Probe for trypanosomes detection, application thereof and trypanosomes detection method
By using reverse linear hybridization methods and a variety of specially designed trypanosome probes in trypanosome detection, the limitations of the prior art in detecting chronic, mixed infections and new species are solved, and high sensitivity and specific detection effects are achieved.
Patent Information
- Application Number
- CN202411738828.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-03-14
AI Technical Summary
Existing trypanosome detection methods have limitations in detecting chronic infections, mixed infections and new species, especially PCR technology has false positive reactions when detecting multiple complex mixed infections of multiple samples simultaneously.
Reverse linear hybridization (RLB) method is used to combine a special trypanosome detection probe. The probe covers a variety of trypanosomes and is detected through the 18S rRNA gene to achieve high sensitivity and specificity.
High sensitivity and specific detection of trypanosomes is achieved, and the species can be determined, mixed infections can be distinguished, and new species can be discovered. It is simple to operate and has low cost, and the results are easy to interpret.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of trypanosome detection, and in particular to a trypanosome detection probe, an application thereof and a trypanosome detection method. Background Art
[0002] At present, there are three main methods for detecting trypanosomes: etiological detection, serological detection and molecular biological detection. Etiological detection is often difficult to detect chronic cases with low infection rates and difficult to identify the species of the worms. It is even more limited in cases of mixed infection. Serological methods have certain advantages in detecting a large number of samples, but it is difficult to exclude cross-reactions between different species, and there are often false positives and missed detections. In addition, there are no serological detection kits for some infected trypanosomes. Molecular biological detection technology is based on PCR and is currently the most commonly used molecular diagnostic method for detecting blood protozoa. Compared with the first two, it has high sensitivity and strong specificity, and can effectively detect early infection and cases of worm immunity. However, PCR has certain limitations when detecting multiple samples and multiple complex mixed infections at the same time. At the same time, PCR also has false positive reactions in the detection of trypanosomiasis.
[0003] Reverse linear hybridization (RLB) can detect multiple samples with multiple probes at the same time, and has incomparable advantages over other detection methods in detecting mixed infection samples. In recent years, RLB has been successfully applied to the discovery and identification of new species. For example, if a sample only has a genus-specific probe hybridization signal, but no positive signal with all the specific probes on the hybridization membrane, it indicates that the sample may be a new species or a new subspecies. Therefore, the RLB method has incomparable advantages in detecting parasites, especially blood parasite pathogens. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a probe for trypanosome detection, its application and trypanosome detection method, wherein the probe covers a variety of trypanosomes and is suitable for the preparation of trypanosome detection products. At the same time, the RLB method is used for trypanosome detection, which has high sensitivity and good specificity, and can determine the species, distinguish mixed infections, and discover new species.
[0005] In order to solve the above technical problems, the first aspect of the present invention is to provide a probe for trypanosome detection, comprising one or more of the following probe sequences, wherein the probe sequences are as follows:
[0006] T-genus probe: 5′-GCGTCAGAGGTGAAATTC-3′;
[0007] T. lewisi probe: 5′-TCGCAGAGAGTGCTTCCGT-3′;
[0008] T.simiae probe: 5'-ACACTTCCGTGTGCGTTTCGG-3';
[0009] T. conorhini probe: 5′-TCGGTGGCGCCGTGTTGTGTGT-3′;
[0010] T. dinoisii probe: 5′-CCATCTTCACGCGTCGCCTT-3′;
[0011] T.cruzi probe: 5'-ATGTTGGTTTTCTGTGTGTGTG-3';
[0012] T. theileri probe: 5′-CCATCTCTCCGGCGCCTT-3′;
[0013] T. rangeli probe: 5′-ACGGTAGTGCTGCTGTATTGTT-3′;
[0014] T. vespertilionis probe: 5′-CACGTGTGTCACACGCGCCCT-3′;
[0015] T.vivax probe: 5′-ACCGCGGCATTCCTCTCGGA-3′.
[0016] In the present invention, the probe is preferably designed based on the 18S rRNA gene, wherein the T-genus probe is a genus-specific probe, and other probes are designed, for example, based on the V7V8 hypervariable region. The designed probe has good specificity and high sensitivity, and can be used to detect trypanosomes and can distinguish mixed infections. When the genus-specific probe has hybridization points, while other probes have no hybridization points, new species can also be discovered. It should be noted that the 5' end of the probe can be modified with an amino group, for example, to facilitate subsequent applications, such as fixation with a carrier (membrane), and when modified with N-methyl-(trifluoroacetamidohexylcyanoethyl, N, N-diisopropylphosphoramidite [TFA]-C6), it can be bound to the Biodyne C membrane.
