Uracil-DNA glycosylase mediated multiple multi-cross substitution amplification system for identifying brucella ovisTechnical Field
The invention relates to the technical field of biological medicine detection, relates to a POCT (point of care testing) rapid nucleic acid detection system, and in particular relates to a uracil-DNA glycosylase mediated multiple multi-cross displacement amplification system for identifying Brucella melitensis.
Background
Brucellosis (simply called brucellosis) is a chronic infectious disease that is caused by brucellosis (Brucella) infection and is widely prevalent in people and animals around the world. The main clinical characteristics of the disease are chronic and recurrent attacks, which are manifested by long-term fever (such as maltese fever, mediterranean fever, direct brothers fever, etc.), sweating, arthritis, hepatosplenomegaly, etc. About 50 thousands of new cases of spread disease annually are worldwide. Animal infection mainly involves livestock such as sheep, cattle and pigs, and often results in abortion and infertility, orchitis and sterility in females. Currently, brucella is classified into 10 or more organism species such as brucella ovis (b.melitensis), brucella bovis (b.abortus), brucella suis (b.suis) and the like according to pathogenicity of brucella and preferred host. Of these, b.melitensis isolated in maltese in 1887 was the pathogen in most cases of brucellosis and was also the most pathogenic species in brucella. In addition, due to the characteristics of various and non-specific clinical symptoms of the cloth disease, misdiagnosis is easily caused and the optimal treatment time is delayed. Thus, early, precise identification of b.melitensis from Brucella (Brucella spp.) is particularly important for preventing and controlling brucellosis.
Isolation and culture of brucella from a sample suspected of being a patient remains one of the standard methods for laboratory diagnosis and identification of brucella infection. However, since brucella grows slowly (3-4 days), it takes more than two weeks to confirm the results, making it difficult to meet the early diagnosis needs. Based on this, there is a need to develop a method for rapidly detecting brucella to achieve the purpose of early diagnosis. Conventional serological tests include SAT, RBT, CFT, ELISA and the like. Although serological tests are characterized by rapid speed, they have low sensitivity, high false positive rate and complex workflow and none of them can be used alone to diagnose brucella infection (usually in combination), which increases the complexity of the detection procedure. Therefore, to meet the need for early diagnosis, there is an urgent need to develop a novel method for detecting and identifying brucella that is simplified, sensitive, specific and easy to use.
Currently, nucleic acid amplification assays (NAATs), such as the Polymerase Chain Reaction (PCR) and its derivative techniques, improve the sensitivity and specificity of pathogen detection. Especially, the research and development of the classical real-time fluorescent quantitative PCR (real-time PCR) technology greatly improves the efficiency of nucleic acid detection and plays a vital role in diagnosing infectious diseases such as bubbly disease, leptospirosis, tuberculosis and the like. However, detection methods based on PCR technology are limited by reliance on PCR or fluorescent quantitative PCR thermocyclers, which prevents the popularization and application of such technologies in laboratories in areas of scarce resources.
As described above, in order to overcome these shortcomings, researchers have developed techniques based on isothermal NAATs in recent years, including Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), multiple cross-substitution amplification (MCDA), and the like. MCDA, as a novel NAATs method, has a higher sensitivity (about 10-fold) than LAMP and the amplification process is simpler in dependence on the reaction enzyme than RPA method (only Bst DNA polymerase is required). At this stage, MCDA-based NAAT technology has been used for various pathogen detection (e.g., bacterial, viral, and other explosive infection events). However, as with most thermostated NAATs techniques, traditional product validation methods can only recognize single targets, such as visualization dyes (hydroxy naphthol blue and SYBR Green I), real-time turbidity, and agarose gel electrophoresis. Such single-target detection not only reduces NAATs detection efficiency but also increases the complexity of the detection process (multiple reactions are required for multi-target detection). At the same time, these verification methods have difficulty in accurately distinguishing between specific and non-specific amplifications. Therefore, the novel triple nano biosensor (AuNPs-LFA) can be designed to effectively overcome the defect of single-target detection. However, MCDA in combination with AuNPs-LFA biosensors requires open lid detection, which increases the risk of aerosol contamination.
Based on the problems existing in the prior art, there is a need to develop a novel method and system for detecting brucella and brucella ovis species which can aim at a plurality of target genes and simultaneously overcome the risk of aerosol pollutants.
