Dual digital PCR (Polymerase Chain Reaction) kit for monkey foamy virus and monkey T cell tropism virus type I and detection method thereof

Abstract

The invention discloses a monkey foamy virus and monkey T cell tropism virus type I dual digital PCR kit and a detection method thereof, and relates to the technical field of biological detection, the kit comprises the following components: a reagent for extracting pathogen genome DNA; by designing the specific primers and probes for the monkey T cell tropism virus type I and the monkey foamy virus, accurate identification and detection of the two viruses are realized, cross reaction and leak detection are avoided, an optimized micro-droplet digital PCR amplification system and specific reaction conditions are utilized, a standard curve does not need to be established, and the detection accuracy is greatly improved. According to the method, the initial quantity of target molecules is directly and absolutely quantified, the limitation that a traditional PCR method depends on a standard substance and a Ct value is overcome, interference of human factors is reduced, the double microdroplet type digital PCR technology is adopted, two viruses are detected in the same reaction system at the same time, the detection efficiency is greatly improved, and compared with a traditional single detection method, a large amount of time and cost are saved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and specifically relates to a dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I and a detection method thereof. Background Art

[0002] Simian T cell tropic virus type I (STLV-1) was first discovered in the serum of Japanese macaques in 1982. It belongs to retroviruses, and its genome is single-stranded RNA, which is highly similar to human T-lymphotropic virus type I. In the natural environment, the infection range of STLV-1 is extremely wide. Almost all Old World monkeys and great apes, such as cynomolgus monkeys, rhesus monkeys, baboons and other commonly used experimental monkeys, are easily infected. After being infected with this virus, the immune system of monkeys will be damaged, showing pathological changes in immune organs and disorders of immune functions. Moreover, infected macaques often have no obvious clinical symptoms, but can act as virus carriers to spread the virus, seriously interfering with the accuracy and reliability of experiments.

[0003] Simian foamy virus (SFV) belongs to the subfamily Spumaretrovirinae of the family Retroviridae. It is the retrovirus with the longest known genome at present. It is widely present in Africa, the Americas and Asia. Its natural hosts are mainly non-human primates. Monkeys can carry SFV asymptomatically throughout their lives and spread it among the exposed population, which may cause human central nervous system degenerative diseases. In the field of vaccine production, monkey kidney cells are important cell matrices. For example, rhesus monkey kidney cells are used for the production of live attenuated poliovirus vaccine, and African green monkey kidney cells are used for the production of rabies vaccine. However, the infection rate of SFV in the monkey population is as high as 70%. Monkey kidney cells used for vaccine production should come from monkeys negative for SFV antibodies. Therefore, it is crucial to establish an effective SFV detection method.

[0004] Currently, the detection methods for simian T cell tropic virus type I include serological methods and nucleic acid detection methods. Serological methods, such as immunofluorescence, neutralization tests, etc., usually need to be combined with immunoblotting for diagnosis, with cumbersome operations, and due to the latency of the virus, it is easy to miss detections. Among nucleic acid detection methods, conventional PCR and nested PCR are prone to produce false positive results; although fluorescence quantitative PCR has high sensitivity and good specificity, it relies on standard curves and Ct values and has limitations in the detection of some complex samples. Although droplet digital PCR has advantages such as not requiring a standard curve and being able to perform absolute quantification, the droplet digital PCR detection technology for simian T cell tropic virus type I has not been reported previously. In addition, most of the existing detection technologies can only detect one pathogen each time, with low detection efficiency and unable to meet the actual needs. Therefore, it is of great significance to develop a dual digital PCR detection method and kit for simian foamy virus and simian T cell tropic virus type I. Summary of the Invention

[0005] The purpose of the present invention is to make up for the shortcomings of the prior art, and provide a dual digital PCR kit for monkey foamy virus and monkey T cell tropism virus type I and a detection method thereof, which can provide droplet digital PCR primers and probes with strong specificity and high sensitivity for accurately detecting monkey T cell tropism virus type I; at the same time, a droplet digital PCR method for detecting the virus is established, and its application is clarified. The method does not require the construction of a standard curve, belongs to endpoint detection, does not rely on Ct value and amplification efficiency, can effectively overcome the influence of PCR inhibitors, can accurately distinguish samples with small concentration differences, and has higher accuracy, precision and repeatability. In addition, the present invention aims to establish a dual droplet digital PCR method for simultaneously detecting monkey T cell tropism virus type I and monkey foamy virus to improve detection efficiency.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: a dual digital PCR kit for simian foamy virus and simian T cell tropism virus type I, the kit comprising the following components:

