Primer probe composition for detecting bacillus anthracis and application thereof

Abstract

The invention provides a primer probe composition for detecting bacillus anthracis and application thereof, and relates to the technical field of biological detection, the primer probe composition comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 36, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 46 and a specific fluorescent probe. The designed primer probe composition can be used for distinguishing bacillus anthracis from pathogenic bacteria such as bacillus subtilis, bacillus thuringiensis, bacillus coagulans, bacillus licheniformis, bacillus cereus, bacillus megatherium and bacillus lentus, and has high specificity; the sensitivity can reach 5 * 100 copies, the primer probe for detecting the bacillus anthracis based on the RPA is provided, and the technical problem that an RPA primer for detecting the bacillus anthracis lacks in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a primer-probe composition for detecting anthrax Bacillus and an application thereof. Background Art

[0002] Anthrax is a very serious zoonotic infectious disease caused by Bacillus anthracis, affecting almost all mammals, including humans. The disease occurs all over the world and persists for a long time in some countries or specific regions. Ruminants are highly sensitive to anthrax and often develop rapid and fatal septicemia.

[0003] In order to quickly and accurately detect Bacillus anthracis, researchers have developed many qPCR detection methods, which mainly target three regions (BA5345, PL3 and BA5357) on the Bacillus anthracis genome and several virulence genes carried on two plasmids pX01 and pX02. However, the three regions of BA5345, PL3 and BA5357 are located in the prophage λBa03 region and have very high genetic instability. Plasmids pX01 and pX02 have also been found to exist in some Bacillus cereus and Bacillus thuringiensis. Therefore, all of the above methods have the possibility of false detection and missed detection. Olga Bassy et al. designed a new qPCR method for the E4 sequence on the Bacillus anthracis genome. Since the E4 sequence is highly specific in the genome of Bacillus anthracis, it can detect Bacillus anthracis specifically in the laboratory.

[0004] Although the qPCR method can well detect anthrax Bacillus in the laboratory, its use depends on professional laboratories and operators. In general, anthrax is mainly prevalent in pastoral animals such as cattle, horses, and sheep. The sampling, sample delivery, and testing process is too long, which is not conducive to the rapid implementation of epidemic prevention and control measures. Therefore, the development of a simple, highly accurate and rapid on-site detection method will provide great help in the prevention and control of anthrax.

[0005] Recombinase polymerase amplification (RPA) is a new isothermal nucleic acid amplification technology that can realize direct reading of test results in a portable constant temperature amplification fluorescence detector. The detection time and convenience are better than the traditional PCR method, and it has broad application prospects in the rapid detection of animal diseases. However, due to the great difference between the RPA method and the traditional PCR method, it has higher requirements for primers and probes. In addition, primers and probes are the most critical factors affecting the RPA method. Different primers directly affect the amplification speed and detection sensitivity, thereby affecting on-site detection. At present, there is no bioinformatics software that can predict the efficiency of RPA primers and probes, and it can only be screened through experiments. Therefore, the primer design of RPA is more difficult.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] One of the purposes of the present invention is to provide a primer-probe composition for detecting Bacillus anthracis, so as to solve the technical problem of the lack of RPA primers for detecting Bacillus anthracis in the prior art.

[0008] A second object of the present invention is to provide the use of the above-mentioned primer-probe combination in preparing a product for detecting or assisting in the detection of Bacillus anthracis, preparing a product for detecting whether a sample contains Bacillus anthracis, preparing a product for identifying and distinguishing Bacillus anthracis from other pathogens in a sample, preparing a product for diagnosing or assisting in the diagnosis of a disease caused by Bacillus anthracis, or preparing a product for screening a disease caused by Bacillus anthracis.

[0009] A third object of the present invention is to provide a reagent for detecting Bacillus anthracis.

[0010] A fourth object of the present invention is to provide a kit for detecting Bacillus anthracis.

[0011] A fifth object of the present invention is to provide the use of the above-mentioned reagent or the above-mentioned kit in preparing a product for detecting or assisting in the detection of Bacillus anthracis, preparing a product for detecting whether a sample contains Bacillus anthracis, preparing a product for identifying and distinguishing Bacillus anthracis from other pathogens in a sample, preparing a product for diagnosing or assisting in the diagnosis of a disease caused by Bacillus anthracis, or preparing a product for screening a disease caused by Bacillus anthracis.

