LAMP primer set for identifying bactrocera minax and application thereof

Abstract

The application discloses a LAMP primer set for identifying Bactrocera minax and application thereof. The LAMP primer set is composed of primer-F3, primer-B3, primer-FIP and primer-BIP, and the nucleotide sequences are shown in sequence 1-sequence 4 in order. The LAMP primer set can be applied to identifying whether the to-be-tested fruit fly is or is a candidate of Bactrocera minax, and can also be applied to detecting whether the to-be-tested sample contains or is a candidate of containing Bactrocera minax. The LAMP primer set has high specificity and high sensitivity, and can realize simple, rapid and accurate detection. The application has great application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a LAMP primer set for identifying Bactrocera minax and application thereof. BACKGROUND

[0002] Bactrocera minax (Enderlein) belongs to the order Diptera, the family Tephritidae and the genus Bactrocera, and is one of the most serious pests of citrus. With the increase of domestic citrus planting area and the increasing frequency of trade of citrus fruits, the occurrence and damage of Bactrocera minax are expanding.

[0003] Bactrocera minax is a specific parasite of citrus trees, and its larvae feed on the locules of the damaged fruits and occasionally on the seeds, causing a fluff-like state in the later stage of the damaged fruits. The larvae can move between the locules and feed horizontally. The early hatched larvae are in large numbers, causing the damaged fruits to turn yellow prematurely, resulting in fruit drop and decay. The pest not only seriously reduces the yield and quality of citrus, but also greatly discourages the enthusiasm of farmers to plant citrus. Meanwhile, the purchase of damaged fruits by consumers causes panic and serious economic losses. Bactrocera minax is mainly spread through the trade of fruits with larvae in the damaged fruits, and the adults also have a certain flying ability and transmission capacity. Therefore, how to effectively, quickly and accurately identify Bactrocera minax is particularly important for pest monitoring in the field of citrus fruit trade and pest identification in the field of plant quarantine.

[0004] At present, the identification methods of Bactrocera minax mainly include traditional morphological identification and molecular identification technology. Bactrocera minax and Bactrocera nox are morphologically similar, and the identification of larvae lacks obvious identification characteristics. The identification of adult Bactrocera minax and adult Bactrocera nox can be distinguished by the characteristics of the ovipositor of female insects, but the identification of male insects only has the thoracic bristles as the identification characteristic, which requires professional morphological knowledge and is difficult to identify morphologically. Although the existing PCR molecular identification technology can achieve rapid and accurate identification, it requires expensive equipment and has high identification cost, which is not suitable for popularization and application in basic departments.

[0005] Loop-mediated isothermal amplification (LAMP) is a nucleic acid denaturation and automatic cycle chain displacement nucleic acid amplification reaction under isothermal conditions using a Bst DNA polymerase with chain displacement activity and cascade nucleic acid amplification function. LAMP designs four specific primers for six regions of the target sequence, and uses a DNA polymerase with chain displacement function to continuously replicate and amplify DNA at a constant temperature. In order to improve the reaction efficiency, two loop primers can be added to the reaction system to bind to the stem-loop structure, start chain displacement synthesis, and cycle replication. LAMP is a simple and efficient nucleic acid isothermal amplification technology, which can amplify reaction products in a short time, has higher sensitivity, and is simple to operate without special instruments. The detection result can be observed and judged by naked eye, and is suitable for popularization and application in basic departments. In LAMP, primers are the key factors that determine the sensitivity and specificity of the detection results. SUMMARY

[0006] The technical problem to be solved by the present application is how to effectively and quickly and accurately identify the citrus fruit fly.

[0007] To solve the above technical problems, the present application first provides a primer set for identifying or assisting in identifying the citrus fruit fly.

