Double-stranded probe-assisted loop-mediated isothermal amplification system for detecting brucella

Abstract

The invention relates to the technical field of pathogen detection, relates to a POCT (point-of-care testing) rapid nucleic acid detection system, and in particular relates to a double-stranded probe-assisted loop-mediated isothermal amplification system for detecting brucella. According to the system, an amplification primer and a detection double-stranded probe are designed aiming at a Brucella specific gene bcsp31, and loop-mediated isothermal amplification (LAMP) and a double-stranded probe (TP) based on foot-mediated strand displacement are integrated to form a TP-LAMP unit of the scheme. The amplification result can be monitored in real time through a PCR luminoscope, and can also be visually identified through a nano biosensor (AuNPs-LFB). The method is rapid, sensitive, specific and easy to use, can solve the technical problems of non-ideal detection speed, specificity and sensitivity of a brucella POCT detection technology in the prior art, can be used as a valuable brucella rapid identification tool, and has ideal application and popularization prospects.

Description

Technical Field

[0001] The invention relates to the technical field of pathogen detection, and in particular to a double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella. Background Art

[0002] Brucellosis (abbreviated as brucellosis) is a chronic infectious disease caused by Brucella infection and is widely prevalent around the world. Its main clinical characteristics are chronic and frequent recurrences. Common symptoms include long-term fever (such as Malta fever, Mediterranean fever, Gibraltar fever, etc.), sweating, arthritis and hepatosplenomegaly. There are about 500,000 new cases of brucellosis worldwide each year. The main infected animals include livestock such as sheep, cattle and pigs, which often cause miscarriage and infertility in female animals, and orchitis and infertility in male animals. At present, Brucella can be divided into more than ten species according to its pathogenicity and host type, including Brucella melitensis, Brucella abortus and Brucella suis. Because the clinical symptoms of brucellosis are diverse and non-specific, it is easy to lead to misdiagnosis, thereby delaying the best treatment time. Therefore, early and accurate detection of Brucella is extremely critical for the prevention and control of brucellosis.

[0003] Isolation and cultivation of Brucella from samples of suspected brucellosis patients remains the gold standard for laboratory diagnosis and identification of Brucella infection. However, due to the slow growth rate of Brucella (usually 3 to 4 days), confirmation of results often requires more than two weeks, making it difficult to meet the needs of early diagnosis. Therefore, it is urgent to develop a method for rapid detection of Brucella to achieve the purpose of early diagnosis. Conventional serological detection methods, such as SAT, RBT, CFT and ELISA, although they have the advantages of rapid detection, have low sensitivity, high false positive rate, and complex operation procedures. They are usually not used alone to diagnose Brucella infection (need to be used in combination with other methods), which further increases the complexity of detection. Therefore, in order to meet the requirements of early diagnosis, it is urgent to develop a simplified, sensitive, specific and easy-to-use new Brucella detection method.

[0004] Currently, nucleic acid-based molecular detection methods have greatly improved the sensitivity and specificity of pathogen detection, such as polymerase chain reaction (PCR) and real-time fluorescence PCR, which have played an important role in the diagnosis of infectious diseases such as brucellosis and tuberculosis. However, PCR technology has certain requirements for reaction monitoring instruments, such as thermal cyclers, which hinders the application of this technology in resource-poor areas. In recent years, researchers have developed a variety of nucleic acid constant temperature amplification technologies, such as loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), rolling circle amplification (RCA) technology, etc. Among them, LAMP is a rapid, sensitive, and simple isothermal amplification method that does not require temperature cycling and can amplify 10 DNA fragments within 10 minutes through DNA displacement polymerase. 9 The LAMP assay is a promising and attractive alternative to PCR for the detection of multiple copies of target nucleic acid. It can be used for POC detection of pathogens including Brucella. However, since LAMP assays rely on nonspecific signal transduction methods such as fluorescent dye insertion, pH-responsive dye changes, or colorimetric dyes, they cannot distinguish between LAMP amplicons and byproducts, and their sensitivity and specificity are challenged. Therefore, a simple and sequence-specific detection method is urgently needed to address the above limitations.

