescent quantitation kit Realtime-PCR
As can be seen from Table 4: the commercial fluorescent quantitative detection kit has good repeatability (Table 4), and the coefficient of variation Cv of each concentration gradient is less than 1.45%.
The above results illustrate that: the stability of the prepared Reaction mixer is equivalent to that of a commercial detection kit, the stability of freeze-dried powder is slightly low, and a protective agent can be added.
(4) Sensitivity evaluation
By 1X 1012To 1X 100Sensitivity test is carried out on copied/mu L plasmid, and the lowest detection of the Reaction mixer freeze-dried powder and the Reaction mixer method which is prepared at present can reach 1 x 101The plasmid with copy/. mu.L can generate an amplification curve when the concentration is more than or equal to 10 copies/. mu.L (see figure 9 and figure 10), and the lowest detection limit of the commercial fluorescent quantitative kit is 1 x 102Copy/. mu.L (see FIG. 11), which shows that the Realtime-PCR detection method of the present invention has very high sensitivity, 10 times higher than commercial kits.
While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.
SEQUENCE LISTING
<110> Henan Zhongze bioengineering Co., Ltd, Zheng Zhou university
<120> molecular probe for detecting African swine fever virus and Realtime-PCR detection method
<130> 2019
<160> 4
<170> PatentIn version 3.2
<210> 1
<211> 258
<212> DNA
<213> ASFV
<400> 1
cgtatccgat cacattacct attattaaaa acatttccgt aactgctcat ggtatcaatc 60
ttatcgataa atttccatca aagttctgca gctcttacat acccttccac tacggaggca 120
atgcgattaa aacccccgat gatccgggtg cgatgatgat tacctttgct ttgaagccac 180
gggaggaata ccaacccagt ggtcatatta acgtatccag agcaagagaa ttttatatta 240
gttgggacac ggattacg 258
<210> 2
<211> 28
<212> DNA
<213> Artificial Synthesis
<400> 2
ccaacccagt ggtcatatta acgtatcc 28
<210> 3
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 3
cgtatccgat cacattacc 19
<210> 4
<211> 18
<212> DNA
<213> Artificial Synthesis
<400> 4
cgtaatccgt gtcccaac 18
Claims (3)
1. An African swine fever virus detection kit is characterized by comprising a molecular probe and RT-PCR reaction liquid freeze-dried powder;
the nucleotide sequence of the molecular probe is as follows:
5'-X-CCAACCCAGTGGTCATATTAACGTATCC-3' -Y, wherein X is a fluorescence reporter group, and Y is a fluorescence quenching group;
the preparation method of the RT-PCR reaction solution freeze-dried powder comprises the following steps:
(1) preparing a Reaction Mixer;
every 11.5mL Reaction Mixer consisted of 5pmol of the molecular probe 500. mu.L, TaqMan interaction mix 6250. mu.L, 50pmol of the primer ASFV-For 500. mu.L, 50pmol of the primer ASFV-Back 500. mu.L and Nuclear-free water 3750. mu.L;
the nucleotide sequence of the primer ASFV-For is shown in SEQ ID NO. 3; the nucleotide sequence of the primer ASFV-Back is shown in SEQ ID NO. 4;
(2) subpackaging the prepared Reaction Mixer into 10 mL-capacity ampoule according to the amount of 1mL in each ampoule, and freeze-drying in a dark place to obtain powder;
(3) the obtained powder is stored at-40 to-80 ℃.
2. The African swine fever virus detection kit of claim 1, wherein the fluorescent reporter group is any one of FAM, HEX, TET.
3. The African swine fever virus detection kit of claim 1, wherein the fluorescence quenching group is TAM-RA or BHQ.
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| CN110423847A (en) * | 2019-07-31 | 2019-11-08 | 北京市动物疫病预防控制中心 | A kind of primer sets, kit and the method for African swine fever virus LAMP fluorescence detection |
| CN110468239A (en) * | 2019-09-22 | 2019-11-19 | 山东森芃生物科技有限公司 | A kind of quick Q-PCR detection method of freeze-dried type African swine fever virus and kit |
| CN111304361A (en) * | 2019-11-04 | 2020-06-19 | 浙江大学 | Kit for detecting African swine fever virus and method for detecting African swine fever virus |
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