AU2020101818A4 - A novel LAMP method, primer set and kit for SNP detection - Google Patents

A novel LAMP method, primer set and kit for SNP detection Download PDF

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AU2020101818A4
AU2020101818A4 AU2020101818A AU2020101818A AU2020101818A4 AU 2020101818 A4 AU2020101818 A4 AU 2020101818A4 AU 2020101818 A AU2020101818 A AU 2020101818A AU 2020101818 A AU2020101818 A AU 2020101818A AU 2020101818 A4 AU2020101818 A4 AU 2020101818A4
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primer
nucleotide sequence
salmonella
loop
detecting
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Hongchao Gou
Ming LIAO
Qijie Lin
Haiyan Shen
Junping Wen
Jianmin Zhang
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South China Agricultural University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The invention discloses a novel LAMP method capable of detecting SNP, comprising the following steps: (1) extracting DNA as a template to be detected; (2) designing a loop primer probe modified by a a fluorescent group and a fluorescence quenching group, a pair of inner primers and a pair of outer primers the loop primer probe has an RNA base bound to a DNA mutation site; (3)Establishing a reaction system, adding a template to be detected into the reaction system, and detecting whether reactants generate fluorescent signals, wherein if fluorescent signals are generated, target DNA exists in the template to be detected, and if fluorescent signals are not generated, no target DNA exists in the template to be detected.Accordingly, the present invention also discloses a primer set and a reagent kit for detecting Salmonella Pullorum based on the above method. The detection method of the present invention has the advantage of high specificity and high sensitivity.

Description

A novel LAMP method, primer set and kit for SNP detection
TECHNICAL FIELD
[01] The invention relates to the technical field of gene detection, in particular to a novel LAMP method, a primer set and a kit capable of detecting SNP.
BACKGROUND
[02] Single nucleotide polymorphism (SNP) mainly refers to a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, that is, a single base difference in the DNA sequence. In nature, SNP has many characteristics such as large number, wide distribution and stable heredity, and is directly related to many diseases. It is very important for the detection and analysis of SNP in the research of new drugs, exploration of drug use, clinical test and gene mutation diagnosis.
[03] At present, the reported SNP detection technologies include high throughput sequencing technology, capillary electrophoresis technology, flow fluorescence hybridization, denaturing high-performance liquid chromatography detection, allele-specific oligonucleotide fragment analysis, In this method of the pyrosequencing technology, the ARMS-technology and the HRM technology, for example, the detection period is long, the sensitivity is low, the specificity is not strong enough, the operation is complex at high cost, or the method is easy to cause pollution. Therefore, it is necessary to invent a SNP detection method which can better overcome the defects of the mainstream methods.
[04] In the loop-mediated isothermal amplification (LAMP) technique, a plurality of specific primer are designed for multiple region of a target gene, by using a DNA polymerase (Bst enzyme) having strand displacement activity, the nucleic acid amplification reaction can be completed. Compared with the traditional isolation and identification, it has the advantages of short cycle, high sensitivity and high specificity. The LAMP primer set generally consists of inner primers FIP (F lc + F2) and BIP (B1c + B2), outer primers F3 and B3, and loop primers LoopF / LoopB. The loop primer is designed between Fc and F2c or between Blc and B2c, and plays a role of greatly improving the isothermal amplification efficiency on the premise that the LAMP normal extension reaction is initiated. However, the initial LAMP technique could only amplify certain gene fragments to a extent and could not detect SNPs.
[05] Salmonella Pullorum (S. Pullorum) is a member of the genus Salmonella of the Enterobacteriaceae family, which, together with Salmonella Gallinarum and Salmonella Enteritidis, belongs to the Salmonella D serum group. Pullorum disease caused by Salmonella Pullorum is a serious systemic disease, which can spread vertically, and its clinical symptoms include weight loss, reduced egg laying and impaired reproductive ability, which seriously affects the production of laying hen. The mortality rate of 2-3-week-old chickens is very high; Salmonella Gallinarum infection had an effect on birds of any age, mainly observed in adult chickens, and could spread vertically. The main clinical symptoms were a sudden decrease in food intake, lethargy, fever, yellow-green diarrhea, and rapid death. Salmonella Enteritidis is the leading cause of acute gastroenteritis, with typical symptoms after infection including fever, diarrhea and vomiting, which are sometimes difficult to distinguish in clinical production.
