Kit for detecting non-human primate tuberculosis and application thereof

Abstract

The invention discloses a kit for detecting non-human primate tuberculosis and application thereof, and relates to the technical field of biology, the kit comprises a specific primer probe combination, a whole blood stimulant, a one-step RT-qPCR reaction solution and a nucleic acid extraction reagent; through the specific primer probe combination designed according to the characteristics of the non-human primate CXCL10 gene and the internal reference ACTB gene, cross-species universal detection can be achieved, reagents do not need to be independently designed according to different species, the application range is widened, the two probes are used for marking different fluorophores and corresponding quenching groups respectively, and the detection sensitivity is high. In the amplification process, double-channel synchronous detection can be realized, signal interference is avoided, the specificity and stability of detection are guaranteed, a whole blood stimulation process is matched with a one-step RT-qPCR reaction system, the operation steps are simplified, the loss and error in the sample treatment process are reduced, meanwhile, the whole detection period is shortened, the actual requirement of rapid screening is met, and the detection efficiency is improved. And a high-quality detection basis is provided for subsequent result interpretation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a kit for detecting tuberculosis in non-human primates and its application. Background Technology

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which has long threatened human public health and safety, and also seriously affects the survival of non-human primates. Non-human primates are highly similar to humans in anatomy, physiology and immunology, and are indispensable animal models for tuberculosis research. In recent years, there have been multiple outbreaks of tuberculosis in non-human primates in captivity, quarantine and wild environments around the world, involving multiple types of mycobacteria. The potential risk of zoonotic transmission is becoming increasingly prominent, and there is an urgent need for the timeliness and accuracy of related biosafety monitoring.

[0003] However, existing detection technologies have many limitations and are difficult to meet practical application needs. Traditional tuberculin skin tests are easily affected by external factors, resulting in unreliable results. Although bacterial culture has high specificity, the process is time-consuming and cannot achieve rapid screening. Modern interferon-gamma release tests are cumbersome to operate, and a single indicator cannot comprehensively reflect the body's immune status. Automated molecular diagnostic technologies have high requirements for equipment and cost, which is not conducive to large-scale promotion. Serological tests can only identify antibodies in the later stages of infection, and their effectiveness in detecting early or latent infections is limited. In addition, there is a lack of cross-species universal detection tools for commonly used non-human primates, and existing detection schemes have not formed a suitable rapid stimulation and accurate interpretation system. Nucleic acid extraction reagents have not been optimized for the characteristics of whole blood samples after stimulation, which further restricts the efficiency and accuracy of detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a kit for detecting tuberculosis in non-human primates and its application. This invention utilizes a combination of specific primers and probes designed to target the characteristics of the CXCL10 gene and the internal reference ACTB gene in non-human primates, enabling universal detection across species without the need for separately designed reagents for different species, thus broadening the application scope. The two probes are labeled with different fluorescent groups and corresponding quenching groups, enabling simultaneous dual-channel detection during amplification, avoiding signal interference, and ensuring the specificity and stability of the detection. The whole blood stimulation process, combined with a one-step RT-qPCR reaction system, simplifies the operation steps, reduces sample processing losses and errors, and shortens the overall detection cycle, meeting the practical needs of rapid screening and providing a high-quality detection foundation for subsequent result interpretation.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On the one hand, a kit for detecting tuberculosis in non-human primates, the kit comprising: a specific primer-probe combination, whole blood stimulant, a one-step RT-qPCR reaction solution and a nucleic acid extraction reagent; The specific primer-probe combination is designed for the non-human primate CXCL10 gene and the internal reference ACTB gene, including the CXCL10 detection combination and the ACTB internal reference detection combination; The whole blood irritants include bovine tuberculin bPPD, avian tuberculin aPPD, and a positive control mitogen; The one-step RT-qPCR reaction solution provides the enzyme and ionic environment required for reverse transcription and amplification reactions; The nucleic acid extraction reagent extracts total RNA from stimulated whole blood samples.

