Dual TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for brucella

Abstract

The invention relates to the technical field of bacterial detection, and discloses a dual TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for Brucella, which comprises the following steps: designing specific primers and probes according to nucleic acid sequences of Brucella galU and Omp31, and constructing a recombinant plasmid standard substance containing galU and Omp31 full-length fragments; optimizing a dual TaqMan fluorescent quantitative PCR reaction system, wherein the total reaction system is 20 [mu] L; establishing a standard curve and verifying specificity, sensitivity and repeatability; a sample is detected by using the dual TaqMan fluorescent quantitative PCR reaction system, and the brucella infection condition is judged and bovine species and non-bovine species brucella are distinguished by detecting galU and Omp31 genes; the detection method provided by the invention is strong in specificity, high in sensitivity and good in repeatability, can simultaneously realize general detection of the Brucella and differential diagnosis of bovine species / non-bovine species Brucella, is wide in application range, and provides efficient and convenient technical support for clinical detection of the Brucella and purification of animal brucellosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacterial detection, more particularly, it relates to a dual TaqMan fluorescence quantitative PCR detection method for Brucella. BACKGROUND

[0002] Brucellosis (Brucellosis for short) is one of the widespread and harmful zoonoses caused by Brucella infection. For a long time, Brucellosis has brought immeasurable losses to the economy of animal husbandry, and has brought serious hidden dangers to the safety of animal products and national biological safety. Recent studies have found that there are 12 species and 34 biotypes of Brucella, among which the ovine, bovine and porcine Brucella are the most harmful to humans and animals. After animal infection, it can cause clinical symptoms such as reproductive system disorders mainly including abortion, orchitis, arthritis, etc. Human infection is mostly transmitted from animals, and symptoms such as fever, infertility, arthritis, spondylitis, and nerve damage often occur. 16%-25% of the world's population are threatened by the disease, and the global human incidence is about 2.1 million. The global economic losses caused by the disease are about 3-4 billion US dollars per year. Implementing animal Brucellosis purification is an important path for global Brucellosis prevention and control, and is also the ultimate goal of Brucellosis prevention and control. Among the existing Brucellosis diagnosis technologies in China, most are based on serological methods, such as the tube agglutination test (SAT), the rose bengal plate agglutination test (RBT), the enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT), and the colloidal gold rapid detection test paper.

[0003] Serological detection is a commonly used diagnostic method for human and animal Brucellosis detection, but due to the limitations of specificity, sensitivity, application range, etc., it is currently impossible to make accurate judgments, and it cannot be used for differential diagnosis. Etiological detection is a key method for Brucellosis diagnosis, especially bacterial isolation, which is the "gold standard" for Brucellosis diagnosis. However, bacterial isolation requires BSL-3 laboratory conditions and is difficult to achieve. Therefore, a more sensitive and convenient Brucella etiological detection method is needed to overcome the limitations of detection methods based on bacterial culture and antigen-antibody combination. At the same time, the combined application of etiological and serological detection methods will greatly improve the accuracy of animal Brucellosis detection, and is an urgently needed detection method for animal Brucellosis purification. SUMMARY

[0004] Compared with serological detection, molecular biology diagnosis technology is widely used in pathogen infection detection due to its strong specificity, high sensitivity and other characteristics. With the development of molecular biology, PCR-based pathogen detection technology is also widely used in animal brucellosis diagnosis. For example, the PCR detection method based on BCSP31 gene can not only accurately diagnose human and animal brucellosis, but also detect animal products and environmental pollution. Real-time fluorescent quantitative PCR (qPCR) technology is a nucleic acid quantitative technology developed on the basis of PCR qualitative technology, which is a method widely used in nucleic acid detection and quantification. TaqMan qPCR method is based on the use of specific primers and probes, which are combined with target DNA sequences and cut off during the amplification process to produce a fluorescent signal. Compared with dye qPCR, it has stronger specificity. In previous studies, it was found that galU is a highly conserved virulence gene of Brucella, which can be used for bacterial detection of Brucella; outer membrane protein 31 gene (Omp31) is a conserved gene of non-bovine Brucella, which can be used to distinguish bovine Brucella and other species of Brucella. On this basis, the full-length fragment recombinant plasmid is constructed based on the nucleic acid sequences of galU and Omp31, and a sensitive and specific Brucella TaqMan double fluorescent quantitative PCR detection method is established, which is used for accurate detection of Brucella infection in humans and animals, and contaminated animal products and environment, and combined with existing serological detection methods to provide scientific, accurate and efficient detection technology for animal brucellosis purification in China.