[06] Early and rapid detection and identification of Salmonella Pullorum is the basis of prevention, control and purification of the disease. The traditional isolation and identification method has a long cycle, and it goes through the processes of pre enrichment, selective enrichment, biochemical identification, serological identification, etc. Moreover, slide agglutination test is easy to produce nonspecific reaction and lack of sensitivity. Since Salmonella Pullorum, Salmonella Gallinarum and Salmonella Enteritidis all belong to the D serogroup and have very similar antigen expressions, it is often wrong to rely purely on the antigen expression in slide agglutination test. Therefore, the invention of a high-efficiency, strong specificity, high sensitivity of the detection of Salmonella Pullorum has important public health significance.
SUMMARY
[07] The object of the present invention is to provide a novel LAMP method for detecting SNPs, which is characterized by strong specificity and high sensitivity.
[08] The purpose of the present invention is to propose a novel LAMP method based on SNP detection for detecting Salmonella Pullorum in a primer set and a kit, which has the characteristics of accurately and rapidly detecting Salmonella Pullorum.
[09] In order to achieve that purpose, the invention adopts the follow technical scheme:
[010] A novel LAMP method for SNP detection comprises the steps:
(1) DNA was extracted as the template to be detected.
(2) Designing a fluorescent group and a fluorescence quenching group modified loop primer probe, a pair of inner primers and a pair of outer primers, the loop primer probes having an RNA base bound to a DNA mutation site;
(3) In this method, establishing a reaction system, adding a template to be detected into the reaction system, and detecting whether reactants generate fluorescent signals, wherein if fluorescent signals are generated, target DNA exists in the template to be detected, and if fluorescent signals are not generated, no target DNA exists in the template to be detected. Further, the loop primer probe is modified by a fluorescent group and a quenching group;
[011] The reaction system contains RNase H2 enzyme, and when the loop primer probe is combined with the DNA mutation site, the RNase H2 enzyme is activated to cut, so that the fluorescent group and the quenching group are separated. Further, the inner primer consists of a forward inner primer FIP and a reverse inner primer BIP, and the outer primer consists of the forward outer primer F3 and the reverse outer primer B3.
[012] Further, the reaction system comprises a buffer, magnesium sulfate, dNTP, Bst enzyme, Rnase H2 enzyme, loop primer probe, inner primer and outer primer.
[013] Further, the reaction temperature of the reaction system is 58°C to 62°C .
[014] A primer set for detecting Salmonella Pullorum based on the above detection method, comprising:
[015] Nucleotide sequence of forward primer F3:
5'-AGGAACAATGAAGCTACCATA-3';
[016] Nucleotide sequence of reverse primer B3:
5'-GGCAGTGATGTTCCACAAT-3';
[017] Nucleotide sequence of the forward inner primer FIP:
5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAATT GGATCG-3';
[018] Nucleotide sequence of reverse inner primer BIP:
5'-ACCTGCAACAGCTTTAATAGAAAGCGAATACTGCATCAAGTG ATGAG-3';
[019] The nucleotide sequence of the loop primer probe:
5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT Eclipse-3'
[020] The primer set was targeted against the rfbS gene of Salmonella Pullorum.
[021] The kit for detecting Salmonella Pullorum based on the above detection method comprises a reaction solution, which includes a loop primer probe, a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, and a reverse outer primer B3;
[022] Nucleotide sequence of forward lateral primer F3:
5'-AGGAACAATGAAGCTACCATA-3';
[023] Nucleotide sequence of reverse lateral primer B3:
5'-GGCAGTGATGTTCCACAAT-3';
[024] Nucleotide sequence of the forward medial primer FIP:
5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAATT GGATCG-3';
[025] Nucleotide sequence of reverse inner primer BIP:
5'-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGTG ATGAG-3';
[026] The nucleotide sequence of the loop primer probe:
5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT Eclipse-3'
[027] The primer set is targeted at the rfbS gene of Salmonella Pullorum.
