Double Taqman detection probe primer group for trichina and sarcocystis and application of double Taqman detection probe primer group

Abstract

The invention discloses a double Taqman detection probe primer group for trichina and sarcocystis and application of the double Taqman detection probe primer group. Relates to the technical field of parasite detection. The invention provides a detection probe primer group, a kit and application. According to the detection method established by the invention, an amplification curve has a good linear relationship; the sensitivity is high; the specificity is high; dNA detection on 12 parasites such as toxoplasma gondii, ascaris suum, cryptosporidium and pulmonary nematode does not have an amplification curve; agarose gel electrophoresis shows that the molecular mass of the obtained PCR product strip accords with expectation. The kit has the characteristics of simplicity in operation, high specificity and high sensitivity, can quickly and efficiently detect trichina and sarcocystis, and has guiding significance in quick laboratory diagnosis of infection of the trichina and sarcocystis.

Description

Technical Field

[0001] The invention relates to the technical field of parasite detection, and more particularly to a double Taqman detection probe primer set for Trichinella spiralis and Sarcocystis and an application thereof. Background Art

[0002] Trichinellosis and sarcocystosis are both zoonotic infectious diseases that seriously endanger the health of humans and animals, have a serious impact on public health and the economy, and also pose huge challenges to animal husbandry production and food safety.

[0003] Currently, humans generally lack knowledge about trichinellosis and sarcocystosis. The two have many similarities in clinical symptoms, and when the infection is mild, it is not easy to be detected during the quarantine process.

[0004] Therefore, it is necessary to establish a more sensitive, rapid and specific detection method to detect these two diseases.

[0005] Therefore, whether it is possible to provide a dual Taqman detection probe primer set for Trichinella spiralis and Sarcocystis and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a dual Taqman detection probe primer set for Trichinella spiralis and Sarcocystis and its application. The dual Tapman probe detection technology is mainly applied to the detection of Trichinella spiralis and Sarcocystis, aiming to establish a simpler, faster, more sensitive and more specific detection method, which provides a reference for the detection of trichinellosis and Sarcocystis, and also helps to effectively prevent and control these two diseases and promote the development of animal husbandry.

[0007] Specifically, specific primer probes were designed based on the 18SrRNA gene sequences of Trichinella spiralis and Sarcocystis, the primer probes were tested using three different reaction products, the optimal combination and concentration of the primer probes were screened, the gradient was set and the optimal annealing temperature was screened, and sensitivity and specificity tests were performed, and a dual TaqMan probe detection method for Trichinella spiralis and Sarcocystis was established.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A dual Taqman detection probe primer set for Trichinella spiralis and Sarcocystis, comprising the following primer and probe sequences:

[0010] Trichinella spiralis-F3: 5'-ACTTCTTAGAGGGACTGGCAAA-3', as SEQ ID No.1

[0011] As shown;

[0012] Trichinella spiralis-R3: 5'-GTGCAGCCCCGGACAT-3', as shown in SEQ ID No. 2;

[0013] Trichinella spiralis-T3: 5′-CGAAGGGCATCACAGACCTGTTATTG-3′, as shown in SEQ ID No. 3;

[0014] Sarcocystis-F2: 5'-GAGTATGGTCGCAAGGCTGAA-3', as shown in SEQ ID No. 4;

[0015] Sarcocystis-R2: 5'-CGTGTTGAGTCAAATTAAGCCG-3', as shown in SEQ ID No. 5;

[0016] Sarcocystis-T2: 5'-CTTAAAGGAATTGACGGAAGGGCAC-3', as shown in SEQ ID No.6;

[0017] Among them, Trichinella-T3 was 5′ labeled with HEX and 3′ labeled with BDQ1;

[0018] Sarcocystis-T2 is 5' labeled with FAM and 3' labeled with BDQ1.

[0019] The present invention also provides a detection kit comprising the detection probe primer set.

[0020] Preferred: Reaction system: Template 1.0 μl, Premix Ex Taq TM 5 μl, 10 μM primer F / R 0.8 μl, 10 μM Probe 0.4 μl, and ddH2O to 10 μl.

[0021] Preferred reaction conditions: pre-denaturation at 95°C for 30s, fluorescence detection at 95°C for 10s → 60°C for 30s, 40 cycles, and FAM and HEX dual-channel detection.

