Primers and methods for double PCR detection of Heterozoan japonensis and Heterozoan brevis

Abstract

The present invention relates to a dual PCR detection method for simultaneously detecting Japanese trematode and short-intestinal trematode, comprising the following steps: firstly extracting DNA of Japanese trematode and short-intestinal trematode and designing specific primers of the two trematodes, then optimizing the dual PCR reaction system and reaction conditions, and finally determining the best PCR reaction system and reaction conditions. The specificity and sensitivity of the established method and the feasibility of the clinical sample detection method are verified according to the best conditions. In addition, the present invention also discloses a specific primer sequence for simultaneously detecting Japanese trematode and short-intestinal trematode. Experimental verification shows that the primer sequence disclosed by the present invention can simultaneously identify Japanese trematode and short-intestinal trematode, has the advantages of simple and rapid operation, intuitive results, high sensitivity and specificity, etc., and lays a foundation for epidemiological investigation and disease prevention and control of Japanese trematode and short-intestinal trematode.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture and animal quarantine, and in particular to a double PCR detection method for short-bowel trematode and Japanese trematode. In addition, the present invention also relates to primers for simultaneously detecting short-bowel trematode and Japanese trematode. Background Art

[0002] Diplodiscus japonicus and Diplodiscus mehrai both belong to the Diplodiscidae family and the Diplodiscus genus. They are two important parasites in the intestinal tract of frogs and are widely found in frogs in my country. The infection rates of Diplodiscus japonicus and Diplodiscus mehrai in forest frogs in Heilongjiang Province alone can reach 14.19% and 22.38%, respectively, which poses a huge threat to the frog breeding industry. At present, the main method for distinguishing and detecting Diplodiscus japonicus and Diplodiscus mehrai is morphological identification. However, since the morphologies of the two are extremely similar, it requires researchers with rich clinical experience to distinguish them. Moreover, morphological identification often relies on subjective judgment, which is prone to misjudgment. Therefore, relying on morphological identification of these two trematodes is greatly limited.

[0003] With the development of molecular biology technology, PCR technology has been widely used in the field of rapid detection of pathogens. Compared with conventional detection technology, this method not only shortens the detection time, but also saves manpower and material resources, and provides a reliable basis for the rapid diagnosis of pathogens. Mitochondrial DNA (Mitochondria DNA, mtDNA) has maternal inheritance, small genome, relatively fast evolution speed and other characteristics, and is therefore suitable as a molecular marker for parasite molecular classification, species identification and system evolution. There is currently no report on the detection of Japanese trematode and short-intestinal trematode by dual PCR method at home and abroad. Therefore, the present invention intends to establish a dual PCR detection method for Japanese trematode and short-intestinal trematode based on mitochondrial genome DNA sequence, which eliminates the problems of false detection, missed detection and time-consuming in conventional detection, and can intuitively detect the presence of Japanese trematode and short-intestinal trematode from the gene level simultaneously, which lays the foundation for epidemiological investigation and disease prevention and control of two trematodes, and has very important research significance. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a multiplex PCR primer and design method for simultaneously detecting Japanese trematode and short-intestinal trematode. The method can quickly and accurately detect these two morphologically similar trematodes, and provide a technical method for the identification, separation, epidemiological investigation and other research of Japanese trematode and short-intestinal trematode.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] The invention provides double PCR primers for Japanese trematode and short-intestinal trematode, Japanese trematode specific primers: DJ-F: 5'-TTTGTAATGATAATGCGATGT-3', DJ-R: 5'-AACCGAAACAATAAACTACAC-3'; short-intestinal trematode specific primers: DM-F: 5'-GGGTTGTGGGCTCTGATTTGT-3', DM-R: 5'-ATAACCTAGTTCGCTAATTCC-3'.