[0005] Based on the problems existing in the existing technology, there is an urgent need to develop a new Brucella-specific POCT detection technology to achieve rapid, specific and ultra-sensitive detection of Brucella. Summary of the invention

[0006] The object of the present invention is to provide a double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella, so as to solve the technical problems of unsatisfactory detection speed, specificity and sensitivity of Brucella POCT detection technology in the prior art. This technical scheme designs a new type of Brucella detection technology, called double-stranded probe-assisted loop-mediated isothermal amplification technology (TP-LAMP) mediated by foot-mediated strand displacement reaction. Due to its high programmability and sequence specificity, foot-mediated DNA strand displacement reaction (TMSD) can be used as a condition for specific detection of LAMP amplification products. The TP-LAMP method combines isothermal amplification, DNA strand displacement reaction with real-time fluorescence analysis and nanobiosensor (AuNPs-LFB) to solve the problems existing in the current LAMP technology in Brucella detection, that is, the present invention provides a method for detecting Brucella based on a double-stranded probe-assisted loop-mediated isothermal amplification system mediated by DNA strand displacement reaction.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella, comprising a TP-LAMP unit; the TP-LAMP unit comprises an amplification primer for amplifying a bcsp31 gene, and a detection probe for detecting an amplification product of the bcsp31 gene;

[0009] The amplification primers are composed of F3 with a nucleotide sequence as shown in SEQ ID NO.1, B3 with a nucleotide sequence as shown in SEQ ID NO.2, BIP with a nucleotide sequence as shown in SEQ ID NO.3, and FIP* with a nucleotide sequence as shown in SEQ ID NO.4; a tag protein is modified at the 5′ end of FIP*;

[0010] The detection probe consists of TP-A with a nucleotide sequence as shown in SEQ ID NO.5 and TP-B with a nucleotide sequence as shown in SEQ ID NO.6; the 3' end of TP-A is modified with a fluorescent quenching group, and the 5' end of TP-B is modified with a fluorescent group.

[0011] Furthermore, the label protein is biotin; the fluorescence quenching group is a black hole quenching group; and the fluorescent group is carboxyfluorescein.

[0012] Furthermore, a double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella also includes a nanosensor for detecting the amplification product of the bcsp31 gene.

[0013] Furthermore, the nanosensor comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorbent pad; a test line and a quality control line are arranged on the nitrocellulose membrane.

[0014] Furthermore, the conjugate pad is coated with streptavidin coupled with gold nanoparticles; the test line is fixed with anti-fluorescein isothiocyanate antibody; and the quality control line is fixed with bovine serum albumin coupled with biotin.

[0015] Furthermore, the TP-LAMP unit also includes Bst DNA polymerase, reaction buffer, and dNTP; the reaction buffer contains betaine, MgSO 4 , KCl, (NH 4 ) 2 SO4, Tris-HCl, Tween-20.

[0016] Further, the working concentrations of FIP* and BIP were 1.6 μM, the working concentrations of F3 and B3 were 0.4 μM, and the working concentrations of TP-A and TP-B were 0.2 μM.

[0017] Furthermore, the operating temperature of the TP-LAMP unit is 60° C. and the operating time is 60 minutes.

[0018] Furthermore, the detection limit of the TP-LAMP unit in real-time fluorescence assay is 5 fM.

[0019] Furthermore, the detection limit of the nanosensor is 5 fM.

[0020] The technical principle and beneficial effects of this technical solution are:

[0021] The present invention designs a novel LAMP amplification detection system and a nano-biosensor (AuNPs-LFB) based on the principle of foot-mediated DNA strand displacement reaction and loop-mediated isothermal amplification (Loop-MediatedIsothermalAmplification, LAMP) amplification reaction. The technical solution uses the sequence specificity of the DNA strand displacement reaction to design a detection probe, which can distinguish single-base mismatches at the reaction site. The system uses the primer combination of LAMP to amplify the bcsp31 gene, and detects the obtained gene amplification product. The detection of the amplification product includes not only a double-linked probe for identifying the amplification product, but also a nano-sensor. In the presence of amplification primers and double-stranded probes and the action of DNA polymerase, the genome to be tested isothermally amplified, and if the target gene is present, a double-stranded amplification product is formed. During the amplification process, the double-stranded probe can perform specific fluorescence detection on the product (which can be monitored in real time by a conventional PCR fluorescence instrument in the prior art). In addition, the detection of the amplification product can also be achieved by using a nano-biosensor detection.

