EDNA-based carposina moth species identification method and primer composition used by same

Abstract

The invention discloses an eDNA (enhanced deoxyribonucleic acid)-based carposinia moth species identification method and a primer composition used by the same. The invention belongs to the technical field of biology, and particularly relates to an eDNA-based carpomoth species identification method and a primer composition used by the same. The identification method comprises the following steps: 1) collecting an environment eDNA sample from a to-be-detected environment; 2) extracting DNA in the environment eDNA sample to obtain a DNA sample solution; and 3) taking the DNA sample solution as a template, carrying out PCR amplification by using a primer composition for identifying the carpoworm species, and judging a PCR product. The primer composition comprises a primer group A, a primer group B and a primer group C, wherein the nucleotide sequences of the primer group A are SEQ ID No. 1 and SEQ ID No. 2; the nucleotide sequences of the primer group B are as shown in SEQ ID No.3 and SEQ ID No.4. The method is simple and convenient in sampling and low in cost, and has a potential application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an eDNA-based method for identifying fruit moth species and a primer composition used therein. Background Art

[0002] Fruit borers include pear borer and peach borer. Both Grapholitha molesta and Carposina sasakii belong to the family Carposinidae of the order Lepidoptera. They are widely distributed and mainly harm the fruits of many fruit trees, including apples, dates, pears, peaches, plums, apricots, and hawthorns. They pose a great threat to fruit production and cause serious economic losses to fruit farmers. Both are key targets for prevention and control. However, the external morphology of the larvae of the two species is very similar, so it is very difficult to accurately identify the two species of borers during field surveys and population dynamics monitoring.

[0003] Environmental DNA (eDNA) refers to the collection of materials left by organisms in the surrounding environment, including the skin, mucus, saliva, sperm, secretions, eggs, feces, urine, blood, roots, leaves, fruits, pollen and remains of larger organisms. It can determine whether a species exists without directly observing or capturing the complete organism, thus expanding the scope of identification for port quarantine. This technology combines DNA barcoding technology, extracting DNA from environmental samples, amplifying it using designed specific primers, and performing agarose gel electrophoresis and sequencing on the amplified products for species identification.

[0004] At present, pest identification requires observation of the insect body, and investigators need to have traditional external morphological recognition and identification experience. In addition, some molecular identification methods have the disadvantages of high technical requirements, difficult operation steps, long time consumption, and high cost. Therefore, it is urgent to develop a method for identifying pests based on eDNA extraction technology that does not require insect bodies, is non-destructive, sensitive, efficient, and accurate. Summary of the invention

[0005] The main problem to be solved by the present invention is how to efficiently and accurately identify the types of fruit borers in the environment.

[0006] In order to solve the above problems, the present invention provides a method for identifying or assisting in identifying fruit moth species.

[0007] The present invention provides a method for identifying or assisting in identifying a fruit borer species, wherein the fruit borer is a pear borer (Grapholitha molesta) and / or a peach borer (Carposina sasakii), comprising the following steps:

[0008] 1) Collect environmental eDNA samples from the environment to be tested;

[0009] 2) extracting DNA from the environmental eDNA sample to obtain a DNA sample solution;

[0010] 3) Using the DNA sample liquid as a template, PCR amplification is performed using a primer set for identifying fruit moths, and determining whether the environment to be detected contains the fruit moths according to the size of the PCR amplification product in accordance with the following method:

[0011] If the PCR amplification product contains a DNA fragment of 250-500bp, pear borer exists in the environment to be detected; if the PCR amplification product contains a DNA fragment greater than 100bp and less than 250bp, peach borer exists in the environment to be detected; if the PCR amplification product contains a DNA fragment of 250-500bp and a DNA fragment greater than 100bp and less than 250bp, pear borer and peach borer exist in the environment to be detected.

[0012] The primer set for identifying fruit moth includes primer set A and primer set B, wherein primer set A is composed of a single-stranded DNA with a nucleotide sequence of SEQ ID No.1 and a single-stranded DNA with a nucleotide sequence of SEQ ID No.2; and primer set B is composed of a single-stranded DNA with a nucleotide sequence of SEQ ID No.3 and a single-stranded DNA with a nucleotide sequence of SEQ ID No.4.

[0013] In the above method, the 250-500 bp DNA fragment is a 288 bp DNA fragment, and the DNA fragment larger than 100 bp and smaller than 250 bp is 191 bp.

[0014] The nucleotide sequence of the 288 bp DNA fragment may be SEQ ID No. 5 in the sequence listing.

[0015] The nucleotide sequence of the 191 bp DNA fragment may be SEQ ID No. 6 in the sequence listing.

