CN113604606A - African swine fever LAMP detection primer group, kit and detection method - Google Patents
African swine fever LAMP detection primer group, kit and detection method Download PDFInfo
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Abstract
The invention provides an African swine fever LAMP detection primer group, a kit and a detection method, wherein 6 primer groups P1-P6 are designed for a target sequence according to a relative conserved region of a P54 gene, degenerate basic groups are introduced into partial primers, any one group of the degenerate basic groups can be used for detecting variant strains at home and abroad, the detection rate and the specificity are greatly improved, and the detection method has good application value for early detection and early prevention of African swine fever viruses, and has the advantages of simple operation, low cost, short reaction period and the like.
Description
Technical Field
The invention belongs to the technical field of African swine fever virus detection, and particularly relates to an African swine fever LAMP detection primer group, a kit and a detection method.
Background
African Swine Fever (ASF) is an acute, febrile and high-contagious disease caused by African swine fever virus, the clinical symptoms and pathological changes of the ASF are similar to those of acute swine fever, high fever, skin congestion, abortion, edema and organ bleeding are shown, the fatality rate reaches up to 100%, the disease is specified as a type of animal disease in China, the disease is classified as an A type animal disease by the world animal health organization, and the disease is highly valued by various countries and researched as an animal exotic disease. African swine fever is abused worldwide, posing a great threat to the breeding industry. In the absence of commercial vaccines, rapid and reliable laboratory diagnosis is critical for the prevention and control of disease.
The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, and has been successfully applied to the detection of partial pathogens, viruses and fungi due to the characteristics of simplicity, rapidness, strong specificity, high sensitivity and the like.
The current african swine fever virus uses LAMP detection as an example:
the patent application document with the application number of CN201610023649.4 provides a loop-mediated isothermal amplification method for rapidly detecting African swine fever virus, and LAMP primer design is carried out on the gene sequence of the African swine fever virus K205R, and the efficiency is greatly reduced without loop primers;
the patent application document with the application number of CN201910592331.1 provides an LAMP method for specifically detecting African swine fever viruses, wherein a primer combination of the LAMP method is designed aiming at a conserved gene B646, and the conserved gene can find that mutations exist in a plurality of African swine fever viruses after actual sequence comparison, so that the detection rate is greatly reduced, and the omission is easy.
Disclosure of Invention
The invention provides an LAMP detection primer group for African swine fever, the target sequence of the LAMP detection primer group is a relatively conserved region of an African swine fever P54 gene, degenerate basic groups are introduced into partial primers, the detection rate and specificity are greatly improved, and domestic and international variant strains can be detected.
Specifically, the sequence of the LAMP detection primer group for African swine fever is as follows:
(1) primer set P1;
the sequence of F3 is shown in SEQ ID NO: 1 is shown in the specification;
the sequence of B3 is shown in SEQ ID NO: 2 is shown in the specification;
the sequence of FIP is shown in SEQ ID NO: 3 is shown in the specification;
the sequence of BIP is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of LF is shown in SEQ ID NO: 5 is shown in the specification;
the sequence of LB is shown in SEQ ID NO: 6 is shown in the specification;
(2) primer set P2;
the sequence of F3 is shown in SEQ ID NO: 7 is shown in the specification;
the sequence of B3 is shown in SEQ ID NO: 8 is shown in the specification;
the sequence of FIP is shown in SEQ ID NO: 9 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 10 is shown in the figure;
the sequence of LF is shown in SEQ ID NO: 11 is shown in the figure;
the sequence of LB is shown in SEQ ID NO: 12 is shown in the specification;
(3) primer set P3;
the sequence of F3 is shown in SEQ ID NO: 13 is shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 14 is shown in the figure;