[0022] In summary, the present invention has developed a Brucella detection system based on a toehold-mediated probe (TP)-assisted LAMP amplification (TP-LAMP) and a nanobiosensor (AuNPs-LFB) and applied it to the specific detection of Brucella in isolated bacteria and whole blood samples. This technical solution solves the technical problems of the unsatisfactory detection speed, specificity and sensitivity of Brucella POCT detection technology in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The TP-LAMP amplification and detection principle of the present invention (LAMP amplification and double-stranded DNA probe strand displacement-mediated specific detection; A: schematic diagram of detection concept; B: amplification and detection flow chart).

[0024] Figure 2 This is the LAMP amplification principle of the present invention.

[0025] Figure 3The TP-LAMP confirmation test results of Example 2 of the present invention (A: TP-LAMP real-time turbidity monitoring confirmation test; B: gel electrophoresis confirmation test, M is DL1000 DNA Marker, the positive reaction template is plasmid, and the blank control uses enzyme-free water instead of template; C: amplification effect of different template concentrations).

[0026] Figure 4 The TP-LMAP confirmation test results of Example 2 of the present invention (A: TP-LAMP fluorescence experiment confirmation test; B: gel electrophoresis confirmation test, M is DL1000 DNA Marker, the positive reaction template is plasmid, and the blank control uses enzyme-free water instead of template; C: AuNP-LFB confirmation experiment).

[0027] Figure 5 The results of the TP-LAMP optimal temperature optimization test of Example 3 of the present invention (AC: amplification temperatures are 60° C., 63° C., and 65° C.; D: fluorescence signal-to-background ratio corresponding to different temperatures).

[0028] Figure 6 The results of the TP-LAMP specific probe screening test of Example 4 of the present invention (A: TP-LAMP double-stranded probe detection principle diagram; BC: real-time fluorescence detection results of four groups of probes; D: Ct values ​​of four groups of probes).

[0029] Figure 7 The results of the TP-LAMP probe sequence specificity test of Example 5 of the present invention (A: schematic diagram of probe mutation; B: mutation probe detection result).

[0030] Figure 8 The TP-LAMP sensitivity test results of Example 6 of the present invention (the plasmid concentrations are 5fM, 10fM, 100fM, 1pM, 10pM, and 100pM, respectively; and the blank control is enzyme-free water).

[0031] Fig. 9 This is the TP-LAMP detection specificity test result of Example 7 of the present invention (sheep and cattle species are representative Brucella isolates as positive individual photos, distilled water and non-Brucella strains including Staphylococcus aureus, Escherichia coli, Mycobacterium leprae, Shigella, Klebsiella pneumoniae, Listeria monocytogenes, and Pseudomonas aeruginosa are used as control groups to verify the specificity of the method, and enzyme-free water is used as a blank control).

[0032] Fig.10 This is the TP-LAMP isolated strain application test of Example 8 of the present invention (AB: 19 Brucella isolated strains).

[0033] Fig.11This is a whole blood sample application test of TP-LAMP according to Example 9 of the present invention (AC: 18 whole blood samples were evaluated and tested). DETAILED DESCRIPTION

[0034] The following is further described in detail through specific implementation methods:

[0035] Embodiment 1:

[0036] (1) Basic principles of TP-LAMP detection

[0037] This technology has developed a Brucella detection system that combines double-stranded probe-assisted (based on toehold-mediated probe, TP) loop-mediated isothermal amplification (TP-LAMP) with nanobiosensor (AuNPs-LFB). The technical principle of TP-LAMP is as follows Figure 1 The main detection includes LAMP-mediated nucleic acid amplification reaction, double-stranded probe-mediated strand displacement detection reaction and nanobiosensor (AuNPs-LFB)-mediated amplification reaction detection.

