Establishment and application of PCR (Polymerase Chain Reaction) detection method for detecting brucella S19

Abstract

The invention discloses establishment and application of a PCR (Polymerase Chain Reaction) detection method for detecting brucella S19, which is characterized in that a specific interval of a brucella S19 genome and a brucella A19 genome is analyzed by adopting high-throughput Mauve genome colinearity, and a specific primer is designed. The PCR detection primer of the Brucella S19 vaccine strain is screened out, and the PCR detection method for differential diagnosis of the Brucella S19 vaccine strain is established. The PCR method disclosed by the invention has the characteristics of clinical convenience and rapid diagnosis, can be widely used in clinical application of Brucella S19 differential diagnosis, and has better market prospect and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and particularly relates to a specific PCR detection method for Brucella abortus S19 vaccine strain based on genomic collinearity analysis, which is used to quickly distinguish the S19 vaccine strain from other Brucella strains infections, and provides technical support for the diagnosis of brucellosis, vaccine effect evaluation and epidemiological investigation. Background Art

[0002] Brucella is a Gram-negative facultative intracellular parasite, which can cause zoonotic brucellosis and pose a serious threat to public health and animal husbandry. Currently, there are more than 10 known Brucella species in the world, among which the S19 strain of Brucella abortus is a widely used attenuated live vaccine strain. However, traditional serological detection methods (such as agglutination test) cannot distinguish vaccine immunity from natural infection, which is prone to misdiagnosis; while the bacterial culture method is time-consuming, has low sensitivity, and requires a large number of live bacteria in the sample.

[0003] Although existing veterinary Brucella vaccines (such as S, M5-90, A19, etc.) are effective, their residual virulence and serological interference problems limit the disease purification. All existing attenuated live vaccines can infect multiple animals and cannot be distinguished from each other. Therefore, there is an urgent need for a rapid, specific and sensitive molecular detection method to accurately identify multiple or vaccine strains and wild virus infections, and provide a reliable basis for disease prevention and control.

[0004] In the existing brucellosis prevention and control technologies, there are mainly the following two problems:

[0005] 1. Clinically, the current methods for controlling brucellosis mainly rely on large-scale vaccination of susceptible animals and early disease diagnosis. There are as many as 9 live Brucella vaccines currently used in cattle and sheep breeding, and these live vaccines all have the risk of mutual infection among multiple animals. There are serious cross-reactions between the sera immunized with different vaccines, and multiple vaccines cannot be distinguished and detected from each other, and it is even more impossible to distinguish between vaccine immunity and wild virus infection well. This is the key bottleneck problem in the brucellosis control and eradication plan.

[0006] 2. So far, there is still no good technical means to distinguish and diagnose vaccine immunity and wild virus infection, and it is even more impossible to efficiently and rapidly distinguish and diagnose the same species of Brucella strains. To achieve the difference between strains of the same species of Brucella, only more expensive and time-consuming and laborious high-throughput sequencing methods can be used for differential analysis.

[0007] The key scientific problem of the lack of vaccine differential diagnosis for Brucella in the above-mentioned aquaculture industry, and the bottleneck technical problems causing this practical production scientific problem have led to the backward diagnosis of brucellosis, resulting in the widespread prevalence and incidence of brucellosis at the present stage. The current diagnostic technology cannot meet the clinical needs, and ultimately leads to significant economic losses of brucellosis to the aquaculture industry at the present stage. Summary of the Invention

[0008] Aiming at the problems of insufficient specificity, low sensitivity and inability to distinguish vaccine strains existing in the existing Brucella detection technology, the present invention screens specific gene fragments of Brucella strain S19 through genomic collinearity analysis, designs primers and optimizes reaction conditions to establish a rapid and highly specific S19 vaccine strain differential PCR detection method. The present invention is realized as follows: screening and identifying the differential diagnosis target of Brucella pathogen S19, and establishing an S19 differential PCR detection method, including the following steps:

[0009] Step 1: Obtain the genomic sequences of Brucella abortus S19 (GCF_000018725.1) and A19 (GCF_003290345.1) from the NCBI database, and use Mauve software for collinearity analysis to screen out the S19 specific interval (such as Gap5 segment);