Disclosure of Invention
The invention aims to provide a uracil-DNA glycosylase mediated multiple multi-cross replacement amplification system for identifying Brucella melitensis, which is used for solving the technical problem that the existing detection method is difficult to perform isothermal nucleic acid amplification test on multiple targets of Brucella melitensis and Brucella melitensis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A uracil-DNA glycosylase mediated multiple cross-over displacement amplification system for the identification of brucella ovis, comprising a gene amplification unit for simultaneous amplification of a bcsp31 gene and a BMEII0466 gene, and a detection unit for detection of a gene amplification product obtained from the gene amplification unit; the gene amplification unit comprises an MCDA-based primer combination for amplifying the bcsp31 gene and an MCDA-based primer combination for amplifying the BMEII0466 gene.
By adopting the technical scheme, the technical principle is as follows:
The gene amplification unit based on the MCDA principle firstly amplifies the bcsp31 gene and the BMEII0466 gene in the sample, enlarges the copy number of the target fragment, then uses the detection unit to detect the amplified product, and judges whether Brucella melitensis (B.melitensis) or Brucella sp.) is contained in the sample by detecting whether the bcsp31 gene and the BMEII0466 gene exist. Because of the various and nonspecific clinical symptoms of the cloth disease, misdiagnosis is easily caused and the optimal treatment time is delayed. The technical scheme can realize early and accurate identification of the B.melitensis from Brucella (Brucella spp.), and effectively prevent and control the Brucella.
Further, the detection unit is a triple nano biosensor; the triple nano biosensor comprises a sample pad, a bonding pad, a nitrocellulose membrane and a water absorption pad which are sequentially fixed on a back plate; and a detection line 1, a detection line2 and a quality control line are sequentially arranged on the nitrocellulose membrane. The biosensor can realize the visualization of the detection result, can intuitively observe the experimental result, and does not need an expensive PCR product detection instrument.
Further, the binding pad is coated with gold nanoparticle coupled streptavidin; the detection line 1 is fixed with an anti-fluorescein isothiocyanate antibody, the detection line 2 is fixed with an anti-digoxin antibody, and the quality control line is fixed with biotin-coupled bovine serum albumin. The gold nanoparticle coupled streptavidin can be combined with biotin in an amplification product, so that the visual marking of a target substance is realized; the detection line can capture amplification products containing carboxyfluorescein or amplification products containing digoxin; the quality control line captures the streptomycin avidin which is not coupled with the nano particle combined with the target substance.
Further, the MCDA-based primer combination for amplifying bcsp31 gene includes:
bcsp-F1：GCGAAGTGACGATGATGA；
bcsp-F2：CATAGGAAGCTTGAGGCC；
bcsp-CP1*：FAM-AGGCGGCGAATGGCATAGTCATTCTCAAGATGACCTAGCA；
bcsp-CP2：GCTTGTCGGCAAGCGATTGTATCAACCACCGCATTCCAT；
bcsp-C1：AGGCGGCGAATGGCATAGTC；
bcsp-C2：GCTTGTCGGCAAGCGATTGTA；
bcsp-D1*：Biotin-GGGTTTCCTGGATGTGAAGA；
bcsp-D2：CTTTGGGAAAATCCAGAA；
bcsp-R1：AACGCAGCCCAAAACCCTGC；
bcsp-R2：TCGTTTCAGTCGGCTCTGGC；
wherein: FAM is 5 'carboxyfluorescein labeled and Biotin is 5' Biotin labeled.
Further, the MCDA-based primer combinations for amplifying BMEII0466 gene include:
BMEII-F1：TGAAAGAAGCGGCGAAAT；
BMEII-F2：AGCGGCAGCATTATCCGG；
BMEII-CP1*：Dig-TCATTGAAACTGCCGATGCGATGTATCAGCTTGCCGCCGATC；
BMEII-CP2：AGCTGGTATCAGCTTGCCGCACCTGTGGCAAAAAGCACG；
BMEII-C1：TCATTGAAACTGCCGATGCGAT；
BMEII-C2：AGCTGGTATCAGCTTGCCGC；
BMEII-D1*：Biotin-ATTGCGCAGGCGCAAAGCC；
BMEII-D2：AGATCAGGGCAATGCAAGC；
BMEII-R1：GGCCATGCCGAGGCCCTT；
BMEII-R2：CGCAATCTGGAAAAGGCCA；
Wherein: dig is 5 'digoxin label and Biotin is 5' Biotin label.