[0038] TP-LAMP detection mainly includes BstDNA polymerase-mediated amplification reaction, double-stranded probe-assisted amplification product detection reaction, and the final POCT detection (Point-of-Care Testing) mediated by a nanobiosensor (AuNPs-LFB). First, a set of four primers, including two outer primers (F3 and B3) and two inner primers (FIP and BIP), were designed based on the Brucella-specific gene Bscp31 (Genbank accession number M20404), which encodes a 31kDa surface protein in all Brucella species and biological species. Biotin was labeled at the 5′ end of the FIP primer, and the new primer was named FIP* for POCT determination. The technical reaction system adopts a single-pot one-step reaction (25μL). The system contains two reaction units, amplification and detection. A large number of amplicons are generated when the amplification reaction is started. The probe and the amplicon undergo a strand displacement reaction at the same time, releasing a fluorescent signal to achieve real-time detection. After the reaction is completed, take a drop and add it to the sensor strip to achieve portable POC detection.

[0039] (2) Reagents, structures and instruments involved in the embodiments of the present invention:

[0040] Bst DNA polymerase (2.0) and reaction buffer were purchased from Bio-Tech Co., Ltd. The bacterial genomic DNA extraction kit was purchased from Xi'an Tianlong Technology Co., Ltd. dATP (100 mM), dTTP (100 mM), dCTP (100 mM) and dGTP (100 mM) were purchased from Bio-Tech Co., Ltd. The whole blood DNA extraction kit was purchased from QIAGEN, Germany.

[0041] PCR thermal cycler was purchased from Hangzhou Biori Technology Co., Ltd. High-speed refrigerated centrifuge (3-18K) was purchased from Sigma, Germany Electrophoresis equipment and gel imaging system were purchased from Bio-Rad, USA Oscillating constant temperature metal bath was purchased from Beijing Jinhao Pharmaceutical Co., Ltd. Real-time turbidity meter (LA-500) was purchased from Eiken, Japan.

[0042] (3) The method involved in the embodiment of the present invention:

[0043] Genomic extraction: A total of 28 pathogens were used to evaluate the performance of the TP-LAMP+AuNPs-LFB method, including 21 Brucella and 7 non-Brucella pathogens. The genomic nucleic acids of the above pathogens were extracted using a bacterial genomic DNA extraction kit or a viral qEx-DNA / RNA extraction kit. Nineteen Brucella isolates and 18 whole blood samples were used to evaluate the applicability of TP-LAMP and the combined AuNPs-LFB method in the detection of clinical samples. The genomic DNA of the isolated strains was extracted using the rapid extraction method: 200 μL of culture (picking colonies and adding TE buffer) was added to an enzyme-free EP tube, and 100 μL of DNA extraction solution was added. Subsequently, it was heated and boiled for 8 minutes, centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken as an amplification template (stored at -20°C). The genomic DNA extraction of whole blood samples was carried out according to the instructions of the kit. 25 μL reaction system of TP-LAMP: The system used a one-pot reaction, which contained 12.5 μL 2× reaction buffer (containing 2M betaine, 16mM MgSO 4 , 40 mM KCl, 20 mM (NH 4 ) 2SO4, 40mM Tris-HCl (pH 8.8), 0.2% Tween-20), 3μL dNTP mixture (10mM), 1μL 2.0Bst DNA polymerase, 1.6μM each of FIP* and BIP, 0.4μM each of F3 and B3, 0.2μM each of TP-A and TP-B, 1μL DNA plasmid or extracted nucleic acid sample, add double distilled water (DW) to 25μL. Incubate the reaction tube at 60℃ for 60 minutes and then terminate at 80℃ for 5 minutes. During the amplification process, it can be monitored by a real-time turbidity meter, and the TP-LAMP amplification reaction can be verified by gel electrophoresis and nanobiosensor (AuNPs-LFB).