[0016] In the above method, the environmental eDNA sample is a water sample, and the step 2) includes filtering the water sample through a filter membrane and then extracting the DNA in the environmental eDNA sample.

[0017] In the above method, the environmental eDNA sample is a water sample.

[0018] In the above method, when the environmental eDNA sample is a water sample, the step 2) includes filtering the water sample with a filter membrane and then extracting the DNA in the environmental eDNA sample.

[0019] In the above method, the filter membrane is a 0.45 μm cellulose acetate filter membrane.

[0020] In the above method, the primer annealing condition used in the PCR amplification is annealing at 55° C. for 30 seconds.

[0021] In the above method, the PCR reaction system (25 μL) is 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 12.5 μL 2×Taq PCR Mastermix, 9.5 μL ddH2O, and 1.0 μL DNA.

[0022] In the above method, the qPCR amplification program includes: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0023] The present invention also provides a primer composition for identifying fruit borers, the primer composition comprising a primer group A and a primer group B, the nucleotide sequence of the primer group A is shown as SEQ ID No.1 and SEQ ID No.2; the nucleotide sequence of the primer group B is shown as SEQ ID No.3 and SEQ ID No.4.

[0024] The present invention also provides a fruit moth detection kit, comprising the primer combination as described above.

[0025] The present invention also provides the use of the primer combination in any of the following:

[0026] (1) Detect or assist in the detection of pear borer and peach borer;

[0027] (2) preparing products for detecting or assisting in detecting pear borer and peach borer;

[0028] (3) Amplification of pear borer and peach borer DNA;

[0029] (4) Preparing products for amplifying DNA of pear borer and peach borer.

[0030] The present invention also provides the use of the primer composition or the kit described above in an environmental pollution monitoring, food safety testing product or disease diagnosis device.

[0031] In the present invention, when the primer set described in this article is used for PCR, when the size of the obtained PCR product is 288bp (the nucleotide sequence is SEQ ID No.5 in the sequence list), the result is judged to be positive for pear borer, that is, pear borer exists in the environment to be detected; when the size of the obtained PCR product is 191bp (the nucleotide sequence is SEQ IDNo.6 in the sequence list), the result is judged to be positive for peach borer, that is, peach borer exists in the environment; when the 288bp and 191bp bands do not exist in the PCR product, it means that pear borer and peach borer do not exist in the environment to be detected.

[0032] The present invention proposes a method for identifying fruit moth species and a primer composition used therefor for environmental samples. By preparing environmental samples related to fruit moths, sterile water is used to elute the fruit, the damaged part of the fruit is soaked in a measuring cup for 15-30 minutes, water samples are collected, the collected water samples are filtered, eDNA is extracted using a kit, and the extracted eDNA is subjected to PCR using a developed primer composition, and agarose gel electrophoresis and sequencing analysis are performed. The present invention does not require investigators to have traditional species morphological recognition and identification capabilities, and sampling is simple, efficient, low-cost, highly sensitive, and will not cause harm to the target species. In addition, identification can also be performed without finding the insect body. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the relationship between environmental DNA concentration and different filter membranes.

[0034] Figure 2 It is the agarose gel electrophoresis of the PCR products of the environmental DNA samples. Lane M is D2000 Marker; Lane 1 is the electrophoresis result of the pear borer extracted by eDNA technology; Lane 2 is the electrophoresis result of the peach borer extracted by eDNA technology; Lane 3 is the electrophoresis result of the pear borer body; Lane 4 is the electrophoresis result of the peach borer body; Lanes 5 and 6 are the electrophoresis results of the citrus fruit fly extracted by eDNA technology; Lanes 7 and 8 are the electrophoresis results of the citrus fruit fly body; Lanes 9 and 10 are the electrophoresis results of normal and intact apples extracted by eDNA technology; N1 and N2 are ddH2O. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0037] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0038] The pear borer in the following examples was purchased from the Fruit Research Institute of the Chinese Academy of Agricultural Sciences.

[0039] The peach borer in the following examples was purchased from the Fruit Research Institute of the Chinese Academy of Agricultural Sciences.

[0040] The citrus fruit fly in the following examples is preserved by this laboratory and has been recorded in: Li H, Li Z, Zhao Z. Egg-Associated Germs Induce Salicylate Defenses but Not Render Plant Against a Global Invasive Fruit Fly Effectively [J]. Journal of Agricultural and Food Chemistry, 2023, 71 (15): 6023-6031. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0041] The following examples were processed using SPSS11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference.