[0005] The application provides a Brucella double TaqMan fluorescent quantitative PCR detection method, which comprises the following steps:

[0006] Step 1: specific primers and probes are designed according to the nucleic acid sequences of Brucella galU and Omp31, wherein the galU primers and probes are used for detecting all species of Brucella, and the Omp31 primers and probes are used for distinguishing bovine Brucella from other species of Brucella;

[0007] Step 2: a recombinant plasmid standard containing galU and Omp31 full-length fragments is constructed;

[0008] Step 3: the double TaqMan fluorescent quantitative PCR reaction system is optimized, and the total reaction system is 20 μL;

[0009] Step 4: a standard curve is established and specificity, sensitivity and repeatability are verified;

[0010] Step 5: the double TaqMan fluorescent quantitative PCR reaction system is used to detect samples, and the Brucella infection is determined and bovine and non-bovine Brucella are distinguished by detecting galU and Omp31 genes.

[0011] Preferably, the primer sequence designed for the galU gene in step 1 is as follows:

[0012] galU-F: 5'-GAACGGGGGAAAACCGAAGA-3'

[0013] galU-R: 5'-AGTGCAAAAGGTTCGTTGCC-3'

[0014] galU-P: FAM-CAGCAGGTGCCGCTTGGCCTC-BHQ.

[0015] Preferably, the primer sequence designed for the Omp31 gene in step 1 is as follows:

[0016] Omp31-F: 5'-TTCCGCCCCTACTGCTGCT-3'

[0017] Omp31-R: 5'-CCGGAAACCTGTTCGTTGTC-3'

[0018] Omp31-P: HEX-ACGCCGGTTACGCAGGCGGCAAGTT-BHQ1.

[0019] Preferably, the specific steps for constructing the recombinant plasmid standard in step 2 are as follows:

[0020] Step 21: Amplify the galU and Omp31 genes respectively using the nucleic acid of the sheep Brucella as a template;

[0021] Step 22: Perform reverse PCR amplification on the puC19 plasmid sequence to obtain a linearized fragment;

[0022] Step 23: Insert the galU and Omp31 sequences into the puC19 plasmid through seamless cloning;

[0023] Step 24: Transform into competent cells, pick colonies for PCR identification and sequencing verification;

[0024] Step 25: Extract the recombinant plasmid and calculate the copy number.

[0025] Preferably, the optimized double TaqMan fluorescent quantitative PCR reaction system in step 3 is as follows:

[0026] SuperReal fluorescent quantitative premix reagent 10 μL, galU-F / R and Omp31-F / R primers each 1 μL, galU-P and Omp31-P probes each 0.25 μL, template 3 μL, and deionized water 2.5 μL;

[0027] The final concentration of the primer is 500nM, and the final concentration of the probe is 125nM.

[0028] Preferably, the PCR reaction condition in step 3 is as follows: pre-denaturation at 95℃ for 15min; denaturation at 95℃ for 3s, annealing and extension at 57℃ for 30s, and a total of 40 cycles.

[0029] Preferably, in step 4, the standard curve is established by 10 times of continuous dilution of the recombinant plasmid standard, and the concentration range is 1.93×103-1.93×109copies·μL-1.

[0030] Preferably, the detection and judgment method in step 5 is as follows:

[0031] Step 51: galU positive, Omp31 positive: non-bovine brucella infection;

[0032] Step 52: galU positive, Omp31 negative: bovine brucella infection;

[0033] Step 53: galU negative: brucella negative.

[0034] Preferably, the minimum detection concentration of nucleic acid of the brucella double TaqMan fluorescent quantitative PCR detection method is 0.1-100fg·μL-1.

[0035] Preferably, the minimum detection limit of the brucella double TaqMan fluorescent quantitative PCR detection method is 1.93×100copies·μL-1, the intra-batch coefficient of variation is 0.02%-0.55%, and the inter-batch coefficient of variation is 0.45%-1.28%.

[0036] The application of a brucella double TaqMan fluorescent quantitative PCR detection method is used for detecting brucella infected human and animals, and brucella contaminated animal products and environmental samples.