[028] Further, the reaction solution comprises a buffer solution of 2.5[l, magnesium sulfate of 2.0tl and dNTP of 2[1 with a Bst enzyme of 1.0u1 and Rnase H2 enzyme of 0.1 l as well as a loop primer probe of 0.5tl; FIP + BIP4 l & F3 + B30.5 pl.
[029] The beneficial effects of the present invention:
[030] According to the novel LAMP amplification rule, the invention provides a loop-mediated isothermal amplification method based on loop primer probe mononucleotide complementary activator cleavage for SNP detection. The principle of the detection method is to target the gene to be detected containing SNP, and design a reporter fluorescent group and quenching fluorescent group modified loop primer probe. RNA base which can be combined with DNA mutation site is designed at the corresponding site of the probe. when the two are specifically complementary and combined, Rnase H2 enzyme is activated to cut, and the reporter fluorescent group and quenching fluorescent group are separated to generate fluorescent signals; No fluorescence signal is generated when no specific binding occurs. The principle of generating fluorescence signal based on the cleavage of single nucleotide complementary activating enzyme has very good specificity, can complete the specific detection of SNP, and has very good sensitivity.
2. The novel LAMP method capable of detecting SNP of the present invention has the characteristics of high reaction efficiency, and the detection can be completed within 40 minutes.
3. The novel LAMP method capable of detecting SNP of the present invention has strong specificity, which can be accurately detected from 22 kinds of Salmonella and 9 kinds of other pathogenic bacteria, it is highly sensitive, and the detection limit is 21 copies. The results were better than those of PCR.
4. According to the published complete gene sequence of Salmonella from NCBI and relevant data, it is found that the Salmonella rfbS gene is a specific DNA sequence with single nucleotide polymorphism (SNP). At position 237 the Pullorum is guanine, while other serotypes and other strains, such as Salmonella Gallinarum and Salmonella Enteritidis,, are adenine, which do not contain the gene or are highly variable. According to the single nucleotide polymorphism of Salmonella Pullorum at the 237th site, a loop-mediated isothermal amplification primer set based on loop primer probe-single nucleotide complementary activator cleavage and an experimental method are designed to detect Salmonella Pullorum.. A reporter fluorescent group and quenching fluorescent group modified loop primer probe was designed to target the rfbS gene of Pullorum spp. In this loop primer probe, the RNA base complementary to the DNA base at the 237th position of the rfbS gene is designed, and when the two are specifically complementary to each other, the activate Rnase H2 enzyme is cleaved, the reporter fluorescent group and the quenching fluorescent group are separated. In this invention, the fluorescence signal is generated, and when the specific binding does not occur, the fluorescent signal is not generated, so that the aim of accurately and quickly detecting the Salmonella Pullorum is achieved.
BRIEF DESCRIPTION OF THE FIGURES
[031] FIG. 1 is a schematic diagram of a novel LAMP method for SNP detection according to the present invention;
[032] FIG. 2 is a graph of amplification results of a basic reaction system;
[033] FIG. 3 is a graph of reaction temperature optimization results;
[034] FIG. 4 is a graph showing the results of a specificity evaluation experiment;
[035] FIG. 5 is a graph showing the results of sensitivity evaluation experiments;
[036] FIG. 6 is a graph showing the results of identification of clinically isolated samples.
DESCRIPTION OF THE INVENTION
[037] The technical solution of the present invention is further described below with reference to the drawings and specific embodiments.
[038] Example 1 A Novel LAMP Method for SNP Detection
[039] The novel LAMP method capable of detecting SNP of this embodiment comprises the following steps:
(1) DNA was extracted as the template to be detected.
(2) Designing a reporter fluorescent group and quenching fluorescent group loop primer probe, a pair of inner primer and a pair of outer primer, the loop primer probes having an RNA base bound to a DNA mutation site;
(3) In this method, establishing a reaction system, adding a template to be detected into the reaction system, and detecting whether reactants generate fluorescent signals, wherein if fluorescent signals are generated, target DNA exists in the template to be detected, and if fluorescent signals are not generated, no target DNA exists in the template to be detected.
