RPA-CRISPR / Cas12a visual rapid detection system for fruit flies of guava and application of RPA-CRISPR / Cas12a visual rapid detection system

Abstract

The invention discloses an RPA-CRISPR / Cas12a visual rapid detection system for fruit flies of guava and an application of the RPA-CRISPR / Cas12a visual rapid detection system. Based on a recombinase polymerase amplification technology, a rapid identification method of the fruit fly guavas is established, and further through optimization of a chromogenic reaction system and reaction conditions, an RPA-CRISPR / Cas12a visual rapid detection system of the fruit fly guavas is established. The recombinase polymerase amplification technology for guava fruit fly identification can achieve rapid identification of eggs, larvae, pupae and adult individuals of guava fruit flies, has the advantages of being rapid, efficient, high in specificity, high in sensitivity, low in cost, easy and convenient to operate and free of special instruments, is suitable for being used on site, is easy to popularize in the basic level, and has wide application prospects. Reliable technical basis can be provided for prevention and control of guava fruit flies, and the method has important significance on tropical fruit and vegetable industry protection, foreign organism invasion prevention and control and international trade.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an RPA-CRISPR / Cas12a visual rapid detection system for guava fruit flies and applications thereof. Background Art

[0002] Guava fruit fly, Bactrocera correcta (Bezzi), belongs to Diptera, Tephritidae, Bactrocera, and is listed as a quarantine pest in my country. Its invasion prevention and control has received special attention. Guava fruit fly has a wide range of hosts and can harm more than 60 species of plants in 30 families, mainly tropical and subtropical fruit and vegetable plants, including guava (Psidium guajava), wax apple (Syzygiumsamarangense), mango (Mangifere indica), cherry (Muntigia calabura), cashew (Anacardiumoccidentale), etc. In the main occurrence areas of guava fruit fly, its damage rate to the host can reach 60% to 80%, seriously affecting the yield and quality of tropical fruits and vegetables. In recent years, guava fruit flies are often intercepted in imported fruits and vegetables in my country; at the same time, the insect is monitored and causes harm in some provinces and cities in southern my country; its entry quarantine and field management have received more attention, so there is an urgent need to develop quarantine identification and field monitoring technologies suitable for the front line.

[0003] The main way guava fruit flies cause harm is that female insects lay eggs under the host fruit peel, and the hatched larvae eat inside the fruit, causing mold and fungal infections, resulting in large-scale rot and fall of the fruit, causing the affected fruit to lose its economic value. In my country, guava fruit flies were first reported in Yuanjiang, Yunnan in 1982, and then gradually spread to surrounding provinces and cities. The latest data in 2022 show that guava fruit flies were monitored and found in Sanya, Hainan Province. As an important place for my country to expand its opening up and actively promote global economic integration, Hainan's international import and export trade, especially the trade in high-quality tropical fruits, will undoubtedly grow further, but it will also bring greater challenges to my country's customs in preventing the invasion of alien species.

[0004] At present, the identification of guava fruit flies is mainly based on traditional morphological identification and molecular biological identification technology. However, during entry and exit quarantine, customs officers often intercept fruit flies in non-adult stages (eggs, larvae and pupae), which makes it difficult to identify their species only by morphology. It takes a long time to raise non-adult stages to adults and then identify their species, which seriously affects the speed of port quarantine and thus affects trade. Existing molecular identification technologies are mainly PCR and real-time fluorescence PCR technologies. Although accurate identification can be achieved, they rely on expensive instruments and equipment, and the identification cost is high. They do not meet the current port and on-site quarantine requirements. Therefore, it is necessary to develop a recombinase polymerase amplification identification technology for guava fruit flies.

[0005] RPA (recombinase polymerase amplification) is an isothermal amplification technology based on simulating the T4 phage nucleic acid replication mechanism. The RPA system requires two specific primers to identify the target gene, relying on three enzymes: Bacillus subtilis DNA polymerase (IBsu), recombinase uvsX and single-stranded DNA binding protein gp32, as well as the loading factor uvsY of the auxiliary recombinase uvsX. It completes the exponential amplification of the target fragment in 20 to 40 minutes at a constant temperature of 37°C to 42°C. It has been widely used in the efficient detection of human, animal and plant pathogens. The V-type CRISPR effector protein Cas12a has trans-cutting activity. When the target fragment, Cas12a nuclease and crRNA co-exist in the system, a ternary complex is formed to arbitrarily cut the fluorescent reporter molecule in the system. Under the irradiation of a blue light (wavelength of 450nm), the RPA amplification results can be observed by the naked eye. The RPA visualization detection system combined with CRISPR-Cas12a (RPA-CRISPR / Cas12a) has the advantages of rapidity, sensitivity, specificity, and portability, and can meet the needs of rapid and accurate on-site detection. Summary of the invention

[0006] The present invention firstly provides a primer set for identifying or assisting in identifying guava fruit flies.