[0017] In order to solve the above technical problems, the second aspect of the present invention is to provide the use of the trypanosome detection probe in the preparation of a trypanosome detection product. The trypanosome detection product can be, for example, a detection kit, etc. It can be understood that the trypanosome detection product prepared by using the trypanosome detection probe of the present invention at least has all the beneficial effects of the trypanosome detection probe.
[0018] In order to solve the above technical problems, the third aspect of the present invention is to provide a method for detecting trypanosomes, which uses the trypanosome detection probe of the present invention. The method is a reverse linear hybridization method, which specifically comprises the following steps:
[0019] S1. Amplification: amplify the target fragment of the 18s rRNA gene of the collected trypanosome sample, and the amplification primer is labeled with biotin;
[0020] S2, solid membrane: bind one or more probes to the membrane;
[0021] S3, hybridization: applying the amplified product of step S1 to the membrane of step S2 in a direction perpendicular to the direction of the probe so that the amplified product hybridizes with the probe at the intersection;
[0022] S3, reading the test results: after the membrane after S3 hybridization is rinsed, incubated and rinsed again, a color developing substance is added and the test results are read;
[0023] The order of steps S1 and S2 can be adjusted or performed simultaneously.
[0024] In the present invention, the reverse linear hybridization method is preferably used, which has good specificity, high sensitivity, simple operation, low cost, and easy results to interpret. Combined with the probe provided by the present invention, it can accurately and effectively determine the trypanosome infection and is conducive to the discovery of new species of trypanosomes.
[0025] In a specific scheme, in step S1, touchdown PCR is used to amplify the target fragment, and the amplification program is: 1 cycle: 37°C for 5 minutes; 1 cycle: 94°C for 10 minutes; 2 cycles: 94°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 63°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 61°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 59°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 57°C for 30 seconds, 72°C for 30 seconds; 40 cycles: 94°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds; one cycle: 72°C for 10 minutes.
[0026] Because specific amplification of trypanosomes is relatively difficult, touchdown PCR was used, which has the advantages of increasing specificity and reducing nonspecific amplification. Touchdown PCR is conducive to the amplification and accumulation of amplicons with the highest primer-template complementarity by using an annealing temperature higher than the calculated Tm value in the initial cycle. In subsequent cycles, the annealing temperature is gradually lowered until it reaches the normal annealing temperature. After the previous rounds of amplification, the content of the target amplicon increases, and its content is much higher than any nonspecific products or primer dimers, thereby ensuring the amplification of the target fragment and reducing the interference of nonspecific amplification results. Combined with the subsequent experiments with specific probes, the specificity of the experiment is further enhanced.
[0027] In a specific embodiment, the sequence of the amplification primer in step S1 is:
[0028] 18S-F 5'-GGTAATTCCCAGCTCCAAAAGCGT-3'
[0029] 18S-R(5'Biosg)-TCCTTGAAGAATGCCTTCGCTGT-3'.
[0030] In this specific embodiment, the amplification primer is a universal primer of 18s rRNA gene, and is labeled with biotin at its 5' end.
[0031] In a specific embodiment, before binding the probe to the membrane, the membrane is activated to facilitate binding of the probe.