[0012] A sixth object of the present invention is to provide a method for detecting or assisting in detecting Bacillus anthracis for non-therapeutic and non-diagnostic purposes.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0014] In a first aspect, the present invention provides a primer-probe composition for detecting Bacillus anthracis, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO:36, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:46, and a specific fluorescent probe;

[0015] The structure of the specific fluorescent probe is SEQ ID NO: 20-tetrahydrofuran residue-SEQ ID NO: 21, and the 3' end of SEQ ID NO: 21 is blocked and modified.

[0016] Furthermore, the molar ratio of the upstream primer, the downstream primer and the specific fluorescent probe is 11:11:2.

[0017] Furthermore, the T base upstream of the tetrahydrofuran residue is labeled with a fluorescent reporter group, and the T base downstream of the tetrahydrofuran residue is labeled with a fluorescent quencher group, and the distance between the fluorescent reporter group and the fluorescent quencher group is 2 to 4 nucleotides;

[0018] Preferably, the fluorescent reporter group is selected from at least one of Fam, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705;

[0019] The fluorescence quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA;

[0020] The 3' end of SEQ ID NO. 21 is connected to C3-spacer.

[0021] In a second aspect, the present invention provides the use of the primer-probe combination described above in any of the following:

[0022] A1. Application in the preparation of products for detecting or assisting in the detection of Bacillus anthracis;

[0023] A2. Use in the preparation of products for detecting whether a sample contains Bacillus anthracis;

[0024] A3. Use in the preparation of products for identifying and distinguishing Bacillus anthracis and other pathogens in samples;

[0025] A4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis;

[0026] A5. Application in the preparation of products for screening diseases caused by Bacillus anthracis.

[0027] In a third aspect, the present invention provides a reagent for detecting Bacillus anthracis, comprising the above-mentioned primer-probe combination.

[0028] In a fourth aspect, the present invention provides a kit for detecting Bacillus anthracis, comprising the above-mentioned primer-probe combination or the above-mentioned reagent.

[0029] Furthermore, other reagents required for isothermal amplification of the recombinase are also included.

[0030] In a fifth aspect, the present invention provides the use of the above reagent or the above kit in any of the following:

[0031] B1. Use in detecting or assisting in detecting Bacillus anthracis or in preparing products for detecting or assisting in detecting Bacillus anthracis;

[0032] B2. Use in detecting whether a sample contains Bacillus anthracis or in preparing a product for detecting whether a sample contains Bacillus anthracis;

[0033] B3. Use in identifying and distinguishing Bacillus anthracis from other pathogens in samples or in preparing products for identifying and distinguishing Bacillus anthracis from other pathogens in samples;

[0034] B4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis;

[0035] B5. Application in screening for diseases caused by Bacillus anthracis or in preparing products for screening for diseases caused by Bacillus anthracis.

[0036] In a sixth aspect, the present invention provides a method for detecting or assisting in the detection of Bacillus anthracis for non-therapeutic and non-diagnostic purposes, comprising performing recombinase isothermal amplification on a sample using the above-mentioned primer-probe combination, the above-mentioned reagent or the above-mentioned kit, and determining whether Bacillus anthracis is present in the sample based on a fluorescent signal.

[0037] Furthermore, the reaction temperature of the recombinase isothermal amplification is 35-41°C, preferably 39°C;

[0038] Preferably, the reaction time of the recombinase isothermal amplification is 20 min.

[0039] The primer-probe composition for detecting anthrax Bacillus provided by the present invention can distinguish anthrax Bacillus from pathogens such as Bacillus subtilis, Bacillus thuringiensis, Bacillus coagulans, Bacillus licheniformis, Bacillus cereus, Bacillus megaterium and Bacillus lentus, and has high specificity; and the sensitivity can reach 5×10 0The invention provides a primer probe for detecting anthrax Bacillus based on RPA, which solves the technical problem of lack of RPA primers for detecting anthrax Bacillus in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 A result diagram of gel electrophoresis after RPA amplification after different upstream primers and downstream primers are paired is provided for Example 1 of the present invention;

[0042] Figure 2 The RPA amplification results of different probes provided in Example 2 of the present invention;