[0007] The primer set provided by the present invention includes a primer pair consisting of primer Bc-1F and primer Bc-1R;

[0008] The primer Bc-1F is as follows a1) or a2):

[0009] a1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing;

[0010] a2) a single-stranded DNA molecule having the same function as sequence 1 after one or more nucleotides are substituted and / or deleted and / or added;

[0011] The primer Bc-1R is as follows a3) or a4):

[0012] a3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing;

[0013] a4) A single-stranded DNA molecule having the same function as sequence 2 after one or several nucleotides are substituted and / or deleted and / or added.

[0014] In the above primer set, the molar ratio of the primer Bc-1F to the primer Bc-1R is 1:1.

[0015] Furthermore, the primer set also includes crRNA;

[0016] The crRNA is as follows a5) or a6):

[0017] a5) a single-stranded DNA molecule shown in Sequence 3 in the sequence listing;

[0018] a6) A single-stranded RNA molecule having the same function as sequence 3, wherein one or several nucleotides are substituted and / or deleted and / or added.

[0019] Furthermore, the primer set also includes a probe. In a specific embodiment of the present invention, the nucleotide sequence of the probe (single-stranded DNA molecule) is 5'-TTATT-3', one end of the probe is labeled with a FAM fluorescent group, and the other end is labeled with a BHQ quenching group.

[0020] The present invention further provides a new use of the primer set.

[0021] The present invention provides the use of the above primer set in any one of the following b1)-b8):

[0022] b1) Identify or assist in the identification of guava fruit flies;

[0023] b2) Identifying or assisting in the identification of whether the fruit fly to be tested is the guava fruit fly;

[0024] b3) Detecting or assisting in detecting whether the sample to be tested contains guava fruit flies;

[0025] b4) distinguishing or assisting in distinguishing guava fruit flies from other fruit flies;

[0026] b5) preparing products for identifying or assisting in the identification of guava fruit flies;

[0027] b6) preparing a product for identifying or assisting in identifying whether the fruit fly to be tested is the guava fruit fly;

[0028] b7) preparing products for detecting or assisting in detecting whether a sample to be tested contains guava fruit flies;

[0029] b8) preparing products that differentiate or assist in differentiating the guava fruit fly from other fruit flies.

[0030] The present invention also provides a kit containing the above primer set; the function of the kit is any one of the following c1)-c4):

[0031] c1) Identify or assist in the identification of guava fruit flies;

[0032] c2) identifying or assisting in identifying whether the fruit fly to be tested is the guava fruit fly;

[0033] c3) detecting or assisting in detecting whether the sample to be tested contains guava fruit flies;

[0034] c4) Differentiate or assist in differentiating the guava fruit fly from other fruit flies.

[0035] Further, the kit may also include other reagents for identifying or distinguishing guava fruit flies. In the present invention, the other reagents for identifying or distinguishing guava fruit flies may be Primer Free Rehydration buffer, MgOAc, Cas12a protein, 10×Cas12a Reaction Buffer, the above-mentioned probe and the above-mentioned crRNA.

[0036] Furthermore, the kit may also include a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA of the guava fruit fly), as well as a blue light and a yellow filter for observing the results of the color development reaction.

[0037] The preparation method of the above-mentioned kit also belongs to the protection scope of the present invention.

[0038] The preparation method of the kit is as follows d1) or d2):

[0039] d1) mixing the above primers according to proportion;

[0040] d2) Each of the above primers and crRNA is packaged separately.

[0041] The present invention also provides a method for identifying or assisting in identifying whether a fruit fly to be tested is a guava fruit fly.