[0044] (4) Primer design

[0045] According to the principle of LAMP amplification, LAMP primers were designed for the Bcsp31 gene (Table 1). According to the design principles of nanobiosensors (AuNPs-LFB) and fluorescence detection, Biotin (biotin) was modified at the 5′ end of FIP*, FAM (carboxyfluorescein) was modified at the 5′ end of the TP-B chain (detection primer, i.e., probe), and BHQ1 (Black Hole Quencher 1, fluorescence quencher, black hole quenching group) was modified at the 3′ end of the TP-A chain (detection primer, i.e., probe). The HPLC purification grade primers used in the present invention were synthesized and modified by Shanghai Shenggong Biotechnology Co., Ltd. In addition, a partial target fragment of the Bcsp31 gene was synthesized and cloned into a pUC57 vector to prepare a plasmid template. The preparation, extraction, and quantification processes were all completed by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The synthesized plasmid was diluted continuously to prepare a multiple dilution gradient (5fM, 10fM, 100fM, 1pM, 10pM, 100pM).

[0046] Table 1: TP-LAMP amplification primers and detection primers used in the present invention (mer: monomeric unit)

[0047]

[0048]

[0049] (5) Preparation of Nanobiosensor (AuNPs-LFB)

[0050] The preparation of the nanobiosensor (AuNPs-LFB) of this technology adopts the method in the prior art and is commissioned to Tianjin Huidexin Technology Development Co., Ltd. The AuNPs-LFB biosensor (size 64mm×4mm) consists of four parts, namely, the sample pad, conjugate pad, nitrocellulose membrane (NC membrane) and absorbent pad are fixed on the plastic back plate in sequence using plastic adhesive. Streptavidin coupled with gold nanoparticles is coated on the conjugate pad. The detection line and control line are set on the nitrocellulose membrane in sequence. Anti-fluorescein isothiocyanate antibody (concentration of 0.15mg / ml) is fixed on the detection line (TL), and biotin-coupled bovine serum albumin (biotin-BSA, concentration of 2.5mg / ml) is fixed on the quality control line (CL). TL and CL are separated by a gap of 5mm.

[0051] Example 2: TP-LAMP Confirmation Test

[0052] The feasibility of the TP-LAMP method was verified by a confirmation experiment. To evaluate the feasibility of the standard LAMP primers, the amplification process was first monitored by real-time turbidity. The turbidity results showed that the designed primers were effective. Gel electrophoresis analysis also showed the feasibility of the primers ( Figure 3 AB). To further evaluate the sensitivity of the turbidity analysis, the lowest concentration that could be detected was determined to be 10 pM ( Figure 3 C). Figure 3 The TP-LAMP system and reaction conditions used in the experimental study are shown in Example 1, and the concentration of the DNA template is controlled, wherein: Figure 3 The concentration of the DNA template used in the Target groups A and B was specifically 1 nM, while no DNA template was added to the Control group.

[0053] The products of TP-LAMP were analyzed by real-time fluorescence and agarose gel electrophoresis, and the results confirmed the feasibility of TP-LAMP ( Figure 4 AB). Figure 4 The TP-LAMP system and reaction conditions used in the experimental study are shown in Example 1, wherein: Figure 4 The concentration of DNA template used in the Target groups A, B and C was specifically 1 nM, while no DNA template was added to the Control group. The product was further verified using the nanobiosensor strip AuNPs-LFB, and the results showed that AuNPs-LFB can be used for TP-LAMP reaction amplification detection. The results showed that primers targeting the Bscp31 gene were feasible for TP-LAMP determination of Brucella.

[0054] Example 3: TP-LAMP amplification temperature optimization test

[0055] In order to enhance the compatibility between LAMP and TP probes, the combined reaction was performed at different temperatures (60°C, 63°C, and 65°C) to obtain the best detection results. The results showed that the strongest signal intensity was obtained when the reaction temperature was 60°C. Therefore, all subsequent experiments were performed at 60°C ( Figure 5 ). More specifically, Figure 5 The TP-LAMP system and reaction conditions used in the experimental study are shown in Example 1. The concentration of the DNA template is 1 nM, and different reactions are controlled at 60° C., 63° C., and 65° C. to optimize the amplification temperature.