[0043] Figure 3 The RPA amplification results after pairing different upstream primers, downstream primers and probes provided in Example 3 of the present invention;

[0044] Figure 4 The RPA amplification results after pairing different upstream primers, downstream primers and probes provided in Example 4 of the present invention;

[0045] Figure 5 The RPA amplification results of different primer concentrations and probe concentrations provided in Example 5 of the present invention;

[0046] Figure 6 Different Mg provided by Example 6 of the present invention 2+ RPA amplification results of concentration;

[0047] Figure 7 The RPA amplification results using different Bacillus as templates provided in Example 7 of the present invention;

[0048] Figure 8 This is the sensitivity test result of the primer-probe combination provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0049] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0050] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0051] In one aspect, the present invention provides a primer-probe composition for detecting Bacillus anthracis, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO:36, a downstream primer having a nucleotide sequence as shown in SEQ ID NO:46, and a specific fluorescent probe;

[0052] The structure of the specific fluorescent probe is SEQ ID NO: 20-tetrahydrofuran residue-SEQ ID NO: 21, and the 3' end of SEQ ID NO: 21 is blocked and modified.

[0053] The primer-probe combination provided by the present invention is designed according to the nucleotide sequence shown in SEQ ID NO: 1, and can distinguish Bacillus anthracis from pathogenic bacteria such as Bacillus subtilis, Bacillus thuringiensis, Bacillus coagulans, Bacillus licheniformis, Bacillus cereus, Bacillus megaterium and Bacillus lentus, and has high specificity; and the sensitivity can reach 5×10 0 The invention provides a primer probe for detecting anthrax Bacillus based on RPA, which solves the technical problem of lack of RPA primers for detecting anthrax Bacillus in the prior art.

[0054] Specifically, the blocking modification is to modify the blocking group, and any modification group that can prevent the polymerase from synthesizing new bases can be selected.

[0055] In some specific embodiments, the T base upstream of the tetrahydrofuran residue is labeled with a fluorescent reporter group, and the T base downstream of the tetrahydrofuran residue is labeled with a fluorescent quencher group, and the distance between the fluorescent reporter group and the fluorescent quencher group is 2 to 4 nucleotides.

[0056] Specifically, the fluorescent reporter group modified by the probe can be any fluorescent group, and the fluorescent quenching group modified on the probe can be any group sufficient to quench the corresponding fluorescent group. In some specific embodiments, the fluorescent reporter group is selected from at least one of Fam, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705. The fluorescent quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA. The 3' end of the SEQ ID NO: 21 is connected to C3-spacer.

[0057] In order to further improve the amplification efficiency, in some specific embodiments, the molar ratio of the upstream primer, the downstream primer and the specific fluorescent probe is 11:11:2.

[0058] According to another aspect of the present invention, there is also provided the use of the primer-probe combination in any of the following:

[0059] A1. Application in the preparation of products for detecting or assisting in the detection of Bacillus anthracis;

[0060] A2. Use in the preparation of products for detecting whether a sample contains Bacillus anthracis;

[0061] A3. Use in the preparation of products for identifying and distinguishing Bacillus anthracis and other pathogens in samples;

[0062] A4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis;

[0063] A5. Application in the preparation of products for screening diseases caused by Bacillus anthracis.

[0064] The product may be a reagent, a test kit, or other detection products.

[0065] According to another aspect of the present invention, there is provided a reagent for detecting Bacillus anthracis, comprising the above primer-probe combination.

[0066] According to another aspect of the present invention, a kit for detecting Bacillus anthracis is provided, comprising the above primer-probe combination or the above reagent.

[0067] In some specific embodiments, other reagents required for isothermal amplification of the recombinase are also included.

[0068] According to another aspect of the present invention, there is also provided the use of the above reagent or the above kit in any of the following:

[0069] B1. Use in detecting or assisting in detecting Bacillus anthracis or in preparing products for detecting or assisting in detecting Bacillus anthracis;

[0070] B2. Use in detecting whether a sample contains Bacillus anthracis or in preparing a product for detecting whether a sample contains Bacillus anthracis;

[0071] B3. Use in identifying and distinguishing Bacillus anthracis from other pathogens in samples or in preparing products for identifying and distinguishing Bacillus anthracis from other pathogens in samples;

[0072] B4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis;

[0073] B5. Application in screening for diseases caused by Bacillus anthracis or in preparing products for screening for diseases caused by Bacillus anthracis.