[0042] The method for identifying or assisting in identifying whether the fruit fly to be tested is the guava fruit fly provided by the present invention is as follows S1) or S2):

[0043] Said S1) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the above primer pair; after the recombinase polymerase amplification reaction is completed, judging whether the fruit fly to be tested is the guava fruit fly by detecting the reaction product: if the reaction product shows a clear and bright band near 270bp after electrophoresis, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the fruit fly to be tested is not or is not a candidate for the guava fruit fly;

[0044] The S2) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, taking the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification with the above-mentioned primer pair; after the recombinase polymerase amplification reaction is completed, adding the reaction product to a color development reaction system for color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the fruit fly to be tested is the guava fruit fly: if the color development reaction system is green, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the color development reaction system is colorless and transparent, the fruit fly to be tested is not or is not a candidate for the guava fruit fly.

[0045] The present invention also provides a method for detecting or assisting in detecting whether a sample to be tested contains guava fruit flies.

[0046] The method for detecting or assisting in detecting whether a sample to be tested contains guava fruit flies provided by the present invention is as follows T1) or T2):

[0047] Said T1) comprises the following steps: extracting the nucleic acid of the sample to be tested, taking the nucleic acid of the sample to be tested as a template, and using the above-mentioned primer pair to perform recombinase polymerase amplification; after the recombinase polymerase amplification reaction is completed, judging whether the sample to be tested contains the guava fruit fly by detecting the reaction product: if the reaction product shows a clear and bright band near 270bp after electrophoresis, the sample to be tested contains or is a candidate to contain the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the sample to be tested does not contain or is a candidate to not contain the guava fruit fly;

[0048] The step T2) comprises the following steps: extracting nucleic acid of the sample to be tested, taking the nucleic acid of the sample to be tested as a template, and performing recombinase polymerase amplification using the above primer pair; after the recombinase polymerase amplification reaction is completed, adding the reaction product into a color development reaction system for color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the sample to be tested contains the guava fruit fly: if the color development reaction system is green, the sample to be tested contains or the candidate contains the guava fruit fly; if the color development reaction system is colorless and transparent, the sample to be tested does not contain or the candidate does not contain the guava fruit fly.

[0049] Finally, the present invention also provides a method for distinguishing or assisting in distinguishing guava fruit flies from other fruit flies.

[0050] The method for distinguishing or assisting in distinguishing guava fruit flies from other fruit flies provided by the present invention is as follows U1) or U2):

[0051] The U1) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the above primer pair; after the recombinase polymerase amplification reaction is completed, determining whether the fruit fly to be tested is the guava fruit fly or other fruit flies by detecting the reaction product: if the reaction product shows a clear and bright band near 270 bp after electrophoresis, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the fruit fly to be tested is or is a candidate for other fruit flies;

[0052] The method (U2) comprises the following steps: extracting the nucleic acid of the fruit fly to be detected, using the nucleic acid of the fruit fly to be detected as a template, and performing recombinase polymerase amplification using the above primer pair; after the recombinase polymerase amplification reaction is completed, adding the reaction product to a color development reaction system for color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the fruit fly to be detected is the guava fruit fly or other fruit flies: if the color development reaction system is green, the fruit fly to be detected is or is a candidate for the guava fruit fly; if the color development reaction system is colorless and transparent, the fruit fly to be detected is or is a candidate for other fruit flies.

[0053] In any of the above methods, the recombinase polymerase amplification reaction system can be specifically system 1, with a total volume of 25 μL, consisting of 12.5 μL Primer Free Rehydration buffer, 2.5 μL DNA template, 1 μL primer Bc-1F, 1 μL primer Bc-1R, 1.25 μL MgOAc and 6.75 μL sterile ultrapure water.

[0054] In any of the above methods, the color development reaction system can specifically be system 2, with a total volume of 50 μL, consisting of 5 μL 10×Cas12a Reaction Buffer, 200nM Cas12a, 400nM crRNA, 1 μM ssDNA fluorescent probe, and 2 μL reaction product, and the rest is made up to 50 μL with sterile ultrapure water.

[0055] In the above method, the recombinase polymerase amplification reaction conditions may be: constant temperature at 37-39° C. for 5-20 min, preferably constant temperature at 37° C. for 15 min.

[0056] In the above method, the color development reaction conditions may be: constant temperature at 37° C. for 10-30 min, preferably constant temperature at 37° C. for 15 min.

[0057] In the above method, the nucleic acid is genomic DNA.