Further, the gene amplification unit further comprises dATP, dCTP, dGTP, dUTP. The scheme replaces dTTP in dNTP mixture used by mMCUDA reaction system with dUTP to ensure that mMCUDA amplified products all carry uracil bases which can be recognized and hydrolyzed by UDG enzyme, thereby achieving the purpose of preventing and eliminating potential pollutants.
Further, the gene amplification unit further comprises a UDG enzyme. The mMCUDA reaction system introduces UDG enzyme which can recognize and hydrolyze uracil base in pollutant, thus realizing prevention and elimination of possible aerosol pollution.
Further, the gene amplification unit further comprises Bst DNA polymerase, bst reaction buffer, and MgSO4.
Further, the working temperature of the gene amplification unit was 65℃and the time was 60 minutes.
Further, the detection limit was 7.5 fg/reaction.
In conclusion, the invention designs a novel amplification system and a triple AuNPs-LFA biosensor according to the principle of MCDA amplification. Based on the antibody capturing principle of AuNPs-LFA biosensor, two sets of MCDA primers targeting bcsp31 and BMEII0466 gene conservation regions are designed, and are modified by carboxyfluorescein (FAM), digoxin (Dig) and Biotin (Biotin) respectively. The invention develops a multi-target nucleic acid amplification system (called mMCUDA) integrating UDG enzyme-mediated multiplex mMCDA amplification and AuNPs-LFA biosensor, and applies the multi-target nucleic acid amplification system to accurately identify B.melitensis from Brucella. In addition, deoxyuridine triphosphate (dUTP) -based thermosensitive uracil-DNA glycosylase (UDG) -mediated amplification of MCDA is effective in eliminating certain amounts of aerosol contaminants.
Drawings
FIG. 1 shows the mMCUDA amplification principle (cleavage reaction and mMCDA amplification; A: brucella specific gene MCUDA amplification; B: brucella specific gene MCUDA amplification) of example 1 of the present invention.
Fig. 2 shows the design principle of the AuNPs-LFA biosensor in example 1 of the present invention.
FIG. 3 shows the mMCUDA detection scheme (A: UDG enzyme-mediated mMCUDA amplification of target DNA; B: auNPs-LFA detection against mMCUDA amplicon) according to example 1 of the present invention.
FIG. 4 shows the results of the s-and M-MCUDA-proof test of example 2 of the present invention (A1-C1: bcsp-sMCUDA-proof test; A2-C2: BMEII-sMCUDA-proof test; A3-C3: mMCUDA-proof test; 1 (A1-C3) positive reaction (plasmid; 2 (A1-C3) positive reaction (Brucella melitensis isolate), 3 (A1-C3) negative control (Listeria monocytogenes), 4 (A1-C3) negative control (environmental sample), 5 (A1-C3) blank control (enzyme-free water; M: DL2000 DNA MARKER).
FIG. 5 shows the result of mMCUDA-preselected temperature-optimized test (1-1 to 12-1: positive reaction, amplification temperature: 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃,70 ℃ and 1-2 to 12-2: blank control, amplification temperature: 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃ and 70 ℃ in this order) in example 3 of the present invention.
FIG. 6 shows the results of mMCUDA-best temperature optimization test (A: amplification temperature 64 ℃; B: amplification temperature 65 ℃; C: amplification temperature 66 ℃;1-7 (A, B and C); plasmid concentration 5X 104、5×103、5×102、5×101、5×100、5×10-1 and 5X 10-2 fg;8 (A, B and C); enzyme-free water control) in this order) according to example 3 of the present invention.
FIG. 7 shows the result of mMCUDA optimum time test (A: amplification time 40 minutes; B: amplification time 50 minutes; C: amplification time 60 minutes; D: amplification time 70 minutes; 1-8 (A, B, C and D): plasmid concentration 5X 105、5×104、5×103、5×102、5×101、5×100、5×10-1 and 5X 10-2 fg;9 (A, B, C and D): enzyme-free water blank) in this order according to example 3 of the present invention.
