Mycobacterium multiplex PCR (Polymerase Chain Reaction) detection kit and method
By designing multiple PCR detection methods for specifically amplifying Mycobacterium genes, the problem of distinguishing bovine, human and avian Mycobacterium is solved, and efficient and accurate tuberculosis diagnosis is achieved.
Patent Information
- Application Number
- CN202510603843.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to effectively distinguish between bovine, human and avian Mycobacterium, which leads to difficulty in diagnosis of tuberculosis. Especially when the skin test is positive after BCG vaccination, it is impossible to accurately distinguish the infection type.
Multiple PCR detection methods were designed to specifically amplify the 16sRNA of the Mycobacterium genus, the Mycobacterium tuberculosis group_16810 gene, the Mycobacterium bovis Rv1506c gene and the Mycobacterium avian group_32724 gene, and PCR amplification was performed using specific primer pairs, and the results were detected by electrophoresis.
It realizes the accurate distinction between bovine, human and avian Mycobacterium, provides high-quality amplification products, ensures the accuracy and reliability of the test results, and is suitable for the diagnosis of tuberculosis.
Smart Images
Figure CN120230873A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of mycobacterium detection, and specifically relates to a multiplex PCR detection kit and method for differentiating Mycobacterium bovis, Mycobacterium tuberculosis, and Mycobacterium avium. Background Art
[0002] Bovine tuberculosis (bTB) is a chronic granulomatous disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC). Human tuberculosis is mainly caused by Mycobacterium tuberculosis. However, 3% - 10% of cases are caused by Mycobacterium bovis. Animal tuberculosis caused by Mycobacterium bovis has been found in multiple hosts, including domestic and wild animals, which may become new sources of infection and promote the continuous transmission of the pathogen. Due to the global distribution of this pathogen, it has been reported that significant economic losses have been caused to livestock production systems worldwide, and wildlife conservation efforts have also been negatively affected. More importantly, human tuberculosis cases caused by Mycobacterium bovis infection are becoming increasingly common, attracting great attention globally. In 2023, there were 10.8 million tuberculosis cases and 1.25 million deaths due to tuberculosis worldwide. Although most of these cases are caused by Mycobacterium tuberculosis, approximately 142,000 new cases and 12,500 deaths are caused by Mycobacterium bovis. Mycobacterium bovis is called "zoonotic tuberculosis", and due to the lack of reporting in endemic countries and limited laboratory capabilities, the number of human Mycobacterium bovis cases may be higher.
[0003] Mycobacterium bovis has 99.95% genomic similarity with Mycobacterium tuberculosis, which causes great difficulties in clinically differentiating the two bacteria. The tuberculin intradermal hypersensitivity test measures delayed-type hypersensitivity by intradermal injection of purified protein derivative of PPD. However, when tested after receiving the Bacillus Calmette-Guérin (BCG) vaccine, the skin test usually shows positive. Therefore, additional tests are needed to determine whether a person has active Mycobacterium tuberculosis disease and its specific mycobacterium. The interferon gamma release assay (IGRA) is usually used to distinguish BCG vaccination from infection. After an IGRA positive result, it can be confirmed that the patient has been infected, but it cannot determine whether the patient is infected with Mycobacterium tuberculosis or Mycobacterium bovis. Therefore, it is necessary to establish a detection method that can distinguish the two as a supplementary test for clinical diagnosis. Summary of the Invention
[0004] The purpose of the present invention is to find target genes through pan-genome analysis to provide a multiplex PCR detection method that can distinguish Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium avium, so as to achieve the identification of clinical Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium avium, and provide a reference for the diagnosis of tuberculosis.
