CN111893210A - LAMP-TaqMan rapid detection kit for African swine fever virus and application - Google Patents
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Abstract
The invention discloses a LAMP-TaqMan rapid detection kit for African swine fever virus, which comprises LAMP TaqMan Mix mixed liquor, LAMP TaqMan Assay reaction liquid, positive control and negative control. The LAMP TaqMan Mix mixed solution comprises: bst 5.0DNA polymerase mixture; the LAMP TaqMan Assay reaction solution comprises: primer: ASFV P54F 3, ASFV P54B3, ASFV P54 loopF, ASFV P54loopR, ASFV P54 FIP, ASFV P54BIP, and fluorescent Probe P54FAM Probe. The primers and probes correspond to a highly conserved fragment of the p54 gene of African swine fever virus. The invention also discloses a method for preparing the African swine fever virus LAMP-TaqMan rapid detection kit and a method for detecting a sample by using the African swine fever virus LAMP-TaqMan rapid detection kit. The detection time limit of the kit is 25min, and the kit has a good application prospect.
Description
Technical Field
The invention relates to the field of prevention and control of important epidemic diseases of livestock and poultry, in particular to the technical field of pathogen molecular diagnosis, and particularly relates to an African swine fever virus LAMP-TaqMan rapid detection kit, a preparation method and a use method.
Background
China is the first major country of pig raising production in the world, practitioners are tens of millions, the scale of breeding and the consumption are the first in the world, and the pig raising industry is a tightly-tied country-based demon. However, a sudden onset of African swine fever causes great economic loss to the swine industry in China. Since the African swine fever epidemic situation is reported for the first time in Shenyang in 8 months in 2018, the African swine fever confirmed diagnosis cases continue to exist in pig farms in other provinces in China, and the African swine fever epidemic situation is reported in more than 30 provinces until 4 months in 2019, so that the epidemic situation is spread rapidly, and the animal epidemic prevention and control task in China is still very severe. At present, the epidemic situation of the African swine fever is effectively treated in time under the force coordination of governments at all levels and all departments, and the prevention and control work achieves remarkable effect. However, no effective vaccine is available for the African swine fever, the epidemic disease transmission path is complicated, the virus forms a certain pollution surface in China, the traditional production, circulation and consumption modes are difficult to change fundamentally in a short time, and the complexity and the long-term property of the prevention and control of the African swine fever are determined. In view of this, emergency implementation schemes for epidemic situations of African swine fever (2019 edition) are provided in agricultural rural areas to guide various places to treat the epidemic situations and improve the emergency treatment capability of the epidemic situations.
The African swine fever has high propagation speed, high morbidity and high mortality, has clinical symptoms similar to classical swine fever, highly pathogenic blue ear disease, swine erysipelas and other epidemic diseases, and has no effective vaccine. Once epidemic situation occurs, pigs in an epidemic site are completely killed, pigs in an epidemic area are isolated and monitored, and a threat area is strictly controlled. Therefore, the rapid and accurate diagnosis of the epidemic situation is the premise and the basis for formulating scientific prevention and control measures, controlling the spread of the epidemic situation and maintaining the stable development of the pig industry, and the establishment of a sensitive, specific, rapid and reliable diagnosis method is urgent.
Loop-mediated isothermal amplification (LAMP) has the advantages of high sensitivity, high specificity, simplicity in operation, low equipment requirement and the like, and is widely applied to epidemic detection, food and cosmetic detection and import and export rapid diagnosis. LAMP technology involves the risk of non-specific amplification and thus false positives. Therefore, the TaqMan probe technology and the LAMP technology are combined, so that the problems can be effectively solved, and the African swine fever virus detection prospect is good.
Disclosure of Invention
In order to solve the problem of rapid diagnosis of the African swine fever, the invention provides an LAMP amplification method based on a TaqMan probe for detecting the African swine fever virus, and further establishes an LAMP-TaqMan kit of the African swine fever virus and a using method thereof.
Based on the purpose, the LAMP-TaqMan kit for the African swine fever virus provided by the invention comprises LAMPTaqMan Mix mixed liquor, LAMP TaqMan Assay reaction liquor, positive control and negative control. Wherein, the LAMPTaqMan Mix mixed solution comprises: bst 5.0DNA polymerase mixture; the LAMP TaqMan Assay reaction solution comprises: LAMP primer: ASFV P54F 3, ASFV P54B3, ASFV P54 loopF, ASFV P54loopR, ASFV P54 FIP, ASFV P54BIP, and TanMan fluorescent Probe P54FAM Probe.