[0010] Step 2: Design two pairs of specific primers (Ba.c2.gap0m4.F5 / R5 and Ba.c1.gap0m7.F7 / R7), wherein Ba.c2.gap0m4.F5 / R5 can amplify the S19 specific fragment (249bp) and the A19 fragment (954bp) to achieve differential diagnosis. And optimize the PCR reaction system and conditions: establish a 20μL reaction system: 10μL 2×Rapid Taq MasterMix, 1μL of each upstream and downstream primer, 1μL of template, and make up with ddH2O. Optimized conditions: annealing temperature 60°C, extension time 20s, cycling program: pre-denaturation at 95°C for 3min, 35 cycles (95°C for 15s, 60°C for 15s, 72°C for 20s), final extension at 72°C for 5min. At the same time, it is found that the designed primers can not only amplify specific bands with Brucella strains, but also no specific bands are produced with other important bacterial pathogens of cattle and sheep infected through the respiratory tract, proving that the S19 differential PCR method established by the present invention has good specificity and can be used for the establishment and application of the S19 differential diagnosis PCR method;

[0011] Step 3: To verify the sensitivity of the primers in the present invention patent, the above amplified fragments are respectively constructed on the pMD19-T vector to prepare plasmid standards. After calculating the gene copy number, perform 10-fold serial dilution, and take 1×10 7 、~1×10 0The positive plasmid of 1 copies / μL was used for subsequent experiments. According to the PCR system and reaction conditions described in Step 2, the sensitivity of the specific primers was analyzed. The detection primer sensitivity of S19 was 10 2 copies / μL, and the sensitivity of the detection primers designed for the gene of A19 was 10

[0012] copies / . The results showed that the sensitivity of the identified specific primers was 10 - 100 copies / μL, with high sensitivity, and could be used for the detection of S19.

[0013] Step 4: To establish a Brucella S19 differential PCR method, the effects of specific identification extension time and annealing temperature on the PCR detection method were explored. To explore the effects of extension time and annealing temperature on the established PCR detection method, other conditions were relaxed, and the variables were only the extension time and annealing temperature; the optimized Brucella S19 differential PCR method of this invention patent determined the optimal PCR system as follows: 20 μL reaction system: 10 μL 2×Rapid Taq Master Mix, 1 μL each of the upstream and downstream primers, 1 μL of the template, and supplemented with ddH2O. Optimization conditions: annealing temperature 60°C, extension time 20 s, cycling program: pre-denaturation at 95°C for 3 min, 35 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 20 s), and final extension at 72°C for 5 min. The entire PCR reaction process was 1 hour and 11 minutes, with good practical value for clinical detection.

[0014] A Brucella S19 differential PCR detection primer, the primers are Ba.c2.gap0m4.F5 and Ba.c2.gap0m4.R5, and their nucleotide sequences are respectively:

[0015] Ba.c2.gap0m4.F5: 5'-TGACACGCGGCATATAACCAA-3;

[0016] Ba.c2.gap0m4.R5: 5'-TTATCAAGATCGCCATCGACT-3'.

[0017] The application of the above S19 differential PCR detection primer in the preparation of products for simultaneous detection of S19 and A19.

[0018] The application of the above differential PCR detection primer in the preparation of products for diagnosing diseases caused by S19 infection.

[0019] In the above application, the product is a kit.

[0020] A detection kit, comprising the above-mentioned PCR detection primers and PCR detection reagents.

[0021] A differential PCR detection method for detecting Brucella S19, using the above primers to perform PCR detection on S19;

[0022] The PCR system is as follows: the total volume is 20 μL (10 μL of 2×PCR Mix, 1 μL of each of the upstream and downstream primers, 1 μL of the template), and the PCR reaction program is: 95°C, 3 min; 95°C, 15 s, 60°C, 15 s, 72°C, 20 s, for 35 cycles; 72°C, 5 min; the entire PCR reaction process takes 1 hour and 11 minutes.