[0006] Furthermore, the sequence of the specific primer-probe combination is as follows: CXCL10 detection combination: upstream primer SEQ ID NO:1: 5'-CTGACTCTAAGTGGTATTCAAGGAA-3'; downstream primer SEQ ID NO:2: 5'-AGACCTTGGATTAACAGGTTGA-3'; probe SEQ ID NO:3: 5'-FAM-TCTCAAGAACTGTACGCTGTACCTGCA-BHQ1-3'; ACTB internal reference detection combination: upstream primer SEQ ID NO:4: 5'-AGCACGATGAAGATCAAGATCA-3'; downstream primer SEQ ID NO:5: 5'-GATGGAAGGGCCAGACTC-3'; probe SEQ ID NO:6: 5'-VIC-ACTCCTGCTTGCTGATCCACATCTGCT-MGB-3'.

[0007] Furthermore, the probe SEQ ID NO:3 of the CXCL10 detection combination is labeled with a FAM fluorescent group at its 5' end and a BHQ1 quencher group at its 3' end; the probe SEQ ID NO:6 of the ACTB internal reference detection combination is labeled with a VIC fluorescent group at its 5' end and an MGB quencher group at its 3' end.

[0008] Furthermore, the final concentration of bovine tuberculin bPPD and avian tuberculin aPPD used in the whole blood stimulant is 10 µg / mL; the positive control mitogen is a mixture of PWM / PMA.

[0009] Furthermore, the reaction system for the one-step RT-qPCR is 20 μL, specifically composed of: 10 μL 2× reaction buffer, 0.4 μL of 10 μM CXCL10 upstream primer, 0.4 μL of 10 μM ACTB upstream primer, 0.4 μL of 10 μM CXCL10 downstream primer, 0.4 μL of 10 μM ACTB downstream primer, 0.2 μL of 10 μM CXCL10 probe, 0.2 μL of 10 μM ACTB probe, 5 μL RNA template, and the remainder being RNase-free water; wherein the final concentrations of the CXCL10 upstream primer, ACTB upstream primer, CXCL10 downstream primer, and ACTB downstream primer are all 0.4 μM, and the final concentrations of the CXCL10 probe and ACTB probe are both 0.2 μM.

[0010] Furthermore, the nucleic acid extraction reagent includes a lysis buffer, a washing buffer, and an elution buffer; the lysis buffer contains a detergent, proteinase K, and an RNase inhibitor; the washing buffer contains isopropanol and ethanol; and the elution buffer is RNase-free water or TE buffer; wherein the detergent is one or a combination of two of Triton X-100 and SDS; and the RNase inhibitor is RNAsin or Superasin.

[0011] On the other hand, a method for applying a reagent kit for detecting tuberculosis in non-human primates, the specific steps of which are as follows: In vitro stimulation of whole blood: Non-human primate heparin-anticoagulated whole blood to be tested was taken, and negative control, aPPD stimulant, bPPD stimulant and positive control were added respectively, and incubated at 37°C for 4 hours; the negative control was a reaction system without any stimulant, the aPPD stimulant was avian tuberculin, the bPPD stimulant was bovine tuberculin, and the positive control was a PWM / PMA mixture; RNA extraction: After incubation, total RNA was extracted from whole blood samples using nucleic acid extraction reagents; Amplification detection: One-step RT-qPCR was used to amplify and detect the CXCL10 and ACTB genes in the extracted total RNA using a specific primer and probe combination. The reaction was performed on a real-time quantitative PCR instrument, and the FAM and VIC channel signals were collected. Result determination: Using 2 -ΔΔCt The relative expression level of CXCL10 mRNA was calculated using a method, and the results were determined based on a two-dimensional composite diagnostic criterion.

[0012] Furthermore, in the amplification detection, the one-step RT-qPCR adopts a standard reaction procedure, specifically: reverse transcription at 50°C for 30 minutes, pre-denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing extension at 60°C for 1 minute.

[0013] Furthermore, in the result determination, the two-dimensional composite diagnostic criteria are as follows: the absolute response level threshold Cut-off 1 for bPPD is set to 2.70, and the differential response level, i.e., the threshold Cut-off 2 for the difference between the bPPD stimulus value and the aPPD stimulus value, is set to 1.46; when the absolute response level of bPPD in a sample is >2.70 and the differential response level is >1.46, it is determined to be positive; when these conditions are not met and the absolute response level of bPPD is <2.21, or the absolute response level of bPPD is >2.70 but the differential response level is <1.46, it is determined to be negative.