[0042] Embodiment 1, the method for rapid identification of fruit borer moth

[0043] 1. Preparation of environmental samples

[0044] In the laboratory, simulate the outdoor scene, place a fresh-keeping box in the incubator, select Fuji apples of similar size and without obvious bumps, put them in the fresh-keeping box, and then place the egg papers containing pear borer and peach borer, as well as the egg papers of citrus fruit fly on the apples. The relevant parameters of the incubator are set as follows: temperature is 25±1℃, humidity is 70%, and light conditions: dark conditions = 16:8h. Cultivate for 14 days. These two apples are damaged apples. At the same time, uninoculated apples are set as controls, and 5 apples are treated for each.

[0045] 2. Elution and collection of fruit pulp samples damaged by fruit borers

[0046] The affected apples were dissected, and the flesh of the affected apples was soaked in sterile water for 15-30 minutes. The measuring cup was sterilized, and 0.5L of water sample was collected to obtain environmental eDNA samples inoculated with both pear borer and peach borer and environmental eDNA samples inoculated with citrus fruit fly. At the same time, the flesh of uninoculated apples was treated in the same way to obtain uninoculated environmental eDNA samples.

[0047] 3. Collect water samples for filtration

[0048] After collecting the water samples in step 2 (environmental eDNA samples inoculated with pear borer and peach borer, environmental eDNA samples inoculated with citrus fruit fly and non-inoculated environmental eDNA samples), in order to speed up the filtration efficiency, two layers of filter membranes were used for filtration. The upper filter membrane was replaced each time it was blocked, and the lower filter membrane was not replaced. The filter membranes were collected to obtain environmental eDNA sample filters of pear borer and peach borer and environmental eDNA sample filters of citrus fruit fly. The upper filter membrane used a mixed fiber filter membrane (MCE) with a diameter of 47 mm and a pore size of 10 μm, and the lower filter membrane used a glass fiber filter membrane (GF) with a diameter of 47 mm and pore sizes of 1.2 μm, 0.8 μm, and 0.45 μm, respectively. The eDNA samples were extracted from 0.5L of water samples, and the filtration time was calculated. The filtration time of different lower filter membranes for mixed environmental DNA samples inoculated with pear borer and peach borer is shown in Table 1.

[0049] The results showed that different filter membranes and pore sizes had different filtration times. In glass fiber filter membranes, the filtration times for pore sizes of 1.2μm, 0.8μm, and 0.45μm were 5.05min, 6.33min, and 12.54min, respectively; in mixed fiber filter membranes, the filtration times for pore sizes of 1.2μm, 0.8μm, and 0.45μm were 5.40min, 15.57min, and 34.96min, respectively; in acetate fiber filter membranes, the filtration times for pore sizes of 1.2μm, 0.8μm, and 0.45μm were 18.85min, 28.63min, and 34.96min, respectively. In 1.2μm, the filtration time of glass fiber filter membrane, mixed fiber filter membrane, and acetate fiber filter membrane is 5.05min, 5.40min, and 18.85min respectively; in 0.8μm, the filtration time of glass fiber filter membrane, mixed fiber filter membrane, and acetate fiber filter membrane is 6.33min, 15.57min, and 28.63min respectively; in 0.45μm, the filtration time of glass fiber filter membrane, mixed fiber filter membrane, and acetate fiber filter membrane is 12.54min, 34.96min, and 43.26min respectively. Therefore, the smaller the pore size in the same filter membrane, the longer the filtration time. The acetate fiber filter membrane takes the longest time in the same pore size, and the glass fiber takes the shortest time.

[0050] After filtration, each filter membrane was rolled up separately, placed in a 2 mL centrifuge tube, and stored at -20°C for eDNA extraction. The sand core filter device was cleaned with 75% ethanol and sterile water before and after use.

[0051] Table 1. Filtration time of different membranes

[0052]

[0053] 4. DNA extraction of eDNA samples using DNA extraction kit

[0054] The DNeasy Blood and Tissue Kit of QIAGEN Co., Ltd. of Germany was used to extract eDNA. The preparation of DNA sample solution of environmental eDNA samples of pear borer and peach borer was taken as an example. The specific method is as follows:

[0055] (1) Take out the centrifuge tube containing the mixed environmental eDNA sample filter membrane of pear borer and peach borer from -20°C, thaw the mixed environmental eDNA sample filter membrane of pear borer and peach borer, add 800 μL ATL Buffer and 80 μL Proteinase K, and place it in a constant temperature water bath at 56°C for 3 h.

[0056] (2) Add 400 μL of AL Buffer to the centrifuge tube, heat in a 56°C water bath for 20 min, centrifuge at 12,000 rpm for 30 s, and then transfer the supernatant to a new centrifuge tube.