[0056] Example 4: TP-LAMP probe screening.

[0057] The high sequence specificity of the strand displacement reaction ensures that TP-LAMP should also have high sequence specificity. To demonstrate this design, the foot-mediated reaction mechanism ( Figure 6 A), four different groups of probes were carefully designed according to the different sites of the ring product (Table 2). The best probe was selected by real-time fluorescence analysis. The experimental method can be found in Example 2. In the presence of the target nucleic acid, the best was determined by detecting the recovery value of the FAM fluorescence signal intensity labeled on the detection chain B (TP-B4). The experimental results showed that probes TP-1 / 2 / 3 produced fluorescence signals at the beginning of the reaction, probably because the three groups of probes were thermodynamically unstable at the reaction temperature and could not maintain a stable double-stranded structure, while the fourth probe (TP-4) showed a stable double-stranded structure before the amplification reaction, and then produced a strong fluorescence signal after 30 minutes of reaction ( Figure 6 BC). In the absence of target input, no increased fluorescence leakage was observed, and the Ct values ​​of different probes also showed that the fourth group of probes TP-4 was the best choice ( Figure 6 D). Therefore, the 4th probe (TP-4) is selected as the final probe of Brucella TP-LAMP detection. Although the combination of the above-mentioned probes is all designed according to the reaction mechanism, in the process of actual testing, it is found that only TP-4 can form stable double-stranded chains, ensures the accuracy of detection, and obtains unexpected technical effect.

[0058] Table 2: Candidate primers for TP-LAMP probe screening used in the present invention (mer: monomeric unit)

[0059]

[0060] Example 5: TP-LAMP sequence specificity assay

[0061] To demonstrate the sequence specificity of this design, mutations were introduced into the probe recognition site (Table 3, only point mutations, others the same as the original TP-B4 probe), and two mutant probes were constructed to perform the same experiment ( Figure 7 ), the experimental method can be found in Example 1. The results showed that the mutation probe did not generate a fluorescent signal, indicating that the TP-LAMP of the present technology has a strong sequence specificity.

[0062] Table 3: Probe mutation primers used in the present invention (mer: monomeric unit; double underlines indicate mutation sites)

[0063]

[0064] Example 6: TP-LAMP sensitivity test

[0065] In order to evaluate the detection sensitivity of the TP-LAMP method, the plasmid containing the Bscp31 gene was diluted to test the sensitivity of the TP-LAMP assay. Referring to the technical parameter settings of Example 1, after repeated tests, Figure 8 As shown, real-time fluorescence assay can detect plasmid concentrations up to 5 fM ( Figure 8 A), the detection limit is lower than the result of the above turbidity test, and the detection sensitivity is better. If the reaction time is sufficient, the detection concentration may be lower. Similarly, gel electrophoresis can also detect up to 5fM ( Figure 8 B). The presence of TL and CL on LFB indicated a positive TP-LAMP result for the Bscp31 gene. The sensitivity of the TP-LAMP assay using LFB was consistent with the results of gel electrophoresis detection and real-time fluorescence experiments, confirming that the TP-LAMP assay can be used for high-sensitivity detection of the Brucella-specific gene Bscp31.

[0066] Example 7: TP-LAMP detection specificity test

[0067] In the present invention, take sheep and cattle as representative Brucella isolated strains as positive independent photo, take distilled water and non-brucella strains as control group verify the specificity (table 4) of the method including Staphylococcus aureus, Escherichia coli, Mycobacterium leprae, Shigella, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa.Shown in the result, only Brucella produces fluorescent signal, and control group does not have signal ( Fig. 9 A). In addition, Brucella strains produced two red lines in the detection area of ​​LFB, while for non-Brucella strains and DW, only CL appeared in the detection area of ​​LFB, indicating that only Brucella spp. was amplified ( Fig. 9B) These results confirmed that TP-LAMP not only accurately detected all Brucella strains, but also had no cross-reaction with other non-Brucella strains, proving that its specificity was 100%.