[0005] The technical solution adopted by the present invention is as follows:
[0006] A kit for multiplex PCR for detecting mycobacteria, characterized by comprising:
[0007] A primer pair 16sRNA-F and 16sRNA-R for specifically amplifying the 16sRNA gene of the mycobacterium genus; a primer pair group_16810-F and group_16810-R for specifically amplifying the group_16810 gene of Mycobacterium tuberculosis; a primer pair Rv1506c-F and Rv1506c-R for specifically amplifying the Rv1506c gene of Mycobacterium bovis; a primer pair group_32724-F and group_32724-R for specifically amplifying the group_32724 gene of Mycobacterium avium;
[0008] The nucleotide sequences of each primer are respectively:
[0009] 16sRNA-F, 5'-ACGGTGGGTACTAGGTGTGGGTTTC-3';
[0010] 16sRNA-R, 5'-TCTGCGATTAGCGACTAAGACTTCA-3';
[0011] group_16810-F, 5'-ATGCCCCAGCAGACCCTTGCC-3';
[0012] group_16810-R, 5'-TCAGCCCGAGCGCCGATTGT-3';
[0013] Rv1506c-F, 5'-GTCGCCGCTCCCAAAAATTAC-3';
[0014] Rv1506c-R, 5'-GGAGAGCGCCGTTGTAGG-3';
[0015] group_32724-F, 5'-ATGGTAAGCGTTGTGGGTAAAAACAC-3';
[0016] group_32724-R, 5'-TCAACGGCCGTCCGAAGC-3';
[0017] In a preferred embodiment of the present invention, it is characterized in that:
[0018] The size of the target fragment amplified by the primer pair 16sRNA-F and 16sRNA-R is 575bp;
[0019] The size of the target fragment amplified by the primer pair group_16810-F and group_16810-R is 360 bp;
[0020] The size of the target fragment amplified by the primer pair Rv1506c-F and Rv1506c-R is 126 bp;
[0021] The size of the target fragment amplified by the primer pair group_32724-F and group_32724-R is 264 bp;
[0022] On the other hand, the present invention also relates to a multiplex PCR detection method for detecting Mycobacterium 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene. The method is for non-diagnostic purposes and is characterized by including the following steps:
[0023] Step 1, preparing a PCR template based on the bacteria to be detected;
[0024] Step 2, downloading 7005 strains of Mycobacterium tuberculosis, 175 strains of Mycobacterium bovis, 39 strains of Mycobacterium avium tuberculosis and other mycobacteria, a total of 7304 strains from NCBI. All genomic sequences are downloaded from NCBI in nucleotide FASTA format. All analyzed genomic sequences are automatically annotated using Prokka v1.14 software. Roary v3.11.2 is used to establish a pan-genome for the output of Prokka with default settings. The presence / absence profiles of all genes in all samples are converted into a 0 / 1 matrix with a local script. Then, core genes (present in ≥99% of the target genomes) and specific genes are screened using this matrix. Specific genes for bovine, human, and avian tuberculosis are screened according to the following criteria: present in 99% of the target strains and absent in non-target strains. Candidate target gene sequences are extracted using Tbtools, and the candidate specific genes are aligned with the NCBI nucleotide sequence database using BLAST to ensure their specificity;
[0025] Step 3, preparing a specific primer set, and preparing a specific primer set using the primers described in any one of claims 1 or 2 of the patent;
[0026] Step 4, performing a PCR amplification reaction. Based on the PCR template obtained in Step 1, a PCR reaction system including the primer set is established using the specific primer set obtained in Step 2, and then the PCR amplification reaction is performed using this PCR reaction system under predetermined PCR reaction parameter conditions to obtain a PCR amplification product;
[0027] Step 5: Perform electrophoresis detection. Electrophoresis detection is carried out on the amplification product to obtain an electrophoresis result, and it is determined whether the Mycobacterium genus gene 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene appear according to the electrophoresis result.
[0028] In a preferred embodiment of the present invention, it is characterized in that:
[0029] The PCR reaction system in Step 4 includes:
[0030] 2.5 μL of 10×Butter, 0.15 μL of rTaq polymerase with a concentration of 5 U / μL, 2 μL of dNTP with a concentration of 2.5 mM, 2 μL of Mg2+ with a concentration of 25 mM, 1 μL of the PCR template, 11.6 μL of ultrapure water, and the primer set.
[0031] In the primer set, the volume of each of the primer pairs 16sRNA-F and 16sRNA-R is 2 μL, the volume of each of the primer pairs group_16810-F and group_16810-R is 0.5 μL, the volume of each of the primer pairs Rv1506c-F and Rv1506c)-R is 1 μL, and the volume of each of the primer pairs group_32724-F and group_32724-R is 1 μL.
[0032] In a preferred embodiment of the present invention, it is characterized in that:
[0033] When preparing the specific primer set, the concentration of each primer used in the primer set is 10 μM.
