Specific primer and probe for detecting bovine tuberculosis and kit composed of specific primer and probe

Abstract

The invention provides a specific primer and probe for detecting bovine tuberculosis and a kit composed of the specific primer and probe, and belongs to the technical field of biological detection. Specific primer pairs and probes are designed for cattle CXCL10, IFN-gamma and GAPDH genes, and quantitative detection of the transcription level of CXCL10 and IFN-gamma in cattle in-vitro whole blood is realized by using a qPCR technology, so that cattle tuberculosis is diagnosed. The method greatly shortens the detection time (incubation only needs 4 hours), simplifies the operation process, eliminates the system error in the sample adding process by using the GAPDH gene as the internal reference, and improves the accuracy of the detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a specific primer for detecting bovine tuberculosis, a probe and a kit composed of the specific primer and the probe. BACKGROUND

[0002] Mycobacterium bovis is the main pathogen causing bovine tuberculosis (bTB), which is a zoonotic pathogen that can infect humans and cause serious public health risks. The disease causes significant economic losses in global livestock industry (including milk yield reduction, culling of sick animals, trade restrictions, etc.), and is difficult to eradicate by conventional means. At present, China classifies bovine tuberculosis as a class II animal disease, and compulsory quarantine and purification are required, so efficient and accurate detection technology is urgently needed to support the prevention and control work.

[0003] Interferon-Gamma Release Assay (IGRA) is the current internationally recognized mainstream diagnostic method for bovine tuberculosis, which is based on the detection of host cell immune response to tuberculosis-specific antigens. Although IGRA has high specificity and sensitivity, its technical process has a significant time delay defect, and antigen stimulation needs to be incubated for 16-24 hours, followed by ELISA / ELISPOT detection.

[0004] IP-10 is IFN-gamma chemokine 10 (also known as CXCL10), which is the latest discovered molecular marker for human tuberculosis. It can be detected in tuberculosis granulomas and is involved in the delayed hypersensitivity reaction caused by mycobacterial antigens. When the lymphocytes of tuberculosis patients come into contact with tuberculosis mycobacterial antigens again, IP-10 and IFN-gamma can be transcribed in a short time, and the detection of the transcription levels of the two can greatly shorten the diagnosis time of bovine tuberculosis.

[0005] The current detection method for bovine tuberculosis, such as Interferon-Gamma Release Assay (IGRA), has high specificity and sensitivity, but its technical process has a significant time delay defect, and the operation is complicated and time-consuming, which cannot meet the requirements of rapid and efficient detection. SUMMARY

[0006] The purpose of the present application is to provide a method for simply, rapidly and efficiently detecting bovine tuberculosis.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a set of primers and probe combinations for detecting bovine tuberculosis, which are as follows:

[0009] CXCL10-F: 5'-GATCTCCTCGAACACGGAAAG-3';

[0010] CXCL10-R: 5'-ACACAGGGAAACAGTTGTAGAG-3';

[0011] CXCL10-P: 5'-ACTGCACTACTGATAAGGATGGACCAGA-3';

[0012] IFN-γ-F: 5'-GATCTGGATTCTGAGCCACTAC-3';

[0013] IFN-γ-R: 5'-GCCAGGTATAAGGTGAGATGAG-3';

[0014] IFN-γ-P: 5'-TGTGTCAGGCTATATGACTTGTGCCC-3';

[0015] GAPDH-F: 5'-CAGCGACACTCACTCTTCTAC-3';

[0016] GAPDH-R: 5'-CCTGTTGCTGTAGCCAAATTC-3';

[0017] GAPDH-P: 5'-TTGCCCTCAACGACCACTTTGTCA-3'.

[0018] The application provides application of the primer and probe combination in preparation of a kit for detecting bovine tuberculosis.

[0019] Preferably, the probe is modified by a fluorescent reporter group and a fluorescent quencher group;

[0020] The fluorescent reporter group of the CXCL10 gene and the IFN-γ gene is FAM; the fluorescent reporter group of the GAPDH gene is VIC; and the fluorescent quencher group is BHQ1.