[0037] The brucella double TaqMan fluorescent quantitative PCR detection method has the following advantages:

[0038] 1. Strong specificity: no cross reaction with nucleic acid of other gram-negative bacteria, which can be used as a general brucella pathogen detection method, and can effectively distinguish bovine brucella and other species of brucella, and realize differential diagnosis.

[0039] 2. High sensitivity: the minimum detection limit of the recombinant plasmid standard reaches 1.93×100copies·μL-1, the minimum detection concentration of nucleic acid is 0.1-100fg·μL-1, and the sensitivity is obviously higher than that of conventional PCR and rose agglutination test.

[0040] 3. Good repeatability: the intra-batch and inter-batch test results are stable, and the coefficient of variation is less than 2%, which ensures the reliability of the detection results.

[0041] 4. Wide application range: suitable for detection of Brucella infection in human, animals and contaminated animal products and environmental samples, and verified effective through clinical deer serum, sheep serum and sheep spleen samples.

[0042] 5. High detection efficiency: compared with PCR and the tiger red agglutination test, the sensitivity is 100%, 81.92% and 51.47% respectively, and the detection rate of the double TaqMan qPCR method is significantly improved.

[0043] 6. Breakthrough technical limitations: overcome the limitations of bacterial culture requiring BSL-3 laboratory conditions, and provide an operable Brucella pathogenic diagnosis means for a routine laboratory.

[0044] 7. Support disease prevention and control: provide scientific, accurate and efficient technical support for clinical detection, epidemiological investigation and purification of animal brucellosis, which has important significance for the purification of animal brucellosis in China.

[0045] The detection method provided by the present application has strong specificity, high sensitivity and good repeatability, can simultaneously realize universal detection and differential diagnosis of bovine and non-bovine Brucella, has wide application range, and provides efficient and convenient technical support for clinical detection of Brucella and purification of animal brucellosis. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a PCR amplification diagram of the present application;

[0047] Figure 2 is an Omp31 and galU standard curve of the present application;

[0048] Figure 3 is a specific test result of the present application;

[0049] Figure 4 is a sensitivity test result of the present application. DETAILED DESCRIPTION

[0050] The subject matter described herein will now be discussed with reference to example implementations. It should be understood that the discussion of these implementations is merely meant to provide a better understanding of the subject matter described herein and can be changed without departing from the scope of the present specification. Various processes or components can be omitted, substituted, or added according to desired implementations. Additionally, features described in some examples can be combined in other examples.

[0051] Example 1

[0052] In this embodiment, a double TaqMan fluorescent quantitative PCR detection method for Brucella is provided, which comprises the following steps:

[0053] Step 1: Design specific primers and probes according to the nucleic acid sequences of Brucella galU and Omp31, wherein the galU primers and probes are used to detect all species of Brucella, and the Omp31 primers and probes are used to distinguish B. abortus from other species of Brucella;

[0054] Step 2: Construct recombinant plasmid standards containing full-length fragments of galU and Omp31;

[0055] Step 3: Optimize the double TaqMan fluorescent quantitative PCR reaction system, and the total reaction system is 20 μL;

[0056] Step 4: Establish a standard curve and verify specificity, sensitivity, and repeatability;

[0057] Step 5: Use the double TaqMan fluorescent quantitative PCR reaction system to detect samples, and determine Brucella infection and distinguish B. abortus from non-B. abortus by detecting galU and Omp31 genes.

[0058] Wherein:

[0059] The primer sequence designed in step 1 for the galU gene is:

[0060] galU-F: 5'-GAACGGGGGAAAACCGAAGA-3'

[0061] galU-R: 5'-AGTGCAAAAGGTTCGTTGCC-3'

[0062] galU-P: FAM-CAGCAGGTGCCGCTTGGCCTC-BHQ.

[0063] The primer sequence designed in step 1 for the Omp31 gene is:

[0064] Omp31-F: 5'-TTCCGCCCCTACTGCTGCT-3'

[0065] Omp31-R: 5'-CCGGAAACCTGTTCGTTGTC-3'

[0066] Omp31-P: HEX-ACGCCGGTTACGCAGGCGGCAAGTT-BHQ1.

[0067] The specific steps for constructing the recombinant plasmid standard in step 2 are as follows:

[0068] Step 21: Amplify the galU and Omp31 genes respectively using the nucleic acid of B. melitensis as a template;

[0069] Step 22: Reverse PCR amplification of puC19 plasmid sequence to obtain linearized fragment;

[0070] Step 23: Insert galU and Omp31 sequences into puC19 plasmid by seamless cloning;

[0071] Step 24: Transform into competent cells, pick colonies for PCR identification and sequencing verification;

[0072] Step 25: Extract recombinant plasmid and calculate copy number.