[0058] In any of the above-mentioned applications, kits or methods, the fruit flies to be tested may specifically be eggs, larvae, pupae or adults of the fruit flies to be tested.

[0059] In any of the above-mentioned applications or kits or methods, the samples to be tested include various tropical and subtropical economic crops that can be harmed by guava fruit flies, such as guava, mango, peach, rambutan, sapodilla, jujube, rose rambutan, tropical almond, citrus, pepper and other fruits and vegetables.

[0060] In any of the above-mentioned applications, kits or methods, the other fruit flies may be at least one of the following fruit flies: peach fruit fly Bactrocera zonata, citrus fruit fly Bactrocera dorsalis, carambola fruit fly Bactrocera carambolae, citrus fruit fly Bactrocera minax, citrus fruit fly Bactrocera tsuneonis, and South Asian fruit fly Zeugodaccus tau.

[0061] In any of the above applications, kits or methods, the guava fruit fly Bactrocera correcta can be guava fruit fly Bactrocera correcta from different geographical populations, including guava fruit fly Bactrocera correcta from Sanya, Hainan, Xishuangbanna, Yunnan, Jinghong, Yunnan, Dali, Yunnan, Baoshan, Yunnan, Hyderabad, India, Bangkok, Thailand, and Phan Thiet, Vietnam.

[0062] The present invention is based on recombinase polymerase amplification technology, establishes a rapid identification method for guava fruit flies, and by optimizing the color development reaction system and reaction conditions, according to the ssDNA fluorescent probe being cut by Cas12a during the color development reaction, the characteristics of changing from colorless and transparent to green, establishes RPA-CRISPR / Cas12a visualization rapid detection system. The recombinase polymerase amplification technology for the identification of guava fruit flies of the present invention can realize the rapid identification of eggs, larvae, pupae, and adult individuals of guava fruit flies, with the advantages of fast, efficient, strong specificity, high sensitivity, low cost, simple operation, and no need for special instruments, suitable for the scene, easy to promote at the grassroots level, can provide reliable technical basis for the prevention and control of guava fruit flies, and is of great significance to grain storage protection, prevention and control of alien biological invasion and international trade. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The results of the primer pair Bc-1F / R detection in step 2 of Example 1. Wherein, M: D2000 DNA marker, N: ddH 2O, channels 1-7 are the guava fruit fly, peach fruit fly, citrus fruit fly, carambola fruit fly, citrus fruit fly, citrus fruit fly and South Asian fruit fly, respectively.

[0064] Figure 2 The visualization detection results of step 2 in Example 2. Among them, channels 1-7 are respectively guava fruit flies, peach fruit flies, citrus fruit flies, carambola fruit flies, citrus fruit flies, citrus fruit flies, and South Asian fruit flies, N: ddH 2 O.

[0065] Figure 3 It is the specific detection result in Example 3. Wherein, M: D2000 DNA marker, N:ddH 2 O, channels 1-11 are respectively the guava fruit fly in Sanya, Hainan, the guava fruit fly in Xishuangbanna, Yunnan, the guava fruit fly in Jinghong, Yunnan, the guava fruit fly in Dali, Yunnan, the guava fruit fly in Baoshan, Yunnan, the guava fruit fly in Hyderabad, India, the guava fruit fly in Bangkok, Thailand, the guava fruit fly in Phan Thiet, Vietnam, the peach fruit fly, the citrus fruit fly, and the carambola fruit fly.

[0066] Figure 4 The sensitivity test results in Example 4. Among them, the sensitivity of channels 1-6 are 100.0ng / μL, 10.0ng / μL, 1.0ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, N:ddH 2 O. DETAILED DESCRIPTION

[0067] 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.

[0068] 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.

[0069] The blood / cell / tissue genomic DNA extraction kit in the following examples is a product of Tiangen Biotech (Beijing) Co., Ltd. Primer Free Rehydration buffer and MgOAc are both from TwistAmp Basic Kit, a product of TwistDx Ltd. in the UK. Cas12a and 10×Cas12a Reaction Buffer are products of Megazyme.

[0070] The numbers, species names, and collection location information of the tested fruit fly samples in the following examples are shown in Table 1. The samples were all placed in absolute ethanol and stored at -20°C for later use.