The LAMP primer and the LAMP probe correspond to a highly conserved segment of the African swine fever virus p54 gene, and the nucleotide sequence is as follows:
the LAMP Primer is designed according to the conserved sequence of the P54 gene by software Primer Explorer V4, and preferably, the LAMP Primer sequence is ASFV P54F 3: 5'-GTACCTCTAAACCGGCTG-3' and ASFV P54B 3: 5'-CAAGGAGTTTTCTAGGTCTTT-3', ASFV P54 LoopF: 5'-TAACTGGGTTGTCCGTGA-3' and ASFV P54 LoopR: 5'-CACAAACAATGTCGGCTATTG-3', ASFV P54 FIP: 5'-CGCCAGTTGCCATGACTAGCAACAAACAGACCAGCAA-3' and ASFV P54 BIP: 5'-AGTCACTACTCAGAACACTGCTTGCGTATAGGTGTTTCTTT-3' are provided.
The TaqMan fluorescent Probe should be free of non-specific complementary region and fluorescently labeled, preferably, the TanMan fluorescent Probe P54FAM Probe: 5 '- (FAM) ACCTGCGGCCGCGAGTGCTCATCCGACTGAGCCTT(BHQ) -3', FAM being a fluorescence reporter gene, BHQ being a quenching fluorophore.
The content of each component of the detection kit is as follows: 2000. mu.l of LAMP TaqMan Mix mixture, 400. mu.l of LAMP TaqMan assay reaction solution, 400. mu.l of positive control and 400. mu.l of negative control.
The LAMP-TaqMan detection method of the African swine fever virus comprises the following steps: adding a sample to be detected and components of the African swine fever virus LAMP-TaqMan detection kit according to the following mixture ratio: 10.0 mu l of LAMP TaqMan Mix mixed solution, 2.0 mu l of LAMPTaqMan Assay reaction solution, 0.5-2.0 mu l of sample to be detected and ddH2And (3) supplementing the total amount of O to 20.0 mu l, carrying out constant-temperature reaction at 65 ℃ for 25-30 min, preferably 30min, collecting FAM fluorescent signals by using constant-temperature fluorescent equipment or a standard quantitative PCR instrument, judging that a sample with an ascending curve is a positive result, and judging that the curve is not ascending as a negative result. Wherein the sample to be tested is selected from live pig blood, tonsil, secretion or excretion, or any tissue of lung, spleen, liver and lymph node of dead pig; and grinding the non-liquid components of the sample to be detected to homogenate, centrifuging and taking the supernatant as a detection sample.
The LAMP-TaqMan detection method for the African swine fever virus can be used for quickly and accurately detecting various clinical samples, is simple in preparation method, is beneficial to industrial production, and lays a foundation for prevention and control of the epidemic situation of the African swine fever.
Drawings
FIG. 1 is a graph of linear amplification using pEt28a-p54 standard plasmid as a template in the present invention.
FIG. 2 is a graph of linear amplification using African swine fever virus nucleic acid samples as templates in the examples of the present invention.
FIG. 3 is a graph of linear amplification curve using African swine fever swine blood sample as template in the present invention.
FIG. 4 is a linear amplification curve diagram of spleen samples of killed African swine fever as a template in the present invention.
FIG. 5 is a graph of linear amplification curve using lung sample of swine of African swine fever dead as a template in the present invention.
FIG. 6 is a graph of linear amplification curve using a sample of the liver of a swine from African swine fever disease dead as a template in an embodiment of the present invention.
FIG. 7 is a linear amplification curve diagram of a lymph node sample of a swine dead of African swine fever disease as a template in an embodiment of the present invention.
FIG. 8 is a graph of linear amplification using a healthy pig blood sample as a template in accordance with an embodiment of the present invention.
FIG. 9 is a graph of linear amplification using a healthy pig spleen sample as a template in accordance with an embodiment of the present invention.
FIG. 10 is a graph of linear amplification using a healthy pig lung sample as a template in accordance with an embodiment of the present invention.
FIG. 11 is a linear amplification curve diagram using porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, and porcine circovirus type 2 nucleic acid samples as templates in the embodiment of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1:
in the embodiment, the LAMP-TaqMan kit for African swine fever virus provided by the invention is used for detecting the sensitivity of the LAMP-TaqMan kit by taking different concentrations of pEt28a-p54 standard plasmids as templates. LAMP TaqMan Mix 10.0. mu.l, LAMP TaqMan Assay 2.0. mu.l, pEt28a-p54 standard plasmid different dilutions (i.e. 5X 105Copies/. mu.l, 5X 104Copies/. mu.l, 5X 103Copy/. mu.l, 500 copies/. mu.l, 50 copies/. mu.l, 5 copies/. mu.l, 0.5 copies/. mu.l) 2.0. mu.l, ddH2O was replenished to 20.0. mu.l, incubated at 65 ℃ for 30min, 30 cycles of FAM fluorescence signal (FIG. 1) were collected using a standard quantitative PCR instrument, and the results showed that the detection limit of the p54 gene standard plasmid was 5 copies and the reaction time was 25 min.