[0007] Reagents for extracting pathogenic genomic DNA;

[0008] Specific primers and probes for monkey T cell tropism virus type I, the nucleotide sequence of the upstream primer is 5'-TTCCCAGGGTTTGGACAAAG-3', the nucleotide sequence of the downstream primer is 5'-GARCATAGTCCCCCRGAGATG-3', the nucleotide sequence of the probe is 5'-FAM-ACAGTCTCCAAACACGTAGACTGGGTATCC-BHQ1-3', the primers and probe are designed for the tax gene sequence of monkey T cell tropism virus type I, and the amplified fragment is 101bp;

[0009] Specific primers and probes for simian foamy virus, the nucleotide sequence of the upstream primer is 5'-TGGTCTCCTKYTGTWGGC-3', the nucleotide sequence of the downstream primer is 5'-TGTTGCCAAGATGGTCCAAAA-3', the nucleotide sequence of the probe is 5'-Cy5-CGACCACGBTGGCATAARCCT-BHQ1-3', which is designed based on the gene sequence of simian foamy virus, and the amplified fragment is 139bp;

[0010] Premix required for droplet digital PCR amplification;

[0011] The positive control is a recombinant plasmid containing monkey T cell tropism virus type I and monkey foamy virus specific amplified fragments.

[0012] Furthermore, the reagents for extracting pathogenic genomic DNA adopt conventional methods and reagents for extracting DNA from common primate experimental animal samples. Common primate experimental animal samples include rhesus monkey cytomegalovirus, monkey varicella virus, measles virus, monkey retrovirus type D and monkey foamy virus samples. After optimization, operational errors are reduced to ensure that the purity and integrity of the extracted DNA or RNA meet the requirements of subsequent droplet digital PCR detection.

[0013] Furthermore, the design process of the primers and probes for monkey T cell tropism virus type I includes multiple rounds of comparison analysis of tax gene sequences to screen out sequence regions with high specificity. Experimental verification shows that the primers and probes accurately identify the nucleic acid of monkey T cell tropism virus type I in a complex nucleic acid environment, have no obvious cross-reaction with other common primate virus nucleic acids, and stably amplify a 101bp target fragment in a droplet digital PCR reaction.

[0014] Furthermore, the primers and probes for simian foamy virus are designed based on the simian foamy virus POL gene published in GenBank using Primer Premier 5 software. After the design is completed, the length and GC content parameters of the primers and probes are adjusted through multiple rounds of experimental optimization to enable them to identify the nucleic acid of simian foamy virus in different sample types, including nucleic acids extracted from samples such as blood and tissues, and amplify a 139bp specific fragment in a droplet digital PCR reaction system.

[0015] Furthermore, the droplet digital PCR amplification premix contains the buffer, dNTPs, and polymerase components necessary for the PCR reaction. The concentration and ratio of each component are optimized through experiments to provide a stable chemical environment for the binding and amplification of primers and templates in the droplet digital PCR reaction.

[0016] Furthermore, the recombinant plasmid in the positive control is precisely cloned during the construction of the specific amplified fragments of monkey T cell tropism virus type I and monkey foamy virus. The recombinant plasmid is used as a positive reference in the detection process of the kit. Each time it is used in an experiment, the effectiveness of the entire detection system is verified by synchronous detection. If the test result of the positive control is normal, it indicates that the detection system is running well. If an abnormality occurs, it indicates that there is a problem in the detection process, which should be checked and corrected in time.

[0017] Furthermore, the kit also includes a negative control, which is a sample that does not contain monkey T cell tropism virus type I and monkey foamy virus nucleic acid. In each detection experiment, the negative control is detected simultaneously with the sample to be detected and the positive control. The detection result of the negative control is positive, indicating that there is contamination in the experimental process and re-testing is required.

[0018] The present invention also provides a technical solution. A detection method for a dual digital PCR kit of simian foamy virus and simian T-cell tropic virus type I includes the following steps:

[0019] First, extract the pathogen genomic DNA from common primate experimental animal samples;

[0020] Then, add specific primers, probes, digital PCR premix, and template DNA to the reaction system in proportion;

[0021] Next, in a ddPCR amplifier, generate oil droplets and perform amplification according to the program maintained by the amplification conditions;

[0022] After the amplification is completed, put the chip into a scanner, use the software GeneCount Analysis System to read the copy number of DNA, and judge whether the sample contains simian T-cell tropic virus type I and simian foamy virus and their contents according to the copy number and in combination with the preset determination criteria.