[0073] The optimized double TaqMan fluorescent quantitative PCR reaction system in step 3 is as follows:

[0074] SuperReal fluorescent quantitative premix reagent 10 μL, galU-F / R and Omp31-F / R primers each 1 μL, galU-P and Omp31-P probes each 0.25 μL, template 3 μL, and deionized water 2.5 μL;

[0075] The final concentration of primers is 500 nM, and the final concentration of probes is 125 nM.

[0076] The PCR reaction conditions in step 3 are as follows: 95°C pre-denaturation for 15 min; 95°C denaturation for 3 s, 57°C annealing and extension for 30 s, a total of 40 cycles.

[0077] In step 4, the recombinant plasmid standard was diluted by 10 times in succession, and the standard curve was established using the concentration range of 1.93×103-1.93×109copies·μL-1.

[0078] The detection judgment method in step 5 is as follows:

[0079] Step 51: galU positive, Omp31 positive: non-bovine brucella infection;

[0080] Step 52: galU positive, Omp31 negative: bovine brucella infection;

[0081] Step 53: galU negative: brucella negative.

[0082] The minimum detection concentration of the brucella double TaqMan fluorescent quantitative PCR detection method is 50 fg·μL-1.

[0083] The minimum detection limit of the brucella double TaqMan fluorescent quantitative PCR detection method is 1.93×100copies·μL-1, the intra-batch coefficient of variation is 0.35%, and the inter-batch coefficient of variation is 0.9%.

[0084] Example 2

[0085] The difference between this embodiment and embodiment 1 is that:

[0086] The minimum detection concentration of the Brucella double TaqMan fluorescent quantitative PCR detection method is 0.1 fg·μL-1.

[0087] The minimum detection limit of the Brucella double TaqMan fluorescent quantitative PCR detection method is 1.93x100 copies·μL-1, the within-run coefficient of variation is 0.02%, and the between-run coefficient of variation is 0.45%.

[0088] Example 3

[0089] The difference between this embodiment and embodiment 1 is that:

[0090] The minimum detection concentration of the Brucella double TaqMan fluorescent quantitative PCR detection method is 100 fg·μL-1.

[0091] The minimum detection limit of the Brucella double TaqMan fluorescent quantitative PCR detection method is 1.93x100 copies·μL-1, the within-run coefficient of variation is 0.55%, and the between-run coefficient of variation is 1.28%.

[0092] Example 4

[0093] 1. Materials and methods

[0094] 1.1 Nucleic acid

[0095] The nucleic acids of Brucella and other gram-negative bacteria required by the institute were stored in the laboratory.

[0096] 1.2 Main reagents and instruments

[0097] TIANprep Mini Plasmid Kit plasmid extraction kit, TIANgel Purification Kit agarose gel DNA recovery kit, SuperReal fluorescent quantitative premix reagent, Beijing Tiangen Biochemical Technology Co., Ltd.; ClonExpress MultiS One Step Cloning Kit seamless cloning kit, XL10 chemically competent cells, 2x Phanta Max Master Mix high-fidelity enzyme premix, Novozyme Biopharmaceutical Technology Co., Ltd.; Primers and probes were synthesized by Shanghai Sangon Biological Engineering Technology Service Co., Ltd.

[0098] QuantStudio 3 Real-Time PCR System, NanoDrop Micro-volume Spectrophotometer, Dry Heater, Thermo Fisher Scientific; FIRE READER XSD-56-20M Gel Imaging System, uvitec; Constant Temperature Incubator, Shanghai Santeng Instrument Co., Ltd.; SCI1000-G Gradient PCR Instrument, Silekt Co., Ltd.

[0099] 1.3 Primer and probe design

[0100] According to the reference sequence of the galU (Gene ID: 29595478) and Omp31 (Gene ID: 29595531) genes and the reference sequence of the puC19 plasmid of the Brucella melitensis 16M strain in GenBank, three pairs of primers with homologous arms and two pairs of specific primers and probes were designed using the NCBI primer-BLAST software (Table 1), and were synthesized by Shanghai Biotechnology Co., Ltd.