[040] In step (2), the loop primer probe is modified by a fluorophore and a quench group; in the step (3), the reaction system comprises a Rnase H2 enzyme, which is activated to cleave when the loop primer probe is bound to a DNA mutation site, the fluorescent group and the quenching group are separated.
[041] In accordance with the LAMP amplification rule, the present invention propose a loop-mediated isothermal amplification method for SNP detection based on loop primer probe-single nucleotide complementary activation enzyme cleavage. The principle of the detection method is to target the gene to be detected containing SNP, and design a loop primer probe modified by fluorescent group. In the correspond position of the probe, an RNA base capable of binding to a DNA mutation position is designed, as shown in FIG. 1, when the two are specifically and complementarily bound, the activated Rnase H2 enzyme undergoes cleavage, the reporter fluorescent group and the quench fluorescent group are separated, a fluorescence signal is generated; no fluorescence signal occurs when specific binding does not occur. Based on the principle of single nucleotide complementary activator (SNPs) cleavage to generate fluorescence signal, the method has very good specificity, which can detect SNP specifically, and has good sensitivity.
[042] Specifically, the inner primer is a forward inner primer FIP and a reverse inner primer BIP, and the outer primer is the forward outer primer F3 and the reverse outer primer B3. FIP, BIP, F3 and B3 constitute an inner primer set and an outer primer set in LAMP amplification.
[043] Specifically, the reaction system includes a buffer, magnesium sulfate, dNTP, Bst enzyme, Rnase H2 enzyme, loop primer probe, inner primer, and outer primer.
[044] Specifically, the reaction temperature of the reaction system is 58°C-62°C. In this reaction temperature range, the reaction can be smoothly performed, and most preferably the reaction temperature of the reaction system is 61°C.
[045] Example 2 Primer set for detection of Salmonella Pullorum
[046] The primer group of this example is a primer group for detecting Salmonella Pullorum based on the detection method of Example 1, and includes:
[047] Nucleotide sequence of forward lateral primer F3:
5'-AGGAACAATGAAGCTACCATA-3';
[048] Nucleotide sequence of reverse lateral primer B3:
5'-GGCAGTGATGTTCCACAAT-3';
[049] Nucleotide sequence of the forward medial primer FIP:
5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAATT GGATCG-3';
[050] Nucleotide sequence of reverse inner primer BIP:
5'-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGTG ATGAG-3';
[051] The nucleotide sequence of the loop primer probe:
5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT Eclipse-3'
[052] where Eclipse is a fluorescence quenching group.
[053] The primer set was directed against the rfbS gene of Salmonella Pullorum.
[054] The rfbS gene of Salmonella Pullorum was detected by a new LAMP method which can detect SNP. the results showed that the method was rapid and accurate.
[055] Example 3 Kit for Detection of Salmonella Pullorum
[056] In that kit for detect Salmonella Pullorum according to the detection method of example 1, the reagent kit comprise a reaction solution, The reaction solution includes a loop primer probe, a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, and a reverse outer primer B3;
[057] Nucleotide sequence of forward lateral primer F3:
5'-AGGAACAATGAAGCTACCATA-3';
[058] Nucleotide sequence of reverse lateral primer B3:
5'-GGCAGTGATGTTCCACAAT-3';
[059] Nucleotide sequence of the forward medial primer FIP:
5'_GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAATT GGATCG-3';
[060] Nucleotide sequence of reverse inner primer BIP:
5'-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGTG ATGAG-3';
[061] The nucleotide sequence of the loop primer probe:
5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT Eclipse-3'
[062] The primer set was targeted against the rfbS gene of Salmonella Pullorum.
[063] the reaction solution comprises a buffer solution of 2.5tl, magnesium sulfate of 2.0tl and dNTP of 2[1 with a Bst enzyme of 1.0u1 and Rnase H2 enzyme of 0. 1 as well as a loop primer probe of 0.5 l; FIP + BIP4 l & F3 + B30.5 1d.
[064] When the kit of this example was used for detection, 2.5tl of bacteria template was added to the reaction solution, and 251 could not be obtained by adding ultrapure water, that is, the reaction system was a 25 [ system, and the reaction temperature was 58 °C -62 °C.