[0022] The present invention also provides the use of the above detection probe primer set, or any one of the above detection kits in non-diagnostic and therapeutic detection of Trichinella spiralis and Sarcocystis.

[0023] Preferred: used in animal husbandry and production.

[0024] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses a dual Taqman detection probe primer set for Trichinella spiralis and Sarcocystis and its application, and the technical effect achieved is that the amplification curve of the established detection method has a good linear relationship; high sensitivity; strong specificity; no amplification curve for the detection of 12 parasite DNAs such as Toxoplasma gondii, Ascaris suum, Cryptosporidium, and Lungworm; agarose gel electrophoresis shows that the molecular mass of the obtained PCR product band is consistent with the expectation. It has the characteristics of simple operation, strong specificity, and high sensitivity, and can quickly and efficiently detect Trichinella spiralis and Sarcocystis, which has guiding significance for the laboratory rapid diagnosis of these two parasitic infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The accompanying drawing is a graph showing the amplification curve of the Trichinella spiralis probe provided by the present invention.

[0027] Figure 2 The attached drawing is a graph showing the amplification curve of the Sarcocystis probe provided by the present invention.

[0028] Figure 3 The attached figure is a graph showing the test results of the Trichinella spiralis template in the dual detection probe 1 provided by the present invention.

[0029] Figure 4 The attached figure is a diagram showing the test results of the Sarcocystis template in the dual detection probe 1 provided by the present invention.

[0030] Figure 5 The attached figure is a graph showing the test results of the Trichinella spiralis template in the dual detection probe 2 provided by the present invention.

[0031] Figure 6 The attached figure is a diagram showing the test results of the Sarcocystis template in the dual detection probe 2 provided by the present invention.

[0032] Figure 7 The accompanying drawing is a sensitivity amplification curve diagram of Trichinella spiralis provided by the present invention.

[0033] Figure 8 The accompanying drawing is a sensitivity amplification curve diagram of Sarcocystis provided by the present invention.

[0034] Fig. 9 The accompanying drawing is a standard curve diagram of Trichinella spiralis provided by the present invention.

[0035] Fig.10 The accompanying drawing is a standard curve diagram of Sarcocystis provided by the present invention.

[0036] Fig.11 The accompanying drawing is a diagram of repeated tests of the minimum detection amount of Trichinella spiralis provided by the present invention.

[0037] Fig.12 The accompanying drawing is a diagram of repeated tests of the minimum detection amount of Sarcocystis provided by the present invention.

[0038] Fig.13 The accompanying drawing is a graph showing a specific amplification curve for detecting Trichinella spiralis provided by the present invention.

[0039] Fig.14 The accompanying drawing is an amplification curve diagram for specific detection of Sarcocystis provided by the present invention.

[0040] Fig.15 The accompanying drawing is an electrophoresis diagram of the amplified product of the Trichinella sample provided by the present invention; wherein, M is a relative molecular mass standard 2000bp DNA Ladder; 1 to 3 are positive controls; 4 is Toxoplasma gondii, 5 is Ascaris suum, 6 is Cryptosporidium, 7 is Lungworm, 8 is Cysticercus, 9 is Gastrostrongylus, 10 is Acanthocephalus, 11 is Kidney nematode, 12 is Nodular nematode, 13 is Coccidia, 14 is Whipworm, 15 is Fasciola zingiberensis, and 16 to 17 are negative controls.

[0041] Fig.16 The accompanying drawing is an electrophoresis diagram of the amplified products of the Sarcocystis sample provided by the present invention, wherein M is a relative molecular mass standard 2000bp DNA Ladder; 1-2 are positive controls; 3 is Toxoplasma gondii, 4 is Ascaris suum, 5 is Cryptosporidium, 6 is Lungworm, 7 is Cysticercus, 8 is Gastrostrongylus, 9 is Acanthocephalus, 10 is Kidney nematode, 11 is Nodular nematode, 12 is Coccidia, 13 is Whipworm, 14 is Fasciola zingiberensis, and 15-16 are negative controls.

[0042] Fig.17 The accompanying drawing is a diagram of the sequencing results of Trichinella spiralis provided by the present invention.