[0014] Furthermore, the lowest detection limit of the application method is 1.0 × 10⁻⁶. 3 Copy / mL.

[0015] Compared with existing technologies, this reagent kit for detecting tuberculosis in non-human primates and its application have the following advantages: I. This invention utilizes a combination of specific primers and probes designed to target the characteristics of the CXCL10 gene and the internal reference ACTB gene in non-human primates. This enables universal detection across species, eliminating the need for separately designed reagents for different species and broadening the application scope. The two probes are labeled with different fluorescent groups and corresponding quenching groups, enabling simultaneous dual-channel detection during amplification, avoiding signal interference, and ensuring the specificity and stability of the detection. The whole blood stimulation process, combined with the one-step RT-qPCR reaction system, simplifies the operation steps, reduces sample processing losses and errors, and shortens the overall detection cycle, meeting the practical needs of rapid screening and providing a high-quality detection foundation for subsequent result interpretation.

[0016] II. This invention employs a dual-dimensional composite diagnostic standard to capture specific reaction signals triggered by Mycobacterium tuberculosis infection while effectively eliminating cross-reaction interference from environmental mycobacteria, thus improving the accuracy of result determination. The nucleic acid extraction reagent consists of lysis buffer, washing buffer, and elution buffer. These components work synergistically to efficiently break down cells and degrade contaminating proteins, while protecting RNA from degradation throughout the process. This effectively removes impurities from the sample, ensuring the integrity and purity of the extracted total RNA. This design, combined with the specific primer-probe combination and optimized reaction system, creates a synergistic effect, guaranteeing high sensitivity of the detection method and enabling accurate identification of early or low-level infection states. This provides reliable support for the prevention and control of tuberculosis in non-human primates and related research.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 A flowchart illustrating the application method of a reagent kit for detecting tuberculosis in non-human primates; Figure 2 A trend diagram of relative expression levels of CXCL10 mRNA under different stimulation times; Figure 3 The standard curve and sensitivity analysis diagram of the CXCL10 mRNA RT-qPCR detection method are shown. Figure 4 This is a frequency distribution map of the absolute response levels of bPPD in clinical samples. Figure 5 This is a frequency distribution map of the differential response levels in clinical samples (bPPD group - aPPD group). Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example 1: In a laboratory setting for the clinical diagnosis of tuberculosis in rhesus monkeys, clinical diagnosis was conducted on rhesus monkeys under observation after artificial infection with Mycobacterium tuberculosis. This assessed the infection status and disease progression of the monkeys, providing reliable data support for subsequent research such as the study of tuberculosis pathological mechanisms and the evaluation of vaccine development effectiveness. The detection kit used included a specific primer-probe combination, whole blood stimulant, one-step RT-qPCR reaction solution, and nucleic acid extraction reagents, with each component adapted to the genetic characteristics and detection requirements of rhesus monkeys. The specific primer-probe combination was designed for the rhesus monkey CXCL10 gene and the internal control ACTB gene. The CXCL10 detection combination consists of upstream primer SEQ ID NO:1, downstream primer SEQ ID NO:2, and probe SEQ ID NO:3, while the ACTB internal control detection combination consists of upstream primer SEQ ID NO:4, downstream primer SEQ ID NO:5, and probe SEQ ID NO:6. The CXCL10 probe is labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher group at the 3' end, while the ACTB probe is labeled with a VIC fluorescent group at the 5' end and an MGB quencher group at the 3' end, enabling simultaneous detection through dual channels and avoiding signal interference. Whole blood stimulants include bovine tuberculin bPPD, avian tuberculin aPPD, and a mixture of PWM / PMA. The final concentration of bPPD and aPPD is 10 µg / mL, which can specifically induce the expression of CXCL10 mRNA in immune cells infected with Mycobacterium tuberculosis. The one-step RT-qPCR reaction solution integrates key components such as reverse transcriptase, Taq enzyme, and dNTPs, providing the enzyme and ionic environment required for reverse transcription and amplification reactions. The 20μL reaction system specifically includes 10μL 2× reaction buffer, 0.4μL of 10μM CXCL10 upstream primer, 0.4μL of 10μM ACTB upstream primer, 0.4μL of 10μM CXCL10 downstream primer, 0.4μL of 10μM ACTB downstream primer, 0.2μL of 10μM CXCL10 probe, 0.2μL of 10μM ACTB probe, and 5μL RNA template. The remaining volume is made up with RNase-free water. The final concentration of each primer is 0.4μM, and the final concentration of each probe is 0.2μM. The nucleic acid extraction reagent consists of a lysis buffer, a washing buffer, and an elution buffer. The lysis buffer contains Triton X-100 detergent, proteinase K, and RNAsin inhibitor, which can efficiently break down immune cells, degrade impurities and proteins, and protect RNA from degradation. The washing buffer contains isopropanol and ethanol, which can effectively remove impurities such as proteins and salts from the sample. The elution buffer is RNase-free water, which can ensure the purity and integrity of the extracted RNA.