FIG. 8 shows the results of mMCUDA% decontamination test (A: mMCUDA reaction (containing UDG enzyme), B: mMCUDA reaction (without UDG enzyme), 1-15 (A and B) simulated contaminant mass 1×10-8、1×10-9、1×10-10、1×10-11、1×10-12、1×10-13、1×10-14、1×10-15、1×10-16、1×10-17、1×10-18、1×10-19、1×10-20、1×10-21 and 1X 10-22 g/. Mu.L, 16 (A and B) enzyme-free water blank) in this order, according to the present invention, in example 4.
FIG. 9 shows the result of mMCUDA sensitivity test in example 5 of the present invention (1-8: plasmid concentration 5X 105、5×104、5×103、5×102、5×101、5×100、5×10-1 and 5X 10-2 fg in order; 9: enzyme-free water blank).
FIG. 10 shows the result of mMCDA (no UDG enzyme) sensitivity test of example 5 of the present invention (1-8: plasmid concentration 5X 105、5×104、5×103、5×102、5×101、5×100、5×10-1 and 5X 10-2 fg in order; 9: no enzyme water blank).
FIG. 11 shows mMCUDA specific test results (1: plasmid; 2, vaccine strain M5;3, brucella ovis 16M, 4, brucella ovis Ether; brucella melitensis 5, brucella melitensis 6, brucella melitensis 7, brucella melitensis 8, brucella melitensis 9, brucella melitensis 10, brucella melitensis 11, brucella melitensis 25, streptococcus pneumoniae 26, streptococcus pneumoniae 27, leuconostoc 28, pseudomonas aeruginosa 29, brucella melitensis 14, brucella melitensis 15, brucella melitensis 16, brucella suis 1330 17, brucella melitensis S2, brucella melitensis 18, brucella suis quality control material, mycobacterium tuberculosis H37Rv 19, mycobacterium tuberculosis H37Ra 20, mycobacterium tuberculosis 21, brucella melitensis 22, mycobacterium marmorale 23, mycobacterium bovis 24, listeria, staphylococcus aureus 25, streptococcus pneumoniae 26, streptococcus pneumoniae 27, escherichia coli 28, pseudomonas aeruginosa 29, pseudomonas aeruginosa 30, brucella orientalis 31, neissimago 37, neisseria 37, brucella hemangiosis 38, and Neisseria 37, and a control strain of the Brucella nucifera 35, and a. 37, respectively.
FIG. 12 shows the mMCUDA primer set of comparative example 1 of the present invention (A-C: three bcsp-MCUDA primer set; D-F: three BMEII-MCUDA primer set; pos: positive reaction (plasmid), neg: blank (no enzyme water), and the third bcsp-MCUDA primer (C) and the third BMEII-MCUDA primer (F) were selected as the optimal primers for mMCUDA test according to the result of the screening test).
Detailed Description
Example 1:
(1) mMCUDA principle of detection
The technical scheme develops a multi-target nucleic acid amplification system (called mMCUDA) with integrated UDG enzyme-mediated multi-cross displacement amplification (mMCDA) and triple nano-biosensor (AuNPs-LFA), and the technical principle of mMCUDA is shown in figures 1 and 2 in detail.