[0043] Fig.18 The attached figure is a diagram of the sequencing results of Sarcocystis provided by the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0045] The embodiment of the invention discloses a double Taqman detection probe primer set for Trichinella spiralis and Sarcocystis and application thereof.

[0046] The experimental reagents and materials not mentioned in the examples are all commercially available, and the experimental equipment not mentioned are all conventional experimental equipment, such as: 6× Loading Buffer, Fermentas (MBI), USA; Premix Ex Taq TM (ProbeqPCR), Bao Bioengineering (Dalian) Co., Ltd.; Entrans 2X qPCR Probe Master Mix, Abotek Biotechnology Co., Ltd.; 2×GUeasy antibody-modified Taq probe qPCR Master Mix, Anhui General Biotechnology Co., Ltd.; DL2000 DNA Marker, Bao Bioengineering (Dalian) Co., Ltd.; 2x PCR TaqMix, Beijing Mylabo Medical Technology Co., Ltd.; agarose, Biowest, Spain; RNase-free H2O, Walgene, South Korea; 50TAE, BIONEER, South Korea; GelRed, biotium, USA; Real Time PCR 384-well plate, Roche, Germany; 10μl / 200μl / 1000μl pipette tips, AXYGEN, USA; 1.5ml / 0.5ml centrifuge tubes, AXYGEN, USA.

[0047] 2720 ​​PCR instrument, ABI, USA; system electrophoresis Mupid (Mupid 2plus), TAKARA, Japan; image system (EUV-LDUV), Korea Biotech, South Korea; centrifuge (MICRO 17TR), Hanil, South Korea; clean bench, Suzhou Purification Company; 2.5μl / 10μl / 100μl / 200μl / 1000μl pipette, Eppendorf, Germany; Eppendorf epMotion 5070 automatic sample loading workstation, Eppendorf, Germany; autoclave (LS-B50L), Binjiang, Jiangyin, China; Freezer Big (DW-40L262), Haier, Qingdao; mixer vortex (Voltex-Genie2), SI, USA; oven dry (DHG-9240A) blast drying oven, Shanghai Yiheng Company; Balance (BT-214) balance, Denver, USA; Roche 480II real-time fluorescence quantitative PCR system, Roche, Germany.

[0048] I will not go into details here.

[0049] Example 1

[0050] Primer probe design

[0051] According to the gene sequences of Sarcocystis and Trichinella spiralis published on NCBI, and according to the design principles of primers and probes for TaqMan probe fluorescence PCR detection, the conserved sequences of 18SrRNA genes were artificially synthesized and linked into the vector. The synthesis company was Anhui General Biological Co., Ltd., the primer synthesis company was Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and the probe synthesis company was Shanghai Shenggong Biotechnology Co., Ltd. (Tables 1 and 2).

[0052] Table 1 Trichinella spiralis primer and probe sequence information

[0053]

[0054] Table 2 Primer and probe sequence information of Sarcocystis

[0055]

[0056]

[0057] Primer probe screening and validation

[0058] Three different reaction products test primer probe

[0059] Take a Roche 384-well plate and number it separately. The template is 5×10 3 For plasmid samples with a copy / μl, prepare the system according to different products (Table 3, Table 4 and Table 5). Mix well and place in a fluorescence quantitative PCR instrument. Reaction conditions: 95℃30s pre-denaturation, 95℃10s→60℃30s (fluorescence detection), 40 cycles, select FAM and HEX dual channel detection.

[0060] Table 3 Entrans 2X qPCRProbe MasterMix

[0061]

[0062] Table 4 Takara Premix Ex Taq TM (Probe qPCR)

[0063]

[0064]

[0065] Table 5 Universal 2×GUeasy Antibody Modified Taq Probe qPCR Master Mix

[0066]

[0067] Experimental results: Comparison of CT values, amplification curves and fluorescence signal intensities of the same template and the same gene ( Figure 1 and Figure 2 ), Takara Biotechnology (Dalian) Co., Ltd.: Takara Premix Ex Taq TM (Probe qPCR) amplification effect is the best. All subsequent experiments used Takara amplification reagents.

[0068] The amplification effects of the three sets of Sarcocystis probes were similar, but the T3 probe showed a false positive, so T3 was abandoned. The T3 probe amplified the most ideal CT value for Trichinella spiralis, while the T4 and T5 probes had larger CT values ​​and low fluorescence signals, so T4 and T5 were abandoned. Therefore, the T1 and T2 probes for Sarcocystis were combined with the T3 probe for Trichinella spiralis.