[0057] (3) Add 400 μL of anhydrous ethanol to the centrifuge tube in step (3), gently invert it upside down to mix it, and then transfer the mixture to the adsorption column in batches, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid until the mixture is completely filtered.

[0058] (4) Add 500 μL AW1 Buffer to the adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid.

[0059] (5) Add 500 μL AW2 Buffer to the adsorption column, centrifuge at 12,000 rpm for 3 min, and discard the waste liquid.

[0060] (6) Transfer the adsorption column to a new centrifuge tube, add 100 μL AE Buffer for elution, and collect the eluate to obtain the DNA sample solution.

[0061] (7) To increase the concentration, step (6) can be repeated for a second elution.

[0062] (8) The extracted DNA (DNA sample solution) was tested for its concentration and A using an ultra-micro spectrophotometer. 260 / A 280 Value, stored at -20℃. Figure 1 , Table 2. Figure 1 As shown in Tables 1 and 2, the concentration of eDNA extracted by the 0.45 μm cellulose acetate filter is higher, and the quality of the extracts from the CA and MCE filters is higher, so the 0.45 μm cellulose acetate filter was selected.

[0063] The environmental eDNA sample filter of Bactrocera dorsalis was also extracted using the above method to obtain the Bactrocera dorsalis DNA sample solution for subsequent experiments.

[0064] Table 2. A of different filter membranes 260 / A 280

[0065] type <![CDATA[A 260 / A 280 -1]]> <![CDATA[A 260 / A 280 -2]]> <![CDATA[A 260 / A 280 -3]]> Glass fiber filter-1.2μm 2.15 2.15 1.98 Mixed fiber membrane-1.2μm 1.99 1.83 2.51 Cellulose acetate membrane - 1.2μm 2.05 2.03 1.99 Glass fiber filter-0.8μm 1.84 2.04 2.12 Mixed fiber filter-0.8μm 1.83 2.06 1.98 Cellulose acetate membrane - 0.8μm 1.93 1.85 1.92 Glass fiber filter-0.45μm 2.11 2.00 2.07 Mixed fiber membrane-0.45μm 2.00 1.94 1.85 Cellulose acetate filter - 0.45 μm 1.99 1.81 1.97

[0066] 5. PCR identification of eDNA samples

[0067] The specific primer set A (GM-F / GM-R) was designed using the coi gene of Grapholitha molesta (the nucleotide sequence of the coi gene is SEQ ID No. 7) as the reference gene to amplify the pear borer, and the specific primer set B (CS-F / CS-R) was designed using the reference genome of the peach borer (Carposina sasakii, Genbank accession number: HM377790.1) to amplify the peach borer. The primer information is shown in Table 3.

[0068] Table 3. Specific primers for pear borer and peach borer

[0069]

[0070] Each DNA sample solution was subjected to two PCR amplifications (PCR amplification of pear borer and PCR amplification of peach borer). The two PCR amplification systems were identical except for the upstream primer and the downstream primer. The upstream primer in the PCR amplification of pear borer was the upstream primer GM-F, and the downstream primer was the downstream primer GM-R; the upstream primer in the PCR amplification of peach borer was the upstream primer CS-F, and the downstream primer was the downstream primer CS-R.

[0071] Single-plex PCR verification was used, and the PCR reaction system (25 μL): 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 12.5 μL 2×Taq PCR Mastermix (company product number), 9.5 μL ddH2O, and 1.0 μL DNA (DNA sample solution).

[0072] The PCR amplification program was as follows: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0073] 5 μL of each PCR product obtained above was electrophoresed in 1.0% agarose gel for 40 min (120 V), and observed and photographed using a gel imaging system.

[0074] Agarose gel electrophoresis Figure 2 .Depend on Figure 2 It can be seen that: lane M is D2000 Marker; lane 1 is the electrophoresis result of pear borer extracted by eDNA technology; lane 2 is the electrophoresis result of peach borer extracted by eDNA technology; lane 3 is the electrophoresis result of pear borer body extraction; lane 4 is the electrophoresis result of peach borer body extraction; lanes 5 and 6 are the electrophoresis results of citrus fruit fly extracted by eDNA technology; lanes 7 and 8 are the electrophoresis results of citrus fruit fly body extraction; lanes 9 and 10 are the electrophoresis results of normal and intact apples extracted by eDNA technology; N1 and N2 are ddH2O.