the sequence of FIP is shown in SEQ ID NO: 15 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 16 is shown in the figure;
the sequence of LF is shown in SEQ ID NO: 17 is shown;
the sequence of LB is shown in SEQ ID NO: 18 is shown in the figure;
the degenerate base Y in the primer sets of groups (1) to (3) represents C or T;
(4) primer set P4;
the sequence of F3 is shown in SEQ ID NO: 19 is shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 20 is shown in the figure;
the sequence of FIP is shown in SEQ ID NO: 21 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 22;
the sequence of LF is shown in SEQ ID NO: 23 is shown;
the sequence of LB is shown in SEQ ID NO: shown at 24;
(5) primer set P5;
the sequence of F3 is shown in SEQ ID NO: 25 is shown;
the sequence of B3 is shown in SEQ ID NO: 26 is shown;
the sequence of FIP is shown in SEQ ID NO: 27 is shown;
the sequence of BIP is shown as SEQ ID NO: 28 is shown;
the sequence of LF is shown in SEQ ID NO: 29 is shown;
the sequence of LB is shown in SEQ ID NO: 30 is shown in the figure;
(6) primer set P6;
the sequence of F3 is shown in SEQ ID NO: 31, shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 32 is shown;
the sequence of FIP is shown in SEQ ID NO: 33;
the sequence of BIP is shown as SEQ ID NO: 34;
the sequence of LF is shown in SEQ ID NO: 35 is shown in the figure;
the sequence of LB is shown in SEQ ID NO: shown at 36.
The invention also provides application of the LAMP detection primer in preparation of an African swine fever detection reagent.
The invention also provides an African swine fever detection kit, which comprises any group of LAMP detection primer groups.
The invention also provides an LAMP detection method for detecting African swine fever, which comprises the following steps:
(1) extracting genome DNA of a sample to be detected to prepare a template to be detected;
(2) adding the template to be detected into the kit,
the reaction system in the kit is 25 mu l, wherein, 2.5 mu l of 10 multiplied by Isotermal Amplification Buffer, 3.5 mu l of 10mM dNTPs and 100mM MgSO41.5. mu.l, 10 XPrimers 2.5. mu.l, Bst2.01. mu.l, pig spleen DNA (containing ASFV) 1. mu.l, H2O 13μl；
The reaction procedure in the kit is reaction at 65 ℃ for 40min, and inactivation at 95 ℃ for 1min to finish the reaction. The LAMP Primer Mix is prepared by 16 μ L of FIP/BIP Primer of 100 μmol/L, 2 μ L of F3/B3 Primer of 100 μmol/L, 4 μ L of LF/LB Primer of 100 μmol/L and ddH2O is complemented to 100 mu L;
(3) and drawing a nucleic acid amplification curve according to the reaction result.
The beneficial effects are as follows:
according to the invention, 60 African swine fever sequences at home and abroad are compared according to the African swine fever sequence in NCBI, a conserved sequence is searched in the whole genome range of the African swine fever, and finally, a relative conserved region of a P54 gene is used as a target gene for ASFV detection to design 6 primer groups P1-P6, wherein degenerate bases are introduced into partial primers, the detection rate and specificity are greatly improved, and domestic and international variant strains can be detected.
Drawings
FIG. 1 is a LAMP gene amplification curve of the primer set P1 provided by the present invention;
FIG. 2 is a LAMP gene amplification curve of the primer set P2 provided by the present invention;
FIG. 3 is a LAMP gene amplification curve of the primer set P3 provided by the present invention;
FIG. 4 is a LAMP gene amplification curve of the primer set P4 provided by the present invention;
FIG. 5 is a LAMP gene amplification curve of the primer set P5 provided by the present invention;
FIG. 6 is a LAMP gene amplification curve of the primer set P6 provided by the present invention;
FIG. 7 is a graph showing the results of a sensitivity test of the primer set P1 provided in the present invention;
FIG. 8 is a graph showing the results of a sensitivity test of the primer set P2 provided in the present invention;
FIG. 9 is a graph showing the results of a sensitivity test of the primer set P3 provided in the present invention;
FIG. 10 is a graph showing the results of a sensitivity test of the primer set P4 provided in the present invention;
FIG. 11 is a graph showing the results of a sensitivity test of the primer set P5 provided in the present invention;
FIG. 12 is a graph showing the results of a sensitivity test of the primer set P6 provided in the present invention;
FIG. 13 is a diagram showing the result of the experiment on the LAMP detection (primer set P1-P6) of ASFV virus-containing samples according to the present invention.