[0069] Optimal primer-probe combination and concentration screening

[0070] Take a Roche 384-well plate and number it separately. The template is 5×10 3 Copies / μl of plasmid samples were used with Takara Premix Ex Taq TM (Probe qPCR). Reaction system: template 1.0μl, Premix Ex Taq TM (Probe qPCR) 5μl, primer F / R 0.8μl, Probe 0.4μl, ddH2O to 10μl, mix well, and place in a fluorescence quantitative PCR instrument. Reaction conditions: 95℃30s pre-denaturation, 95℃10s→60℃30s (fluorescence detection), 40 cycles, select FAM and HEX dual channel detection.

[0071] Two sets of dual detection primers were combined: dual detection probe 1 (Sarcocystis T1 + Trichinella T3), dual detection probe 2 (Sarcocystis T2 + Trichinella T3). Each set of detection primers was prepared with the following ratio system (Table 6), and then 5×10 3 Copies / μl of plasmid sample template

[0072] Table 6 Reaction system

[0073]

[0074] Experimental results: The test results of dual detection probe 1 (Sarcocystis T1 + Trichinella T3) are shown in Figure 3 ,4 ; Double detection probe 2 (Sarcocystis T2 + Trichinella T3) test results are shown in Figure 5 , 6 Referring to the CT value and amplification curve, the dual detection probe 2 (Sarcocystis T2 + Trichinella T3) had the best amplification effect, and the dual detection probe 1 (Sarcocystis T1 + Trichinella T3) had the problem of reduced fluorescence signal intensity.

[0075] Double detection probe 2 (Sarcocystis T2 + Trichinella spiralis T3) was selected as the final experimental probe primer, and the primers involved in subsequent experiments were all this combination;

[0076] The comparison results of primer concentrations showed that 1.0μM, 0.6μM and 0.4μM had insufficient fluorescence signal intensity or increased CT values, respectively. The fluorescence signal intensity of the primer at 0.8μM concentration was the most ideal, and the CT value was also very close, so this primer probe concentration was determined as the final experimental system (Table 7).

[0077] Table 7 Final experimental system

[0078]

[0079] Screening of the optimal annealing temperature of primers and probes

[0080] Take Roche 384-well plates, number them respectively, and use Takara Premix Ex Taq TM (Probe qPCR), reaction system: template 1.0μl, Premix Ex Taq TM (Probe qPCR) 5μl, primer F / R 0.8μl, Probe 0.4μl, ddH2O to 10μl. Mix well and place in fluorescent quantitative PCR instrument. Reaction conditions: 95℃30s pre-denaturation, 95℃10s→60℃30s (fluorescence detection), 40 cycles, select FAM and HEX dual channel detection. The system remains unchanged, the annealing temperature is set to 58℃, 59℃, 60℃, 61℃, 62℃, respectively, and experiments are carried out respectively to compare the experimental results.

[0081] Experimental results: By comparing the CT values ​​and fluorescence intensities at annealing temperatures of 58°C, 59°C, 60°C, 61°C, and 62°C, it can be found that when the annealing temperature is 60°C, the CT value is the lowest and the fluorescence intensity is the highest. The annealing temperature of 60°C is selected as the set temperature for the amplification experiment.

[0082] Sensitivity test

[0083] Take Roche 384-well plates, number them separately, and use Takara Premix Ex Taq TM(Probe qPCR), reaction system: template 1.0μl, Premix Ex Taq TM (Probe qPCR) 5μl, 10μM primer F / R 0.8μl, 10μM Probe 0.4μl, ddH2O to 10μl. Mix well and place in fluorescence quantitative PCR instrument. Reaction conditions: 95℃30s pre-denaturation, 95℃10s→60℃30s (fluorescence detection), 40 cycles, select FAM and HEX dual channel detection.

[0084] The two standards were respectively 6 The initial concentration was diluted 10-fold to 1 × 10 copies / μl 0 , perform fluorescence quantitative PCR amplification reaction. After the reaction is completed, the system automatically generates a kinetic curve of the amplification reaction of the starting template number according to the change law of the fluorescence value.