MMCUDA detection consisted essentially of UDG enzyme (thermosensitive uracil-DNA glycosylase) -mediated cleavage reaction (step 1 of FIGS. 1A and B), dUTP-based mMCDA amplification (steps 2-6 of FIGS. 1A and B), and AuNPs-LFA-mediated amplification product validation (FIG. 2). First, when the reaction system was premixed, incubated at 37℃for 10 minutes, potential uracil base-carrying contaminants were removed (i.e., aerosol contamination was prevented and eliminated) by UDG cleavage (FIGS. 1A and B, step 1). The UDG enzyme has only cleavage activity for DNA amplification products carrying uracil bases, and does not cleave free dUTP and uracil-free nucleic acid templates in mMCUDA reaction systems (step 1 of FIGS. 1A and B). Subsequently, mMCUDA amplification was performed under the drive of mMCDA primers and Bst DNA polymerase (step 2 of fig. 1A and B). dTTP is replaced by dUTP in dNTP mixture of mMCUDA reaction system to ensure that mMCUDA amplified products all carry uracil base which can be recognized and hydrolyzed by UDG enzyme, thereby achieving the purpose of preventing and eliminating potential pollutants. Next, CP1 x-FAM or CP1 x-Dig may bind to D1 x-biotin in mMCUDA reactions to form a large number of FAM-amplicon-biotin and Dig-amplicon-biotin complexes (steps 3-6 of fig. 1A and B). After appropriate amounts of mMCUDA amplicon and buffer were added to the wells, these complexes were captured by anti-FITC antibody and anti-Dig antibody embedded on NC membrane (steps 1-3 of FIG. 2). Finally, there may be five potential detection results on the AuNPs-LFA biosensor, including negative (fig. 2, R1), positive (fig. 2, R2, targeting b.melitensis), positive (fig. 2, R3, targeting b.melitensis), positive (fig. 2, R4, targeting other members of the genus brucella, excluding b.melitesis), and invalid results (fig. 2, R5). As shown in FIG. 3, the entire detection procedure of mMCUDA method includes DNA preparation (FIG. 3A, step 1), UDG-mediated cleavage reaction (FIG. 3A, step 2), mMCUDA amplification (FIG. 3A, step 3), and AuNPs-LFA validation (FIG. 3B, steps 1-3).
(2) Reagents, structures and instrumentation according to embodiments of the invention:
Bst DNA polymerase (6.0), 10 Xreaction buffer, thermosensitive uracil-DNA glycosylase (UDG) were purchased from Biyun Tian Biotechnology Co. Bacterial genomic DNA and viral qEx-DNA/RNA extraction kits were purchased from Sian Tianlong technologies Inc. dUTP (100 mM), dATP (100 mM), dCTP (100 mM) and dGTP (100 mM) were purchased from Sieimer technologies Co. Whole blood DNA extraction kit was purchased from QIAGEN, germany.
PCR thermal cycler was purchased from Hangzhou Bori technologies Co. A high-speed cryocentrifuge (3-18K) was purchased from Sigma, germany. Electrophoresis apparatus and gel imaging systems were purchased from Bio-Rad, inc. of America. The oscillating constant temperature metal bath was purchased from Beijing Jinhao pharmaceutical Co., ltd. Real-time turbidity meter (LA-500) was purchased from Eiken corporation, japan.
(3) The method according to the embodiment of the invention comprises the following steps:
Genome extraction: a total of 37 pathogens were used to evaluate the specificity of the mMCUDA methods, including 17 brucella and 20 non-brucella pathogens. The pathogen genomic nucleic acid is extracted using a bacterial genomic DNA extraction kit or a viral qEx-DNA/RNA extraction kit. Furthermore, the applicability of the mMCUDA method in testing clinical samples was assessed using 44 brucella isolates and 54 whole blood samples. Extracting and separating strain genome DNA by a rapid extraction method: 200. Mu.L of the culture (picked colonies were added to TE buffer) was added to the enzyme-free EP tube, and 100. Mu.L of the DNA extract was added. Subsequently, the mixture was boiled for 8 minutes with heating, centrifuged at 12000rpm for 5 minutes, and the supernatant was used as an amplification template (-20 ℃ C.) for preservation. Whole blood sample genomic DNA extraction was performed with reference to kit instructions.
MMCUDA. Mu.L of reaction system: 1 μL Bst DNA polymerase (40U), 1.5 μL MgSO4 (100 mM), 2.5 μL reaction buffer (10×), 1.4mM dATP,1.4mM dCTP,1.4mM dGTP,1.4mM dUTP,1 μL UDG enzyme (1U), 0.17 μM bcsp-F1 and bcsp-F2,0.34 μM bcsp-C1, bcsp-C2, bcsp-R1, bcsp-R2, bcsp-D1 and bcsp-D2,0.68 μM bcsp-CP1 and bcsp-CP2,0.23 μM BMEII-F1 and BMEII-F2,0.46 μM BMEII-C1, BMEII-C2, BMEII-R1, BMEII-R2, BMEII-D1 and BMEII-CP2, 0.92 μM BMEII-CP1, 1.5 μL or genomic DNA or pure plasmid or DNA samples were added to 25 μL of the solution. After pre-incubation of the reaction tube for 10 minutes at 37 ℃, reaction was carried out for 60 minutes at 65 ℃. Listeria monocytogenes genomic DNA and test environmental samples were used as negative controls and enzyme-free water was used as blank controls, respectively. Finally, the amplified products were verified by AuNPs-LFA biosensor, real-time turbidimeter and 1.5% agarose gel electrophoresis.