[0074] According to another aspect of the present invention, a method for detecting or assisting in detecting Bacillus anthracis for non-therapeutic and non-diagnostic purposes is also provided, comprising performing recombinase isothermal amplification on a sample using the above-mentioned primer-probe combination, the above-mentioned reagent or the above-mentioned kit, and determining whether Bacillus anthracis is present in the sample based on a fluorescent signal.

[0075] Compared with the existing PCR technology, this method only takes 20 minutes, with a short detection time; and no expensive detection equipment is required. Compared with the RPA test strip method, this method does not need to open the cover after amplification, which minimizes the risk of aerosol contamination. This method provides strong technical support for the prevention and control of Bacillus anthracis and has a very broad application prospect.

[0076] The samples include environmental samples or animal tissues and / or organs used as food.

[0077] The system for isothermal amplification of the recombinase is 50 μL, and the reaction system is shown in Table 1.

[0078] Table 1 Recombinase isothermal amplification system

[0079] Reagents Volume (μL) RPA Lyophilized Enzyme 1 tube 10 μmol / L upstream primer 3.3 10μmol / L downstream primer 3.3 10μmol / L specific fluorescent probe 0.6 <![CDATA[0.5M of Mg 2+ > 1.4 RPA Reaction Buffer 29.5 Nucleic acid to be tested 1 <![CDATA[ddH2O]]> 10.9

[0080] In some specific embodiments, the reaction temperature of the recombinase isothermal amplification is 35-41°C, preferably 39°C;

[0081] In some specific embodiments, the reaction time of the recombinase isothermal amplification is 20 min.

[0082] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0083] In the following examples, the recombinase isothermal amplification (RPA amplification) was performed using the TwistAmp exo kit (manufacturer: TwistDx, model: TAEXO02KIT).

[0084] Example 1 Primary screening of RPA amplification primers

[0085] 1. Design of primary candidate primer sets

[0086] Using E4 sequence (nucleotide sequence shown in SEQ ID NO: 1) as template, a primer set with a fixed length of 32 bp was designed, and each primer in the set was separated by 10 bp. The upstream primer set was F1 to F7, and the downstream primer set was R1 to R8, as shown in Table 2. The following sequences were sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

[0087] Table 2 Primary candidate primer sets

[0088]

[0089]

[0090] 2. RPA Amplification

[0091] The primers in Table 2 were amplified by recombinase polymerase isothermal amplification using the TwistDx RPA kit. The total volume of the RPA reaction system was 50 μL, wherein 2.1 μL of the forward primer with a concentration of 10 μmol / L was added, 2.1 μL of the reverse labeled primer was added, 1 μL of the DNA template with a concentration of 2 pg / μL (a plasmid containing the E4 sequence) was added, 29.5 μL of the RPA reaction buffer, and 0.5 M MgCl2 were added. 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0092] First, fix and select F1, and pair it with R1-R7 primers for amplification. After comparing the amplification efficiency of these primer pairs, fix the best amplification primer among the R primers, pair it with F1-F7 primers for amplification, and compare the amplification effects.

[0093] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0094] Detection of RPA products: Use the PCR product recovery kit to recover the RPA amplification product, then use 2% agarose gel electrophoresis, and finally use EB staining and observe under ultraviolet light. The results are as follows Figure 1 As shown, A is the RPA amplification result after fixing the upstream primer F1 and then pairing F1 with the downstream primers in pairs; B is the RPA amplification result after fixing the downstream primer R2 and then pairing R2 with the upstream primers in pairs. Figure 1 From A in the figure, we can see that R2 pairing amplification efficiency is better. Figure 1 From B, we can see that F5 has better amplification efficiency.

[0095] Primary screening results: F5 and R2 are the primer pair with the highest amplification efficiency in this round.