[0101] Table 1 Primer and probe sequences

[0102]

[0103] 1.4 Construction and identification of recombinant plasmid

[0104] The galU and Omp31 genes and the puC19 plasmid sequence were amplified by reverse PCR using the nucleic acid sequence of the Brucella melitensis 16M strain as a template. The amplification products were separated by agarose gel electrophoresis, and the target nucleic acid fragments were recovered. The galU and Omp31 sequences were inserted into the puC19 plasmid by seamless cloning, transformed into BL21 competent cells, and then cultured overnight on ampicillin LB solid medium. After single colony PCR identification, the cells were transferred to LB liquid medium for expansion culture. The bacterial liquid was extracted for plasmid extraction, and then sent to Shanghai Biotechnology Co., Ltd. for sequencing. The plasmid with correct nucleic acid sequence after sequencing was named puC19-galU&Omp31. The plasmid concentration was measured using an ultramicro spectrophotometer, the copy number was calculated, and the plasmid was diluted with deionized water to a final concentration of 1.93 x 10-1.93 x 109copies·μL-1, and stored at -20℃ for standby. Calculation formula: Plasmid copy number (copies·μL-1) = 6.02 x 1023x 10-9x plasmid concentration (ng·μL-1) / plasmid base length x 660

[0105] 1.5 Double qPCR reaction system optimization and standard curve establishment

[0106] The primer and probe concentration was optimized by square matrix method. The total system was 20 μL, the SuperReal fluorescent quantitative premix reagent was 10 μL, the final concentration of primer was 500 nM, 250 nM, 125 nM, and the final concentration of probe was 250 nM, 125 nM, 62.5 nM, 3 μL of 1.93×106 copies·μL-1 recombinant plasmid, and 2.5 μL of deionized water.

[0107] The recombinant plasmid puC19-galU&Omp31 with a concentration of 1.93×109 copies·μL-1 was continuously diluted by 10 times, and the concentrations of 1.93×103-1.93×109 copies·μL-1 were used as templates. Each concentration was repeated for 3 times, and double qPCR amplification was performed. The standard curve was plotted with the logarithmic value of plasmid concentration as the abscissa and the cycle threshold (Ct) as the ordinate.

[0108] 1.6 Specificity test

[0109] The DNA of Brucella melitensis 16M strain, Brucella abortus A19 strain, Acinetobacter, Clostridium perfringens, enteropathogenic Escherichia coli, meningitis-causing Escherichia coli, and Pasteurella multocida were used as templates, and deionized water was used as negative control. The optimized double qPCR reaction system was used for amplification to detect the specificity of the method.

[0110] 1.7 Sensitivity test

[0111] The recombinant plasmid puC19-galU&Omp31 was continuously diluted by 10 times, i.e. 1.93×100-1.93×104 copies·μL-1, and the plasmid at 8 concentrations was used as template. The optimized double qPCR reaction system was used for amplification, and deionized water was used as negative control to detect the sensitivity of the method.

[0112] 1.8 Minimum nucleic acid detection concentration test

[0113] The nucleic acids of Brucella melitensis 16M strain, Brucella abortus A19 strain, and Brucella suis S2 strain were continuously diluted by 10 times, i.e. 1 ng·μL-1-0.1 fg·μL-1, and the 8 concentrations were used as templates. The optimized double qPCR reaction system was used for amplification, and deionized water was used as negative control to determine the minimum nucleic acid detection concentration.

[0114] 1.9 Reproducibility test

[0115] The 1.93 x 104, 1.93 x 105, 1.93 x 106copies·μL-1three concentrations of recombinant plasmid puC19-galU&Omp31 were selected as templates, each concentration was repeated for 3 times, after double qPCR amplification, the coefficient of variation within the group was calculated. Each gradient was repeated for 3 times in 3 different batches, and the coefficient of variation between groups was calculated. According to the formula: The coefficients of variation within and between groups were calculated.

[0116] 1.10 Clinical sample detection and compliance test

[0117] The 52 deer sera and 19 sheep sera collected in clinic were detected by the Rose Bengal plate agglutination test, and the negative and positive brucellosis were determined. The nucleic acid was extracted, and the nucleic acid of 38 sheep spleens infected with Brucella was extracted. The double TaqMan qPCR method established in this study was used for detection, and the general Brucella PCR detection method of national standard was used for re-detection, and the coincidence rate of the two methods was compared and analyzed.