[0007] A double PCR detection method for detecting Heterozoa japonicus and Heterozoa brevis, comprising the following specific steps:

[0008] 1. Extraction of insect DNA; 2. Primer design and screening; 3. Optimization of dual PCR reaction conditions; 4. Verification of dual PCR amplification specificity; 5. Verification of dual PCR amplification sensitivity; 6. Feasibility verification of clinical sample detection method.

[0009] In step 1, the method for extracting the genomic DNA of Japanese trematode and short-intestinal trematode is as follows: the Japanese trematode and short-intestinal trematode bodies identified by morphology are stored in 70% ethanol, and the genomic DNA of the two trematodes is extracted separately using the tissue genomic DNA extraction kit of TIANGEN Company according to the instructions, and stored at -20°C for later use.

[0010] In step 2, the dual PCR primer design and screening of the Japanese heavy disc fluke and short intestinal heavy disc fluke are specifically as follows: the biological software DNASTARLasergene 8.0 and Oligo 7.0 are used to compare and analyze the interspecific differences of the mitochondrial genome DNA sequences of the two trematodes, select the region with lower homology, and design the specific primers of the Japanese heavy disc fluke and short intestinal heavy disc fluke. The primers used in the final experiment are screened to ensure that the designed primers can specifically amplify the target product of the corresponding worm species, and in the process of implementing multiple PCR, there is no interference between multiple pairs of primers, that is, there is no homology, complementarity, and no hairpin structure is formed. The final determined primer sequence is shown in Table 1.

[0011] Table 1 Information of specific primer sequences for Diplodiscus japonicus and Diplodiscus mehrai

[0012]

[0013] In step 3, the specific method for optimizing the PCR reaction conditions is as follows: the reaction system is 50 μL, including 5 μL of 10×Ex Buffer, 4 μL of dNTP, 1 μL of upstream and downstream primers, and the amount and ratio of the two pairs of primers are adjusted according to the brightness of the double PCR target band, 2 μL of DNA template, 0.2 μL of Ex Taq DNA polymerase, and ddH2O is added to adjust the total volume to 50 μL. The concentrations of the two pairs of primers (100nmol / L, 200nmol / L, 300nmol / L, 400nmol / L, 500nmol / L) and annealing temperatures (46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃) were optimized, and the optimal multiplex PCR reaction system was finally determined as follows: 5μL of 10×Ex Buffer, 4μL of dNTPs, 1μL each of the upstream and downstream primers of the two trematode metacercariae, 1μL each of the primers of the two trematodes (200nmol / L primers for Japanese trematode and 400nmol / L primers for short-bowel trematode), 2μL each of DNA templates, 0.2μL of Ex Taq DNA polymerase, and ddH2O were added to adjust the total volume to 50μL. The optimal PCR reaction conditions were: pre-denaturation at 92°C for 2 min; denaturation at 92°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles; and final extension at 72°C for 7 min.

[0014] In step 4, the specific method for verifying the specificity of the multiplex PCR amplification is as follows: using two pairs of primers of Pleurogenoides japonicus and Pleurogenoides short-bowel trematode at the same time, using Pleurogenoides japonicus, Pleurogenoides short-bowel trematode, a combination of two templates, and Pleurogenoides japonicus, Glypthelmins jilinensis, and Dolichosaccus huabeiensis as templates, and amplifying according to the above PCR reaction system and conditions to evaluate the specificity of the method.

[0015] In step 5, the PCR sensitivity verification is specifically as follows: the DNA mass concentration of Japanese trematode and short-bowel trematode is determined by Nano-Drop 2000 ultra-micro spectrophotometer, uniformly diluted to the same mass concentration, and then diluted in multiples to a mass concentration of 10% of the original solution. -1 ~10 -6 Multiplex PCR was performed according to the above conditions to evaluate the sensitivity of the method.

[0016] In step 6, the feasibility verification of the clinical sample detection method is specifically as follows: extracting total genomic DNA from the worm samples morphologically identified as Japanese trematode and short-bowel trematode, and detecting them according to the above-mentioned PCR reaction system and conditions.