[0096] Example 2 Screening of RPA probes

[0097] 1. Probe Design

[0098] The sequence between F5 and R2 was used as a template and the probe was designed according to the principle of RPA fluorescent probe design. The specific sequence is shown in Table 3, where F is the fluorescent group dT-Fam, H is the THF (tetrahydrofuran residue) site, Q is the quenching group dT-BHQ1, and B is the blocking group C3-spacer. The following sequence was sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

[0099] Table 3 Candidate probe sequences

[0100]

[0101]

[0102] 2. Probe Screening

[0103] The probes in Table 3 were amplified by recombinase polymerase isothermal amplification using the TwistDx RPA-nfo kit. The total volume of the RPA reaction system was 50 μL, wherein 2.1 μL of the forward primer F5 with a concentration of 10 μmol / L, 2.1 μL of the reverse labeled primer R2, 0.6 μL of the probe with a concentration of 10 μmol / L, 1 μL of the DNA template with a concentration of 2 pg / μL (a plasmid containing the E4 sequence), 29.5 μL of the RPA reaction buffer, and 0.5 M MgCl2 were added. 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0104] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0105] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 2 As shown in Figure 2, probe P3 has the highest detection efficiency.

[0106] Example 3 Secondary screening of RPA primers

[0107] 1. Probe Design

[0108] On the basis of the best primer pair F5 and R2 in Example 1, the primer lengths were kept unchanged and the primers were shifted by about 2 bp to obtain new upstream primer sets and downstream primer sets, and the sequences were sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. For specific sequences, refer to Table 4.

[0109] Table 4 Second level candidate primer sets

[0110]

[0111]

[0112] 2. RPA Amplification

[0113] The primers in Table 4 were amplified by recombinase polymerase isothermal amplification using the TwistDx RPA-nfo kit. The total volume of the RPA reaction system was 50 μL, wherein 2.1 μL of the forward primer at a concentration of 10 μmol / L, 2.1 μL of the reverse labeled primer, 0.6 μL of the probe P3 at a concentration of 10 μmol / L, 1 μL of the DNA template (plasmid containing the E4 sequence) at a concentration of 20 fg / μL, 29.5 μL of the RPA reaction buffer, and 0.5 M MgCl2 were added. 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0114] First, fix and select F5, and pair it with R21-R23, R2, and R24-R27 primers for amplification. After comparing the amplification efficiency of these primer pairs, fix the best amplification primer among the R primers, pair it with F51-F53, F5, and F54-F57 primers for amplification, and compare the amplification effects.

[0115] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0116] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 3 As shown, A is the RPA amplification result after fixing the upstream primer F5 and then pairing F5 with the downstream primers in pairs, 1 to 8 are R21 to R23, R2, and R24 to R27 respectively; B is the RPA amplification result after fixing the downstream primer R2 and then pairing R2 with the upstream primers in pairs, 1 to 8 are F51 to F53, F5, and F54 to F57 respectively. Figure 3 From A, we can see that the R2 reaction signal has the fastest starting time and the strongest signal, and R2 is the best downstream primer; Figure 3 From B, we can see that the F57 reaction signal has the fastest onset time and the strongest signal, and F57 is the best upstream primer.

[0117] Secondary screening results: F57 and R2 are the primer pair with the highest amplification efficiency in this round.

[0118] Example 4: Tertiary Screening of RPA Primers

[0119] 1. Probe Design

[0120] On the basis of the best primer pair F57 and R2 in Example 3, the 5' end position of the primer was kept unchanged, and the primer was shortened or extended by 1 bp, thus obtaining a new upstream primer set and downstream primer set, and the sequence was sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. For specific sequences, refer to Table 5.

[0121] Table 5 Third level candidate primer sets

[0122]

[0123]

[0124] 2. RPA Amplification

[0125] The primers in Table 5 were amplified by recombinase polymerase isothermal amplification using the TwistDx RPA-nfo kit. The total volume of the RPA reaction system was 50 μL, wherein 2.1 μL of the forward primer with a concentration of 10 μmol / L, 2.1 μL of the reverse labeled primer, 0.6 μL of the probe P3 with a concentration of 10 μmol / L, 1 μL of the DNA template with a concentration of 2 fg / μL (a plasmid containing the E4 sequence), 29.5 μL of the RPA reaction buffer, and 0.5 M MgCl2 were added. 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0126] First, fix and select F57, and pair it with R201, R202, R2, and R203-R207 primers for amplification. After comparing the amplification efficiency of these primer pairs, fix the best amplification primer among the R primers, pair it with F571, F572, F57, and F573-F577 primers for amplification, and compare the amplification effects.