[065] Taking the detection of Salmonella Pullorum as an example, the reliability of the novel LAMP method capable of detecting SNP of the present invention is verified.
1. Materials and methods
1.1 strain
[066] Salmonella Pullorum, Salmonella Gallinarum, Salmonella Enteritidis, Salmonella Corvallis, Salmonella Derby, Salmonella Rissen, Salmonella London, Salmonella Weltevreden, Salmonella Typhimurium, Salmonella Albany, Salmonella Panama, Salmonella Cerro Salmonella Kentucky, Salmonella Havana, Salmonella Mbandaka, Salmonella Infantis, Salmonella Goldcoast ,Salmonella Meleagridis,
Salmonella Argona, Salmonella Indiana, Salmonella Saintpaul, Salmonella Choleraesuis, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria iuanuii, Shigella, Riemerella anatipestifer, Campylobacterjejuni, and Campylobacter coli, including the standard strain and the strain identified and preserved by the Key Laboratory of Zoonosis Prevention and Control in Veterinary College of South China Agricultural University.
1.2 Main reagents
[067] LB broth, LB agar, XLT4 agar basis, buffer protein peptone aqueous solution (BPW), Tetrathionate Broth Base (TTB), brain heart extract broth (BHI), etc. were purchased from Guangdong Huankai Microbiology Technology Co., Ltd. BST enzyme, magnesium sulfate, Buffer, etc. (New England Biological Laboratory Company); Rnase H2 enzyme (Integrated DNA Technologies); dNTP (Beijing Transgen Biotech Co., Ltd.); Primers were synthesized by Bioengineering (Shanghai) Co., Ltd., and ultrapure water was prepared by Elix100 purified water plant (Millipore, USA).
1.3 Experiment
1.3.1 Isolation of bacterial genomic DNA
[068] Each bacterial genomic DNA template was prepared by thermal lysis method and used for the following specificity evaluation experiment and sensitivity experiment evaluation.
[069] The DNA templates include Salmonella Pullorum (2 strains), Salmonella Gallinarum, Salmonella Enteritidis, Salmonella Corvallis, Salmonella Derby, Salmonella Rissen, Salmonella London, Salmonella Weltevreden, Salmonella Typhimurium, Salmonella Albany, Salmonella Panama,Salmonella Cerro, Salmonella Kentucky, Salmonella Havana, Salmonella Mbandaka, Salmonella Infantis, Salmonella Goldcoast, Salmonella Meleagridis, Salmonella Argona, Salmonella Indiana ,
Salmonella Saintpaul, Salmonella Choleraesuis, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria iuanuii, Shigella, Riemerella anatipestifer, Campylobacterjejuni, and Campylobactercoli.
1.3.2 Construction of standard plasmid
[070] According to the accession number of rfbS gene of Salmonella Pullorum: GenBank: LK931482.1, and that of Salmonella AF442573, PCR primers were designed on both sides of the two genes. rfbS F:AATATCACCATGTACAAACTCAAAG, rfbS R:ATCGTGTAGTGGGTGAGT. The full-length rfbS genes of Salmonella Pullorum and Salmonella Gallinarum were amplified by PCR.
[071] The amplified product was verified by electrophoresis experiment, recovered by using a commercial kit (Omega), ligated with a T-vector overnight, transformed the ligated product into DH5a competent cells, and the competent cells were expanded and cultured. The DNA was extracted for electrophoretic testing and sequencing validation, while the plasmid was extracted as described in the commercial kit. The concentration of the plasmid was determined with a spectrophotometer and converted into the number of copies, and then stored at -20 c for use.
1.3.3 Primer set
[072] In accordance with that rfbS gene of Salmonella Pullorum, the reaction primer were designed as follow:
[073] Nucleotide sequence of forward lateral primer F3:
5'-AGGAACAATGAAGCTACCATA-3';
[074] Nucleotide sequence of reverse lateral primer B3:
5'-GGCAGTGATGTTCCACAAT-3';
[075] Nucleotide sequence of the forward medial primer FIP:
5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAATT GGATCG-3';
[076] Nucleotide sequence of reverse inner primer BIP:
5-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGTGA TGAG-3';
[077] The nucleotide sequence of the loop primer probe:
5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT Eclipse-3'
1.3.4 Establishment of basic reaction system
[078] Using the constructed standard plasmids of Salmonella Pullorum and Salmonella Gallinarum, setting the fluorescence collection to 1 cycle / min for 60 cycles, adjusting the ratio of each component of the reagent, The basic reaction system was established and the reaction temperature was optimized.