[0068] Table 4: Pathogens used in the present invention (GZCDC: Guizhou Provincial Center for Disease Control and Prevention)

[0069]

[0070]

[0071] Example 8: TP-LAMP isolated strain application test

[0072] In order to verify the feasibility of TP-LAMP for detecting isolates, 19 Brucella isolates were used for evaluation. The results showed that except for the distilled water control group, the 19 Brucella isolates showed fluorescence signals ( Fig.10 A), and all genomic DNA of Brucella strains generated CL and TL lines ( Fig.10 B) The results of LFB sensor are consistent with the fluorescence results, indicating that TP-LAMP is highly practical in Brucella detection.

[0073] Example 9: TP-LAMP whole blood sample application test

[0074] In order to verify the practicality of TP-LAMP for whole blood samples, 18 whole blood samples were evaluated ( Fig.11 A). Of the 18 whole blood samples tested by TP-LAMP, 1 (5.56%) was positive for Brucella and 17 (94.44%) were negative for Brucella. The results of real-time fluorescence analysis and LFB test were consistent ( Fig.11 BC). These data are consistent with the traditional culture biotechnology method, indicating that the diagnostic accuracy of the TP-LAMP assay is 100% compared with the culture biotechnology method. These results indicate that TP-LAMP is a simpler, faster, and more applicable assay than the culture biotechnology method.

[0075] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella, characterized in that: The invention comprises a TP-LAMP unit; the TP-LAMP unit comprises an amplification primer for amplifying the bcsp31 gene, and a detection probe for detecting the amplification product of the bcsp31 gene; The amplification primers are composed of F3 with a nucleotide sequence as shown in SEQ ID NO.1, B3 with a nucleotide sequence as shown in SEQ ID NO.2, BIP with a nucleotide sequence as shown in SEQ ID NO.3, and FIP* with a nucleotide sequence as shown in SEQ ID NO.4; a tag protein is modified at the 5′ end of FIP*; The detection probe consists of TP-A with a nucleotide sequence as shown in SEQ ID NO.5 and TP-B with a nucleotide sequence as shown in SEQ ID NO.6; a fluorescent quenching group is modified at the 3' end of TP-A, and a fluorescent group is modified at the 5' end of TP-B.

2. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 1, characterized in that: The label protein is biotin; the fluorescence quenching group is a black hole quenching group; and the fluorescent group is carboxyfluorescein.

3. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 2, characterized in that: Also included is a nanosensor for detecting the amplified product of the bcsp31 gene.

4. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 3, characterized in that: The nanosensor comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorbent pad; a test line and a quality control line are arranged on the nitrocellulose membrane.

5. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 4, characterized in that: The conjugate pad is coated with streptavidin coupled with gold nanoparticles; the test line is fixed with anti-fluorescein isothiocyanate antibody; and the quality control line is fixed with bovine serum albumin coupled with biotin.

6. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 1, characterized in that: The TP-LAMP unit also includes Bst DNA polymerase, reaction buffer, and dNTP; the reaction buffer contains betaine, MgSO4, KCl, (NH4)2SO4, Tris-HCl, and Tween-20.

7. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 6, characterized in that: The working concentrations of FIP* and BIP were 1.6 μM, the working concentrations of F3 and B3 were 0.4 μM, and the working concentrations of TP-A and TP-B were 0.2 μM.

8. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 7, characterized in that: The operating temperature of the TP-LAMP unit is 60° C. and the operating time is 60 minutes.

9. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 8, characterized in that: The lower limit of detection of the TP-LAMP unit in real-time fluorescence assay was 5 fM.

10. A double-stranded probe-assisted loop-mediated isothermal amplification system for detecting Brucella according to claim 3, characterized in that: The lower limit of detection of the nanosensor is 5 fM.

Images