[0021] The kit is used as follows:

[0022] (1) placing the sample to be detected in a culture tube containing stimulating antigen, and shaking the culture tube up and down violently for 30-40 seconds, so that the sample to be detected covers the whole tube wall and foam appears on the tube wall;

[0023] (2) then immediately placing it vertically upward in a water bath box at 35-40°C for incubation for 3-5 hours, extracting RNA, and then reverse transcribing into cDNA;

[0024] (3) the cDNA obtained in step (2) is divided into two parts, one part is subjected to qPCR reaction by using the primer probe composition of the CXCL10 and GAPDH, and the other part is subjected to qPCR reaction by using the primer probe composition of the IFN-γ and GAPDH, and the results of the reactions are used to judge the positivity of the sample to be detected.

[0025] The present application is directed to the bovine CXCL10, IFN-γ and GAPDH genes, and specific primer pairs and probes are designed, and the transcription levels of the bovine CXCL10 and IFN-γ in the whole blood in vitro are quantitatively detected by using the qPCR technology, so that the bovine tuberculosis is diagnosed. The present application can greatly shorten the detection time (only 4 hours of incubation is needed), simplify the operation process, eliminate the systematic error in the sample adding process by using the GAPDH gene as an internal reference, and improve the accuracy of the detection results.

[0026] Compared with the prior art, the present application is based on the qPCR technology, and the primers and probes are designed and the detection method is established for the bovine CXCL10, IFN-γ and GAPDH genes, and the purpose of detecting the bovine tuberculosis is achieved by calculating the Ct value of the results, and the present application has the following beneficial effects:

[0027] (1) rapid detection: the whole blood is stimulated and incubated for 4 hours, and then RNA extraction and detection are performed.

[0028] (2) simple operation: after the extracted RNA is reverse transcribed, it is added to the prepared qPCR reaction system, and the results can be determined by calculating the Ct value, and compared with the frequent incubation, plate washing and detection in the ELISA process, the process is simple and fast.

[0029] (3) the qPCR system established in the present application can quickly and sensitively detect the results, and has good accuracy and stability. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 the amplification efficiency of the CXCL10 primer probe;

[0031] Figure 2 the amplification efficiency of the IFN-γ primer probe;

[0032] Figure 3 the amplification efficiency of the GAPDH primer probe;

[0033] Figure 4 the comparison of 10 bovine tuberculosis positive detection results;

[0034] Figure 5 the comparison of 10 bovine tuberculosis negative detection results. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present application will be described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0036] Example 1

[0037] According to the bovine CXCL10 (NM_001046551.2), IFN-γ (NM_174086.1) and GAPDH (NM_001034034.2) genes downloaded from NCBI, the specific primer probe was designed by using Primer Premier 5 software.

[0038] Table 1 primer and probe sequence information table

[0039]

[0040] Among them, the 5' end of the probe for detecting the CXCL10 gene and the IFN-γ gene is labeled with a FAM fluorescent group, the 5' end of the probe for detecting the GAPDH gene is labeled with a VIC fluorescent group, and the 3' end of all probes is labeled with a BHQ1 quenching group.

[0041] Example 2: amplification efficiency of primer probe

[0042] The plasmids of bovine CXCL10, IFN-γ and GAPDH genes were synthesized by experiment, and the synthesis steps were as follows:

[0043] (1) According to the gene specificity primer designed according to the pUC19 vector and the gene source, the bovine whole blood mRNA was used as the template for PCR experiment;

[0044] (2) The PCR product recovered by gel was connected to the pUC vector, and the DH5α chemically competent cells were used to transform the vector connection product, and the LB solid medium plate containing ampicillin antibiotic was used for screening;

[0045] (3) Eight single colonies were extracted for culture, and the bacteria were shaken for 4-6h, and PCR amplification was carried out, and the brightest band was selected by 1.0% agarose gel electrophoresis detection, 200ul of which was taken to GenScript Biotech (Shanghai) Co., Ltd. for sequencing, and the positive recombinant plasmid was extracted according to the sequencing results, and the positive recombinant plasmid was used as a standard for constructing real-time fluorescent quantitative PCR.