[079] The basic reaction system was a 25 [ system, in which there were a buffer (Buffer) 2.541, a magnesium sulfate 2.0u1, a dNTP 2uL, a Bst enzyme (DNA polymerase) 1.0 1and a Rnase H2 enzyme 0.1l1; a loop primer probe (Loop) 0.51. FIP+BIP 4tl , F3+B3 0.51 , Bacterial template 2.5 1, ultrapure water for
replenishment.
[080] The amplification results of the basic reaction system are shown in FIG. 2, in which, line I is the amplification result of the standard plasmid containing Salmonella Pullorum, line II is the expansion result of standard vector containing Salmonella Gallinarum Line III is a negative control for amplification of the template reaction system without bacteria. It can be seen from Figure 2 that only the standard plasmid of Salmonella Pullorum was amplified, but that of Salmonella Gallinarum was not amplified, the experimental results proved that the principle was feasible, and the basic reaction system was successfully established.
[081] In this reaction system containing the standard plasmid of Salmonella Pullorum, different reaction temperatures were set for amplification, and the reaction temperature was optimize, as shown in FIG. 3, wherein line a is reaction temperature of 62 °C, line b is reaction temp. of 61 °C. C line is the reaction temperature of 60 °C, d line is 59 °C, e line is 58 °C, f line is 63 °C -65 °C, negative control. Take the reaction temperature with the minimum ct value, and the optimal reaction temperature is 61 °C.
1.3.5 Specificity evaluation experiment
[082] Using the genomic DNA template of 32 bacterial strains extracted from 1.3.1 and using the reaction system and reaction temperature established by 1.3.4, the specificity evaluation experiment was performed. the experimental conditions were as follows: The basic reaction system was established and the reaction temperature was optimized using 1.3.4. The results are shown in FIG. 4, in which line a and line b are the results of fluorescence detection of Salmonella Pullorum, and line c is the result of fluorescent detection of other serotype Salmonella and other species of bacteria, and the negative control. As can be seen from FIG. 4, the novel LAMP method capable of detecting SNP of the present invention can specifically detect Salmonella Pullorum.
1.3.6 Sensitivity evaluation test
[083] After calculation, the copy number of the plasmid of Salmonella Pullorum constructed in 1.3.2 was 2.1 x 100 copies, and the 10-4-10-° "dilution plasmid was used in the amplification experiment. The experimental conditions were as follows: 1.3.4 to establish basic reaction system and optimize reaction temperature. The results of sensitivity evaluation are shown in Figure 5. In FIG. 5, line a is a 2.1 x 10 copy number amplification curve, line b is 2.1 x 10' copy number expansion curve, and line c is a 2.1 x 104 copy number cultivation curve and line d is a 2100 copy number development
curve. Line e is a 210 copy number amplification curve, line f is a 21 copy number expansion curve, and line g is a 2.1 copy number and negative control expansion curve.
[084] 1.3.7 Identification of clinically isolated samples
[085] In clinic, 27 strains of Salmonella Pullorum were isolated, and that DNA template were extracted respectively, which used for amplification experiments. The experimental condition are as follows: 1.3.4 to establish the basic reaction system and optimize the reaction temperature; The results showed that all 27 strains of Salmonella Pullorum were rapidly detected within 40 min.
[086] The technical principles of the present invention have been described above in connection with specific embodiments. These descriptions are merely intended to explain the principles of the present invention and are not to be construed in any way as limiting the scope of the invention. Based on the explanation herein, those skilled in the art can associate with other embodiments of the present invention without any creative effort, and these embodiments will fall within the scope of the invention.
[087] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.
[088] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims (1)

  1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
    1. A novel LAMP method capable of detecting SNPs is characterized by comprising the steps:
    (1) DNA was extracted as the template to be detected.