[0017] The dual PCR detection method of the present invention for the detection of Heterozoan spp. and Heterozoan spp. has the following advantages:

[0018] 1. Simple and fast operation: The dual PCR detection method of the present invention can be completed within 3 hours, eliminating the tedious process of morphological identification or gene sequencing, which not only shortens the detection time, but also saves manpower and material resources.

[0019] 2. The results are intuitive: the two pairs of primers designed in the present invention are similar in length, GC content and melting temperature (Tm), which can effectively avoid mismatch between primers and the formation of primer dimers and hairpin structures. The amplified gene fragments are 267 bp and 543 bp, respectively. Therefore, the insect species represented by different bands can be displayed on one electrophoresis gel through agarose gel electrophoresis, and the presence of the two trematodes can be intuitively detected at the gene level.

[0020] 3. High sensitivity and high specificity: The multiplex PCR detection method of the present invention has good sensitivity, and the minimum detection concentration of the two trematode template DNAs can reach 40ng / μL×10 -5 At the same time, this method has high specificity and can specifically amplify the Japanese trematode and the short-bowel trematode, while the amplification results for similar trematode species are all negative, which solves the problem of false detection and missed detection in conventional testing.

[0021] Compared with the prior art, the present invention can simultaneously detect and identify the Japanese trematode and the short-bowel trematode. The method is simple and fast to operate, the results are intuitive, and it has high sensitivity and high specificity, which cannot be met by traditional morphological identification methods and sequencing identification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the double PCR amplification results in the embodiment of the present invention. M: DL 2 000 Marker; 1: double PCR amplification bands of Heterozoa japonicus and Heterozoa brevis; 2: negative control.

[0023] Figure 2 Schematic diagram of the specific results of double PCR amplification in the embodiment of the present invention. M: DL2 000 Marker; 1: templates of Helicoverpa japonicum and Helicoverpa brevicaulis; 2: template of Helicoverpa brevicaulis; 3: template of Helicoverpa japonicum; 4: template of Pseudoparagonimus japonicum; 5: template of Jilin cleavers; 6: template of Longitumosphaeria serrata; 7: negative control.

[0024] Figure 3 It is a schematic diagram of the PCR sensitivity results in the embodiment of the present invention. M: DL 2 000Marker; 1-6: 10 -1 ~10 -6 1: times diluted DNA template; 7: negative control. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further specifically described below through specific embodiments and drawings, but the present invention is not limited to these embodiments.

[0026] The invention provides double PCR primers for Japanese trematode and short-intestinal trematode. Japanese trematode specific primers: DJ-F: 5'-TTTGTAATGATAATGCGATGT-3', DJ-R: 5'-AACCGA AACAATAAACTACAC-3'; short-intestinal trematode specific primers: DM-F: 5'-GGGTTGTGGGCTCTGATTTGT-3', DM-R: 5'-ATAACCTAGTTCGCTAATTCC-3'.

[0027] A dual PCR detection primer and method for Helicoverpa japonicus and Helicoverpa short-bowel trematode, the specific steps comprising:

[0028] 1. Extraction of DNA from parasites; Extraction of genomic DNA from Heterozoan japanese and Heterozoan short-bowel trematodes:

[0029] The genomic DNA was extracted according to the TIANGEN tissue genomic DNA extraction kit as follows:

[0030] (1) The identified adults of H. japonicum and H. shortgut were placed in sterile 1.5 mL Eppendorf tubes respectively;

[0031] (2) After adding sterile ddH2O and washing repeatedly three times, discard the ddH2O;

[0032] (3) Add 200 μL buffer GA and 20 μL Proteinase K and incubate in a 55°C water bath for 1–3 h until digestion is complete;

[0033] (4) Centrifuge at 12,000 rpm for 2 min and transfer the supernatant to another sterile 1.5 mL Eppendorf tube;

[0034] (5) Add 200 μL of buffer GB, mix thoroughly by inversion, and incubate at 70°C for 10 min.