[0023] Regarding the problems existing in the prior art, some creative technical effects brought after solving the problems are as follows. The specific description is as follows:

[0024] First, the present invention conducts high-throughput collinearity analysis on the genomes of S19 and A19. By analyzing the differential regions between the genomes of the two Brucella strains, and by designing specific primers, specific primers for the differential diagnosis of Brucella S19 are screened out, which proves the creativity of this invention patent; there is a high degree of conservation between the genomes of different Brucella strains, and most Brucella genomes have only a small number of single nucleotide polymorphisms. It is difficult to screen specific sequences from a large area of the genome. The present invention uses the differential gene fragments of two attenuated vaccine strains of Brucella abortus, and it is a challenging accidental event to design specific primer sequences that can differentially diagnose a single strain from a very small number of differential fragments, which has great creativity.

[0025] In addition, through optimizing reaction conditions such as extension time and annealing temperature, the present invention patent establishes a differential PCR detection method for S19 for the first time at home and abroad, and conducts sensitivity, specificity and repeatability tests. The PCR detection method established by the present invention has the clinical advantages of strong specificity, good sensitivity and timeliness, and can be used for the rapid differential diagnosis of S19 infection and epidemiological investigation, which has important significance.

[0026] Second, the expected benefits and commercial value after the transformation of the technical solution of the present invention are as follows: Brucellosis is one of the important zoonotic diseases that affect the breeding industry and human health in various countries and regions of the world. After being infected, cattle and sheep mainly show symptoms such as fever and diseases of the reproductive organs, such as abortion, infertility, sterility, and retention of the fetal membranes. Brucella infection in humans can cause damage to multiple systems throughout the body, especially the osteoarticular system, manifested as symptoms such as fever, sweating, general weakness, joint swelling, and muscle pain. In severe cases, the ability to work may be lost. Brucellosis is a major zoonotic disease that has drawn international attention and is a key area of prevention in China. Currently, the Brucella S19 vaccine has been widely used in the cattle breeding industry in many countries around the world, and China has also approved the use of S19 in the breeding industry. The current key problem is that there is no clinical rapid detection method for S19, which will fill the long-term technical gap in Brucella prevention and control. According to the current demand, it has a large market prospect, and the expected benefits and commercial value after transformation are relatively large.

[0027] The technical solution of the present invention fills the technical gaps in the domestic and international industries: This invention patent has established a PCR method for differentiating and detecting the S19 vaccine for the first time at home and abroad, filling the gap that there is no method for differentiating and diagnosing the S19 vaccine strain at home and abroad.

[0028] The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been successful: Clinically, the existing Brucella detection technologies have problems such as insufficient specificity, low sensitivity, and inability to distinguish between vaccine strains and wild strains. This invention has established a rapid and highly specific S19 vaccine strain differentiation PCR detection method, providing technical support for the clinical diagnosis, epidemiological investigation, and scientific prevention and control of the above-mentioned pathogen infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The technical solution of the present invention will be further described below in conjunction with the drawings:

[0030] Figure 1 is the result diagram of the specific difference segments between the S19 and A19 genomes analyzed by high-throughput Mauve genome collinearity analysis;

[0031] Figure 2 is the result diagram of the screening of Brucella S19 differentiation PCR primers;

[0032] Figure 3 is the result diagram of the specificity identification of Brucella S19 differentiation PCR;

[0033] Figure 4 is the result diagram of the sensitivity identification of Brucella S19 differentiation PCR;

[0034] Figure 5 is the result diagram of the optimization of the conditions of the Brucella S19 differentiation PCR detection method;

[0035] Figure 6 It is a diagram showing the test results of clinical samples by the Brucella S19 differential PCR detection method. Specific implementation manners

[0036] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific implementation manners. However, those skilled in the art should understand that the implementation manners described below are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention.

[0037] The Brucella S19 differential PCR detection method provided by the present invention includes the following steps:

[0038] Step 1: Obtain the genomic sequences of Brucella abortus S19 (GCF_000018725.1) and A19 (GCF_003290345.1) from the NCBI database, and use high-throughput Mauve genome collinearity analysis to analyze the specific differential regions of the S19 and A19 genomes. According to the results of the S19 and A19 genome collinearity analysis, design and screen primers Ba.c2.gap0m4.F5 (5'-TGACACGCGGCATATAACCAA-3') and Ba.c2.gap0m4.R5 (5'-TTATCAAGATCGCCATCGACT-3'). See the appendix Figure 1 as shown.