[0022] The application steps of this test kit are as follows: In a sterile laboratory operating room, researchers collected heparin-anticoagulated whole blood from three artificially infected rhesus monkeys with Mycobacterium tuberculosis via venous blood collection. Three mL of blood was collected from each monkey and placed into sterile blood collection tubes labeled with the animal's number. Figure 1As shown, whole blood samples from each rhesus monkey were aseptically divided into four aliquots (0.75 mL each) and transferred into four sterile centrifuge tubes, labeled as negative control, aPPD stimulation tube, bPPD stimulation tube, and positive control tube, respectively. Avian tuberculin aPPD was added to the aPPD stimulation tube using a micropipette, and bovine tuberculin bPPD was added to the bPPD stimulation tube, ensuring a final concentration of 10 µg / mL in both tubes. A PWM / PMA mixture was added to the positive control tube as a positive control. No stimulant was added to the negative control tube, which was kept in the same reaction environment as the other tubes to eliminate interference from non-specific reactions. All centrifuge tubes were incubated at 37°C for 4 hours. Figure 2 As shown, this incubation duration allows the expression of CXCL10 mRNA by infection-related immune cells to reach its peak, ensuring the strength and stability of the detection signal.

[0023] After incubation, remove each centrifuge tube and extract total RNA according to the nucleic acid extraction reagent operating procedure. Add lysis buffer to each centrifuge tube sample, invert and mix well, then let stand at room temperature for 10 minutes to allow the lysis buffer to fully penetrate the cells, break the cell membrane and degrade intracellular proteins. At the same time, the RNase inhibitor in the lysis buffer inhibits endogenous RNase activity throughout the process, protecting RNA from degradation. Then, add an equal volume of washing buffer to each centrifuge tube, vortex to mix, and transfer to the adsorption column. Centrifuge at 8000 rpm for 1 minute, discard the waste liquid in the collection tube, and repeat the washing step twice to thoroughly remove impurities from the sample. Finally, add 50 μL of RNase-free water to the adsorption column, let stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 2 minutes. Collect the eluent to obtain the RNA template for each sample. The purity of the RNA template is detected by a Nanodrop instrument to ensure that the A260 / A280 ratio is between 1.8 and 2.0, which meets the requirements for subsequent amplification and detection.

[0024] In the PCR preparation area, following the established 20 μL reaction system, add the following to a sterile PCR tube sequentially: 10 μL 2× reaction buffer, 0.4 μL 10 μM CXCL10 upstream primer, 0.4 μL 10 μM ACTB upstream primer, 0.4 μL 10 μM CXCL10 downstream primer, 0.4 μL 10 μM ACTB downstream primer, 0.2 μL 10 μM CXCL10 probe, 0.2 μL 10 μM ACTB probe, and 5 μL... After adding RNA template to the remaining volume with RNase-free water, gently mix by pipetting to avoid air bubbles affecting amplification. Transfer the prepared PCR tube to a real-time quantitative PCR instrument and set and start the standard reaction program: reverse transcription at 50℃ for 30 minutes to complete the conversion of RNA to cDNA; pre-denaturation at 95℃ for 10 minutes to activate Taq enzyme and denature the cDNA; followed by 40 cycles of 95℃ denaturation for 15 seconds and 60℃ annealing extension for 1 minute. During the annealing and extension phase, fluorescence signals from the FAM and VIC channels are simultaneously collected, and the amplification curve and Ct value data are recorded in real time. Figure 3 As shown, this detection method has good linearity and amplification efficiency, ensuring the reliability of the detection.