[0035] (6) Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds;

[0036] (7) Add all the solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the liquid in the collection tube;

[0037] (8) Add 500 μL of buffer GD, centrifuge at 12,000 rpm for 1 min, and discard the liquid in the collection tube;

[0038] (9) Add 700 μL of rinse solution PW, centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube;

[0039] (10) Repeat step (9) once;

[0040] (11) Centrifuge at 12,000 rpm for 3 min and discard the waste liquid;

[0041] (12) Place the adsorption column at room temperature for 3 min to completely dry the remaining rinse solution in the adsorption column;

[0042] (13) Place the adsorption column in another sterile 1.5 mL Eppendorf tube, add 200 μL of elution buffer, let stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and collect the eluted DNA into a centrifuge tube;

[0043] (14) The eluted DNA was stored at -20°C for future use.

[0044] 2. Primer design and screening: Primer design and screening for dual PCR of Heterozoan japanese and Heterozoan short-bowel trematodes:

[0045] The mitochondrial whole genome sequences of Helicoverpa japonicum (gene accession number: OL961442) and Helicoverpa brachyentes (gene accession number: OL961441) were downloaded from the NCBI website, and the homology of the whole mitochondrial genome sequences of Helicoverpa japonicum and Helicoverpa brachyentes was compared using the biological software DNASTARLasergene 8.0. The biological software Oligo7.0 was used to design specific primers for the two trematodes in the regions with low homology between the two trematodes, and the designed candidate primers were subjected to Blast comparison in NCBI to determine the specificity of the primers. The final primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primer sequences are shown in Table 1 below.

[0046] Primer design must meet the following requirements:

[0047] (1) The length of the primer is designed to be between 20 and 27 bp. The primer length can be adjusted to avoid the region where primer dimers are formed.

[0048] (2) The 3′ end of the primer is designed at a site with low homology between the two to increase the specificity of each primer;

[0049] (3) The primer itself should not have a complementary sequence, and the GC content of the primer sequence should be 40-60%;

[0050] (4) The difference between the Tm values ​​of the two pairs of primers is controlled within 1°C;

[0051] (5) The bands amplified by the two pairs of primers are controlled within 1000 bp to shorten the detection time and achieve the purpose of rapid detection.

[0052] (6) When designing two pairs of primers, the amplified bands should be significantly different so that they can be distinguished by observing the gel electrophoresis pattern.

[0053] According to the above requirements, the primer information designed in the present invention is shown in Table 1.

[0054] Table 1 Information of specific primer sequences for Diplodiscus japonicus and Diplodiscus mehrai

[0055]

[0056] 3. Optimization of double PCR reaction conditions: Double PCR reaction system and conditions for Helicoverpa japonicum and Helicoverpa short-bowel trematode:

[0057] The specific method of the double PCR reaction conditions is as follows: the reaction system is 50 μL, including 5 μL of 10×Ex Buffer, 4 μL of dNTP, 1 μL of upstream and downstream primers, and the amount and ratio of the two pairs of primers are adjusted according to the brightness of the double PCR target band, 1 μL of DNA template, 0.2 μL of Ex Taq DNA polymerase, and ddH2O is added to adjust the total volume to 50 μL. The concentrations of the two pairs of primers (100 nmol / L, 200 nmol / L, 300 nmol / L, 400 nmol / L, 500 nmol / L) and annealing temperatures (46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C) were optimized. The optimal multiplex PCR reaction system was finally determined as follows: 5 μL of 10×Ex Buffer, 4 μL of dNTP, 1 μL of each primer of the two trematodes (200 nmol / L primer of Japanese trematode and 400 nmol / L primer of short-bowel trematode), 1 μL of each DNA template, 0.2 μL of Ex Taq DNA polymerase, and ddH2O were added to adjust the total volume to 50 μL. The optimal PCR reaction conditions were: pre-denaturation at 92°C for 2 min; denaturation at 92°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles; final extension at 72°C for 7 min. Using the optimized reaction system and conditions, the genomic DNA of the two trematodes was amplified, and the specific target bands of the two trematodes could be amplified simultaneously, such as Figure 1 shown.