[0118] 2 Results

[0119] 2.1 Construction of recombinant plasmid puC19-galU&Omp31

[0120] The nucleic acid sequence of ovine Brucella 16M strain was used as a template, and the target fragments were amplified by PCR using P-galUF / R, P-Omp31-F / R, and puC19-F / R. The results are shown in Figure 1 The target fragments with expected sizes were obtained, which were galU (917 bp), Omp31 (755 bp), and puC19 linearized fragment (2698 bp). The amplified products with homologous arms were connected with the linearized puC19 fragment by seamless cloning to construct the recombinant plasmid, which was named puC19-galU&Omp31. The recombinant plasmid was extracted, and the concentration of the recombinant plasmid was measured to be 90.7 ng·μL-1. According to the calculation, the copy number of puC19-galU&Omp31 recombinant plasmid was 1.93 x 1010copies·μL-1.

[0121] Figure 1 In the PCR amplification:

[0122] M. DNA molecular weight marker; 1. galU; 2. Omp31; 3. puC19 linearized fragment;

[0123] 2.2 Optimization of double qPCR reaction conditions and establishment of standard curve

[0124] The primer and probe concentration were optimized using 1.93 x 106copies^mL-1puC19-galU & Omp31 recombinant plasmid by square method (Table 2), and the total system of duplex qPCR was determined as 20 μL: SuperRealPreMix (Probe) 10 μL, galU-F / R and Omp31-F / R 1 μL each, galU-P and Omp31-P 0.25 μL each, recombinant plasmid template 3 μL, and deionized 2.5 μL. The reaction program was 95℃ for 15 min; 95℃ for 3 s, 57℃ for 30 s, for a total of 40 cycles. The recombinant plasmid puC19-galU & Omp31 was diluted by 10 times successively, and the concentration of 1.93 x 103-1.93 x 109copies^mL-1was used as a template to perform duplex qPCR amplification to draw a standard curve. The results showed that the optimal primer concentration was 500 nM, and the probe was 125 nM (Table 2). The standard curve of galU was y = -3.561x + 44.069, R2= 0.999, and the standard curve of Omp31 was y = -3.38x + 43.455, R2= 0.998 Figure 2

[0125] Table 2 Primer and probe concentration optimization and results

[0126]

[0127] Figure 2 : Omp31 and galU standard curves.

[0128] 2.3 Specificity test

[0129] The DNA of Brucella melitensis 16M strain, Brucella abortus A19 strain, Acinetobacter, Clostridium perfringens, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Pasteurella multocida, Proteus vulgaris, and Pseudomonas aeruginosa was amplified by the duplex qPCR method established in this study. The results Figure 3 ) showed that the method had good specificity. The amplification results of Brucella melitensis 16M strain showed that galU and Omp31 were positive, the amplification results of Brucella abortus A19 strain showed that galU was positive and Omp31 was negative, and the amplification results of other bacterial nucleic acids were all negative.

[0130] Figure 3 : Specificity test results.

[0131] A. Omp31 gene; B. galU gene; 1. Brucella melitensis 16M strain; 2. Brucella abortus A19 strain; 3. Acinetobacter, Clostridium perfringens, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Pasteurella multocida, Proteus vulgaris, and Pseudomonas aeruginosa; N. deionized water.

[0132] ​2.4 Sensitivity test

[0133] The puC19-galU&Omp31 recombinant plasmid was gradient diluted, and 1.93 x 100-1.93 x 104copies·μL-1of the 15 gradient concentrations were subjected to double qPCR amplification. The results (Table 2) showed that the method had high sensitivity, and the minimum detection was 1.93 x 100copies·μL-1. Figure 4

[0134] Figure 4 Sensitivity test results.

[0135] A. Omp31 gene; B. galU gene; 1-5. Concentration of plasmid (104-100copies·μL-1).

[0136] 2.5 Minimum detection concentration test of nucleic acid

[0137] In this study, the nucleic acids of Brucella melitensis M28 strain, Brucella abortus A19 strain and Brucella suis S2 strain were diluted by 10 times successively, and 1 ng·μL-1-0.1 fg·μL-1of the 8 gradients were used as templates. Double qPCR method and BCSP-31 primer were used for PCR amplification. The minimum detection concentration of nucleic acid by double qPCR method was between 0.1-100 fg·μL-1, and the minimum detection concentration of nucleic acid by PCR method was 100 fg·μL-1.