(4) Primer design
According to the MCDA amplification principle, mMCDA primers were designed for the bcsp31 gene (GenBank ID: M20404.1) and the BMEII0466 gene (GenBank ID: CP 026337.1), respectively (Table 1). According to the design principle of the AuNPs-LFA biosensor, FAM (bcsp-CP 1. Times. -FAM, carboxyfluorescein) and Dig (BMEII-CP 1. Times. -Dig, digoxin) are respectively modified at the 5' -ends of the bcsp-CP1 and BMEII-CP 1. Times. -primer. Meanwhile, biotin (bcsp-D1 x-biotin and BMEII-D1 x-biotin, biotin) was modified at the 5' end of the amplification primer D1 x, respectively. HPLC purification grade primers used in the present invention were synthesized and modified by Beijing Tian Yihui biotechnology Co., ltd. In addition, plasmid templates were prepared by synthesizing and cloning a partial target fragment of the bcsp31 gene and BMEII0466 gene into pUC57 vector. The preparation, extraction, quantification and other processes are completed by Beijing Tianyi-Hui-far biotechnology limited company. The synthesized plasmids were serially diluted to prepare a multiple dilution gradient (5×105、5×104、5×103、5×102、5×101、5×100、5×10-1 and 5×10-2 fg).
Table 1: mMCUDA primers (mer: monomeric unit, monomer Unit; nt: nucleotide) used in the present invention
(5) Preparation of triple nano biosensor (AuNPs-LFA)
The preparation of the triple nano biosensor (AuNPs-LFA) adopts the method in the prior art, and entrusts the Tianjin Huidexin technology development Co. The AuNPs-LFA biosensor (size 64mm×4 mm) includes four parts, i.e., a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and a water absorbing pad are sequentially fixed on a plastic back plate using a plastic adhesive. And the binding pad is coated with gold nanoparticle coupled streptavidin. And a detection line and a control line are sequentially arranged on the nitrocellulose membrane. The anti-fluorescein isothiocyanate anti-FITC (with the concentration of 0.15 mg/ml) is fixed on the detection line 1 (TL 1), the anti-digoxin antibody anti-Dig (with the concentration of 0.2 mg/ml) is fixed on the detection line 2 (TL 2), and the biotin-coupled Bovine Serum Albumin (BSA) is fixed on the quality Control Line (CL) with the concentration of 2.5 mg/ml. TL1, TL2 and CL are separated at 5mm intervals.
Example 2: mMCUDA proof of test
The feasibility of the mMCUDA method was verified by a validation test. Single MCUDA (MCUDA) and multiple MCUDA (MCUDA) experiments were performed separately to verify the feasibility of the method design. A single MCUDA, i.e.only one set of primers (directed to the bcsp31 gene or BMEII0466 gene) was added during the amplification stage, the other conditions were the same as in example 1. 1.5. Mu.L of plasmid and Brucella melitensis isolate DNA were used as positive controls, 1.5. Mu.L of Listeria monocytogenes DNA template and test environmental samples were used as negative controls, and 1.5. Mu.L of enzyme-free water was used as a blank. In s-and m-MCUDA positive reactions, the CL, TL1 or TL2 lines on AuNPs-LFA biosensors were red (FIGS. 4A1-A3, 1-2), turbidity values were all greater than 0.1 (turbidity threshold 0.1) (FIGS. 4B1-B3, 1-2), and characteristic gradient-like bands (FIGS. 4C1-C3, 1-2). In the s-and m-MCUDA negative reactions, only the CL lines appear red (FIGS. 4A1-A3, 3-5), the turbidity values are all less than 0.1 (FIGS. 4B1-B3, 3-5), and no characteristic gradient-like bands (FIGS. 4C1-C3, 3-5) are present.