[0023] Furthermore, when the kit is used for the dual droplet digital PCR detection of simian T-cell tropic virus type I and simian foamy virus, the reaction system is 25 μL, and the specific components and dosages are as follows: 10.0 μL of digital PCR premix, 1.0 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.0 μL each of SFV upstream and downstream primers (20 μM), 0.5 μL of SFV probe (20 μM), 0 μL of RNase-free ddH2O, and 5 μL of template DNA.

[0024] Furthermore, the amplification conditions for the droplet digital PCR detection using this kit are as follows: pre-denaturation at 95°C for 15 min; 40 cycles are carried out, and each cycle includes denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 30 s; final extension at 98°C for 10 min, hold at 20°C for 2 min, and the reaction temperature rise and fall rate is 2.0°C / s. These amplification conditions are determined by optimizing the annealing temperature, cycle number, denaturation, and extension time parameters.

[0025] Compared with the prior art, the dual digital PCR kit of simian foamy virus and simian T-cell tropic virus type I and its detection method have the following beneficial effects:

[0026] The present invention realizes the accurate identification and detection of two viruses by designing specific primers and probes for type I simian T cell tropic virus and simian foamy virus, avoiding cross-reaction and missed detection. By using an optimized droplet digital PCR amplification system and specific reaction conditions, it directly performs absolute quantification on the starting amount of the target molecule without establishing a standard curve, overcoming the limitations of traditional PCR methods that rely on standards and Ct values, reducing the interference of human factors. By adopting the dual droplet digital PCR technology to simultaneously detect two viruses in the same reaction system, the detection efficiency is greatly improved, saving a large amount of time and cost compared with traditional single detection methods.

[0027] Other advantages, objectives and features of the present invention will be described to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0029] Figure 1 Establish an amplification map for dual droplet digital PCR STLV-1;

[0030] Figure 2 Establish an amplification map for dual droplet digital PCR SFV;

[0031] Figure 3 Amplification map for optimizing annealing temperature of dual droplet digital PCR (STLV-1);

[0032] Figure 4 Amplification map for optimizing annealing temperature of dual droplet digital PCR (SFV);

[0033] Figure 5 Scatter plot for optimizing the dual droplet digital PCR system (System 1 - System 3: SFV);

[0034] Figure 6 Scatter plot for optimizing the dual droplet digital PCR system (System 1 - System 3: STLV-1);

[0035] Figure 7 Scatter plot for optimizing the dual droplet digital PCR system (System 4 - System 6: SFV);

[0036] Figure 8 Scatter plot for optimizing the dual-droplet digital PCR system (System 4 - System 6: STLV-1);

[0037] Figure 9 Scatter plot for specific detection of dual-droplet digital PCR (STLV-1);

[0038] Figure 10 Scatter plot for specific detection of dual-droplet digital PCR (SFV);

[0039] Figure 11 Scatter plot for detecting the standard curve and the lowest detection limit of dual ddPCR (STLV-1);

[0040] Figure 12 Scatter plot for detecting the standard curve and the lowest detection limit of dual ddPCR (SFV);

[0041] Figure 13 Scatter plot for detecting the repeatability of dual ddPCR (SFV-1);

[0042] Figure 14 Scatter plot for detecting the repeatability of dual ddPCR (SFV);

[0043] Figure 15 Flow chart of the detection method of a dual digital PCR detection method and kit for simian foamy virus and simian T cell tropic virus type I. Detailed implementation manners

[0044] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in combination with the accompanying drawings and preferred embodiments, details the specific implementation manners, structures, features and their effects according to the present invention as follows.

[0045] The present invention provides a dual digital PCR detection method and kit for simian foamy virus and simian T cell tropic virus type I. Unless otherwise specified, the experimental methods used in the following implementation steps are all conventional methods; the materials, reagents, etc. used in the steps can be obtained from commercial channels unless otherwise specified.