[0025] Using the analysis unit built into the real-time fluorescence quantitative PCR instrument, 2 -ΔΔCt The relative expression level of CXCL10 mRNA in each sample was calculated, and the results were determined according to the two-dimensional composite diagnostic criteria: the absolute response level threshold Cut-off 1 for bPPD was set at 2.70, and the differential response level threshold (bPPD stimulation value minus aPPD stimulation value) Cut-off 2 was set at 1.46; if the absolute response level of bPPD in a sample was >2.70 and the differential response level was >1.46, it was judged as positive for tuberculosis; if the above conditions were not met and the absolute response level of bPPD was <2.21, or the absolute response level of bPPD was >2.70 but the differential response level was <1.46, it was judged as negative. The results of this test showed that all three rhesus monkey samples artificially infected with Mycobacterium tuberculosis met the positive criteria, which was completely consistent with the fact of artificial infection. This proves that the kit and detection method can be accurately used for the clinical diagnosis of tuberculosis in rhesus monkeys in the laboratory, providing reliable data support for scientific research.

[0026] In summary, in the clinical diagnosis of tuberculosis in rhesus monkeys in the laboratory, a detection kit containing specific primer and probe combinations, whole blood stimulants, a one-step RT-qPCR reaction solution, and nucleic acid extraction reagents was successfully used to detect tuberculosis in three artificially infected rhesus monkeys through a complete process of whole blood in vitro stimulation, RNA extraction, amplification detection, and result interpretation. The kit components are adapted to the genetic characteristics of rhesus monkeys, dual-channel synchronous detection avoids signal interference, and the optimized reaction system and extraction process ensure RNA purity and amplification efficiency. The dual-dimensional composite diagnostic criteria accurately determined the results. The tests showed that all three rhesus monkeys were positive, completely consistent with the artificial infection, fully demonstrating that the kit and method can accurately assess the infection status and disease progression in rhesus monkeys, providing reliable data support for tuberculosis-related scientific research.

[0027] Example 2: In a scenario of tuberculosis screening in captive cynomolgus macaques, a screening was conducted on 200 cynomolgus macaques at a large-scale non-human primate breeding base. This allowed for the timely identification of potentially infected individuals within the group, enabling the implementation of isolation and control measures to prevent the spread of tuberculosis within the population, thus protecting the health of the cynomolgus macaque population and providing a scientific basis for biosafety management at the breeding base. The detection kit used included a specific primer and probe combination, whole blood stimulant, one-step RT-qPCR reaction solution, and nucleic acid extraction reagents, with each component adapted to the requirements of cynomolgus macaque testing and large-scale screening scenarios. The specific primer-probe combination is designed for the CXCL10 gene and the internal control ACTB gene in cynomolgus monkeys. The CXCL10 detection combination includes upstream primer SEQ ID NO:1, downstream primer SEQ ID NO:2, and probe SEQ ID NO:3. The ACTB internal control detection combination includes upstream primer SEQ ID NO:4, downstream primer SEQ ID NO:5, and probe SEQ ID NO:6. This combination can accurately identify the target gene in cynomolgus monkeys, ensuring detection specificity. The CXCL10 probe uses the FAM-BHQ1 fluorescence quenching system, and the ACTB probe uses the VIC-MGB fluorescence quenching system, which can realize dual-channel simultaneous detection and improve the efficiency of large-scale screening. The whole blood stimulant consists of bovine tuberculin bPPD, avian tuberculin aPPD, and a mixture of PWM / PMA. The final concentration of both bPPD and aPPD is 10 µg / mL, which can effectively stimulate the response of Mycobacterium tuberculosis-specific immune cells in cynomolgus monkeys and generate detectable signals. The one-step RT-qPCR reaction solution is a premixed system that does not require the addition of additional enzymes and reagents, making it convenient to use. Its 20μL reaction system is prepared in a fixed ratio, and the final concentration of each primer and probe meets the detection requirements, which can ensure the high efficiency and repeatability of the amplification reaction and is suitable for large-scale sample detection. Nucleic acid extraction reagents include lysis buffer, washing buffer, and elution buffer. The lysis buffer contains SDS detergent, proteinase K, and superasin inhibitor, which is adapted to the characteristics of stimulated whole blood samples and can process samples quickly and efficiently. The washing buffer contains isopropanol and ethanol, which can remove sample impurities in batches. The elution buffer is TE buffer, which can meet the needs of rapid extraction of high-quality RNA in large-scale screening. Furthermore, RNA is more stable in TE buffer, which facilitates subsequent detection operations.