Example 3: mMCUDA amplification condition optimization
In order to obtain the best detection efficiency, the reaction conditions of mMCUDA method were optimized by setting different temperatures and times, other conditions being referred to in example 1. As shown in FIGS. 5 and 6, the optimal preselected temperature range for mMCUDA amplification was shown to be 64-66℃by temperature optimization experiments. Further optimization of the preselected temperature has found that the amplification efficiency is more stable at 65℃than at 64℃and 66 ℃. In addition, the optimal incubation time for the mMCUDA method was also optimized. At amplification times of 60 and 70 minutes, mMCUDA detected a minimum concentration of plasmid of 7.5 fg/reaction (FIG. 7). Thus, the optimal amplification conditions for the mMCUDA method are 65℃for 60 minutes.
Example 4: mMCUDA pollution abatement Capacity verification test
The experiment was performed with reference to example 1, except that the DNA template in example 1 was replaced with mMCUDA amplification product (mMCUDA product of the previous round of amplification obtained by the method of example 1) to simulate aerosol contamination by the amplification product. The experimental results are shown in FIG. 8, which shows by a simulated contamination test that mMCUDA can eliminate the maximum contaminant droplet caused by mMCUDA amplicon by a mass of about 1.5X10-13 g (concentration 1.0X10-13 g/. Mu.L, 1.5. Mu.L/reaction added). This excellent contamination resistance can significantly reduce the risk of false positives by a factor of 102-105 over similar MCDA-LFB methods (about 1.0X10-15 or 1.0X10-18 g).
Example 5: mMCUDA sensitivity test
In order to evaluate the detection sensitivity of the mMCUDA method, a test was performed on its limit of detection (LoD). Referring to the technical parameter set of example 1, as shown in FIG. 9, through repeated experiments mMCUDA, a minimum concentration of 7.5fg per reaction of plasmid-fold dilution gradient can be detected. The analytical sensitivity was consistent with the control mMCDA method without addition of UDG enzyme (FIG. 10), indirectly confirming the rationality and reliability of the mMCUDA assay protocol.
Example 6: mMCUDA specificity test
In the present invention, representative Brucella melitensis vaccine strain (M5), brucella melitensis 16M, brucella melitensis Ether, brucella melitensis quality control, brucella melitensis isolate, brucella melitensis 544, brucella melitensis isolate, brucella melitensis vaccine strain (A19), brucella melitensis quality control, brucella suis 1330, brucella suis vaccine strain (S2), brucella suis quality control and other 20 strains of non-Brucella pathogens (Table 2) were used. And extracting nucleic acid templates from these strains to evaluate the specificity of mMCUDA methods. The results show that mMCUDA method not only can accurately detect all Brucella strains (including M5, ether, 16M, 544, A19, 1330, S2, corresponding isolates and quality control products) but also can identify Brucella ovis (M5, 16M, ether, isolates and quality control products) at the same time (FIG. 11), and the Brucella strains have no cross reaction with other non-Brucella strains, so that the specificity is 100%.
Table 2: pathogens used in the present invention (NCTC: national collection of Strain, england; ATCC: american type culture Collection; GZCDC: guizhou disease prevention control center; BNCC: north Nanophyte)
| Pathogens | Source(s) | Quantity of |
| Brucella melitensis 16M | NCTC 10094 | 1 |
| Brucella melitensis Ether | NCTC 10509 | 1 |
| Brucella melitensis M5 | Vaccine strain | 1 |
| Brucella melitensis isolated strain | GZCDC | 50 |
| Brucella quality control product for sheep | Quality control product | 1 |
| Brucella bovis 544 | (NCTC 10093) | 1 |
| Brucella bovis A19 | Vaccine strain | 1 |
| Brucella bovis isolated strain | GZCDC | 3 |
| Brucella bovis | Quality control product | 1 |
| Brucella suis 1330 | NCTC 10316 | 1 |
| Brucella suis S2 | Vaccine strain | 1 |
| Brucella quality control product for pig breeds | Quality control product | 1 |
| Mycobacterium tuberculosis H37Rv | ATCC 27294 | 1 |
| Mycobacterium tuberculosis H37Ra | ATCC 25177 | 1 |
| Mycobacterium tuberculosis isolated strain | GZCDC | 1 |
| Mycobacterium marmoreus | ATCC 29571 | 1 |
| Mycobacterium bovis | ATCC 19210 | 1 |
| Listeria monocytogenes | GZCDC | 1 |
| Staphylococcus aureus | GZCDC | 1 |
| Streptococcus pneumoniae | GZCDC | 1 |
| Coli bacterium | GZCDC | 1 |
| Klebsiella pneumoniae | GZCDC | 1 |
| Pseudomonas aeruginosa | GZCDC | 1 |
| Shigella strain | GZCDC | 1 |
| Streptococcus suis (S.suis) | GZCDC | 1 |
| Oriental tsutsugamushi disease | GZCDC | 1 |