[0046] The initial concentrations were 7.03×10 10 copies / μL, 8.98×10 10 copies / μL and 3.92×10 10The plasmid was serially diluted to obtain eight working standards at concentrations of 5 × 10⁻⁶ copies / μL. 8 Copies / μL; Working standard 2, concentration 5×10⁻⁶ 7 Copies / μL; Working standard 3, concentration 5×10⁻⁶ 6 copies / μL; Working standard 4, concentration 5×10⁴ 5 Copies / μL; Working standard 5, concentration 5×10⁻⁵ 4 Copies / μL; Working standard 6, concentration 5×10⁶ 3 copies / μL; Working standard 7, concentration 5×10⁷ 2 Copies / μL; Working standard 8, concentration 5×10⁸ 1 copies / μL.

[0047] Reaction system: 10 μL of 2×Premix Ex Taq (Probe qPCR), 0.8 μL of PCR Forward Primer (10 μM), 0.8 μL of PCR Reverse Primer (10 μM), 0.4 μL of Probe (10 μM), 2 μL of working standard, and water to a final volume of 20 μL.

[0048] The reaction procedure was as follows: pre-denaturation at 95℃ for 30s, PCR reaction at 95℃ for 5s, and amplification at 60℃ for 30s for 40 cycles.

[0049] like Figure 1 , Figure 2 , Figure 3 As shown, the standard curve fitting of the CXCL10 gene is R. 2 The value was 0.998, the amplification efficiency was 104.65%, and the R value of the IFN-γ gene standard curve fitting was [value missing]. 2 The value was 0.999, the amplification efficiency was 105.77%, and the standard curve fitting R for the GAPDH gene was [value missing]. 2 The amplification efficiency was 101.58%, both of which met the requirements for qPCR detection experiments.

[0050] Example 3: Sensitivity Experiment

[0051] The lowest copy number detectable by the primers and probes designed in Example 1 was determined using in vitro transcribed RNA targeting the relevant target. The specific steps are as follows:

[0052] (1) Specific primers containing the T7 promoter were designed based on the detection target and PCR amplification was performed;

[0053] (2) The amplification products were transcribed in vitro using the Novizan T7 HighYield Transcription Kit and purified using VAHTS RNA CleanBeads;

[0054] (3) After detecting the concentration, the copy number was calculated and serial dilutions were performed to obtain 8 working standards with different concentrations, namely Working Standard 1, with a concentration of 1×10⁻⁶. 7 Copies / μL; Working standard 2, concentration 1×10⁻⁶ 6 Copies / μL; Working standard 3, concentration 1×10⁻⁶ 5 copies / μL; Working standard 4, concentration 1×10⁴ 4 copies / μL; 5 working standards, concentration 1×10⁵ 3 copies / μL; Working standard 6, concentration 1×10⁶ 2 copies / μL; Working standard 7, concentration 1×10⁷ 1 Copies / μL; Working standard 8, concentration 1×10⁸ 0 copies / μL;

[0055] (4) Using PrimeScript TM Reverse transcription was performed using an RT reagent kit with gDNA Eraser.

[0056] (5) The reaction system in Example 2 was used for detection. Each group of qPCR was repeated 3 times, and the Ct value was taken as the average value. The results are shown in Table 2.

[0057] Table 2 Sensitivity Detection Experiment

[0058]

[0059] Note: *The Ct value appears only twice in three repetitions, #The Ct value appears only once in three repetitions.

[0060] As shown in Table 2, a stable Ct value can be obtained when the copy number is 10 copies / ul, enabling rapid and sensitive detection results.

[0061] Example 4: Repeatability Test

[0062] Using the plasmid and reaction system constructed in Example 2, the repeatability of the primers and probes designed in Example 1 in multiple experiments was tested. The specific steps are as follows:

[0063] (1) Select 1×10 2 copies / μL, 1×10 4copies / μL, 1×10 6 Plasmids with a copy / μL concentration;

[0064] (2) The reaction system in Example 2 was used to perform 6 replicates in the same batch of experiments, and the coefficient of variation between the data was calculated;

[0065] (3) The reaction system in Example 2 was used to conduct experiments on days 1, 3 and 5. Each experiment was repeated 6 times, and the coefficient of variation between the data of the three experiments was calculated. The results are shown in Table 3.