[0032] The beneficial technical effects of the present invention include at least a rich variety of probes, good specificity, high sensitivity, the use of reverse linear hybridization technology, simple operation, low cost, easy interpretation of results, and the test results can not only determine the species, but also distinguish mixed infections and discover new species. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0034] Figure 1 is a test result diagram of Example 2;
[0035] Figure 2 is a test result diagram of Example 3;
[0036] Figure 3 is a test result diagram of Embodiment 4;
[0037] Figure 4a is a graph of the test results of the animal samples in Example 5;
[0038] Figure 4b is a graph of the test results of the animal samples in Example 5;
[0039] Figure 5 This is a test result diagram of comparative example 1;
[0040] Figure 6a This is a test result diagram of comparative example 2;
[0041] Figure 6b This is a test result diagram of comparative example 2. DETAILED DESCRIPTION
[0042] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0043] In the present invention, the sample is amplified by a touchdown PCR amplification method. The equipment for touchdown PCR amplification is Bio-rad C1000 Touch PCR instrument. The reagents are purchased from TaKaRa (China). The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2:
[0044] Table 1 PCR reaction conditions
[0045] <![CDATA[ddH2O]]> 8.5μ Mix 12.5μl 18S rRNA F 1.0μl 18S rRNA 1.0μl gDNA 2.0μl common 25.0μl
[0046] Table 2 PCR reaction program
[0047] Number of cycles Temperature, time 1 cycle 37℃ for 5 minutes 1 loop: 94℃10min 2 cycles 94°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds; 2 cycles 94°C for 20 seconds, 63°C for 30 seconds, 72°C for 30 seconds; 2 cycles 94°C for 20 seconds, 61°C for 30 seconds, 72°C for 30 seconds; 2 cycles 94°C for 20 seconds, 59°C for 30 seconds, 72°C for 30 seconds; 2 cycles 94°C for 20 seconds, 57°C for 30 seconds, 72°C for 30 seconds; 40 cycles 94°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 1 cycle 72℃ 10 minutes
[0048] The reverse linear hybridization method of the present invention uses a Miniblotter MN45 blotting apparatus and a Biodyne C membrane. The 5' end of the probe is modified with N-methyl-(trifluoroacetamidohexylcyanoethyl, N, N-diisopropylphosphoramidite [TFA]-C6).
[0049] The steps of reverse linear hybridization are as follows:
[0050] Step 1. Probe and Biodyne C membrane
[0051] Step 11, incubate the Biodyne C membrane with freshly prepared EDAC (carbodiimide) with ultrapure water, for example, at a concentration of 16% (w / v), at room temperature for, for example, 10 minutes, in order to activate the Biodyne C membrane; after the incubation, wash with ultrapure water, for example, twice, each time for 2 minutes, and then place in a Miniblotter MN45 blotting instrument, tighten the handle and set aside;
[0052] Step 12: After draining the remaining liquid in the wells of the blotting instrument, add the diluted probes to the wells of different blotting instruments respectively, and add the ink diluted 1:100 with 2x SSPE buffer to the first and last wells for marking. After incubation at room temperature for 2 minutes, drain the solution and ink added to each well of the blotting instrument;
[0053] Step 13, take out the incubated membrane, put it into a freshly prepared sodium hydroxide solution (concentration of 100 mM, volume of 100 mL), continue to incubate for 10 minutes, then rinse with ultrapure water, and then rinse with 2x SSPE / 0.1% SDS at 60°C;
[0054] Step 14, rinse with 20 mM EDTA at pH 8.0 at room temperature for 15 min, for example, to obtain a labeled Biodyne C membrane (i.e., a Biodyne C membrane bound with a probe), and store at 4°C for later use (if stored for a long time, add EDTA at pH 8.0 and a concentration of 20 mM);
[0055] Step 2: Reverse linear hybridization
[0056] Step 21, incubate the labeled Biodyne C membrane in step 4 with 2x SSPE / 0.1% SDS at room temperature for, for example, 5 minutes, and then place it in a blotting apparatus in a direction perpendicular to the probe, place an air cushion under the membrane, and drain the solution in the slot of the blotting apparatus;
[0057] Step 22, adding the amplified product to 2x SSPE / 0.1% SDS, denaturing at 99°C for example, 10 min, and then immediately cooling in ice, adding the cooled product to the slot of the blotting apparatus, placing it horizontally at 42°C for hybridization for example, 60 min;
[0058] Step 23, after the hybridization is completed, the liquid in the well of the blotting apparatus is drained, the hybridization membrane is taken out, and the membrane is rinsed with 2x SSPE / 0.5% SDS at 50° C., for example, twice, each time for 10 minutes;
[0059] Step 24, mix streptavidin-peroxidase conjugate (500 U / ml, 2.5 μL) with 2x SSPE / 0.5% SDS (10 mL), and place the hybridization membrane rinsed in step 23 into the mixture, and incubate at 42° C. for 30 min, for example;
[0060] After the incubation in step 25 and step 24 is completed, the reaction solution is discarded, and 2x SSPE / 0.5% SDS washing solution is added, and the mixture is rinsed at 42° C., for example, twice, each time for 10 minutes, and then rinsed again with 2x SSPE at room temperature, for example, twice, each time for 5 minutes;
[0061] Step 26: Take equal volumes of immunoblotting luminescent solution A and immunoblotting luminescent solution B, mix them evenly, and then add them to the hybridization membrane to react for, for example, 1 minute, and then develop them using Blots-Chemi in a Bio-Rad gel imaging system to read the results.