[0039] Step 2: According to the results of the genomic high-throughput differential interval analysis, use primer design software to design and synthesize S19-specific primers that can be differentiated; a total of 2 pairs of PCR primer sequences are designed and synthesized, and the primer information is shown in Table 1. Use a bacterial genomic DNA extraction kit to extract the positive genomes of S19 and A19 respectively, and dilute them to 0.1 ng / μL respectively, and use them as templates for PCR amplification. Perform 1% agarose gel electrophoresis on the PCR products respectively to observe the specificity and sensitivity of the two pairs of S19 differential detection primers, and screen out the primers with good sensitivity and clear amplified bands. The PCR amplification system is: 1 μL of template, 10 μL of 2×PCR Mix, 7 μL of ddH2O, 1 μL each of the designed upstream and downstream primers (10 μmol·L-1), for a total of 20 μL. The reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 15 s, for 35 cycles; extension at 72°C for 5 min. The amplification results are shown in the appendix Figure 2 .

[0040] Table 1 Primer information

[0041]

[0042] Step 3: Sensitivity is a key link in the PCR technique. To verify the sensitivity of the newly screened and identified target and its specific primers, according to the instruction manual of the Tiangen DNA extraction kit, genomic DNA of S19 and A19 was extracted respectively; using the extracted DNA as a template, the designed primers were used to amplify the target sequence respectively; after the amplified products were recovered and purified, they were ligated to the T vector. The three ligation products were transformed into DH5-α competent cells. After coating the plates, positive clone bacteria were immediately screened and expanded. The target plasmids were extracted for amplification and sequencing identification. After successfully constructing the recombinant plasmid standards, the original copy numbers of the two recombinant plasmid standards can be obtained according to the copy number calculation formula, and then they were diluted to 1×10 8 copies / μL respectively, and then serially diluted 10-fold. Take 1×10 7 、~1×10 0 copies / μL of the positive plasmids for subsequent experiments. Analyze the sensitivity of the specific primers according to the PCR system and reaction conditions described in Step 2. The sensitivity of S19 is 10 1 copies / μL, and the sensitivity of A19 is 10 2 copies / μL. The above results show that the sensitivity of the screened and identified PCR specific primers is between 10-100 copies / μL, indicating that the established PCR method has high sensitivity and can be used for the detection of Brucella, as shown in the appendix Figure 4 shown

[0043] Step 4: The specific primers designed in the present invention are mainly used for the rapid detection of S19. Therefore, in order to establish a Brucella S19 differential PCR method and explore the effects of extension time and annealing temperature on the PCR detection method, the specific implementation plan is as follows:

[0044] 1. Screening of PCR amplification extension time

[0045] The PCR amplification system is: 1 μL of template, 10 μL of 2×PCR Mix, 7 μL of ddH2O, 1 μL each of the designed upstream and downstream primers (10 μmol·L-1), totaling 20 μL. The reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 60, 30, 20, 15, 10 s respectively, 35 cycles; extension at 72°C for 5 min

[0046] 2. Optimal annealing temperature for PCR amplification

[0047] The PCR amplification system is as follows: 1 μL of template, 10 μL of 2×PCR Mix, 7 μL of ddH2O, 1 μL each of the designed upstream and downstream primers (10 μmol·L-1), totaling 20 μL. The reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 50, 50.6, 51.5, 52.6, 54, 55.7, 57.1, 58.2, 59.2, 60°C for 15 s respectively, extension at 72°C for 20 s respectively, with 35 cycles; extension at 72°C for 5 min.