[0066] Table 3 Results of repeatability tests for the qPCR system

[0067]

[0068] As shown in Table 3, the coefficients of variation within and between batches are both less than 5%, indicating that the qPCR system established in this study has good accuracy and stability.

[0069] Example 5: Clinical Sample Testing

[0070] The primer and probe reaction system designed in Example 1 was used to test 20 bovine whole blood samples. Ten samples were negative for bovine tuberculosis (botulism-negative) from bovine tuberculosis-free areas (detected negative by the Vitasoy bovine tuberculosis-specific cellular immunoassay kit), and the other ten were positive for bovine tuberculosis (botulism-positive) from farms with a high prevalence of bovine tuberculosis (detected positive by the Vitasoy bovine tuberculosis-specific cellular immunoassay kit). The specific steps are as follows:

[0071] (1) Take 5 mL of heparin-anticoagulated whole blood sample from the tail vein of the cattle to be tested from each sample, and aliquot it into three culture tubes (provided by the stimulation part of the Bovine Mycobacterium tuberculosis Specific Cell Immunoreactivity Detection Kit) for negative control N (containing PBS), positive control P (containing PMA) and detection T (containing Mycobacterium tuberculosis specific antigen), 1 mL per tube. Then shake the culture tube vigorously up and down for 40 seconds to make the blood cover the entire tube wall and foam appear on the tube wall.

[0072] (2) Then immediately place it upright in a 37°C water bath for 4 hours before use. RNABlood Mini Handbook was used for whole blood RNA extraction. The extracted RNA was then processed using PrimeScript. TM The RTreagent Kit with gDNA Eraser was used for reverse transcription to cDNA.

[0073] (3) Divide the cDNA obtained in step (2) into two portions. Perform qPCR on one portion using the primer and probe combination of CXCL10 and GAPDH, and perform qPCR on the other portion using the primer and probe combination of IFN-γ and GAPDH. Select the Ct value after the qPCR reaction for paired T test. Determine the positive or negative result of the sample according to the calculation method provided based on the reaction results. The results are as follows: Figure 4 , Figure 5 As shown.

[0074] The method for determining gender is as follows:

[0075] N-channel: ΔCtN = Ct value (FAM channel) - Ct value (VIC channel);

[0076] T-tube: ΔCtT = Ct value (FAM channel) - Ct value (VIC channel);

[0077] P-tube: ΔCt P = Ct value (FAM channel) - Ct value (VIC channel);

[0078] T value = ΔCt T - ΔCtN;

[0079] P value = ΔCt P - ΔCtN.

[0080] Table 4 Result Judgment Table

[0081]

[0082] If the p-value is greater than -0.83, it indicates that the immune cells in the blood sample are malfunctioning, and the result will be meaningless.

[0083] The results showed that there was no response to Mycobacterium tuberculosis-specific T cells in the whole blood of bovine tuberculosis-negative cattle, so the transcriptional levels of CXCL10 and IFN-γ did not change significantly before and after stimulation. In contrast, there was a response to Mycobacterium tuberculosis-specific T cells in the whole blood of bovine tuberculosis-positive cattle, so the transcriptional levels of CXCL10 and IFN-γ in the whole blood increased significantly after stimulation with Mycobacterium tuberculosis-specific proteins.

[0084] Comparative Example 1

[0085] VitiAi's Bovine Mycobacterium Tuberculosis Specific Cellular Immunoassay Kit (ELISA) and Thermo Fisher's Bovine Mycobacterium Tuberculosis Gamma Interferon Detection Kit The 2G results showed a compliance rate of over 84% under different parameters.

[0086] This method was compared with the VitiAi Bovine Mycobacterium tuberculosis-specific cellular immunoassay kit (ELISA). The specific steps are as follows:

[0087] (1) Collect 30 whole blood samples from cattle to be tested;

[0088] (2) Each whole blood sample was divided into two small samples (5 mL), and the detection methods of Example 5 and the Bovine Mycobacterium tuberculosis-specific cell immunoreaction detection kit of Vitti were used for detection. Each small sample was then aliquoted into three culture tubes: negative control N (containing PBS), positive control P (containing PMA), and detection T (containing Mycobacterium tuberculosis-specific antigen), 1 mL in each tube, and incubated in an incubator at 37°C for 4 h and 22 h, respectively.