[0138] 2.6 Reproducibility test

[0139] In this study, the puC19-galU&Omp31 recombinant plasmid was diluted by 10 times gradient, and 1.93 x 104-1.93 x 106copies·μL-1of the 3 gradients were subjected to reproducibility test, and the coefficient of variation was calculated. The results (Table 3) showed that the average coefficient of variation within the group was between 0.02%-0.55%, and the coefficient of variation between the groups was between 0.6%-1.8%. It was shown that the method established in this study had good reproducibility.

[0140] Table 3 Reproducibility test results

[0141]

[0142] 2.7 Detection of clinical samples

[0143] ​The established Brucella double TaqMan qPCR detection method was used to detect 52 deer serum samples, 38 sheep spleen samples and 19 serum samples collected from the clinic. The results are shown in Table 4. The qPCR nucleic acid galU positive rate of the clinical samples was 69.75% (83 / 119), the Omp31 detection rate was 68.91% (82 / 119), the PCR positive rate was 57.14% (68 / 119), and the positive rate of the deer serum and sheep serum rose bengal plate agglutination test was 43.21% (35 / 81). The sensitivity of qPCR was higher than that of rose bengal plate agglutination test and PCR. In the qPCR detection, a total of 83 detection samples were positive for Brucella nucleic acid, of which 82 samples were infected with other Brucella, and 1 sample was infected with B. abortus.

[0144] Table 4 Sample detection results

[0145]

[0146] 3 Discussion

[0147] Brucella is a facultative intracellular parasite that can cause animal joint swelling, abortion, and nervous system damage. The galU gene is located on the second chromosome of Brucella and encodes uridine triphosphate-glucose-1-phosphate-uridylyltransferase, which is involved in the indirect regulation of bacterial glycoprotein and glycolipid synthesis in the sugar metabolism pathway. The nucleic acid sequence of Brucella is highly conserved. The Omp31 gene is located on the second chromosome of Brucella, and Omp31 encodes a channel protein and forms a complex with peptidoglycan, which is a heme-binding protein of Brucella and plays a role in the iron metabolism pathway of Brucella. The Omp31 gene nucleic acid sequence is highly conserved in other Brucella species except B. abortus, which is a good molecular detection marker.

[0148] In recent years, TaqMan fluorescence quantitative PCR technology has been widely used in the detection of clinical samples due to its good sensitivity, excellent specificity, and the ability to design multiple primers and probes in the reaction system to diagnose and differentiate multiple pathogens. Currently, there have been studies on Brucella detection methods at home and abroad. Chang Xiujun et al. established a fluorescence quantitative PCR detection method based on Brucella IS711, and the detection rate of human peripheral blood samples was higher than that of blood culture (39% vs. 15%)[8]. Sanni et al. established a TaqMan fluorescence quantitative PCR detection method based on Brucella Omp31 gene, and the sensitivity and specificity (98%, 100%) were higher than those of iELISA (87.5%, 90%) and test tube agglutination test (56%, 75%)

[10] . The authors constructed a TaqMan fluorescent quantitative PCR method to distinguish Brucella biovar by using the nucleic acid site BS1330_II0657 of swine Brucella, and the sensitivity of the method was higher than that of single nucleotide polymorphism analysis (detection limit of 50 fg·μL-1) in detecting swine serum mixed with swine Brucella genome (detection limit of 12.5 fg·μL-1)

[11] . The above-mentioned studies showed excellent sensitivity and excellent specificity in detecting clinical samples and simulated samples by using the fluorescent quantitative PCR method for detecting Brucella or identifying Brucella biovar. In this study, a double TaqMan fluorescent quantitative PCR method for detecting Brucella infection and identifying bovine Brucella was designed by using galU and Omp31 specific fluorescent probes and primers, and the detection results of 119 deer serum samples, sheep serum samples and sheep spleen samples showed good diagnostic and identification effects. Among the 52 deer serum samples, the nucleic acid detection rate of the double TaqMan fluorescent quantitative PCR method was 90.38%, and one deer serum sample infected with bovine Brucella was identified, the PCR nucleic acid detection rate was 88.46%, and the positive rate of the rose bengal agglutination test was 26.92%; among the 29 sheep serum samples, the nucleic acid detection rate of the double TaqMan fluorescent quantitative PCR method was 72.41%, the PCR nucleic acid detection rate was 44.83%, and the positive rate of the rose bengal agglutination test was 72.41%; among the 38 sheep spleen samples, the nucleic acid detection rate of the double TaqMan fluorescent quantitative PCR method was 39.47%, and the PCR nucleic acid detection rate was 23.68%. The sensitivity of the double TaqMan fluorescent quantitative PCR method was higher than that of PCR and the rose bengal agglutination test.