Detailed Description
The technical personnel can modify the technological parameters appropriately to realize the method by taking the contents of the invention as reference. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The porcine pseudorabies virus (PRV), highly pathogenic porcine reproductive and respiratory syndrome virus (also known as porcine reproductive and respiratory syndrome virus, PRRSV), porcine circovirus type 2 (PCV-2), Classical Swine Fever Virus (CSFV) and Porcine Parvovirus (PPV) used in the examples below were given by the military medical academy of sciences.
Example 1, design and synthesis of LAMP primers:
according to the alignment structure of 60 African swine fever viruses, the P54 gene region is determined to be a relatively conservative and screening LAMP primer region (GenBank accession number is MT 358378). A plurality of sets of LAMP amplification primers are designed by utilizing an LAMP primerexplorer online webpage (http:// primerexplorer. jp/lampv5e/index. html), each set of primers respectively aims at 8 relatively conserved regions of a P54 gene, each set of primers comprises a pair of inner primers with the serial number of F3/B3, a pair of outer primers with the serial number of FIP/BIP and a pair of loop primers with the serial number of LF/LB, and 6 sets of primer groups P1-P6 with high sensitivity and specificity are screened by adopting an LAMP method amplification test, wherein the LAMP amplification primers are shown in Table 1.
TABLE 1
Example 2, primer set P1 specific experiment:
the LAMP detection method in example 13, which uses primer set P1, and uses genomic DNA of swine genome (containing ASFV), swine genome, pseudorabies virus (PRV), highly pathogenic porcine reproductive and respiratory syndrome virus (also known as porcine reproductive and respiratory syndrome virus, PRRSV), porcine circovirus type 2 (PCV-2), Classical Swine Fever Virus (CSFV) and Porcine Parvovirus (PPV) as templates, is used to perform LAMP gene amplification, and a gene amplification curve is plotted as shown in fig. 1, which indicates that only african swine fever virus has an amplification curve and no other samples have amplification, indicating that the primer set has better specificity.
F3 in primer set P1 is a degenerate primer, which refers to a mixture of possible primers representing all the different base sequences encoding a single amino acid, the degenerate base Y representing C or T.
Example 3, primer set P2 specific experiment:
the present embodiment differs from embodiment 2 in that: the LAMP gene amplification uses a primer group P2, a gene amplification curve is drawn as shown in figure 2, only African swine fever virus has an amplification curve, and other samples have no amplification, which shows that the LAMP primer has better specificity.
LF in the primer group P2 is degenerate primer, and the degenerate base Y represents C or T.
Example 4, primer set P3 specific experiment:
the present embodiment differs from embodiment 2 in that: the LAMP gene amplification uses a primer group P3, a gene amplification curve is drawn as shown in figure 3, only African swine fever virus has an amplification curve, and other samples have no amplification 21, which shows that the LAMP primer has better specificity.
B3, LF and LB in the primer set P3 are degenerate primers, and the degenerate base Y represents C or T.
Example 5, primer set P4 specific experiment:
the present embodiment differs from embodiment 2 in that: the LAMP gene amplification uses a primer group P4, a gene amplification curve is drawn as shown in figure 4, only African swine fever virus has an amplification curve, and other samples have no amplification, which indicates that the LAMP primer has better specificity.
Example 6, primer set P5 specific experiment:
the present embodiment differs from embodiment 2 in that: the LAMP gene amplification uses a primer group P5, a gene amplification curve is drawn as shown in figure 5, only African swine fever virus has an amplification curve, and other samples have no amplification, which indicates that the LAMP primer has better specificity.