[0048] By optimizing the conditions, the optimal PCR system was determined as follows: total volume of 20 μL (10 μL of 2×Rapid Taq MasterMix, 1 μL each of the determined upstream and downstream primers, 1 μL of template), and the optimal PCR reaction program was: 95°C, 3 min; 95°C, 15 s, 60°C, 15 s, 72°C, 20 s, for 35 cycles; 72°C, 5 min. The PCR reaction process has good practical value for clinical detection. See the appendix Figure 5 as shown. Meanwhile, it was found that in addition to being able to amplify specific bands with Brucella strains, the designed primers did not produce specific bands with other important bacterial pathogens of cattle and sheep infected through the respiratory tract, proving that the S19 differential PCR method established in the present invention has good specificity. See the appendix Figure 3 as shown.

[0049] Step five, using the established differential PCR detection method, perform compliance detection on clinical samples of Brucella isolates. From the detection results, it can be seen that all 43 clinical isolates of Brucella abortus, Brucella melitensis, and Brucella suis can be accurately detected, which is consistent with the expectation. This indicates that the differential PCR technology established in the present invention can be used for the identification and detection of clinical Brucella, with good results. See the appendix Figure 6 as shown.

[0050] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Brucella S19 differential PCR detection primer, characterized in that, The primers are Ba.c2.gap0m4.F5 and Ba.c2.gap0m4.R5, and their nucleotide sequences are respectively: Ba.c2.gap0m4.F5: 5'-TGACACGCGGCATATAACCAA-3; Ba.c2.gap0m4.R5: 5'-TTATCAAGATCGCCATCGACT-3'.

2. The application of the S19 identification PCR detection primer described in claim 1 in the preparation of a product for simultaneously detecting S19 and A19.

3. The application of the identification PCR detection primer described in claim 1 in the preparation of a product for diagnosing diseases caused by S19 infection.

4. The application according to claim 2 or claim 3, characterized in that, The product is a kit.

5. A detection kit, characterized in that, It includes the PCR detection primer and the PCR detection reagent described in claim 1.

6. A PCR detection method for detecting Brucella S19, characterized in that, Using the primer described in claim 1 to perform PCR detection on S19; The PCR system is: the total volume is 20 μL (10 μL of 2×PCR Mix, 1 μL of each upstream and downstream primer, 1 μL of template), and the PCR reaction program is: 95°C, 3 min; 95°C, 15 s, 60°C, 15 s, 72°C, 20 s, 35 cycles; 72°C, 5 min; the entire PCR reaction process is 1 hour and 11 minutes.

7. A method for establishing an S19 differential PCR detection method, characterized in that, It includes the following steps: Step 1, obtain the genomic sequences of Brucella abortus biovar 1 S19 (GCF_000018725.1) and A19 (GCF_003290345.1) from the NCBI database, perform collinearity analysis using the Mauve software, and screen out the S19-specific region; Step 2, design available PCR primer sequences according to the differential fragments; screen out the primer sequences available for differential diagnosis; Step 3, amplify and sequence through the primer sequences to obtain the differential diagnosis marker molecule, and verify the new diagnosis marker through specificity and sensitivity; Step 4, establish and optimize the S19 differential diagnosis PCR method; Step 5, use the established S19 identification PCR method to perform compliance detection on Brucella-positive clinical isolates.

8. The method for establishing the S19 identification PCR detection method according to claim 7, characterized in that, The specific content of step 3 is: The target fragments were respectively constructed on the pMD19-T vector to prepare plasmid standards. After calculating the gene copy number, 10-fold serial dilutions were performed, and positive plasmids with 1×10 7 , ~1×10 0 copies / μL were used for subsequent experiments; Analyze the sensitivity of the specific primer according to the PCR system and reaction conditions described in step 2.

9. The method for establishing the S19 identification PCR detection method according to claim 7, characterized in that, The specific content of step 4 is: Explore the influence of the extension time and annealing temperature on the differential PCR detection method; The optimized S19 differential diagnosis PCR method determines the optimal PCR system as: a 20 μL reaction system: 10 μL of 2×Rapid TaqMaster Mix, 1 μL of each upstream and downstream primer, 1 μL of template, supplemented with ddH2O; optimization conditions: annealing temperature 60°C, extension time 20 s, the cycling program is 95°C pre-denaturation for 3 min, 35 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 20 s), 72°C final extension for 5 min; the entire PCR reaction process is 1 hour and 11 minutes.

Images