[0149] 4CONCLUSION

[0150] Through the above research, the present study established a specific, sensitive and reproducible Brucella double fluorescent quantitative PCR detection method, which can be used for detecting Brucella infection in humans and animals and Brucella-contaminated animal products, and can be combined with serological methods for precise detection of animal Brucellosis and technical support for animal Brucellosis purification.

[0151] The above describes embodiments of the present application, but the embodiments are not limited to the specific implementation described above, which is only illustrative rather than limiting, and those skilled in the art can make more forms of equivalent embodiments under the inspiration of the embodiments, which are all within the protection scope of the embodiments.

Claims

1. A Brucella dual TaqMan fluorescent quantitative PCR detection method, characterized in that, The steps include: Step 1: Design specific primers and probes based on the nucleic acid sequences of Brucella galU and Omp31, wherein the galU primer and probe are used to detect all species of Brucella, and the Omp31 primer and probe are used to distinguish Brucella bovis from other species of Brucella; Step 2: Construct a recombinant plasmid standard containing galU and the full-length fragment of Omp31; Step 3: Optimize the dual TaqMan fluorescent quantitative PCR reaction system, with a total reaction volume of 20 μL; Step 4: Establish a standard curve and verify specificity, sensitivity, and repeatability; Step 5: Detect the sample using the dual TaqMan fluorescent quantitative PCR reaction system, determine the Brucella infection status and distinguish bovine and non-bovine Brucella by detecting galU and Omp31 genes.

2. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The primer sequences designed for the galU gene in step 1 are: galU-F: 5'-GAACGGGGGAAAACCGAAGA-3' galU-R: 5'-AGTGCAAAAGGTTCGTTGCC-3' galU-P:FAM-CAGCAGGTGCCGCTTGGCCTC-BHQ.

3. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The primer sequences designed for the Omp31 gene in step 1 are: Omp31-F: 5'-TTCCGCCCCTACTGCTGCT-3' Omp31-R: 5'-CCGGAAACCTGTTCGTTGTC-3' Omp31-P:HEX-ACGCCGGTTACGCAGGCGGCAAGTT-BHQ1.

4. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The specific steps for constructing the recombinant plasmid standard in step 2 are: Step 21: Using Brucella melitensis nucleic acid as a template, amplify the galU and Omp31 genes respectively; Step 22: Reverse PCR amplifies the puC19 plasmid sequence to obtain a linearized fragment; Step 23: Insert the galU and Omp31 sequences into the puC19 plasmid by seamless cloning; Step 24: Transform into competent cells and pick colonies for PCR identification and sequencing verification; Step 25: Extract the recombinant plasmid and calculate the copy number.

5. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The optimized dual TaqMan fluorescence quantitative PCR reaction system in step 3 is: SuperReal fluorescent quantitative premix reagent 10 μL, galU-F / R and Omp31-F / R primers 1 μL each, galU-P and Omp31-P probes 0.25 μL each, template 3 μL, deionized water 2.5 μL; The final concentration of primers was 500 nM, and the final concentration of probes was 125 nM.

6. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The PCR reaction conditions in step 3 were as follows: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 3 s, and annealing and extension at 57°C for 30 s, for a total of 40 cycles.

7. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, In step 4, a standard curve was established by serially diluting the recombinant plasmid standard 10-fold using a concentration range of 1.93×103-1.93×109 copies·μL-1.

8. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The detection and determination method in step 5 is: Step 51: galU positive, Omp31 positive: non-bovine Brucella infection; Step 52: galU positive, Omp31 negative: Brucella bovis infection; Step 53: galU negative: Brucella negative.

9. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The minimum detection concentration of nucleic acid for Brucella dual TaqMan fluorescence quantitative PCR detection method is 0.1-100 fg·μL-1.

10. A Brucella dual TaqMan fluorescent quantitative PCR detection method according to claim 1, characterized in that, The minimum detection limit of the Brucella dual TaqMan fluorescence quantitative PCR detection method was 1.93×100 copies·μL-1, the intra-assay coefficient of variation was 0.02%-0.55%, and the inter-assay coefficient of variation was 0.45%-1.28%.

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