[0028] The application steps of this test kit are as follows: In the animal clinic at the breeding base, the veterinary team collected blood samples from 200 cynomolgus monkeys in batches. Two mL of heparin-anticoagulated whole blood was collected from each monkey and placed into sterile blood collection tubes labeled with individual numbers, ensuring a one-to-one correspondence between sample numbers and individual monkeys. The whole blood sample from each monkey was then evenly divided into four 0.5 mL portions on a sterile operating table and transferred into four sterile reaction tubes, labeled as negative control, aPPD stimulation, bPPD stimulation, and positive control, respectively, and marked with their corresponding individual numbers. The aPPD stimulation group was then treated using a multichannel pipette. Avian tuberculin aPPD was added to the tubes, and bovine tuberculin bPPD was added to the bPPD stimulation group reaction tubes, ensuring that the final concentration of the stimulant in both stimulation groups was 10 µg / mL. A PWM / PMA mixture was added to the positive control tubes, and the negative control tubes contained no stimulant and were used to verify the effectiveness of the detection system. All reaction tubes were neatly arranged on a test tube rack and placed in a 37°C constant temperature incubator for 4 hours. The incubator temperature was checked regularly to ensure stable incubation conditions and to avoid temperature fluctuations affecting the stimulation effect.

[0029] After incubation, the reaction tubes were transferred in batches to the nucleic acid extraction laboratory for total RNA extraction using the nucleic acid extraction reagents provided with the kit. Lysis buffer was added to each reaction tube, vortexed to mix, and then incubated in a 56°C water bath for 15 minutes to allow the lysis buffer to fully disrupt cell structure and degrade intracellular proteins. The RNase inhibitor in the lysis buffer effectively prevents RNA degradation during extraction. Then, an equal volume of washing buffer was added to each reaction tube, inverted to mix, and then transferred to a multi-well adsorption plate. The plate was centrifuged at 3000 rpm for 5 minutes to adsorb RNA onto the membrane. After discarding the waste liquid, washing buffer was added again and centrifuged once more to remove residual impurities and salts. Finally, 30 μL of TE buffer was added to the adsorption membrane, and after standing at room temperature for 3 minutes, the plate was centrifuged at 5000 rpm for 5 minutes to collect the eluted total RNA. The collected RNA was transferred to sterile centrifuge tubes, labeled, and stored at -20°C for later use to prevent RNA degradation.

[0030] In the preparation area of ​​the PCR laboratory, one-step RT-qPCR reaction systems are prepared in batches according to the number of screening samples. Each component is precisely measured in a fixed ratio: 10 μL 2× reaction buffer, 0.4 μL 10 μM CXCL10 upstream primer, 0.4 μL 10 μM ACTB upstream primer, 0.4 μL 10 μM CXCL10 downstream primer, 0.4 μL 10 μM ACTB downstream primer, 0.2 μL 10 μM CXCL10 probe, and 10 μM... 0.2 μL of ACTB probe and 5 μL of extracted RNA template were added to a total volume of 20 μL with RNase-free water. After gentle mixing, the reaction solution was aliquoted into 384-well PCR plates, 20 μL per well. A blank control well was also included to eliminate contamination. The 384-well PCR plate was sealed with a sealing film and transferred to a real-time quantitative PCR instrument. The amplification program was started: reverse transcription at 50℃ for 30 minutes, pre-denaturation at 95℃ for 10 minutes, followed by 40 cycles of denaturation at 95℃ for 15 seconds and annealing extension at 60℃ for 1 minute. During the cycles, the fluorescence signals of the FAM and VIC channels were collected in real time to generate amplification curves and Ct values.