[0071] Table 1

[0072]

[0073]

[0074] Example 1. Obtaining primer pairs and kits for identifying Bactrocera correcta

[0075] I. Design of primer pairs for identifying Bactrocera correcta

[0076] 1. The present invention designed and synthesized a total of three primer pairs for identifying Bactrocera correcta: Bc-1F / R, Bc-2F / R, and Bc-3F / R. The primer sequences of each primer pair are shown in detail in Table 2.

[0077] Table 2

[0078]

[0079] II. Verification of primer pairs for identifying Bactrocera correcta

[0080] 1. The genomic DNA of the fruit fly samples was extracted using a blood / cell / tissue genomic DNA extraction kit to obtain the genomic DNA of the fruit fly samples.

[0081] The fruit fly samples were Bactrocera correcta numbered 1 in Table 1, Bactrocera zonata numbered 9, Bactrocera dorsalis numbered 10, Bactrocera carambolae numbered 11, Bactrocera minax numbered 12, Bactrocera tsuneonis numbered 13, and Bactrocera tau numbered 14.

[0082] 2. Using the genomic DNA of the fruit fly samples as a template, recombinase polymerase amplification was performed with each primer pair to obtain RPA amplification products.

[0083] The reaction system is 25 μL, consisting of 12.5 μL Primer Free Rehydration buffer, 2.5 μL DNA template, 1 μL front primer, 1 μL rear primer, 1.25 μL MgOAc and 6.75 μL sterile ultrapure water. In the reaction system, the final concentration of the front and rear primers is 0.48 μM.

[0084] Reaction conditions: constant temperature at 37°C for 15 min.

[0085] 3. Perform 1% agarose gel electrophoresis on the RPA amplification product.

[0086] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.

[0087] Test results are shown in Figure 1 Among them, M is DNA Marker, 1 is guava fruit fly, 2 is peach fruit fly, 3 is citrus fruit fly, 4 is carambola fruit fly, 5 is citrus fruit fly, 6 is citrus fruit fly, 7 is South Asian fruit fly, and N is sterile ultrapure water.

[0088] The results showed that the primer pair Bc-1F / R only showed a clear and bright band at 270bp when identifying guava fruit flies, and no band appeared in the negative control. Other primer pairs failed to show high specificity in specific detection. Therefore, the primer pair Bc-1F / R can be used to identify guava fruit flies.

[0089] 3. Preparation of a kit for identifying guava fruit flies

[0090] The kit for identifying Bactrocera guavaensis includes the primer pair Bc-1F / R.

[0091] Example 2: Use of the kit prepared in Example 1 in identifying whether the fruit fly to be tested is or is a candidate for being a guava fruit fly

[0092] 1. Identify whether the fruit fly to be tested is or is a candidate for guava fruit fly by agarose gel electrophoresis results

[0093] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the fruit fly to be tested, and obtain the genomic DNA of the fruit fly to be tested.

[0094] 2. Using the genomic DNA of the fruit fly to be tested as a template, use the primer pair Bc-1F / R to perform recombinase polymerase amplification to obtain the RPA amplification product.

[0095] The reaction system was 25 μL and consisted of 12.5 μL Primer Free Rehydration buffer, 2.5 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 1.25 μL MgOAc, and 6.75 μL sterile ultrapure water. In the reaction system, the final concentrations of the forward and reverse primers were both 0.48 μM.

[0096] Reaction conditions: Incubate at a constant temperature of 37 °C for 15 min.

[0097] 3. Perform 1% agarose gel electrophoresis on the RPA amplification products.

[0098] According to the above method, replace the template with sterile ultrapure water, and keep other steps unchanged as the negative control.

[0099] According to the above method, replace the template with the genomic DNA of Bactrocera correcta, and keep other steps unchanged as the positive control.

[0100] 4. Make the following judgments based on the electrophoresis results:

[0101] When the positive control shows a clear and bright band at around 270 bp and the negative control shows no band, if the tested fruit fly shows a clear and bright band at around 270 bp, then the tested fruit fly is or is a candidate for Bactrocera correcta; if the tested fruit fly does not show a clear and bright band at around 270 bp (such as no band), then the tested fruit fly is not or is not a candidate for Bactrocera correcta;

[0102] When the positive control does not show a clear and bright band at around 270 bp or the negative control shows a band, the identification result of the tested fruit fly is invalid.