[0034] In a preferred embodiment of the present invention, it is characterized in that:
[0035] The predetermined PCR reaction parameter conditions are pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 30 cycles, and further extension at 72°C for 10 min.
[0036] In a preferred embodiment of the present invention, it is characterized in that:
[0037] The specific process of Step 5 is as follows:
[0038] 5 μL of 6×loading buffer is added to the PCR amplification product and mixed evenly to obtain a mixed product. 6 μL of the mixed product is taken and loaded into the wells of a 1.5% agarose gel electrophoresis plate. Electrophoresis is carried out at a voltage of 150 V for 25 min, and a photograph is taken under a gel imaging system to obtain an electrophoresis result, and the detection result is judged according to the electrophoresis result.
[0039] The present invention also relates to the application of the above-mentioned kit in the preparation of a multiplex PCR detection kit for detecting Mycobacterium genus gene 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene.
[0040] The beneficial effects of the present invention are as follows:
[0041] (1) Based on the target genes found in the present invention, Mycobacterium tuberculosis of human type, bovine type, and avian type can be successfully distinguished. The present invention can simultaneously and specifically amplify Mycobacterium genus gene 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene.
[0042] (2) The multiplex primers provided by the present invention have the advantages of not easily forming dimers and hairpin structures, having a uniform Tm value, appropriate primer length and GC content, etc., which are beneficial to obtaining high-quality amplification products and making the detection results accurate and reliable. Description of the Drawings
[0043] Figure 1 It is the result diagram of single PCR amplification. Among them, M. DNA standard DL 2,000; M. DNA standard DL1000; 1. 16sRNA; 2 group_16810; 3. group_32724; 4. Rv1506c; 5. negative control.
[0044] Figure 2 It is the result diagram of optimizing the dNTP dosage of multiplex PCR. Among them, M. DNA standard DL1000; 1-8. 0.4mM, 0.6mM, 0.8mM,.1mM, 1.2mM, 1.4mM, 1.6mM; negative control.
[0045] Figure 3 It is the result diagram of optimizing the 25mM MgCl2 dosage of multiplex PCR. Among them, M. DNA standard DL1000; 1-9. 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM; negative control
[0046] Figure 4 It is the result diagram of optimizing the r Taq enzyme dosage of multiplex PCR. Among them, M. DNA standard DL1000; 1-6. 0.02U; 0.025U; 0.03U; 0.035U; 0.04U; negative control.
[0047] Figure 5It is a graph showing the optimization results of multiplex PCR cycling conditions. Among them, M. DNA standard DL1000; 1 - 13: 24 cycles; 25 cycles; 26 cycles; 27 cycles; 28 cycles; 29 cycles; 30 cycles; 31 cycles; 32 cycles; 33 cycles; 34 cycles; 35 cycles; negative control.
[0048] Figure 6 It is a graph showing the optimization results of multiplex PCR annealing temperature. Among them, M. DNA standard DL1000; 1 - 7. 58℃, 57℃, 56℃, 55℃, 54℃, 53℃; negative control.
[0049] Figure 7 It is a graph showing the results of multiplex PCR specificity test. Among them, M. DNA standard DL1000; 1 - 16: positive control; Staphylococcus aureus ATCC25923; Proteus CMCC49027; Bacillus cereus CMCC67303; Streptococcus uberis SU 883; Pseudomonas aeruginosa An297 Pse11; Haemophilus paragallinarum 008C (clinical isolate); Enterococcus faecalis ATCC29212; Clostridium perfringens ATCC13124; Klebsiella pneumoniae CMCC46117; Salmonella ATCC60049; Listeria monocytogenes CICC21635; Shigella flexneri CMCC51573; Bacillus subtilis ATCC6623; Escherichia coli clinical isolate; negative control.
[0050] Figure 8 It is a graph showing the results of multiplex PCR sensitivity test. Among them, A: Detection results of Mycobacterium tuberculosis sensitivity; B: Detection results of Mycobacterium bovis sensitivity; C: Detection results of Mycobacterium avium sensitivity; M. DNA standard DL1000; 1 - 9: 10ng, 10 - 1 ng, 10 -2 ng, 10 -3 ng, 10 -4 ng, 10 -5 ng, 10 -6 ng, 10 -7 ng; negative control.