[0031] The acquired fluorescence signal data were processed in batches using data analysis software, employing 2 -ΔΔCt The relative expression level of CXCL10 mRNA in each sample was calculated; the test results of each cynomolgus monkey were judged individually according to the two-dimensional composite diagnostic criteria: such as... Figure 4 As shown, when the absolute response level of the sample's bPPD is >2.70, and as... Figure 5 As shown, a differential response level > 1.46 indicates a positive tuberculosis result; a negative result is defined as either a bPPD absolute response level < 2.21 or a bPPD absolute response level > 2.70 but a differential response level < 1.46. Based on the screening results, cynomolgus monkeys that tested positive were immediately isolated and further diagnostic testing was conducted. Negative individuals were managed according to standard feeding and management procedures, with regular follow-up screenings. This screening effort completed tuberculosis testing on 200 cynomolgus monkeys, promptly identifying potential infection risks, effectively preventing the spread of the epidemic within the group, and providing strong support for biosafety management at the breeding base.

[0032] In summary, in the scenario of tuberculosis screening in captive cynomolgus macaques, a detection kit adapted to the needs of large-scale screening was used for 200 cynomolgus macaques in a large-scale breeding base. The kit ensured detection specificity through specific primer and probe combinations, improved operational convenience through a premixed reaction system, and efficiently obtained high-quality RNA through nucleic acid extraction reagents. Combined with whole blood stimulation, amplification detection, and batch data processing, efficient and accurate group screening was achieved. After determining the results based on the dual-dimensional composite diagnostic criteria, positive individuals were promptly isolated, and negative individuals were managed in a standardized manner. This effectively screened potential infection risks and prevented the spread of the epidemic. The kit and method fully demonstrate the practicality and reliability of tuberculosis screening in large-scale non-human primates, providing strong support for biosafety management in breeding bases.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A kit for detecting tuberculosis in a non-human primate, the kit comprising, The kit comprises: a specific primer probe combination, a whole blood stimulant, a one-step RT-qPCR reaction solution, and a nucleic acid extraction reagent; The specific primer probe combination is designed for non-human primate CXCL10 genes and reference ACTB genes, and comprises a CXCL10 detection combination and an ACTB reference detection combination; The whole blood stimulant comprises bovine tuberculin bPPD, avian tuberculin aPPD, and a positive control mitogen; The one-step RT-qPCR reaction solution provides enzymes and ion environments required for reverse transcription and amplification reactions; The nucleic acid extraction reagent extracts total RNA from the stimulated whole blood sample.

2. The kit for detecting tuberculosis in non-human primates according to claim 1, characterized in that, The sequence of the specific primer probe combination is as follows: The CXCL10 detection combination: upstream primer SEQ ID NO: 1: 5'-CTGACTCTAAGTGGTATTCAAGGAA-3'; downstream primer SEQ ID NO: 2: 5'-AGACCTTGGATTAACAGGTTGA-3'; probe SEQ ID NO: 3: 5'-FAM-TCTCAAGAACTGTACGCTGTACCTGCA-BHQ1-3'; The ACTB reference detection combination: upstream primer SEQ ID NO: 4: 5'-AGCACGATGAAGATCAAGATCA-3'; downstream primer SEQ ID NO: 5: 5'-GATGGAAGGGCCAGACTC-3'; probe SEQ ID NO: 6: 5'-VIC-ACTCCTGCTTGCTGATCCACATCTGCT-MGB-3'.

3. A kit for detecting tuberculosis in a non-human primate according to claim 2, wherein The probe SEQ ID NO: 3 of the CXCL10 detection combination is labeled with a FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end; the probe SEQ ID NO: 6 of the ACTB reference detection combination is labeled with a VIC fluorescent group at the 5' end and a MGB quenching group at the 3' end.