The results show that the LAMP-TaqMan kit for the African swine fever virus can finish the reverse reaction within 20min, and has good effect within 25 min.
Example 2:
in this example, the LAMP-TaqMan kit for African swine fever virus according to the present invention was used for African swine feverThe virus nucleic acid sample is used as a template, 10.0 mu l of LAMP TaqMan Mix mixed solution, 2.0 mu l of LAMP TaqMan Assay reaction solution, 2.0 mu l of nucleic acid and ddH2O6.0. mu.l, isothermal reaction at 65 ℃ for 25min, collecting 25 cycles of FAM fluorescence signals using a standard quantitative PCR instrument (FIG. 2), and the result shows that the African swine fever virus is positive.
Example 3:
in the embodiment, the LAMP-TaqMan kit for African swine fever virus provided by the invention is used as a template, and the LAMP TaqMan Mix mixed solution is 10.0 mu l, the LAMP TaqMan Assay reaction solution is 2.0 mu l, the blood is 2.0 mu l, and ddH is added2O6.0. mu.l, isothermal reaction at 65 ℃ for 25min, 25 cycles of FAM fluorescence signal (FIG. 3) collected using a standard quantitative PCR instrument, and the result showed that the blood sample was positive for African swine fever virus.
Example 4:
in the embodiment, the LAMP-TaqMan kit for African swine fever virus provided by the invention takes spleen, lung, liver and lymph node samples of dead African swine fever disease pigs as templates, 10.0 mu l of LAMP TaqMan Mix mixed solution, 2.0 mu l of LAMP TaqMan Assay reaction solution, 2.0 mu l of homogenate supernatant of each sample and 2.0 mu l of ddH2O6.0. mu.l, reacted at 65 ℃ for 25min, 25 cycles of FAM fluorescence signals were collected using a standard quantitative PCR instrument (results for spleen, lung, liver and lymph node samples in FIGS. 4, 5, 6 and 7, respectively), and the results showed that the diseased pig sample was positive for African swine fever virus.
Example 5:
in the embodiment, the LAMP-TaqMan kit for African swine fever virus (AFP) takes healthy pig blood, spleen and lung samples as templates, 10.0 mu l of LAMP TaqMan Mix mixed solution, 2.0 mu l of LAMP TaqMan Assay reaction solution, 2.0 mu l of homogenate supernatant of each sample and ddH2O6.0. mu.l, reacted at 65 ℃ for 25min, and 25 cycles of FAM fluorescence signals were collected using a standard quantitative PCR instrument (FIG. 8, FIG. 9, and FIG. 10 show the results for blood, spleen, and lung samples, respectively), and the results showed that the healthy swine sample was negative to African swine fever virus.
Example 5:
in this example, the LAMP-TaqMan kit for African swine fever virus provided by the invention is used for pig breeding and respiratory synthesisNucleic acid samples of the syndrovirus, the porcine pseudorabies virus and the porcine circovirus type 2 are taken as templates, 10.0 mu l of LAMP TaqMan Mix mixed solution, 2.0 mu l of LAMP TaqMan Assay reaction solution, 2.0 mu l of homogenate supernatant of each sample and ddH2O6.0 μ l, reacting at 65 ℃ for 25min, collecting 25 cycles of FAM fluorescence signals by using a standard quantitative PCR instrument (figure 11), and displaying that each sample is negative to African swine fever virus, which indicates that the kit has good specificity.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> LAMP-TaqMan rapid detection kit for African swine fever virus and application
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Claims (11)
1. An African swine fever virus LAMP-TaqMan detection kit is characterized by comprising LAMP TaqMan Mix mixed liquor, LAMP TaqMan Assay reaction liquid, positive control and negative control.
2. The African swine fever virus LAMP-TaqMan detection kit according to claim 1, wherein the LAMP TaqMan Mix mixture comprises: bst 5.0DNA polymerase mixture; the LAMP TaqMan Assay reaction solution comprises: LAMP primer: ASFV P54F 3 and ASFV P54B3, ASFV P54 loopF and ASFV P54loopR, ASFV 54 FIP and ASFV P54BIP and TanMan fluorescent Probe P54FAM Probe.