[0051] Figure 9 It is a graph showing the test results of clinical samples. Among them, lane 1 is the positive control, lanes 2 - 13 are the genomes of Mycobacterium tuberculosis (human type), lane 14 is the genome of Mycobacterium bovis, and lane 15 is the negative control. Detailed implementation methods
[0052] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For the following embodiments, those without specific technologies or conditions noted are carried out according to the technologies or conditions described in the literature in the field or according to the product specifications; for the reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchases.
[0053] Example 1
[0054] This example provides a primer set for detecting mycobacteria. For primer sets, how to design multiplex primers so that they are not easily formed into dimers and hairpin structures, have a uniform Tm value, appropriate primer length and GC content, are conducive to obtaining high-quality amplification products and preventing the detection results of amplification primers for different genes from interfering with each other, making the detection results accurate and reliable is one of the difficult problems that those skilled in the art urgently need to solve. The method for obtaining the primer set of the present invention includes the following steps:
[0055] (1) Extract the gene sequence using Tbtools software according to the gene ID number.
[0056] (2) Use snapgene software and primerpremier 6.0 to design primer sequences for the reference sequence, and obtain 1 pair of specific primer sequences for the sense strand and antisense strand of each gene. After the primer design is completed, each primer is subjected to Primer-BLAST on the NCBI website to ensure the specificity of the primer. The primer sequences are shown in Table 1.
[0057] Table 1 Primer information for mycobacterium detection
[0058]
[0059]
[0060] Example 2
[0061] This example provides a genomic DNA extraction protocol for mycobacteria. The specific implementation steps are as follows:
[0062] (1) For inactivated Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium, use the Bacterial Genomic DNA Extraction Kit (product number DP302) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract bacterial genomic DNA according to its operation steps.
[0063] (3) Store the extracted bacterial genomic DNA at -20 °C for later use.
[0064] Example 3
[0065] Based on the primer set provided in Example 1, this example provides a reagent for mycobacterium detection, including:
[0066] (1) The primer set shown in Table 1.
[0067] (2) dNTP (10 mM), 10×PCR Buffer (Mg 2+ free), 25 mM MgCl2, rTaq enzyme.
[0068] (3) Negative control product, nuclease-free water.
[0069] (4) A mixed plasmid containing the genomes of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium avium is used as the positive control product.
[0070] Example 4
[0071] Based on the primer set in Example 1, this example provides a single PCR amplification reaction system for mycobacterium detection to verify the feasibility of the primers, and the primer combinations are optimized by the orthogonal test method. The best combination is selected as the initial duplex PCR, and the remaining 2 primer pairs (the initial concentration of each primer is 0.4 μM) are added to the duplex combination to form a triplex PCR. Then the best triplex PCR is selected, and the remaining one primer pair is added to form a quadruplex PCR. The specific reaction system is shown in Table 2.
[0072] (1) After vortexing and briefly centrifuging the prepared reaction system, place it in a PCR instrument and run the program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles, and then extension at 72°C for 10 min.
[0073] (2) After the PCR amplification is completed, take 5 μL of the PCR product and perform electrophoresis on a 1% agarose gel at 150 V for 25 min, and place it in a gel imaging system to observe the results. The results are as Figure 1 shown, and the primers provided by the present invention can specifically amplify the specific genes of mycobacteria.
[0074] (3) The single - PCR amplification of primer group_16810 - F1 / R1 was unsuccessful. Non - specific amplification occurred in the single - PCR of primer group_16810 - F2 / R2. Through orthogonal experiments, it was found that primers 16sRNA - F / R and group_16810 - F / R were the best combination. However, the combination of primer group_16810 - F3 / R3 and 16sRNA - F / R was unsuccessful (no two bands of 575bp and 145bp appeared, only a 575bp band appeared), and the combinations of group - 32724 - F1 / R1 and group - 32724 - F2 / R2 with 16sRNA - F / R and group_16810 - F / R were unsuccessful (only two bands of 575bp and 360bp appeared). Therefore, four pairs of primers with successful combination and specific amplification were finally selected for condition optimization.