[0103] II. Visual identification of whether the tested fruit fly is or is a candidate for Bactrocera correcta

[0104] 1. Using the RPA amplification product sequence of Bactrocera correcta as the target, three crRNAs were designed and synthesized. The crRNA sequences are shown in Table 3. Select the Bc-crRNA1 with the highest amplification efficiency evaluation value as the crRNA for identifying Bactrocera correcta.

[0105] Table 3

[0106]

[0107]

[0108] 2. Extract the genomic DNA of the tested fruit fly using a Blood / Cell / Tissue Genomic DNA Extraction Kit to obtain the genomic DNA of the tested fruit fly.

[0109] 3. Using the genomic DNA of the fruit fly to be tested as a template, use the primer pair Bc-1F / R to perform recombinase polymerase amplification to obtain the RPA amplification product.

[0110] The reaction system is 25 μL, consisting of 12.5 μL Primer Free Rehydration buffer, 2.5 μL DNA template, 1 μL front primer, 1 μL rear primer, 1.25 μL MgOAc and 6.75 μL sterile ultrapure water. In the reaction system, the final concentration of the front and rear primers is 0.48 μM.

[0111] Reaction conditions: 37°C for 15 min.

[0112] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.

[0113] According to the above method, the template was replaced with the genomic DNA of Bactrocera guajavae, and the other steps remained unchanged as a positive control.

[0114] 4. Add the RPA amplification product obtained in step 3 to the color development reaction system, and observe the color of the color development reaction system with the naked eye after the reaction.

[0115] The color reaction system is 50 μL, consisting of 5 μL 10×Cas12a Reaction Buffer, 200 nM Cas12a, 400 nM crRNA (Bc-crRNA1), 1 μM ssDNA fluorescent probe, 2 μL RPA amplification product, and the rest is filled up to 50 μL with sterile ultrapure water. The ssDNA fluorescent probe is a single-stranded DNA molecule with a nucleotide sequence of 5'-TTATT-3', in which the 5' end is labeled with a FAM fluorescent group and the 3' end is labeled with a BHQ1 quenching group.

[0116] Reaction conditions: 37°C for 15 min.

[0117] 5. After completing step 4, observe with the naked eye under blue light and through a yellow filter, and then make the following judgments:

[0118] When the reaction system of the positive control is green and the reaction system of the negative control is colorless and transparent, if the reaction system of the fruit fly to be tested is green, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the reaction system of the fruit fly to be tested is colorless and transparent, the fruit fly to be tested is not or is not a candidate for the guava fruit fly;

[0119] When the reaction system of the positive control does not show green or the reaction system of the negative control shows green, the identification result of the fruit fly to be tested is invalid.

[0120] According to the above method, it is determined whether the fruit fly to be tested is the guava fruit fly numbered 1, the peach fruit fly numbered 9, the citrus fruit fly numbered 10, the carambola fruit fly numbered 11, the citrus fruit fly numbered 12, the citrus fruit fly numbered 13, and the South Asian fruit fly numbered 14 in Table 1.

[0121] Test results are shown in Figure 2 The results showed that only the color reaction system of the guava fruit fly was green, while the color reaction systems of non-guava fruit flies and the negative control were colorless and transparent.

[0122] Example 3, specificity experiment

[0123] The fruit fly 1 to be tested is the guava fruit fly numbered 1.

[0124] The fruit fly 2 to be tested is the guava fruit fly numbered 2.

[0125] The fruit fly 3 to be tested is the guava fruit fly numbered 3.

[0126] The fruit fly 4 to be tested is the guava fruit fly numbered 4.

[0127] The fruit fly 5 to be tested is the guava fruit fly numbered 5.

[0128] The fruit fly 6 to be tested is the guava fruit fly numbered 6.

[0129] The fruit fly 7 to be tested is the guava fruit fly numbered 7.

[0130] The fruit fly 8 to be tested is the guava fruit fly numbered 8.

[0131] The fruit fly 9 to be tested is the peach fruit fly numbered 9.

[0132] The fruit fly 10 to be tested is the fruit fly numbered 10.

[0133] The fruit fly 11 to be tested is the carambola fruit fly numbered 11.

[0134] Each fruit fly to be tested shall undergo the following steps:

[0135] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the fruit fly to be tested.