[0075] Lane 1 is the electrophoresis result of the PCR product of the DNA sample solution of the environmental eDNA sample of the pear borer, and lane 3 is the electrophoresis result of the extracted pear borer. The PCR products of both are a DNA band (DNA fragment) around 250bp. The DNA band (DNA fragment) around 250bp is recovered and sequenced. The results show that the size of the DNA band (DNA fragment) around 250bp is 288bp, and its nucleotide sequence is SEQ ID No. 5 in the sequence list. Lane 2 is the electrophoresis result of the PCR product of the DNA sample solution of the environmental eDNA sample of the peach borer, and lane 4 is the electrophoresis result of the extracted peach borer. The PCR products of both are a DNA band (DNA fragment) greater than 100bp and less than 250bp. The DNA band (DNA fragment) greater than 100bp and less than 250bp is recovered and sequenced. The results show that the size of the DNA band (DNA fragment) greater than 100bp and less than 250bp is 191bp, and its nucleotide sequence is SEQ ID No. 6 in the sequence list. Lanes 5-8, N1, and N2 are all negative controls and show no bands. Therefore, the primers for pear borer and peach borer have good specificity.

[0076] In summary: when the primer set of the present invention is used for PCR, when the size of the obtained PCR product is 288 bp (the nucleotide sequence is SEQ ID No. 5 in the sequence list), the result is judged to be positive for pear borer, that is, pear borer exists in the environment to be detected; when the size of the obtained PCR product is 191 bp (the nucleotide sequence is SEQ ID No. 6 in the sequence list), the result is judged to be positive for peach borer, that is, peach borer exists in the environment; when the 288 bp and 191 bp bands do not exist in the PCR product, that is, pear borer and peach borer do not exist in the environment to be detected.

[0077] The above test results show that the eDNA detection method developed in the present invention can detect pear borer and peach borer at the same time.

[0078] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for identifying or assisting in identifying a fruit borer species, wherein the fruit borer is Grapholithamolesta and / or Carposina sasakii, comprising the following steps: 1) Collect environmental eDNA samples from the environment to be tested; 2) extracting DNA from the environmental eDNA sample to obtain a DNA sample solution; 3) Using the DNA sample liquid as a template, PCR amplification is performed using a primer set for identifying fruit moths, and determining whether the environment to be detected contains the fruit moths according to the size of the PCR amplification product in accordance with the following method: If the PCR amplification product contains a DNA fragment of 250-500bp, pear borer is present in the environment to be detected; if the PCR amplification product contains a DNA fragment greater than 100bp and less than 250bp, peach borer is present in the environment to be detected; if the PCR amplification product contains a DNA fragment of 250-500bp and a DNA fragment greater than 100bp and less than 250bp, pear borer and peach borer are present in the environment to be detected; The primer set for identifying fruit moths comprises primer set A and primer set B. The primer set A consists of a single-stranded DNA having a nucleotide sequence of SEQ ID No.1 and a single-stranded DNA having a nucleotide sequence of SEQ ID No.2; The primer set B consists of a single-stranded DNA having a nucleotide sequence of SEQ ID No.3 and a single-stranded DNA having a nucleotide sequence of SEQ ID No.

4.

2. The method according to claim 1, characterized in that The 250-500 bp DNA fragment is a 288 bp DNA fragment, and the DNA fragment larger than 100 bp and smaller than 250 bp is 191 bp.

3. The method according to claim 2, characterized in that The environmental eDNA sample is a water sample, and the step 2) includes filtering the water sample with a filter membrane and then extracting DNA from the environmental eDNA sample.

4. The method according to claim 3, characterized in that The filter membrane is a 0.45 μm cellulose acetate filter membrane.

5. The method according to any one of claims 1 to 4, characterized in that: The primer annealing condition used in the PCR amplification is annealing at 55° C. for 30 seconds.

6. The method according to claim 5, characterized in that The PCR amplification program includes: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, for a total of 35 cycles; and finally, 72°C for 5 min of extension.

7. A primer composition for identifying or assisting in identifying fruit moths, characterized in that: The primer composition includes primer set A and primer set B, the nucleotide sequence of primer set A is shown as SEQ ID No.1 and SEQ ID No.2; the nucleotide sequence of primer set B is shown as SEQ ID No.3 and SEQ ID No.

4.

8. A fruit moth detection kit, characterized in that: Comprising the composition as claimed in claim 7.

9. Use of the primer combination according to claim 7 in any of the following: (1) Detect or assist in the detection of pear borer and peach borer; (2) preparing products for detecting or assisting in detecting pear borer and peach borer; (3) Amplification of pear borer and peach borer DNA; (4) Preparing products for amplifying DNA of pear borer and peach borer.

10. Use of the primer composition according to claim 7 or the kit according to claim 8 in an environmental pollution monitoring, food safety testing product or disease diagnosis device.

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