[0058] 4. Verification of the specificity of double PCR amplification: Verification of the specificity of double PCR amplification of Heterozoa japonicus and Heterozoa brevis:

[0059] At the same time, two pairs of primers designed by the present invention were used, and the genomic DNA of Japanese trematode, short-intestinal trematode, Japanese trematode + short-intestinal trematode, and Japanese trematode, Jilin cuticle trematode and North China long capsule trematode were used as templates, and amplification was performed according to the above-mentioned PCR reaction system and conditions. At the same time, the two pairs of primers designed by the present invention were used to perform PCR amplification on the templates of Japanese trematode and short-intestinal trematode, and single specific target bands were amplified; at the same time, two pairs of primers were used to perform PCR amplification on the mixed template of Japanese trematode and short-intestinal trematode, and 2 specific target bands were amplified; at the same time, two pairs of primers were used to perform PCR amplification on the templates of Japanese trematode, Jilin cuticle trematode and North China long capsule trematode, and no corresponding bands were amplified, such as Figure 2 Therefore, the dual PCR amplification specificity test of Helicoverpa japonicum and Helicoverpa short-intestinalis confirmed that these two pairs of primers have strong specificity and can be used to simultaneously detect Helicoverpa japonicum and Helicoverpa short-intestinalis.

[0060] 5. Dual PCR amplification sensitivity verification: Dual PCR amplification sensitivity verification of Japanese trematode and short-bowel trematode:

[0061] The mass concentrations of DNA of Japanese trematode and short-bowel trematode were measured by Nano-Drop 2000 ultra-micro spectrophotometer. The mass concentrations of DNA of Japanese trematode were 40.2ng / μL and 52.4ng / μL, so they were uniformly diluted to 40ng / μL. Then the template DNA of the two trematodes was diluted in multiples to 10% of the original solution. -1 ~10 -6 The minimum detection concentration of the two trematode template DNAs can reach 40 ng / μL×10 -5 ,like Figure 3 Therefore, the dual PCR amplification sensitivity test of Helicoverpa japonicum and Helicoverpa shortgut confirmed that this method has high sensitivity.

[0062] 6. Feasibility verification of clinical sample detection method: The dual PCR method of Japanese trematode and short-intestinal trematode established by the present invention was used to detect 32 trematodes isolated from the Northeast Forest Rana. The results showed that there were 21 Japanese trematodes and 11 short-intestinal trematodes. The results of the above detection were consistent with the results of morphological detection. Therefore, the feasibility of the method was confirmed by the dual PCR method for clinical sample detection.

Claims

1. A dual PCR detection primer for Japonagna japonica and Brachyenteroides spp., Japonagna japonica specific primers: DJ-F: 5'-TTTGTAATGATAATGCGATGT -3', DJ-R: 5'-AACCGA AACAATAAACTACAC -3'; Brachyenteroides spp. specific primers: DM-F: 5'-GGGTTGTGGGCTCTGATTTGT -3', DM-R: 5'-ATAACCTAGTTCGCTAATTCC -3'.

2. A non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa shortgut, characterized in that: The specific steps include the following: (1) Extraction of genomic DNA from Heterozoa japonicus and Heterozoa brevis; (2) Optimize the reaction conditions of the primers specific for Japanese trematode: DJ-F: 5'-TTTGTAATGATAATGCGATGT -3', DJ-R: 5'-AACCGA AACAATAAACTACAC -3' and the primers specific for short-bowel trematode: DM-F: 5'-GGGTTGTGGGCTCTGATTTGT -3', DM-R: 5'-ATAACCTAGTTCGCTAATTCC -3', and screen out the best reaction system and reaction conditions; (3) Performing double PCR amplification specificity verification according to the optimal PCR reaction system and reaction conditions in step (2); (4) Perform double PCR amplification sensitivity verification according to the optimal PCR reaction system and reaction conditions in step (3); (5) Conduct feasibility verification on clinical samples according to the established dual PCR method.