[0062] Example 1 Probe design and preparation
[0063] T.lewisi, T.simiae, T.conorhini, T.dinoisii, T.cruzi, T.theileri, T.rangeli, T.vespertilionis, T.vivax gene sequences were downloaded from NCBI. After sequence alignment, the V7V8 hypervariable regions were edited and cut. Primer Primer6.0 was used to design 10 probes, including universal probes (T-genus probes) and species-specific probes. Two probes of each type were designed for sensitivity screening. Highly sensitive probes were retained. If the designed probes were not sensitive, they were redesigned and screened. When the probes were synthesized, their C6 was modified with N-methyl-(trifluoroacetamidohexylcyanoethyl, N, N-diisopropylphosphoramidite [TFA]).
[0064] After sensitivity experiments, the probes finally determined are as follows:
[0065] T-genus probe: 5′-GCGTCAGAGGTGAAATTC-3′;
[0066] T. lewisi probe: 5′-TCGCAGAGAGTGCTTCCGT-3′;
[0067] T.simiae probe: 5'-ACACTTCCGTGTGCGTTTCGG-3';
[0068] T. conorhini probe: 5′-TCGGTGGCGCCGTGTTGTGTGT-3′;
[0069] T. dinoisii probe: 5′-CCATCTTCACGCGTCGCCTT-3′;
[0070] T.cruzi probe: 5'-ATGTTGGTTTTCTGTGTGTGTG-3';
[0071] T. theileri probe: 5′-CCATCTCTCCGGCGCCTT-3′;
[0072] T. rangeli probe: 5′-ACGGTAGTGCTGCTGTATTGTT-3′;
[0073] T. vespertilionis probe: 5′-CACGTGTGTCACACGCGCCCT-3′;
[0074] T.vivax probe: 5′-ACCGCGGCATTCCTCTCGGA-3′;
[0075] Among them, the T-genus probe is a genus-specific probe, and the others are species-specific probes.
[0076] Example 2 Selection of the Optimal Probe Concentration
[0077] The synthesized probe was diluted with 0.5 mM NaHCO 3 (pH 8.4) to concentrations of 200, 400 and 600 pmol / 150 μl to find the optimal probe concentration.
[0078] Sample preparation: synthesize the target sequences of T.lewisi, T.simiae, T.conorhini, T.dinoisii, T.cruzi, T.theileri, T.rangeli, T.vespertilionis, and T.vivax, connect to the T vector, clone into DH5α bacteria, construct plasmids, and sequence the plasmids with the correct sequence as positive reference samples. Prepare the test samples of this example, determine the plasmid concentration of the positive samples, and dilute to 10 ng / ul as experimental samples.
[0079] Evaluation method: Reverse linear hybridization method was used.
[0080] Evaluation results: From the test results of this embodiment ( Figure 1 ) It can be seen that probe A is a specific probe that can detect all samples. There is no difference in the cross-signal between the probe concentrations of 200, 400 and 600 pmol / 150 μl. Therefore, the experimental results are not much different. Therefore, a probe concentration of 200 pmol / 150 μl is selected as the working concentration. Figure 1 In the figure, AM is the probe marker; 1-3 are concentration markers; among them, A is T-genus probe; B is blank; C is T.lewisi probe; D is T.simiae probe; E is T.conorhini probe; F is blank; G is T.dinoisii probe; H is T.cruzi probe; I is T.theileri probe; J is blank; K is T.rangeli probe; L is T.vespertilionis; M is T.vivax probe; 1 is 200pmol / 150μl; 2 is 400pmol / 150μl; 3 is 600pmol / 150μl.
[0081] Example 3 Specificity Assessment
[0082] Probes: 10 probes synthesized in this application, and two species-specific probes of T. congolense and T. evansi reported in the literature.
[0083] Sample preparation: Same as implementation case 2.
[0084] Evaluation method: Reverse linear hybridization method was used, and positive reference samples were mixed for specificity experiment.