Example 7, primer set P6 specific experiment:
the present embodiment differs from embodiment 2 in that: the LAMP gene amplification uses a primer group P6, a gene amplification curve is drawn as shown in FIG. 6, only African swine fever virus has an amplification curve, and other samples have no amplification, which indicates that the LAMP primer has better specificity.
Example 8, primer set P1 sensitivity experiment:
(1) drawing a standard curve: synthesizing real-time fluorescent quantitative primer and plasmid template, and performing gradient dilution on the plasmid to obtain 109，108，107，106，105，104，103，102，101Carrying out fluorescent quantitative PCR on the copied template, and drawing a standard curve;
(2) determining ASFV copy number, using fluorescent quantitative primer to perform ASFV amplification, corresponding the result with the standard curve to obtain ASFV copy number, and performing gradient dilution to obtain 104,103,102,101Copying an ASFV template;
(3) to 104,103,102,101The copies of ASFV were LAMP-amplified, of which 104,103,102All have ideal amplification curves as shown in FIG. 7, and the primer set P1 has good sensitivity.
Example 9, primer set P2 sensitivity experiment:
this example differs from example 8 in that: sensitivity experiments used primer set P2, from the amplification curve as shown in fig. 8: the primer set P2 has good sensitivity.
Example 10, primer set P3 sensitivity experiment:
this example differs from example 8 in that: sensitivity experiments used primer set P3, from the amplification curve as shown in fig. 9: the primer set P3 has good sensitivity.
Example 11, primer set P4 sensitivity experiment:
this example differs from example 8 in that: sensitivity experiments used primer set P4, from the amplification curve as shown in fig. 10: the primer set P4 has good sensitivity.
Example 12, primer set P5 sensitivity experiment:
this example differs from example 8 in that: sensitivity experiments used primer set P5, from the amplification curve as shown in fig. 11: the primer set P5 has good sensitivity.
Example 13, primer set P6 sensitivity experiment:
this example differs from example 8 in that: sensitivity experiments used primer set P6, from the amplification curve as shown in fig. 12: the primer set P6 has good sensitivity.
Example 14 detection of ASFV virus-containing specimen by LAMP technique (primer set P1-P6).
The LAMP reaction system was 25. mu.l, in which 2.5. mu.l of 10 × Isothermal Amplification Buffer, 3.5. mu.l of 10mM dNTPs, and 100mM MgSO41.5. mu.l, 10 XPrimers 2.5. mu.l, Bst2.01. mu.l, pig spleen DNA (containing ASFV) 1. mu.l, H2O13. mu.l. The LAMP reaction program is that the reaction is carried out for 40min at 65 ℃ and the inactivation is carried out for 1min at 95 ℃ to finish the reaction. The LAMP Primer Mix is prepared by 16 μ L of FIP/BIP Primer of 100 μmol/L, 2 μ L of F3/B3 Primer of 100 μmol/L, 4 μ L of LF/LB Primer of 100 μmol/L and ddH2Make up to 100. mu.L of O.
To contain 102LAMP amplification is carried out by using the above system with copy ASFV porcine spleen lysate DNA as a template, and the amplification result is shown in FIG. 13, wherein P1-P3 is 10 pairs2The copy ASFV is well amplified. To contain 102Copies of ASFV were amplified with primers P1-P3 for cycles20-25Ct, while P4-P6 are also preferred, but the number of cycles is slightly greater, about 25-30 Ct.