[0075] Table 2 Single - PCR Amplification Reaction System
[0076]
[0077] Example 5
[0078] Based on the primer sets provided in Example 1 and the reaction system provided in Example 4, the reaction system was optimized respectively according to the following steps:
[0079] (1) Optimize the dosage of dNTP (10mM): Keep the dosages of the rest of the reaction system unchanged. Set the dosages of dNTP (10mM) as 1μL, 1.5μL, 2μL, 2.5μL, 3μL, 3.5μL, 4μL. After vortex - mixing and instant - centrifuging the prepared reaction system, place it in a PCR instrument and run the program: pre - denaturation at 94°C for 3min; denaturation at 94°C for 20s, annealing and extension at 58°C for 30s, extension at 72°C for 30s, final extension at 72°C for 10min, 30 cycles. After the PCR amplification is completed, take 6μL of the PCR product and perform electrophoresis on a 1.5% agarose gel at 150V for 25min, and observe the results in a gel imaging system. The results are as Figure 2 shown, and the best dosage of dNTP (10mM) is 2.5μL.
[0080] (2) Optimization of the dosage of 25 mM MgCl2: With the dosages of the rest reaction system unchanged, the dosages of 25 mM MgCl2 were set as 0.5 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL. After vortexing and mixing the prepared reaction system and performing instantaneous centrifugation, it was placed in a PCR instrument and the program was run: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing and extension at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, for 30 cycles. After the PCR amplification was completed, 6 μL of the PCR product was electrophoresed on a 1.5% agarose gel at 150 V for 25 min, and the results were observed in a gel imaging system. The results are as Figure 3 shown, and the optimal dosage of 25 mM MgCl2 is 2 μL.
[0081] (3) Optimization of the dosage of r Taq enzyme (5 U / μL): With the dosages of the rest reaction system unchanged, the dosages of r Taq enzyme (5 U / μL) were set as 0.1 μL, 0.125 μL, 0.15 μL, 0.175 μL, 0.2 μL. After vortexing and mixing the prepared reaction system and performing instantaneous centrifugation, it was placed in a PCR instrument and the program was run: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing and extension at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, for 30 cycles. After the PCR amplification was completed, 6 μL of the PCR product was electrophoresed on a 1.5% agarose gel at 150 V for 25 min, and the results were observed in a gel imaging system. The results are as Figure 4 shown, and the optimal dosage of r Taq enzyme (5 U / μL) is 0.15 μL.
[0082] In summary, the present invention provides a multiplex PCR amplification reaction system for mycobacterium detection, and the specific reaction system is as described in Table 3.
[0083] Table 3 Multiplex PCR amplification reaction system
[0084]
[0085]
[0086] Example 6
[0087] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the reaction program was optimized respectively according to the following steps:
[0088] (1) Optimize the reaction cycle conditions: According to the reaction system provided in Example 5, after vortexing and mixing the prepared reaction system and performing instantaneous centrifugation, place it in a PCR instrument and run the program: pre-denaturation, 94°C for 3 min; denaturation at 94°C for 20 s, annealing and extension at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min. The cycle conditions are set to 24 - 35 cycles respectively. After the PCR amplification is completed, take 6 μL of the PCR product and perform electrophoresis on a 1.5% agarose gel at 150 V for 25 min, and observe the results in a gel imaging system. The results are as Figure 5 shown, and the optimal reaction cycle conditions of the present invention are 29 - 35 cycles.
[0089] (2) Optimize the reaction annealing temperature: According to the reaction system provided in Example 5, after vortexing and mixing the prepared reaction system and performing instantaneous centrifugation, place it in a PCR instrument and run the program: pre-denaturation, 94°C for 3 min; denaturation at 94°C for 20 s, and the annealing and extension are respectively set to 53°C for 30 s, 54°C for 30 s, 55°C for 30 s, 56°C for 30 s, 57°C for 30 s, 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, and 30 cycles. After the PCR amplification is completed, take 6 μL of the PCR product and perform electrophoresis on a 1.5% agarose gel at 150 V for 25 min, and place it in a gel imaging system to observe the results. The results are as Figure 6 shown, and the optimal annealing condition of the present invention is 58°C for 30 s.
[0090] In summary, the present invention provides a multiplex PCR amplification reaction program for mycobacterium detection. The specific reaction program is: pre-denaturation, 94°C for 3 min; denaturation at 94°C for 20 s, annealing and extension at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, and 30 cycles.