[0136] 2. Using the genomic DNA of the fruit fly to be tested extracted in step 1 as a template, use primer pair Bc-1F / R to perform recombinase polymerase amplification to obtain RPA amplification products.

[0137] The reaction system is 25 μL, consisting of 12.5 μL Primer Free Rehydration buffer, 2.5 L DNA template, 1 μL front primer, 1 μL rear primer, 1.25 μL MgOAc and 6.75 μL sterile ultrapure water. In the reaction system, the final concentration of the front and rear primers is 0.48 μM.

[0138] Reaction conditions: constant temperature at 37°C for 15 min.

[0139] 3. Perform 1% agarose gel electrophoresis on the RPA amplification product.

[0140] According to the above method, the template was replaced with sterile ultrapure water, and other steps remained unchanged as a negative control.

[0141] According to the above method, the template was replaced with the genomic DNA of Bactrocera guajavae, and the other steps remained unchanged as a positive control.

[0142] Test results see Figure 3 The results showed that when the fruit flies to be tested were guava fruit flies, a clear and bright band was present at around 270 bp; when the fruit flies to be tested were not guava fruit flies, no band was present (i.e., no clear and bright band was present at around 270 bp); and the negative control also had no band.

[0143] It can be seen that the use of primer pair Bc-1F / R to identify guava fruit flies has good geographical population universality and inter-species specificity.

[0144] Example 4: Sensitivity experiment

[0145] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the guava fruit fly.

[0146] 2. Take the genomic DNA of the guava fruit fly and dilute it with sterile water to obtain the genomic DNA concentrations of the guava fruit fly at 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL dilution.

[0147] 3. Using the dilution obtained in step 2 as a template, perform recombinase polymerase amplification using primer pair Bc-1F / R to obtain an RPA amplification product.

[0148] The reaction system is 25 μL, consisting of 12.5 μL Primer Free Rehydration buffer, 2.5 μL DNA template, 1 μL front primer, 1 μL rear primer, 1.25 μL MgOAc and 6.75 μL sterile ultrapure water. In the reaction system, the final concentration of the front and rear primers is 0.48 μM.

[0149] Reaction conditions: constant temperature at 37°C for 15 min.

[0150] 4. Add the RPA amplification product obtained in step 3 to the color development reaction system, and observe the color of the color development reaction system with the naked eye after the reaction.

[0151] The color reaction system is 50 μL, consisting of 5 μL 10×Cas12a Reaction Buffer, 200 nM Cas12a, 400 nM crRNA (Bc-crRNA1), 1 μM ssDNA fluorescent probe, 2 μL RPA amplification product, and the rest is filled up to 50 μL with sterile ultrapure water. The ssDNA fluorescent probe is a single-stranded DNA molecule with a nucleotide sequence of 5'-TTATT-3', in which the 5' end is labeled with a FAM fluorescent group and the 3' end is labeled with a BHQ1 quenching group.

[0152] Reaction conditions: constant temperature at 37°C for 15 min.

[0153] If the reaction system is green under blue light, it means that the corresponding genome content in the reaction system can be detected. If the reaction system is colorless and transparent, it means that the corresponding genome content in the reaction system cannot be detected.

[0154] Test results see Figure 4 The results showed that the sensitivity of primer pair Bc-1F / R in identifying guava fruit flies was 1.0×10 -1 ng / μL.

[0155] It can be seen that the primer pair Bc-1F / R has a high sensitivity in identifying guava fruit flies.

[0156] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A primer set for identifying or assisting in identifying guava fruit flies, the primer set comprising a primer pair consisting of primer Bc-1F and primer Bc-1R; The primer Bc-1F is as follows a1) or a2): a1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; a2) a single-stranded DNA molecule having the same function as sequence 1 after one or more nucleotides are substituted and / or deleted and / or added; The primer Bc-1R is as follows a3) or a4): a3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; a4) A single-stranded DNA molecule having the same function as sequence 2 after one or several nucleotides are substituted and / or deleted and / or added.

2. The primer set according to claim 1, Features: The molar ratio of the primer Bc-1F to the primer Bc-1R is 1:

1.

3. The primer set according to claim 1 or 2, Features: The primer set also includes crRNA; The crRNA is as follows a5) or a6): a5) a single-stranded DNA molecule shown in Sequence 3 in the sequence listing; a6) A single-stranded RNA molecule having the same function as sequence 3, wherein one or several nucleotides are substituted and / or deleted and / or added.