[0085] Experimental results: The two standards were respectively 6 The initial concentration was diluted 10-fold to 1 × 10 copies / μl 0 , perform fluorescence quantitative PCR amplification reaction, and after the reaction is completed, the system automatically generates a kinetic curve of the amplification reaction of the starting template number according to the change law of the fluorescence value ( Figure 7 and Figure 8 ).

[0086] According to the changing law of fluorescence value, the system generates regression curve and equation through baseline setting, which are y=-3.989x+38.95, y=-3.334x+36.28, R 2 They are 0.993 and 0.989 respectively, showing a good linear relationship ( Fig. 9 and Fig.10 ).

[0087] The minimum detection amount of Trichinella spiralis is 10 copies / μl sample. The amplification results are as follows after 10 repeats of the test according to the minimum detection amount. All 10 repeats were amplified normally, and there was no amplification in the 4 negative controls ( Fig.11 The minimum detection amount of Sarcocystis is 1 copy / μl sample. The amplification results are as follows after 10 repetitions of the test according to the minimum detection amount. All 10 repetitions were amplified normally, and there was no amplification in the 4 negative controls ( Fig.12 ).

[0088] Specificity test

[0089] Take Roche 384-well plates, number them separately, and use Takara Premix Ex Taq TM (Probe qPCR). Reaction system: template 1.0μl, Premix Ex Taq TM(Probe qPCR) 5μl, 10μM primer F / R 0.8μl, 10μM Probe 0.4μl, ddH2O to 10μl. Mix well and place in fluorescence quantitative PCR instrument. Reaction conditions: 95℃30s pre-denaturation, 95℃10s→60℃30s (fluorescence detection), 40 cycles, select FAM and HEX dual channel detection.

[0090] Experimental results: The positive control showed an amplification curve, while the other parasite DNA showed no amplification curve ( Fig.13 and Fig.14 ), indicating that the established dual TaqMan qPCR detection method for Trichinella spiralis and Sarcocystis has good specificity.

[0091] Agarose gel electrophoresis showed that the molecular weights of the PCR product bands of Trichinella spiralis and Sarcocystis spp. were consistent with the expected molecular weights of 717 bp and 872 bp ( Fig.15 and Fig.16 ).

[0092] Sequence the positive sample product and generate a sequencing result graph ( Fig.17 and Fig.18 ).

[0093] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0094] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A dual Taqman detection probe primer set for Trichinella spiralis and Sarcocystis, characterized in that: The primer and probe sequences are as follows: Trichinella spiralis-F3: 5′-ACTTCTTAGAGGGACTGGCAAA-3′, as shown in SEQ ID No. 1; Trichinella spiralis-R3: 5'-GTGCAGCCCCGGACAT-3', as shown in SEQ ID No. 2; Trichinella spiralis-T3: 5′-CGAAGGGCATCACAGACCTGTTATTG-3′, as shown in SEQ ID No. 3; Sarcocystis-F2: 5'-GAGTATGGTCGCAAGGCTGAA-3', as shown in SEQ ID No. 4; Sarcocystis-R2: 5'-CGTGTTGAGTCAAATTAAGCCG-3', as shown in SEQ ID No. 5; Sarcocystis-T2: 5'-CTTAAAGGAATTGACGGAAGGGCAC-3', as shown in SEQ ID No.6; Among them, Trichinella-T3 was 5′ labeled with HEX and 3′ labeled with BDQ1; Sarcocystis-T2 is 5' labeled with FAM and 3' labeled with BDQ1.

2. A detection kit comprising the detection probe primer set according to claim 1.

3. The detection kit according to claim 2, characterized in that Reaction system: Template 1.0μl, Premix ExTaq TM 5μl, 10μM Primer F / R 0.8μl, 10μM Probe 0.4μl, ddH2O to 10μl.

4. The detection kit according to claim 3, characterized in that Reaction conditions: pre-denaturation at 95°C for 30 s, fluorescence detection at 95°C for 10 s → 60°C for 30 s, 40 cycles, FAM and HEX dual-channel detection.

5. Use of the detection probe primer set according to claim 1, or the detection kit according to any one of claims 2 to 4, in non-diagnostic and therapeutic detection of Trichinella spiralis and Sarcocystis.

6. The use according to claim 5, characterized in that Used in animal husbandry and production.

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