[0085] Evaluation results: Figure 2 As shown, AO is a probe marker, and 1-11 is a sample marker; wherein A is a T-genus probe; B is a T.lewisi probe; C is a T.simiae probe; D is a T.conorhini probe; E is a T.dionnisii probe; F is a T.cruzi probe; G is a T.theileri probe; H is a T.evansi probe; I is a T.rangeli probe; J is a T.vespertilionis probe; K is a blank; L is a T.vivax probe; M is a blank; N is a blank; O is a T.congolense probe; 1 is a mixed sample of a T.evansi and T.congolense positive reference sample; 2 is a T.vivax positive sample ,3 is a positive sample of T.vespertilionis;4 is a positive sample of T.lewisi;5 is a positive sample of T.conorhini;6 is a positive sample of T.theileri;7 is a mixture of positive samples of T.lewisi and T.dionnisii;8 is a mixture of positive samples of T.lewisi and T.dionnisii;9 is a mixture of positive samples of T.lewisi, T.cruzi, T.evansi and T.rangeli;10 is a mixture of positive samples of T.evansi and T.rangeli;11 is a mixture of positive samples of T.dionnisii, T.cruzi and T.evansi, which is consistent with the prepared samples and has good specificity. It can be seen from the test results of this embodiment that probe A is a genus-specific probe that can detect all samples, and probes A, B, C, D, E, F, G, H, and I have good specificity and high sensitivity; J, L, and O have good specificity and slightly weaker sensitivity. These 11 can all distinguish different trypanosomes well, and can distinguish mixed infections.
[0086] Example 4 Reverse linear hybridization method to detect Triatomine bug samples
[0087] Sample preparation: The collected triatomine bug samples were ground whole and DNA was extracted using a blood / tissue genomic DNA extraction kit (Qiagen, Germany). The specific operations were carried out according to the kit's instructions.
[0088] Amplification: Touchdown PCR amplification was used;
[0089] Detection method: Reverse linear hybridization method was used;
[0090] Evaluation results: Figure 3As shown, AL are probe markers: A, T-genus; B, T.lewisi; C, T.simiae; D, T.conorhini; E, T.dionnisii; F, T.cruzi; G, T.theileri; H, T.evansi; I, T.rangeli; J, T.vespertilionis; K, T.vivax; L, T.congolense. 1-23 are samples: GZ1, GZ2, GZ3, GZ6, MM1, MM2, MM3, MM15, SD3, SD4, SD5, SD6, FJ3, FJ4, BT1, BT2, BT3, WZS53, WZS54, HT1, GX1, GX2, GX10 (Note: GZ: Guangzhou, MM: Maoming, SD: Shunde, FJ: Fujian, BT: Baoting, HT: Hongtian 14th Team, WZS: Wuzhishan, GX: Guangxi Zhuang Autonomous Region). The results showed that 2, 3, 6, 7, 8, 13, 14, 18, 19, 22, 23 were mixed infections); 24, positive control; 25, negative control.
[0091] The RLB test results show that RLB results are more intuitive, more accurate, and can quickly detect mixed infections. For example, samples GZ2, GZ3, FJ13, FJ14, etc. are mixed infections, which cannot be seen in the PCR results and can only be distinguished through subsequent cloning and sequencing.
[0092] Example 5 Reverse linear hybridization method to detect animal samples
[0093] Sample preparation: The collected animal blood samples (anticoagulated blood) were used to extract DNA using a blood / tissue genomic DNA extraction kit (Qiagen, Germany). The specific operations were performed according to the kit's operating instructions.
[0094] Amplification: Touchdown PCR amplification was used;
[0095] Detection method: Reverse linear hybridization method was used;
[0096] Evaluation results: Figure 4a and 4b shown.
[0097] Figure 4aMiddle: 1-3 are Hainan chicken blood samples J-11, Hainan chicken blood samples J-12, Hainan chicken blood samples J-13; 4-7 are Hainan cattle blood samples N-1, Hainan cattle blood samples N-2, Hainan cattle blood samples N-3, Hainan cattle blood samples N-4; 8 is blank; 9 is Hainan cattle blood sample N-5; 10, 11 are Zhanjiang rat blood samples S-1, Zhanjiang rat blood samples S-2; 12 is positive control, 13 is negative control. A, T-genus; B, T.lewisi; C, T.simiae; D, T.conorhini; E, T.dionnisii; F, T.cruzi; G, blank; H, T.theileri; I, T.evansi; J, T.rangeli; K, T.vespertilionis; L, T.vivax; M, T.congolense. The results showed that samples 1 and 2 had no hybridization bands with the species-specific probe, indicating that they were new species of Trypanosoma; 3 was negative, 4, 5, 6, and 9 tested positive for T. theileri, 7 was a mixed infection of T. conorhini and T. theileri, 10 was detected as T. lewisi, and 11 was T. conorhini.