[0089] (3) Whole blood samples incubated for 22 hours were tested using the VetiAi Bovine Mycobacterium tuberculosis specific cell immunoassay kit (enzyme-linked immunosorbent assay). The positive and negative results were calculated according to the kit instructions.

[0090] Whole blood samples incubated for 4 hours were tested using the method described in Example 5, and the results were judged as shown in Table 5.

[0091] Table 5. Comparison results between this method and the vitiligo detection kit.

[0092]

[0093] As shown in Table 5, among the 30 samples, this method detected 14 positive samples and 16 negative samples. The VetiAi Bovine Mycobacterium tuberculosis-specific cellular immune response detection kit detected 11 positive samples and 19 negative samples. The overall concordance rate was (11+16) / 30×100%=90%.

[0094] Comparative Example 2

[0095] This method was compared with the in vitro detection method for interferon-gamma (IFN-γ) described in GB / T18645-2020. The specific steps are as follows:

[0096] (1) Collect 25 whole blood samples from cattle to be tested;

[0097] (2) Each whole blood sample was divided into two small samples (5 mL), and the detection methods in Comparative Example 1 and the in vitro detection method for γ-interferon (IFN-γ) described in GB / T18645-2020 were used respectively. The results are shown in Table 6.

[0098] Table 6 Comparison results between our method and the national standard method

[0099]

[0100] As shown in the table above, among the 25 samples, this method detected 24 positive samples and 1 negative sample. The in vitro detection method for γ-interferon (IFN-γ) described in GB / T18645-2020 detected 19 positive samples and 6 negative samples. The overall concordance rate is (19+1) / 25×100%=80%.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer and probe combination for detecting bovine tuberculosis, characterized in that, The primer and probe combination is shown below: CXCL10-F: 5'-GATCTCCTCGAACACGGAAAG-3'; CXCL10-R: 5'-ACACAGGGAAACAGTTGTAGAG-3'; CXCL10-P: 5'-ACTGCACTACTGATAAGGATGGACCAGA-3'; IFN-γ-F: 5'-GATCTGGATTCTGAGCCACTAC-3'; IFN-γ-R: 5'-GCCAGGTATAAGGTGAGATGAG-3'; IFN-γ-P: 5'-TGTGTCAGGCTATATGACTTGTGCCC-3'; GAPDH-F: 5'-CAGCGACACTCACTCTTCTAC-3'; GAPDH-R: 5'-CCTGTTGCTGTAGCCAAATTC-3'; GAPDH-P: 5'-TTGCCCTCAACGACCACTTTGTCA-3'.

2. The use of the primer and probe combination of claim 1 in the preparation of a kit for detecting bovine tuberculosis.

3. The application as described in claim 2, characterized in that, The probe is modified with a fluorescent reporter group and a fluorescent quencher group; The fluorescent reporter groups for the CXCL10 and IFN-γ genes are FAM; the fluorescent reporter group for the GAPDH gene is VIC; and the fluorescent quencher group for all genes is BHQ1.

4. The application as described in claim 3, characterized in that, The method of using the kit is as follows: (1) Place the sample to be tested in a culture tube containing the stimulating antigen, and shake the culture tube vigorously up and down for 30 to 40 seconds so that the sample to be tested covers the entire tube wall and foam appears on the tube wall. (2) Then immediately place it upright in a water bath at 35-40°C for 3-5 hours, extract RNA, obtain RNA of the sample to be tested and reverse transcribe it into cDNA; (3) Divide the cDNA obtained in step (2) into two equal parts. Perform qPCR reaction on one part using the primer and probe combination of CXCL10 and GAPDH, and perform qPCR reaction on the other part using the primer and probe combination of IFN-γ and GAPDH. Determine the positive or negative result of the sample to be tested based on the reaction results.

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