| Mycobacterium leprae | GZCDC | 1 |
| Salmonella bacteria | GZCDC | 1 |
| Pertussis bauter fungus | GZCDC | 1 |
| Neisseria meningitidis | GZCDC | 1 |
| Human cytomegalovirus | BNCC | 1 |
| Influenza virus | GZCDC | 1 |
Example 7: use of mMCUDA methods
To verify the usefulness of the mMCUDA method for detecting clinical isolates or whole blood samples, evaluation tests were performed using 44 Brucella isolates and 54 whole blood samples (Table 3). The results of the 44 isolates showed that 42 were identified as B.melitensis by mMCUDA and 2 were identified as members of Brucella other than B.melitensis (consistent with the initial identification results). In addition, 54 whole blood samples were tested using culture, real-time fluorescent PCR and mMCUDA. The detection result shows that mMCUDA detects 4 positive samples (B.melitensis) and 50 negative samples; 4 positive samples (B.melitensis) and 50 negative samples are detected by a culture method; the real-time fluorescent PCR detects 4 positive samples and 50 negative samples. These data confirm that mMCUDA methods established in the present invention can be a valuable detection and identification tool for brucella infection.
Table 3: results of 3 methods on Whole blood sample detection
Comparative example 1
To find a detection system that can achieve better test sensitivity and accuracy, the inventors designed a large number of primer combinations for testing for both genes. Although there is a certain design guideline for the design of the MCDA primer set, the primer set design according to known information often causes an unsatisfactory amplification effect due to the large number of primers contained in the primer set and the complex situation. The inventors have performed experimental screening of multiple sets of primers synthesized for two target genes, and found that no non-specific amplification was observed with only the primer combination employed in this protocol (see example 1 for details). The three primer combinations synthesized are specifically exemplified below.
The primer combinations used during part of the experiments are now listed in tables 4 and 5. MCDA amplification experiments were performed using the primer combinations of table 1 (third set of primers for both genes), table 4 (first set of primers for both genes) and table 5 (second set of primers for both genes), respectively. The amplification system used was 25. Mu.L of the reaction system of example 1, except that the primer combinations for the two genes were not added simultaneously; the amplification procedure was followed by a reaction at 63℃for 60 minutes after a pre-incubation of 10 minutes at 37 ℃; the template used was 1.5. Mu.L plasmid and the concentration was 5X 107 fg; the negative control was enzyme-free water. The amplification is carried out simultaneously with real-time turbidity detection, a Judgment mode is used for a turbidity detector, the amplification efficiency is mainly examined, and the experimental result is shown in fig. 12. The degree of amplification can be characterized by turbidity, as is conventional in the art, because magnesium pyrophosphate (dNTPs participate in reactions, and lose pyrophosphate, pyrophosphate combines with magnesium ions in the amplification system to produce a precipitate, which is a substance necessary for DNA polymerase activity) is precipitated during the amplification of MCDA.
For the experimental results of fig. 12, the third set of primers adopted in this scheme has the advantages that: the primer combination was not observed to have a non-specific amplification phenomenon (both the first and second primer combinations showed a non-specific amplification phenomenon) for a set reaction time, compared to the first and second primer combinations used in the study. The turbidity threshold is usually set to 0.1, and positive reaction is judged when the turbidity value is larger than 0.1, and negative reaction is judged on the contrary, wherein nonspecific amplification is mainly represented by turbidity signal peaks of negative control and turbidity value is larger than 0.1, while ideal MCDA amplification only represents turbidity signal peaks in samples containing templates, and turbidity values in the negative control are smaller than 0.1. Therefore, the higher reliability of the third set of primers meets the requirement of establishing mMCUDA amplification system, and the reliability and high specificity of the invention are also confirmed.
Table 4: primer combination List (first set of primers) related to this comparative example
Table 5: primer combination List (second set of primers) related to this comparative example
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.