The method obtains 6 sets of LAMP primers with high sensitivity and specificity through screening, has good application value for early detection and early prevention of African swine fever viruses, and has the advantages of simple operation, low cost, short reaction period and the like.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Jilin university
<120> African swine fever LAMP detection primer combination and detection method
<130> 2021
<160> 36
<170> PatentIn version 3.5
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Claims (4)
1. The LAMP detection primer group for the African swine fever is characterized in that: the target sequence is a relative conserved region of African swine fever P54 gene, and the primer group is any one of the following primer groups (1) - (6);
(1) primer set P1;
the sequence of F3 is shown in SEQ ID NO: 1 is shown in the specification;
the sequence of B3 is shown in SEQ ID NO: 2 is shown in the specification;
the sequence of FIP is shown in SEQ ID NO: 3 is shown in the specification;
the sequence of BIP is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of LF is shown in SEQ ID NO: 5 is shown in the specification;
the sequence of LB is shown in SEQ ID NO: 6 is shown in the specification;
(2) primer set P2;
the sequence of F3 is shown in SEQ ID NO: 7 is shown in the specification;
the sequence of B3 is shown in SEQ ID NO: 8 is shown in the specification;
the sequence of FIP is shown in SEQ ID NO: 9 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 10 is shown in the figure;
the sequence of LF is shown in SEQ ID NO: 11 is shown in the figure;
the sequence of LB is shown in SEQ ID NO: 12 is shown in the specification;
(3) primer set P3;
the sequence of F3 is shown in SEQ ID NO: 13 is shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 14 is shown in the figure;
the sequence of FIP is shown in SEQ ID NO: 15 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 16 is shown in the figure;
the sequence of LF is shown in SEQ ID NO: 17 is shown;
the sequence of LB is shown in SEQ ID NO: 18 is shown in the figure;
the degenerate base Y in the primer sets of groups (1) to (3) represents C or T;
(4) primer set P4;
the sequence of F3 is shown in SEQ ID NO: 19 is shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 20 is shown in the figure;
the sequence of FIP is shown in SEQ ID NO: 21 is shown in the figure;
the sequence of BIP is shown as SEQ ID NO: 22;
the sequence of LF is shown in SEQ ID NO: 23 is shown;
the sequence of LB is shown in SEQ ID NO: shown at 24;
(5) primer set P5;
the sequence of F3 is shown in SEQ ID NO: 25 is shown;
the sequence of B3 is shown in SEQ ID NO: 26 is shown;
the sequence of FIP is shown in SEQ ID NO: 27 is shown;
the sequence of BIP is shown as SEQ ID NO: 28 is shown;
the sequence of LF is shown in SEQ ID NO: 29 is shown;
the sequence of LB is shown in SEQ ID NO: 30 is shown in the figure;
(6) primer set P6;
the sequence of F3 is shown in SEQ ID NO: 31, shown in the figure;
the sequence of B3 is shown in SEQ ID NO: 32 is shown;
the sequence of FIP is shown in SEQ ID NO: 33;
the sequence of BIP is shown as SEQ ID NO: 34;
the sequence of LF is shown in SEQ ID NO: 35 is shown in the figure;
the sequence of LB is shown in SEQ ID NO: shown at 36.
2. The LAMP detection primer as set forth in claim 1, and is applied to preparation of African swine fever detection reagents.
3. An African swine fever detection kit is characterized in that: including any one of the LAMP detection primer sets of claim 1.
4. An LAMP detection method for African swine fever comprises the following steps:
(1) extracting genome DNA of a sample to be detected to prepare a template to be detected;
(2) adding the template to be detected into the kit, wherein the reaction system in the kit is 25 mu l, the 10 × Isothermal Amplification Buffer is 2.5 mu l, the 10mM dNTPs is 3.5 mu l, and the 100mM MgSO is MgSO41.5. mu.l, 2.5. mu.l for 10 XPrimers, 1. mu.l for Bst2.0, 1. mu.l for the template to be examined, H2O is 13 mu l;
the reaction procedure in the kit is reaction at 65 ℃ for 40min, and inactivation at 95 ℃ for 1min to finish the reaction. The LAMP Primer Mix is prepared by 16 mu L of FIP/BIP primers of 100 mu mol/L, 2 mu L of F3/B3 primers of 100 mu mol/L, 4 mu L of LF/LB primers of 100 mu mol/L and ddH2O to make up to 100 mu L;
(3) and drawing a nucleic acid amplification curve according to the reaction result.
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