[0098] Figure 4b Among them, samples 1-5 are Shandong bat blood samples B-1, Shandong bat blood samples B-2, Shandong bat blood samples B-3, Shandong bat blood samples B-4, Shandong bat blood samples B-5; 6-9 are Shandong cat blood samples M-1, Shandong cat blood samples M-2, Hainan chicken blood samples J-1, Hainan chicken blood samples J-2; 10 is blank; 11-12 are Hainan chicken blood samples J-3, Hainan chicken blood samples J-4; 13 is a positive control, and 14 is a negative control. A, T-genus; B, T.lewisi; C, T.simiae; D, T.conorhini; E, T.dionnisii; F, T.cruzi; G, blank; H, T.theileri; I, T.evansi; J, T.rangeli; K, T.vespertilionis; L, T.vivax; M, T.congolense. The results showed that 1, 2, 3, 6, and 7 were detected as T. dionnisii; 4 was detected as T. vespertilionis; 8, 9, 11, and 12 had no hybridization bands with the species-specific probe, indicating that they were new species of trypanosomes.
[0099] Comparative Example 1 Detection of Triatomine Bug Samples by Common PCR Detection Method
[0100] Sample preparation: same as Example 4;
[0101] The amplification system, procedure, and detection method were the same as those of the PCR amplification of the RLB detection method, and the primers 18S-F5'-GGTAATTCCAGCTCCAAAAGCGT-3' and 18S-R-TCCTTGAAGAATGCCTTCGCTGT-3' that were not labeled with biotin were used for specific amplification. Figure 5 , where 1-23 are samples: GZ1, GZ2, GZ3, GZ6, MM1, MM2, MM3, MM15, SD3, SD4, SD5, SD6, FJ3, FJ4, BT1, BT2, BT3, WZS53, WZS54, HT1, GX1, GX2, GX10; 24, positive control; 25, negative control. (Note: GZ: Guangzhou, MM: Maoming, SD: Shunde, FJ: Fujian, BT: Baoting, HT: Hongtian 14th Team, WZS: Wuzhishan, GX: Guangxi Zhuang Autonomous Region).
[0102] Compared with Example 4, in the sample detection of Triatomine bugs, in the common PCR amplification, there is only one band, it is impossible to distinguish mixed infection, and it is impossible to identify new species. When mixed infection, it is possible that a kind of insect species is the dominant insect species, and another kind of insect species carries less, which cannot be identified in common PCR, and PCR sequencing cannot be identified, and a large number of clone sequencing is required, and clone sequencing costs a lot of material and manpower. In comparison, RLB results are more intuitive, more accurate, and mixed infection can be found quickly. In addition, the genus probe has a band, and the specific species probe does not have a band, such as, No. 5 sample (MM1), which is suggested to be a new species. After PCR cloning and sequencing, the comparison result shows that it is T.conorhini-like, and the probe position base varies, so only the genus probe has a hybridization signal, and the species-specific probe does not have a hybridization signal.
[0103] Comparative Example 2: Detection of animal samples by conventional PCR detection method
[0104] Sample preparation is the same as in Example 5;
[0105] The amplification system, procedure, and detection method were the same as those of the PCR amplification of the RLB detection method, and the primers 18S-F5'-GGTAATTCCAGCTCCAAAAGCGT-3' and 18S-R-TCCTTGAAGAATGCCTTCGCTGT-3' that were not labeled with biotin were used for specific amplification. Figure 6a and 6b , Figure 6a In the table, 1-11 are J-11, J-13, J-12, N-1, N-2, N-3, N-4, S-1, S-2, and S-3; 12 is a positive control, and 13 is a negative control; Figure 6bAmong them, 1-11 are Shandong bat blood samples B-1, B-2, B-3, B-4, M-1, M-2, B-5, J-1, J-2, J-3, and J-4; 13 is a positive control, and 14 is a negative control.