[0091] Example 7
[0092] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the specific verification steps for the specificity verification of the multiplex PCR provided by the present invention are as follows:
[0093] (1) Extract the genomic DNA of 14 bacteria such as Staphylococcus aureus, Salmonella, and Clostridium perfringens according to the complete genomic DNA extraction protocol in Example 2.
[0094] (2) Using the genomic DNA in the above steps as a template, after vortexing and mixing the prepared reaction system and performing a short centrifugation, place it in a PCR instrument and run the program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, 30 cycles. After the PCR amplification is completed, take 6 μL of the PCR product and perform electrophoresis on a 1.5% agarose gel at 150 V for 25 min, and place it in a gel imaging system to observe the results. The results are as Figure 8 shown.
[0095] (3) The results show that the multiplex PCR provided by the present invention has good specificity and does not cross-react with other bacteria.
[0096] Example 8
[0097] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the sensitivity verification of the multiplex PCR provided by the present invention is as follows:
[0098] (1) Extract the genome of Mycobacterium according to the complete genomic DNA extraction protocol in Example 2.
[0099] (2) Use a NanoDrop spectrophotometer to measure the concentration of the extracted genome, adjust its concentration to 10 ng / μL, and perform serial dilutions step by step. Thus, the genomic DNA added to the corresponding reaction system is 10 ng, 1 ng, 0.1 ng, 0.01 ng, 1 pg, 100 fg, 10 fg, and 1 fg respectively. After vortexing and mixing the prepared reaction system and performing a short centrifugation, place it in a PCR instrument and run the program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, 30 cycles. After the PCR amplification is completed, take 6 μL of the PCR product and perform electrophoresis on a 1.5% agarose gel at 150 V for 25 min, and place it in a gel imaging system to observe the results. The results are as Figure 8 shown
[0100] (3) The results show that the detection sensitivity for Mycobacterium tuberculosis (human type) DNA is 10 pg, the detection sensitivity for Mycobacterium bovis DNA is 1 pg, and the detection sensitivity for Mycobacterium avium DNA is 1 pg.
[0101] Therefore, the multiplex PCR detection kit for Mycobacterium detection provided by the present invention has high detection sensitivity.
[0102] Example 9
[0103] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the clinical sample verification of the multiplex PCR provided by the present invention is as follows:
[0104] (1) Extract the genomes of 12 Mycobacterium tuberculosis (human type) and 1 Mycobacterium bovis according to the complete genomic DNA extraction protocol in Example 2.
[0105] (2) Using the genomic DNA in the above step as a template, after vortexing and mixing the prepared reaction system and performing instantaneous centrifugation, place it in a PCR instrument and run the program: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 20 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, final extension at 72 °C for 10 min, 30 cycles. After the PCR amplification is completed, take 6 μL of the PCR product and perform electrophoresis on a 1.5% agarose gel. Electrophorese at 150 V for 25 min, and place it in a gel imaging system to observe the results. The results are as Figure 9 shown. Lane 1 is the positive control, lanes 2 - 13 are the genomes of Mycobacterium tuberculosis (human type), lane 14 is the genome of Mycobacterium bovis, and lane 15 is the negative control.
[0106] (3) The results show that the multiplex PCR provided by the present invention can specifically detect the group_16810 gene of Mycobacterium tuberculosis, and the detection accuracy rate is 100%; it can specifically detect the Rv1506c gene of Mycobacterium bovis, and the detection accuracy rate is 100%.
Claims
1. A multiplex PCR detection kit for detecting mycobacterium 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene, characterized in that: include: The primer pair 16sRNA-F and 16sRNA-R specifically amplifies the 16sRNA gene of Mycobacterium spp. Primer pair group_16810-F and group_16810-R for specific amplification of Mycobacterium tuberculosis group_16810 gene; primer pair Rv1506c-F and Rv1506c-R for specific amplification of Mycobacterium bovis Rv1506c gene; primer pair group_32724-F and group_32724-R for specific amplification of Mycobacterium avium group_32724 gene; The nucleotide sequences of each primer are: 16sRNA-F, 5'-ACGGTGGGTACTAGGTTGTGGGTTTC-3'; 16sRNA-R, 5'-TCTGCGATTAGCGACTAAGACTTCA-3'; group_16810-F, 5'-ATGCCCCAGCAGACCCTTGCC-3'; group_16810-R,5'-TCAGCCCGAGCGCCGATTGT-3'; Rv1506c-F, 5'-GTCGCCCGCTCCCAAAAATTAC-3'; Rv1506c-R, 5′-GGAGAGCGCCGTTGTAGG-3′; group_32724-F, 5'-ATGGTAAGCGTTGTGGGTAAAAACAC-3'; group_32724-R, 5'-TCAACGGCCGTCCGAAGC-3'.