3. The non-diagnostic dual PCR detection method for Helicoverpa japonicum and Helicoverpa shortgut according to claim 2, characterized in that: In step (1), the method for extracting DNA of the Japanese trematode and the short-intestinal trematode is as follows: the Japanese trematode and the short-intestinal trematode bodies identified by morphology are stored in 70% ethanol, and the genomic DNA of the two trematodes is extracted separately using the tissue genomic DNA extraction kit of TIANGEN Company according to the instructions, and stored at -20°C for later use.

4. The non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa brevicaulis according to claim 2, characterized in that: In step (2), the specific method for optimizing the PCR reaction conditions is as follows: the reaction system is 50 μL, of which 10×Ex Buffer is 5 μL, dNTP is 4 μL, and upstream and downstream primers are 1 μL each. The amount and ratio of the two pairs of primers are adjusted according to the brightness of the multiple PCR target bands. DNA template is 2 μL, Ex Taq DNA polymerase is 0.2 μL, and ddH2O is added to adjust the total volume to 50 μL. The concentrations of the two pairs of primers are 100 nmol / L, 200 nmol / L, 300 nmol / L, 400 nmol / L, 500nmol / L and the annealing temperatures are 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, and 55°C. Finally, the best multiple PCR reaction system is determined.

5. The non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa brevicaulis according to claim 2 or 4, characterized in that: In step (2), the optimal multiplex PCR reaction system is specifically as follows: 5 μL of 10×Ex Buffer, 4 μL of dNTP, 1 μL of each of the two trematode primers (200 nmol / L of the Japanese trematode primer and 400 nmol / L of the short-bowel trematode primer), 1 μL of each of the DNA templates, 0.2 μL of Ex Taq DNA polymerase, and ddH2O are added to adjust the total volume to 50 μL. The optimal PCR reaction conditions are as follows: pre-denaturation at 92°C for 2 min; denaturation at 92°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; and final extension at 72°C for 7 min.

6. The non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa brevicaulis according to claim 2, characterized in that: In step (3), the specificity verification of the multiplex PCR amplification is specifically as follows: using the two pairs of primers in step (2) at the same time, using the genomic DNA of Japanese trematode, short-intestinal trematode, Japanese trematode + short-intestinal trematode, and Japanese pseudopartioglobinuria, Jilin trematode and northern stomata as templates, and amplifying according to the optimal double PCR reaction conditions of step (2) to evaluate the specificity of the method.

7. The non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa brevicaulis according to claim 1, characterized in that: In step (4), the sensitivity verification of the multiple PCR amplification is specifically as follows: using an ultra-micro spectrophotometer to respectively determine the mass concentration of the genomic DNA of the Japanese trematode and the short-bowel trematode, uniformly dilute them to the same mass concentration, and then dilute them in multiple ratios, and the mass concentrations are diluted to 10% of the original solution. -1 ~10 -6 The optimal duplex PCR reaction conditions in step (2) were used to amplify the DNA to evaluate the sensitivity of the method.

8. The non-diagnostic duplex PCR detection method for Helicoverpa japonicum and Helicoverpa brevicaulis according to claim 2, characterized in that: In step (5), the feasibility verification of the clinical sample detection method is specifically as follows: total genomic DNA is extracted from the two trematode samples identified by morphology, amplified according to the optimal double PCR reaction conditions of step (2), and the PCR detection results are compared with the morphological identification results to verify the feasibility of the method for clinical sample detection.

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