3. The African swine fever virus LAMP-TaqMan detection kit LAMP TaqMan Assay reaction solution of claim 2 comprises, wherein the LAMP primer and the probe correspond to a highly conserved segment of the African swine fever virus p54 gene, and the nucleotide sequence is as follows:
4. the African swine fever virus LAMP-TaqMan detection kit LAMP TaqMan assay reaction solution according to claims 2 to 3, wherein the LAMP Primer is designed with software Primer Explorer V4 according to the conserved sequence of P54 gene, preferably the LAMP Primer sequence ASFV P54F 3: 5'-GTACCTCTAAACCGGCTG-3' and ASFVP 54B 3: 5'-CAAGGAGTTTTCTAGGTCTTT-3', ASFV P54 LoopF: 5'-TAACTGGGTTGTCCGTGA-3' and ASFV P54 LoopR: 5'-CACAAACAATGTCGGCTATTG-3', ASFV P54 FIP: 5'-CGCCAGTTGCCATGACTAGCAACAAACAGACCAGCAA-3' and ASFV P54 BIP: 5'-AGTCACTACTCAGAACACTGCTTGCGTATAGGTGTTTCTTT-3' are provided.
5. The African swine fever virus LAMP-TaqMan detection kit LAMPTaqMan Assay reaction solution according to claims 2 to 3, wherein the TaqMan fluorescent Probe has no non-specific complementary region and is fluorescently labeled, preferably, TanMan fluorescent Probe P54FAM Probe: 5 '- (FAM) ACCTGCGGCCGCGAGTGCTCATCCGACTGAGCCTT(BHQ) -3', FAM being a fluorescence reporter gene, BHQ being a quenching fluorophore.
6. The LAMP-TaqMan detection kit for African swine fever virus according to claim 1, wherein the content of each component of the detection kit is as follows: 2000. mu.l of LAMP TaqMan Mix mixture, 400. mu.l of LAMP TaqMan Assay reaction solution, 400. mu.l of positive control and 400. mu.l of negative control.
7. The LAMP TaqMan Assay reaction solution of the African swine fever virus (EHFV) detection kit according to claim 2 comprises the following components, wherein the concentrations of the primer and the probe are respectively: primer: ASFV P54F 3: 2 μ M and ASFV P54B 3: 2 μ M, ASFV P54 LoopF: 8 μ M and ASFV P54 LoopR: 8 μ M, ASFV P54 FIP: 16 μ M and ASFV P54 BIP: 16 μ M and fluorescent Probe P54FAM Probe: 5 μ M.
8. An LAMP-TaqMan detection method of African swine fever virus, which is characterized in that the LAMP-TaqMan detection kit of the African swine fever virus of any claim 1 to 7 is applied, and the detection method comprises the following steps:
adding a sample to be detected and components of the African swine fever virus LAMP-TaqMan detection kit according to the following mixture ratio: 10.0 mu l of LAMPTaqMan Mix mixed solution, 2.0 mu l of LAMP TaqMan Assay reaction solution, 0.5-2.0 mu l of sample to be detected and ddH2And (3) supplementing the total amount of O to 20.0 mu l, carrying out constant-temperature reaction at 65 ℃ for 25-30 min, preferably 30min, collecting FAM fluorescent signals by using constant-temperature fluorescent equipment or a standard quantitative PCR instrument, judging that a sample with an ascending curve is a positive result, and judging that the curve is not ascending as a negative result.
9. The LAMP-TaqMan detection method according to claim 8, wherein the sample to be detected is selected from live pig blood, tonsil, secretion or excretion, and also from any tissue of lung, spleen, liver and lymph node of dead pig; and grinding the non-liquid components of the sample to be detected to homogenate, centrifuging and taking the supernatant as a detection sample.
10. The LAMP-TaqMan detection kit established with other conserved genes of African swine fever virus as claimed in any one of claims 1-9 and application thereof.
11. Use of any one of claims 1 to 9 in other pathogen detection and disease diagnosis methods and control, without significant modification in the relevant field.
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| CN112941234A (en) * | 2021-03-01 | 2021-06-11 | 中国科学院长春应用化学研究所 | Reaction liquid and kit for detecting African swine fever virus and application |
| CN113604606A (en) * | 2021-07-12 | 2021-11-05 | 吉林大学重庆研究院 | African swine fever LAMP detection primer group, kit and detection method |
| CN114592090A (en) * | 2022-02-28 | 2022-06-07 | 华南农业大学 | Dual-fluorescence quantitative PCR detection primer, probe and kit for identifying African swine fever viruses of genes I and II |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941234A (en) * | 2021-03-01 | 2021-06-11 | 中国科学院长春应用化学研究所 | Reaction liquid and kit for detecting African swine fever virus and application |
| CN113604606A (en) * | 2021-07-12 | 2021-11-05 | 吉林大学重庆研究院 | African swine fever LAMP detection primer group, kit and detection method |
| CN114592090A (en) * | 2022-02-28 | 2022-06-07 | 华南农业大学 | Dual-fluorescence quantitative PCR detection primer, probe and kit for identifying African swine fever viruses of genes I and II |
| CN114592090B (en) * | 2022-02-28 | 2024-01-30 | 华南农业大学 | Dual-fluorescence quantitative PCR detection primer, probe and kit for identifying genes I and II of African swine fever virus |
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