[0127] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0128] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 4 As shown, A is the RPA amplification result after fixing the upstream primer F57 and then pairing F57 with the downstream primers in pairs, and 1 to 8 are R201, R202, R2 and R203-R207, respectively; B is the RPA amplification result after fixing the downstream primer R204 and then pairing R204 with the upstream primers in pairs, and 1 to 8 are F571, F572, F57 and F573-F577, respectively.

[0129] Results of the third-level screening: F571 and R204 are the primer pair with the highest amplification efficiency in this round.

[0130] Example 5 Screening of primer and probe concentrations

[0131] 1. Optimal Primer Concentration

[0132] The TwistDx RPA-nfo kit was used for recombinase polymerase isothermal amplification. The total volume of the RPA reaction system was 50 μL. Primers F571 and R204 at a concentration of 10 μmol / L were added to the system according to the following gradient volumes of 1.5 μL, 1.8 μL, 2.1 μL, 2.4 μL, 2.7 μL, 3 μL, 3.3 μL and 3.6 μL, respectively. 0.6 μL of probe P3 at a concentration of 10 μmol / L was added, 1 μL of DNA template at a concentration of 2 fg / μL (plasmid containing E4 sequence) was added, 29.5 μL of RPA reaction buffer, and 0.5 M Mg 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0133] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0134] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 5 As shown in A, it is the effect of different primer concentrations, 1 to 8 are 1.5μL, 1.8μL, 2.1μL, 2.4μL, 2.7μL, 3μL, 3.3μL and 3.6μL respectively. It can be seen that the starting time of primer 7 is the earliest, so the amplification efficiency is the highest when 3.3μL (i.e. 660nM) of primer is added.

[0135] 2. Optimal probe concentration

[0136] Recombinase polymerase isothermal amplification was performed using the TwistDx RPA-nfo kit. The total volume of the RPA reaction system was 50 μL. 3.3 μL of primers F571 and R204 at a concentration of 10 μmol / L were added, 0.6 μL, 0.8 μL, 1 μL and 1.2 μL of probe P3 at a concentration of 10 μmol / L were added, 1 μL of a DNA template (plasmid containing the E4 sequence) at a concentration of 2 fg / μL was added, 29.5 μL of RPA reaction buffer, and 0.5 M Mg 2+ Add 1.4 μL of ddH2O to make up to 50 μL.

[0137] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0138] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 5 As shown in B, the effect of different probe concentrations is shown, 1 is 0.6 μL, 2 is 0.8 μL, and there is no amplification when the addition amount is 1 μL and 1.2 μL. The amplification efficiency is the highest when the probe is added at 0.6 μL (i.e., 120 nM).

[0139] Example 6 Best Mg 2+ Screening of ion concentration

[0140] The recombinase polymerase isothermal amplification was performed using the TwistDx RPA-nfo kit. The total volume of the RPA reaction system was 50 μL. 3.3 μL of primers F571 and R204 at a concentration of 10 μmol / L were added, 0.6 μL of probe P3 at a concentration of 10 μmol / L, 1 μL of DNA template (plasmid containing E4 sequence) at a concentration of 2 fg / μL, 29.5 μL of RPA reaction buffer, and 0.5 M Mg 2+ Add 0.4μL, 0.6μL, 0.8μL, 1μL, 1.2μL, 1.4μL, 1.6μL and 1.8μL respectively and make up to 50μL with ddH2O.

[0141] The RPA amplification reaction procedure is: constant temperature reaction at 39°C and amplification for 20 minutes.

[0142] Detection of RPA products: Use a constant temperature fluorescence detector to record the changes in fluorescence signals in real time, and then compare the start time of each reaction signal and the strength of the signal. Figure 6 As shown, 1 to 8 were 0.4 μL, 0.6 μL, 0.8 μL, 1 μL, 1.2 μL, 1.4 μL, 1.6 μL and 1.8 μL, among which 1 to 4 were not amplified, and 0.5 M Mg 2+ The amplification efficiency is highest when 1.2 to 1.4 μL is added.