    (2) Designing a fluorescent group and a fluorescence quenching group modified loop primer probe, a pair of inner primers and a pair of outer primers, the loop primer probes having an RNA base bound to a DNA mutation site;
    (3) In this method, establishing a reaction system, adding a template to be detected into the reaction system, and detecting whether reactants generate fluorescent signals, wherein if fluorescent signals are generated, target DNA exists in the template to be detected, and if fluorescent signals are not generated, no target DNA exists in the template to be detected.
    2. A novel LAMP method for SNP detection according to claim 1, characterized in that the loop primer probe is modified by a fluorescent group and a quenching group;
    The reaction system contains RNase H2 enzyme, and when the loop primer probe is combined with the DNA mutation site, the RNase H2 enzyme is activated to cut, so that the fluorescent group and the quenching group are separated.
    The novel LAMP method capable of detecting SNP according to claim 1, characterized in that, the inner primer consists of a forward inner primer FIP and a reverse inner primer BIP, and the outer primer consists of the forward outer primer F3 and the reverse outer primer B3.
    The novel LAMP method capable of detecting SNP according to claim 1, characterized in that the reaction system comprises a buffer, magnesium sulfate, dNTP, Bst enzyme, Rnase H2 enzyme, loop primer probe, inner primer and outer primer.
    The novel LAMP method capable of detecting SNP according to claim 1, wherein the reaction temperature of the reaction system is 58°C-62°C.
    6. A novel LAMP method based on the SNP detection according to claim 1 for detecting a primer set of Salmonella Pullorum, characterized by the steps:
    Nucleotide sequence of forward lateral primer F3
    5'-AGGAACAATGAAGCTACCATA-3';
    Nucleotide sequence of reverse lateral primer B3
    5'-GGCAGTGATGTTCCACAAT-3';
    Nucleotide sequence of the forward medial primer FIP:
    5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAA TTGGATCG-3';
    Nucleotide sequence of reverse inner primer BIP:
    5'-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGT GATGAG-3';
    The nucleotide sequence of the loop primer probe:
    5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT-Eclipse-3'
    The primer set is targeted at the rfbS gene of Salmonella Pullorum.
    7. A kit for detecting Salmonella Pullorum based on the novel LAMP method capable of detecting SNP according to claim 1, characterized in that it comprises a reaction solution, the reaction solution includes a loop primer probe, a forward inner primer FIP, a reverse inner primer BIP, a front outer primer F3, and a reverse outer primer B3;
    Nucleotide sequence of forward lateral primer F3
    5'-AGGAACAATGAAGCTACCATA-3';
    Nucleotide sequence of reverse lateral primer B3
    5'-GGCAGTGATGTTCCACAAT-3';
    Nucleotide sequence of the forward medial primer FIP:
    5'-GTCTTCCATAGCAAGCAATAGTGTTCACGACAGAAAATAA TTGGATCG-3';
    Nucleotide sequence of reverse inner primer BIP:
    5'-ACCTGCAACAGCTTTAATAGAAAGGAATACTGCATCAAGT GATGAG-3';
    The nucleotide sequence of the loop primer probe:
    5'-TCTTATGCCTATCAGAGTATT(FAM)AGAG(RNA base)TCTAT-Eclipse-3'
    8. A kit according to claim 7, characterized in that the reaction solution comprises a buffer solution of 2.5t, magnesium sulfate of 2.0tland dNTP of 2[1 with a Bst enzyme of 1.0t1and Rnase H2 enzyme of 0.1 1 as well as a loop primer probe of 0.5[l; FIP + BIP4tl & F3 + B30.5 1.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646905A (en) * 2020-12-30 2021-04-13 华南农业大学 Double visual single nucleotide polymorphism detection method and application thereof
CN113151589A (en) * 2021-02-19 2021-07-23 清华大学 Nested recombinase-polymerase amplification method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646905A (en) * 2020-12-30 2021-04-13 华南农业大学 Double visual single nucleotide polymorphism detection method and application thereof
CN113151589A (en) * 2021-02-19 2021-07-23 清华大学 Nested recombinase-polymerase amplification method and application thereof

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