4. The kit for detecting tuberculosis in non-human primates according to claim 1, wherein In the whole blood stimulant, the final concentrations of bovine tuberculin bPPD and avian tuberculin aPPD are both 10 µg / mL; and the positive control mitogen is a PWM / PMA mixture.

5. The kit for detecting tuberculosis in non-human primates according to claim 1, wherein The reaction system of the one-step RT-qPCR is 20 μL, and the specific components are as follows: 10 μL of 2x reaction buffer, 0.4 μL of 10 μM CXCL10 upstream primer, 0.4 μL of 10 μM ACTB upstream primer, 0.4 μL of 10 μM CXCL10 downstream primer, 0.4 μL of 10 μM ACTB downstream primer, 0.2 μL of 10 μM CXCL10 probe, 0.2 μL of 10 μM ACTB probe, 5 μL of RNA template, and the rest is RNase-free water; wherein the final concentrations of the CXCL10 upstream primer, the ACTB upstream primer, the CXCL10 downstream primer, and the ACTB downstream primer are all 0.4 μM, and the final concentrations of the CXCL10 probe and the ACTB probe are both 0.2 μM.

6. The kit for detecting tuberculosis in non-human primates according to claim 1, wherein The nucleic acid extraction reagent comprises a lysis solution, a washing solution and an elution solution; the lysis solution comprises a detergent, proteinase K and an RNase inhibitor, the washing solution comprises isopropyl alcohol and ethanol, and the elution solution is RNase-free water or a TE buffer; wherein the detergent is one or a combination of two of Triton X-100 and SDS; and the RNase inhibitor is RNAsin or Superasin.

7. A method of using a kit for detecting tuberculosis in a non-human primate, the method being suitable for use with a kit for detecting tuberculosis in a non-human primate according to any one of claims 1 to 6, characterised in that, The specific steps of the application method are as follows: Whole blood in vitro stimulation: taking heparin anticoagulated whole blood of a non-human primate to be tested, adding a negative control, an aPPD stimulant, a bPPD stimulant and a positive control respectively, and incubating at 37 DEG C for 4 hours; the negative control is a reaction system without any stimulant, the aPPD stimulant is avian tuberculin, the bPPD stimulant is bovine tuberculin, and the positive control is a PWM / PMA mixture; RNA extraction: after incubation, a nucleic acid extraction reagent is used to extract total RNA in the whole blood sample; Amplification detection: one-step RT-qPCR is used to amplify and detect the CXCL10 and ACTB genes of the extracted total RNA, and the reaction is carried out on a real-time fluorescent quantitative PCR instrument to collect FAM and VIC channel signals; Results determination: adopt 2 -ΔΔCt The relative expression level of CXCL10 mRNA was calculated by 2 method, and the results were determined based on double-dimensional composite diagnostic criteria.

8. The use of a kit for the detection of tuberculosis in non-human primates according to claim 7, characterized in that, In the amplification detection, the one-step RT-qPCR adopts a standard reaction program, specifically: reverse transcription at 50 DEG C for 30 minutes, pre-denaturation at 95 DEG C for 10 minutes, and then 40 cycles of denaturation at 95 DEG C for 15 seconds, annealing and extension at 60 DEG C for 1 minute.

9. The method of claim 7, wherein the tuberculosis test kit is for non-human primates. In the result determination, the double-dimensional complex diagnostic criteria are as follows: setting the bPPD absolute response level threshold Cut-off 1 as 2.70, and the difference response level threshold Cut-off 2 as 1.46, which is the difference between the bPPD stimulation value and the aPPD stimulation value; when the bPPD absolute response level of the sample is greater than 2.70 and the difference response level is greater than 1.46, it is determined as positive, otherwise, when the bPPD absolute response level is less than 2.21, or the bPPD absolute response level is greater than 2.70 but the difference response level is less than 1.46, it is determined as negative.

10. The method of claim 7, wherein the kit is used for detecting tuberculosis in non-human primates. The minimum detection limit of the application method is 1.0 x 10 3 copies / mL.

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