[0106] Compared with the test results of Example 5, the test results of Example 5 Figure 6a Middle, 3, 4, 5, 6, 9, 10, 11, Figure 6b In 1-7, the results of conventional PCR amplification and sequencing were consistent with the RLB results, e.g. Figure 6a In the middle, 4, 5, 6, and 9 were detected as T. theileria, 10 as T. lewisi, and 11 as T. conorhini; Figure 6b Among them, 1, 2, 3, 6, and 7 were detected as T. dionnisii; 4 was detected as T. vespertilionis; Figure 6a 1, 2, Figure 6b In 8, 9, 10, and 11, the RLB results showed that the genus probe had hybridization points, while the specific probe was hybridized, indicating that the chicken blood sample was other types of trypanosomes or a new species. The target fragment was amplified by PCR, and the sequencing comparison analysis results showed that it was a new species of trypanosome Trypanosome sp. Figure 6a Among them, 7 were mixed infections of T.conorhini and T.theileri. When mixed infections occur, ordinary PCR cannot identify them, and PCR sequencing cannot identify them. A large number of clone sequencing is required, which consumes a lot of material and manpower. In comparison, RLB results are more intuitive and accurate, and can quickly detect mixed infections. At the same time, RLB can also discover new species, which has incomparable advantages over PCR detection.
[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A probe for detecting trypanosomes, characterized in that One or more of the probes comprising the following sequences, the probe sequences are as follows: T-genus probe: 5′-GCGTCAGAGGTGAAATTC-3′; T. lewisi probe: 5′-TCGCAGAGAGTGCTTCCGT-3′; T.simiae probe: 5'-ACACTTCCGTGTGCGTTTCGG-3'; T. conorhini probe: 5′-TCGGTGGCGCCGTGTTGTGTGT-3′; T. dinoisii probe: 5′-CCATCTTCACGCGTCGCCTT-3′; T.cruzi probe: 5'-ATGTTGGTTTTCTGTGTGTGTG-3'; T. theileri probe: 5′-CCATCTCTCCGGCGCCTT-3′; T. rangeli probe: 5′-ACGGTAGTGCTGCTGTATTGTT-3′; T. vespertilionis probe: 5′-CACGTGTGTCACACGCGCCCT-3′; T.vivax probe: 5′-ACCGCGGCATTCCTCTCGGA-3′.
2. The probe according to claim 1, characterized in that The 5' end of the probe was modified with an amino group.
3. The probe according to claim 1, characterized in that The probes were designed based on the 18S rRNA gene, and the T-genus probe was a genus-specific probe.
4. Use of the trypanosome detection probe as described in any one of claims 1 to 3 in the preparation of trypanosome detection products.
5. A method for detecting trypanosomes using the probe according to any one of claims 1 to 3, characterized in that: The detection was performed by reverse linear hybridization, which included the following steps: S1. Amplification: amplify the target fragment of the 18s rRNA gene of the collected trypanosome sample, and the amplification primer is labeled with biotin; S2, solid membrane: bind one or more probes to the membrane; S3, hybridization: applying the amplified product of step S1 to the membrane of step S2 in a direction perpendicular to the direction of the probe so that the amplified product hybridizes with the probe at the intersection; S3, reading the test results: after the membrane after S3 hybridization is rinsed, incubated and rinsed again, a color developing substance is added and the test results are read; The order of steps S1 and S2 can be adjusted or performed simultaneously.
6. The method according to claim 5, characterized in that In step S1, touchdown PCR is used to amplify the target fragment, and the amplification program is: 1 cycle: 37°C for 5 minutes; 1 cycle: 94°C for 10 minutes; 2 cycles: 94°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 63°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 61°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 59°C for 30 seconds, 72°C for 30 seconds; 2 cycles: 94°C for 20 seconds, 57°C for 30 seconds, 72°C for 30 seconds; 40 cycles: 94°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 72°C for 10 minutes.
7. The method according to claim 5, characterized in that The sequence of the amplification primer in step S1 is: 18S-F 5'-GGTAATTCCAGCTCCAAAAGCGT-3' 18S-R(5'Biosg)-TCCTTGAAGAATGCCTTCGCTGT-3'.
8. The method according to claim 5, characterized in that In step S2, before solidifying the film, the film is activated first.