[0143] Example 7 Specificity detection of primers and probes

[0144] The best primers F571 and R204 and probe P3 selected in Example 4 were used to perform primer-probe specificity detection according to the best reaction conditions explored in Examples 5 and 6. The candidate bacteria include: Bacillus subtilis, Bacillus thuringiensis, Bacillus coagulans, Bacillus licheniformis, Bacillus cereus, Bacillus megaterium and Bacillus lentus. After the bacteria were cultured, the respective genomes were extracted using a bacterial genome extraction kit, and then each genome was used as a template for amplification. A plasmid containing the E4 sequence was used to simulate Bacillus anthracis.

[0145] like Figure 7 As shown, 5 is a plasmid containing the E4 sequence, and only the plasmid containing the E4 sequence shows a positive signal, while the other bacteria are negative, which indicates that the specificity of the primers and probes in the present invention is very good.

[0146] Example 8 Sensitivity Detection of Primers and Probes

[0147] The best primers F571 and R204 and probe P3 selected in Example 4 were used to perform sensitivity testing of the primers and probes according to the best reaction conditions explored in Examples 5 and 6. The templates tested were 5*10 0 copies, 5*10 1 copies, 5*10 2 copies, 5*10 3 copies, 5*10 4 copies, 5*10 5 copies and 5*10 6 The concentration of copies.

[0148] The results are as follows Figure 8 As shown, using the methods of Examples 4 to 6, the best detection is 5*10 0 copies of Bacillus anthracis.

Claims

1. A primer-probe composition for detecting Bacillus anthracis, characterized in that: comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 36, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 46, and a specific fluorescent probe; The structure of the specific fluorescent probe is SEQ ID NO: 20-tetrahydrofuran residue-SEQ ID NO: 21, and the 3' end of SEQ ID NO: 21 is blocked and modified.

2. The primer-probe combination according to claim 1, characterized in that The molar ratio of the upstream primer, the downstream primer and the specific fluorescent probe is 11:11:

2.

3. The primer-probe combination according to claim 1, characterized in that: The T base upstream of the tetrahydrofuran residue is labeled with a fluorescent reporter group, and the T base downstream of the tetrahydrofuran residue is labeled with a fluorescent quencher group, and the distance between the fluorescent reporter group and the fluorescent quencher group is 2 to 4 nucleotides; Preferably, the fluorescent reporter group is selected from at least one of Fam, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705; The fluorescence quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA; The 3' end of SEQ ID NO. 21 is connected to C3-spacer.

4. Use of the primer-probe combination according to any one of claims 1 to 3 in any of the following: A1. Application in the preparation of products for detecting or assisting in the detection of Bacillus anthracis; A2. Use in the preparation of products for detecting whether a sample contains Bacillus anthracis; A3. Use in the preparation of products for identifying and distinguishing Bacillus anthracis and other pathogens in samples; A4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis; A5. Application in the preparation of products for screening diseases caused by Bacillus anthracis.

5. A reagent for detecting Bacillus anthracis, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 3.

6. A kit for detecting Bacillus anthracis, characterized in that: The method comprises the primer-probe combination according to any one of claims 1 to 3 or the reagent according to claim 5.

7. The kit according to claim 6, characterized in that Other reagents required for isothermal amplification of the recombinase are also included.

8. Use of the reagent according to claim 5 or the kit according to claim 6 or 7 in any of the following: B1. Use in detecting or assisting in detecting Bacillus anthracis or in preparing products for detecting or assisting in detecting Bacillus anthracis; B2. Use in detecting whether a sample contains Bacillus anthracis or in preparing a product for detecting whether a sample contains Bacillus anthracis; B3. Use in identifying and distinguishing Bacillus anthracis from other pathogens in samples or in preparing products for identifying and distinguishing Bacillus anthracis from other pathogens in samples; B4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by Bacillus anthracis; B5. Application in screening for diseases caused by Bacillus anthracis or in preparing products for screening for diseases caused by Bacillus anthracis.

9. A method for detecting or assisting in detecting Bacillus anthracis for non-therapeutic and non-diagnostic purposes, characterized in that: The method comprises performing recombinase isothermal amplification on a sample using the primer-probe combination according to any one of claims 1 to 3, the reagent according to claim 5, or the kit according to claim 6 or 7, and determining whether Bacillus anthracis exists in the sample according to the fluorescent signal.

10. The method according to claim 9, characterized in that The reaction temperature of the recombinase isothermal amplification is 35-41°C, preferably 39°C; Preferably, the reaction time of the recombinase isothermal amplification is 20 min.

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