[0046] 1. Materials and methods

[0047] Common primate experimental animal pathogens and DNA samples: Five virus strains, including rhesus cytomegalovirus, simian varicella virus, measles virus, simian retrovirus type D, and simian foamy virus, are stored in liquid nitrogen in our laboratory. Genomic DNA / cDNA is extracted from these virus strains for the specificity experiment of the droplet digital PCR method. This step aims to obtain nucleic acids of various virus samples and provide basic materials for the specificity verification of the subsequent detection method, ensuring that the detection method can accurately distinguish the target virus from other common viruses.

[0048] Primer, probe sequence and synthesis: The primer and probe sequences of simian T cell tropism virus type I were derived from its tax gene sequence. The nucleotide sequence of the upstream primer was 5'-TTCCCAGGGTTTGGACAAAG-3', the nucleotide sequence of the downstream primer was 5'-GARCATAGTCCCCCRGAGATG-3', and the nucleotide sequence of the probe was 5'-FAM-ACAGTCTCCAAACACGTAGACTGGGTATCC-BHQ1-3'. The amplified fragment was 101 bp. Based on the simian foamy virus POL gene published in GenBank, Primer Premier 5. Design primers and probes for simian foamy virus droplet digital PCR. The nucleotide sequence of the upstream primer is 5'-TGGTCTCCTKYTGTWGGC-3', the nucleotide sequence of the downstream primer is 5'-TGTTGCCAAGATGGTCCAAAA-3', the nucleotide sequence of the probe is 5'-Cy5-CGACCACGBTGGCATAARCCT-BHQ1-3', and the amplified fragment is 139bp. All primers and probes are synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The precise design of primers and probes is the key to achieving high-specificity detection. Designing primers and probes for specific gene sequences of different viruses can ensure the accurate identification of target viral nucleic acids in complex samples.

[0049] Construction of recombinant plasmids for positive quality control products: The STLV-1 droplet digital PCR amplification target fragment and the SFV droplet digital PCR amplification target fragment were both constructed by Suzhou Hongxun Biotechnology Co., Ltd. As positive quality control products, positive quality control products are used to verify the effectiveness of the entire detection system, ensuring that the detection reagents and instruments and equipment can work normally during each detection process, and providing a reliable reference for the accuracy of the test results.

[0050] Establishment of droplet digital PCR method: The plasmid standard constructed above was used for amplification. The reaction system was 20 μL, including 10.0 μL of digital PCR premix, 1.4 μL each of upstream and downstream primers (20 μM), 0.6 μL of probe (10 μM), 4.0 μL of RNase-free ddH2O, and 5 μL of template DNA. Oil droplets were generated and amplified in a Reagent ddPCR amplifier. The amplification program was as follows: 95 °C for 15 min; 94 °C for 30 s, annealing at 55 °C for 1 min, extension at 72 °C for 30 s, for a total of 40 cycles; 98 °C for 10 min; hold at 20 °C for 2 min. The rate of increase and decrease of the reaction temperature was 2.0 °C / s. After the reaction, the chip was placed in a scanner, and the instrument automatically collected the fluorescence signal. The software GeneCount Analysis System was used to read the copy number of DNA. This reaction system and amplification program were determined through a large number of previous experiments and optimizations, which could ensure the full reaction of primers, probes, and templates, achieve efficient amplification and accurate detection of the target virus nucleic acid, and provide a stable method basis for subsequent experiments.

[0051] Optimization of annealing temperature for droplet digital PCR: The annealing temperature of ddPCR was detected according to the above method. The annealing temperatures were set at 53.8, 55.2, 56.6, 58.0, 59.4, 60.8, 62.2, and 63.7 °C respectively. The primer concentration was 20 μM, and the probe concentration was 10 μM. Experiments were carried out by setting different annealing temperatures to observe their effects on the amplification results.

[0052] Optimization of droplet digital PCR reaction system: Experiments were carried out by setting different reaction systems with a volume of 20 μL.

[0053] System 1: It included 10.0 μL of digital PCR premix, 1.0 μL each of upstream and downstream STLV-1 primers (20 μM), 0.3 μL of STLV-1 probe (20 μM), 1.0 μL each of upstream and downstream SFV primers (20 μM), 0.3 μL of SFV probe (20 μM), 0.4 μL of RNase-free ddH2O, and 5 μL of template DNA.

[0054] System 2: It included 10.0 μL of digital PCR premix, 1.0 μL each of upstream and downstream STLV-1 primers (20 μM), 0.4 μL of STLV-1 probe (20 μM), 1.0 μL each of upstream and downstream SFV primers (20 μM), 0.4 μL of SFV probe (20 μM), 0.2 μL of RNase-free ddH2O, and 5 μL of template DNA.