2. The kit according to claim 1, characterized in that: The size of the target fragment amplified by primer pair 16sRNA-F and 16sRNA-R was 575 bp; The size of the target fragment amplified by the primer pair group_16810-F and group_16810-R was 360 bp; The size of the target fragment amplified by primer pair Rv1506c-F and Rv1506c-R was 126 bp; The primer pair group_32724-F and group_32724-R was used to amplify a target fragment of 264 bp in size.
3. The kit according to claim 1, characterized in that The kit also includes a DNA extraction reagent.
4. The kit according to claim 1, characterized in that The kit also includes a positive quality control product containing a mixed plasmid of three genomes of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium.
5. A multiplex PCR detection method for detecting Mycobacterium 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; Mycobacterium avium gene group_32724 gene, the method is for non-diagnostic purposes, characterized in that: The following steps are involved: Step 1, preparing a PCR template based on the bacteria to be detected; Step 2, screening target genes of Mycobacterium tuberculosis (human type), Mycobacterium bovis, and Mycobacterium avium based on pan-genomic analysis; Step 3, preparing a specific primer set, using the primers described in any one of claims 1 to 4 to prepare a specific primer set; Step 4, performing a PCR amplification reaction, based on the PCR template obtained in step 1, using the specific primer set obtained in step 2 to establish a PCR reaction system including the primer set, and then using the PCR reaction system to perform a PCR amplification reaction under predetermined PCR reaction parameter conditions to obtain a PCR amplification product; Step 5, performing electrophoresis detection, performing electrophoresis detection on the amplified product to obtain an electrophoresis result, and determining whether the Mycobacterium 16sRNA; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; and Mycobacterium avium group_32724 gene are present according to the electrophoresis result.
6. The multiplex PCR detection method according to claim 5, characterized in that: The PCR reaction system of step 3 includes: 2.5 μL of 10× reaction buffer, 0.15 μL of r Taq polymerase at a concentration of 5,000 U / mL, 2 μL of dNTP at a concentration of 2.5 mM each, 2 μL of Mg2+ at a concentration of 25 mM, 1 μL of the PCR template, 11.6 μL of ultrapure water, and the primer set, In the primer set, the volume of primer pair 16sRNA-F and 16sRNA-R is 2 μL each, the volume of primer pair group_16810-F and group_16810-R is 0.5 μL each, the volume of primer pair Rv1506c-F and Rv1506c-R is 1 μL each, and the volume of primer pair group_32724-F and group_32724-R is 1 μL each.
7. The multiplex PCR detection method according to claim 5 or 6, characterized in that: When preparing a specific primer set, the concentration of each primer used in the primer set was 10 μM.
8. The multiplex PCR detection method according to claim 5 or 6, characterized in that: The predetermined PCR reaction parameter conditions are: pre-denaturation at 94° C. for 3 min; denaturation at 94° C. for 20 s, annealing at 58° C. for 30 s, and extension at 72° C. for 30 s, for 30 cycles; and extension at 72° C. for 10 min.
9. The multiplex PCR detection method according to claim 5 or 6, characterized in that: The specific process of step 5 is: 5 μL of 6× loading buffer was added to the PCR amplification product and mixed to obtain a mixed product. 6 μL of the mixed product was spotted in a 1.5% agarose gel electrophoresis plate well. The electrophoresis was performed at a voltage of 150 V for 25 min. The electrophoresis result was obtained by taking a picture under a gel imaging system. The detection result was determined based on the electrophoresis result.
10. Use of the kit according to any one of claims 1 to 4 in the preparation of a multiplex PCR detection kit for detecting Mycobacterium gene 16sRNA gene; Mycobacterium tuberculosis group_16810 gene; Mycobacterium bovis Rv1506c gene; and Mycobacterium avium gene group_32724 gene.