4. A primer set according to any one of claims 1 to 3, Features: The primer set also includes a probe sequence.

5. Use of the primer set according to any one of claims 1 to 4 in any one of the following b1) to b8): b1) Identify or assist in the identification of guava fruit flies; b2) Identifying or assisting in the identification of whether the fruit fly to be tested is the guava fruit fly; b3) Detecting or assisting in detecting whether the sample to be tested contains guava fruit flies; b4) distinguishing or assisting in distinguishing guava fruit flies from other fruit flies; b5) preparing products for identifying or assisting in the identification of guava fruit flies; b6) preparing a product for identifying or assisting in identifying whether the fruit fly to be tested is the guava fruit fly; b7) preparing products for detecting or assisting in detecting whether a sample to be tested contains guava fruit flies; b8) preparing products that differentiate or assist in differentiating the guava fruit fly from other fruit flies.

6. A kit comprising the primer set according to any one of claims 1 to 4; The function of the kit is any one of the following c1)-c4): c1) Identify or assist in the identification of guava fruit flies; c2) identifying or assisting in identifying whether the fruit fly to be tested is the guava fruit fly; c3) detecting or assisting in detecting whether the sample to be tested contains guava fruit flies; c4) Differentiate or assist in differentiating the guava fruit fly from other fruit flies.

7. The method for preparing the kit according to claim 6, comprising the steps d1) or d2) as follows: d1) mixing the primers described in claims 1 to 4 in proportion; d2) packaging each primer and crRNA described in claims 1-4 separately.

8. A method for identifying or assisting in identifying whether a fruit fly to be tested is a guava fruit fly, comprising the following S1) or S2): Said S1) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, judging whether the fruit fly to be tested is the guava fruit fly by detecting the reaction product: if the reaction product shows a clear and bright band near 270 bp after electrophoresis, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the fruit fly to be tested is not or is not a candidate for the guava fruit fly; The step S2) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, adding the reaction product to a color development reaction system for a color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the fruit fly to be tested is a guava fruit fly: if the color development reaction system is green, the fruit fly to be tested is or is a candidate for being a guava fruit fly; if the color development reaction system is colorless and transparent, the fruit fly to be tested is not or is not a candidate for being a guava fruit fly.

9. A method for detecting or assisting in detecting whether a sample to be tested contains guava fruit flies, comprising the following T1) or T2): Said T1) comprises the following steps: extracting the nucleic acid of the sample to be tested, taking the nucleic acid of the sample to be tested as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, judging whether the sample to be tested contains the guava fruit fly by detecting the reaction product: if the reaction product shows a clear and bright band near 270 bp after electrophoresis, the sample to be tested contains or is a candidate for containing the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the sample to be tested does not contain or is a candidate for not containing the guava fruit fly; The step T2) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, adding the reaction product to a color development reaction system for a color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the sample to be tested contains the guava fruit fly: if the color development reaction system is green, the sample to be tested contains or is a candidate for containing the guava fruit fly; if the color development reaction system is colorless and transparent, the sample to be tested does not contain or is a candidate for not containing the guava fruit fly.

10. A method for distinguishing or assisting in distinguishing guava fruit flies from other fruit flies, comprising the following U1) or U2): The method of U1) comprises the following steps: extracting the nucleic acid of the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, determining whether the fruit fly to be tested is the guava fruit fly or other fruit flies by detecting the reaction product: if the reaction product shows a clear and bright band near 270 bp after electrophoresis, the fruit fly to be tested is or is a candidate for the guava fruit fly; if the reaction product does not produce a band after electrophoresis, the fruit fly to be tested is or is a candidate for other fruit flies; The method (U2) comprises the following steps: extracting the nucleic acid of the fruit fly to be detected, using the nucleic acid of the fruit fly to be detected as a template, and performing recombinase polymerase amplification using the primer pair described in claim 1; after the recombinase polymerase amplification reaction is completed, adding the reaction product to a color development reaction system for color development reaction, and after the color development reaction is completed, observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the fruit fly to be detected is the guava fruit fly or other fruit flies: if the color development reaction system is green, the fruit fly to be detected is or is a candidate for the guava fruit fly; if the color development reaction system is colorless and transparent, the fruit fly to be detected is or is a candidate for other fruit flies.

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