[0055] System 3: It contains 10.0 μL of digital PCR premix, 1.0 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.0 μL each of SFV upstream and downstream primers (20 μM), 0.5 μL of SFV probe (20 μM), 0 μL of RNase-free dH2O, and 5 μL of template DNA.

[0056] System 4: It contains 10.0 μL of digital PCR premix, 1.4 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.4 μL each of SFV upstream and downstream primers (20 μM), 0.5 μL of SFV probe (20 μM), 1.2 μL of RNase-free dH2O, and 5 μL of template DNA.

[0057] System 5: It contains 10.0 μL of digital PCR premix, 1.6 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.6 μL each of SFV upstream and downstream primers (20 μM), 0.5 μL of SFV probe (20 μM), 0.8 μL of RNase-free dH2O, and 5 μL of template DNA.

[0058] System 6: It contains 10.0 μL of digital PCR premix, 1.8 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.8 μL each of SFV upstream and downstream primers (20 μM),

[0059] 1.8 μL of SFV probe (20 μM), 0.4 μL of RNase-free dH2O, and 5 μL of template DNA.

[0060] In the Reagent ddPCR amplifier, generate oil droplets and perform amplification according to the program of 95 °C for 15 min; 94 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 30 s, for a total of 40 cycles; 98 °C for 10 min; hold at 20 °C for 2 min. The rate of increase and decrease of the reaction temperature is 2.0 °C / s. After the reaction, put the chip into the scanner, and the instrument automatically collects the fluorescence signal. Use the software GeneCountAnalysis System to read the copy number of DNA.

[0061] Sensitivity and repeatability: The stock solutions of STLV-1 positive control (recombinant plasmid) and SFV positive control (recombinant plasmid) were combined and then serially diluted 10-fold. The theoretical concentrations of each dilution were 185000, 18500, 1850, 185, 18.5, 1.85, and 0.185 copies / μL, respectively. The optimized ddPCR method was used for detection, and each dilution concentration was detected 3 times. The relative standard deviation (RSD) of the detection results at each gradient was calculated to test the accuracy and stability of the method of the present invention. Through this experiment, the detection ability of the detection method for samples at different concentrations can be evaluated, and the results can intuitively reflect the reliability of the detection method, providing an important reference for practical applications.

[0062] Specificity: Using the genomic DNA or cDNA of 5 viruses, namely rhesus cytomegalovirus, simian varicella virus, measles virus, simian retrovirus type D, and simian foamy virus, as templates, the established duplex droplet digital PCR method for simian STLV-1 and SFV was used to detect the above 5 viruses.

[0063] 2. Detection results of PCR method

[0064] Establishment of duplex droplet digital PCR method: The reaction system was 20 μL, including 10.0 μL of digital PCR premix, 1.4 μL of each upstream and downstream primer (20 μM), 0.6 μL of probe (10 μM), 4.0 μL of RNase-free ddH2O, and 5 μL of template (obtained by gradient dilution of positive control). Oil droplets were generated and amplified in a Axygen ddPCR amplifier. The amplification program was: 95°C for 15 min; 94°C for 30 s, annealing at 60°C for 1 min, 72°C for 30 s, extension at 72°C for 30 s, for a total of 40 cycles; 98°C for 10 min; hold at 20°C for 2 min. The rate of increase and decrease of the reaction temperature was 2.0°C / s. After the reaction, the chip was placed in a scanner, and the instrument automatically collected the fluorescence signal. The software GeneCount Analysis System was used to read the copy number of DNA. The results are shown in Figure 1 、 Figure 2 and the following table. This step successfully established a duplex droplet digital PCR detection method, providing a feasible technical means for subsequent experiments and actual detections.

[0065]

[0066] Optimization of duplex droplet digital PCR annealing temperature: The annealing temperature of ddPCR was used to detect plasmid standards according to the above method. The annealing temperatures were set at 51.8, 53.2, 54.6, 56.0, 57.4, 58.3, 60.2, and 61.7°C, respectively. The primer concentration was 20 μM, and the probe concentration was 10 μM. The following table shows that the annealing temperature has no significant effect on the number of oil droplets (total oil droplets, positive oil droplets). CombiningFigure 3 and Figure 4 It can be seen that the amplification effect is the best when the annealing temperature is about 55 °C. This optimized result determines the optimal annealing temperature for subsequent experiments, which helps to improve the sensitivity and accuracy of detection.

[0067]

[0068] Optimization of the droplet digital PCR reaction system: According to the above-set systems, generate oil droplets and perform amplification in the Ruixun ddPCR amplifier. The amplification program is as follows: 95 °C for 15 min; 94 °C for 30 s, annealing at 55 °C for 1 min, 72 °C for 30 s, for a total of 40 cycles; 98 °C for 10 min; hold at 20 °C for 2 min. The reaction temperature rise and fall rate is 2.0 °C / s. After the reaction is completed, put the chip into the scanner, and the instrument automatically collects the fluorescence signal. Using the software Gene Count Analysis System to read the copy number of DNA, it is found that the copy number of system 3 is the highest and the fluorescence signal value is the strongest. Select system 3 as the optimal system. The relevant data and scatter plots are shown in Figures 5 - 8 and the following table. Through system optimization, the reaction system most suitable for detection is determined, further improving the performance of detection.

[0069]

[0070]

[0071] Specific detection results: Using the genomic DNA of 5 viruses, including rhesus cytomegalovirus, simian varicella virus, measles virus, simian retrovirus type D, and simian foamy virus, and the positive control product (recombinant plasmid) as templates, the newly designed primers and probes for simian STLV-1 by droplet digital PCR method are used to detect the above 5 viruses. It can be clearly seen from the following table that positive results are presented when using the positive control product (recombinant plasmid) as the template, while no positive results are detected for the nucleic acids of other pathogens. This result is visually presented in Figure 9 and Figure 10 which shows that the detection method of the present invention has high specificity, can accurately identify simian T cell tropic virus type I and simian foamy virus, effectively exclude the interference of other viruses, greatly reduce the possibility of misjudgment, and provide a strong guarantee for accurate detection of the target virus.

[0072]

[0073] Minimum detection limit detection results: Dilute the positive control product (recombinant plasmid) by 10-fold or 2 gradients. The theoretical concentrations of each dilution factor are 185, 18.5, 9.3, 4.7, 2.3, 1.85 copies / μL respectively. Use the optimized ddPCR method for detection. From the following table and Figure 11 andFigure 12 From the test results, the lowest detection limit is 1.5 copies / μL. This data indicates that the detection method of the present invention has extremely high sensitivity, can detect target viruses at extremely low concentrations, has significant advantages in detecting samples with extremely low virus content, and expands the application scope of this detection method.

[0074]

[0075] Repeatability test results: The positive control product (recombinant plasmid) was diluted by 10-fold gradient. The theoretical concentrations of each dilution were 18500, 1850, 185, and 18.5 copies / μL respectively. The optimized ddPCR was used for detection, and each dilution concentration was detected 3 times. From the following table and Figure 13 、 Figure 14 it can be seen that the relative standard deviation (RSD) of the detection results of each gradient is small, indicating that the detection method of the present invention has good repeatability.

[0076]

[0077]

[0078] This means that when repeatedly detecting the same sample multiple times, relatively stable and consistent results can be obtained, ensuring the reliability and repeatability of the detection results, and providing a stable data basis for multiple detections in practical applications.

[0079] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed technical content to obtain equivalent embodiments with equivalent changes within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A dual digital PCR kit for simian foamy virus and simian T-cell tropic virus type I, characterized in that, The kit includes the following components: Reagents for extracting pathogen genomic DNA; Specific primers and probes for simian T-cell tropic virus type I. The nucleotide sequence of the upstream primer is 5’-TTCCCAGGGTTTGGACAAAG-3’, the nucleotide sequence of the downstream primer is 5’-GARCATAGTCCCCCRGAGATG-3’, and the nucleotide sequence of the probe is 5’-FAM-ACAGTCTCCAAACACGTAGACTGGGTATCC-BHQ1-3’. This primer and probe are designed based on the tax gene sequence of simian T-cell tropic virus type I, and the amplified fragment is 101bp; Specific primers and probes for simian foamy virus. The nucleotide sequence of the upstream primer is 5’-TGGTCTCCTKYTGTWGGC-3’, the nucleotide sequence of the downstream primer is 5’-TGTTGCCAAGATGGTCCAAAA-3’, and the nucleotide sequence of the probe is 5’-Cy5-CGACCACGBTGGCATAARCCT-BHQ1-3’. It is designed based on the simian foamy virus gene sequence, and the amplified fragment is 139bp; Premix for droplet digital PCR amplification; Positive control, which is a recombinant plasmid containing specific amplification fragments of simian T-cell tropic virus type I and simian foamy virus.

2. The dual digital PCR kit for simian foamy virus and simian T-cell tropic virus type I according to claim 1, characterized in that: The reagents for extracting pathogen genomic DNA adopt conventional methods and reagents for extracting DNA from common primate experimental animal samples. Common primate experimental animal samples include rhesus cytomegalovirus, simian varicella virus, measles virus, simian retrovirus type D, and simian foamy virus samples. After optimization, operation errors are reduced to ensure that the purity and integrity of the extracted DNA meet the requirements of subsequent droplet digital PCR detection.

3. The dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 1, characterized in that: The design process of the primers and probes for simian T-cell tropic virus type I includes multiple rounds of alignment and analysis of the tax gene sequence to screen out highly specific sequence regions. Through experimental verification, these primers and probes can accurately identify the nucleic acid of simian T-cell tropic virus type I in a complex nucleic acid environment, without obvious cross-reaction with other common primate virus nucleic acids, and can stably amplify a 101bp target fragment in the droplet digital PCR reaction.

4. A duplex digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 1, characterized in that: The primers and probes for simian foamy virus are designed based on the simian foamy virus POL gene published in GenBank using Primer Premier 5 software. After design, they are optimized through multiple rounds of experiments to adjust the length and GC content parameters of the primers and probes, enabling them to identify the nucleic acid of simian foamy virus in different sample types and amplify a 139bp specific fragment in the droplet digital PCR reaction system.

5. A duplex digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 1, characterized in that: The droplet digital PCR amplification premix contains buffers, dNTPs, and polymerase components necessary for the PCR reaction, and the concentration and ratio of each component have been optimized through experiments.

6. A dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 1, characterized in that: In the recombinant plasmid of the positive control, specific amplification fragments of simian T-cell tropic virus type I and simian foamy virus were precisely cloned during the construction process. This recombinant plasmid serves as a positive reference during the kit detection process. Each time the experiment is conducted, it is used to synchronously detect and verify the effectiveness of the entire detection system. If the detection result of the positive control is normal, it indicates that the detection system is operating well; if it is abnormal, it suggests that there are problems in the detection process.

7. A dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 1, characterized in that: The kit also includes a negative control, which is a sample without the nucleic acids of simian T-cell tropic virus type I and simian foamy virus. In each detection experiment, the negative control is subjected to the detection operation simultaneously with the sample to be detected and the positive control.

8. A detection method for a dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I as described in any one of claims 1 to 7, characterized in that, It includes the following steps: Firstly, extract the pathogen genomic DNA from common primate experimental animal samples; Then add specific primers, probes, digital PCR premix, and template DNA to the reaction system in proportion; Next, in a ddPCR amplifier, generate oil droplets and perform amplification according to the program maintained by the amplification conditions; After the amplification is completed, place the chip into a scanner, and use the software GeneCount Analysis System to read the copy number of DNA. Based on the copy number and combined with the preset determination criteria, determine whether the sample contains simian T-cell tropic virus type I and simian foamy virus and their contents.

9. The detection method of a dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 8, characterized in that: When the kit is used for the dual droplet digital PCR detection of simian T-cell tropic virus type I and simian foamy virus, the reaction system is 25 μL. The specific components and dosages are as follows: 10.0 μL of digital PCR premix, 1.0 μL each of STLV-1 upstream and downstream primers (20 μM), 0.5 μL of STLV-1 probe (20 μM), 1.0 μL each of SFV upstream and downstream primers (20 μM), 0.5 μL of SFV probe (20 μM), 0 μL of RNase-free ddH2O, and 5 μL of template DNA.

10. The detection method of a dual digital PCR kit for simian foamy virus and simian T cell tropic virus type I according to claim 8, characterized in that: The amplification conditions for the droplet digital PCR detection using this kit are as follows: pre-denaturation at 95 °C for 15 min; perform 40 cycles, each cycle including denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 30 s; final extension at 98 °C for 10 min, hold at 20 °C for 2 min, and the reaction temperature rise and fall rate is 2.0 °C / s.

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