[0008] The primer set for identifying or assisting in identifying the citrus fruit fly provided by the present application consists of primer-F3, primer-B3, primer-FIP and primer-BIP;

[0009] The primer-F3 is as follows a1) or a2):

[0010] a1) a single-stranded DNA molecule represented by sequence 1 in the sequence table;

[0011] a2) a single-stranded DNA molecule obtained by substituting and / or deleting and / or adding one or more nucleotides to sequence 1 and having the same function as sequence 1;

[0012] The primer-B3 is as follows a3) or a4):

[0013] a3) a single-stranded DNA molecule represented by sequence 2 in the sequence table;

[0014] a4) a single-stranded DNA molecule obtained by substituting and / or deleting and / or adding one or more nucleotides to sequence 2 and having the same function as sequence 2;

[0015] The primer-FIP is as follows a5) or a6):

[0016] a5) a single-stranded DNA molecule represented by sequence 3 in the sequence table;

[0017] a6) a single-stranded DNA molecule having the sequence 3 with substitution and / or deletion and / or addition of one or several nucleotides and having the same function as sequence 3;

[0018] The primer-BIP is as follows a7) or a8):

[0019] a7) a single-stranded DNA molecule as shown in sequence 4 in the sequence listing;

[0020] a8) a single-stranded DNA molecule having the sequence 4 with substitution and / or deletion and / or addition of one or several nucleotides and having the same function as sequence 4.

[0021] In the above primer set, the molar ratio of the primer-F3, the primer-B3, the primer-FIP and the primer-BIP is 1:1:8:8.

[0022] To solve the above technical problems, the application further provides a new use of the above primer set.

[0023] The application provides an application of the above primer set in any one of the following b1) to b4):

[0024] b1) identifying or assisting in identifying B. cucurbitae;

[0025] b2) identifying or assisting in identifying whether a to-be-tested fruit fly is B. cucurbitae;

[0026] b3) detecting or assisting in detecting whether a to-be-tested sample contains B. cucurbitae;

[0027] b4) distinguishing or assisting in distinguishing B. cucurbitae from other fruit flies.

[0028] To solve the above technical problems, the application further provides a kit containing the above primer set;

[0029] The kit has any one of the following functions c1) to c4):

[0030] c1) identifying or assisting in identifying B. cucurbitae;

[0031] c2) identifying or assisting in identifying whether a to-be-tested fruit fly is B. cucurbitae;

[0032] c3) detecting or assisting in detecting whether a to-be-tested sample contains B. cucurbitae;

[0033] c4) distinguishing or assisting in distinguishing B. cucurbitae from other fruit flies.

[0034] Further, the kit further comprises other reagents for detecting B. dorsalis. In the present application, the other reagents for detecting B. dorsalis are Warm Start LAMP 2xMaster Mix or Warm Start Colorimetric LAMP 2xMaster Mix.

[0035] Still further, the kit further comprises a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA of B. dorsalis).

[0036] The preparation method of the kit also belongs to the protection scope of the present application.

[0037] The preparation method of the kit can be d1) or d2) as follows:

[0038] d1) each primer in the primer set is packaged separately;

[0039] d2) each primer in the primer set is mixed together in proportion.

[0040] In the d2), the primer-F3, the primer-B3, the primer-FIP and the primer-BIP in the primer set are mixed together in a molar ratio of 1:1:8:8.

[0041] To solve the above technical problems, the present application further provides a method for identifying or assisting in identifying whether a to-be-tested fruit fly is B. dorsalis.

[0042] The method for identifying or assisting in identifying whether a to-be-tested fruit fly is B. dorsalis provided by the present application is S1) or S2) as follows:

[0043] The S1) comprises the following steps: extracting nucleic acid of the to-be-tested fruit fly, using the nucleic acid of the to-be-tested fruit fly as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested fruit fly is B. dorsalis is determined by detecting whether the reaction product electrophoresis presents ladder bands; if the reaction product electrophoresis presents ladder bands, the to-be-tested fruit fly is or is a candidate for B. dorsalis; if the reaction product electrophoresis does not present ladder bands, the to-be-tested fruit fly is not or is not a candidate for B. dorsalis.

[0044] The S2) comprises the following steps: extracting nucleic acid of the to-be-tested fruit fly, using the nucleic acid of the to-be-tested fruit fly as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification is completed, whether the to-be-tested fruit fly is B. dorsalis is determined by observing the color of the reaction system; if the reaction system is yellow, the to-be-tested fruit fly is or is a candidate for B. dorsalis; if the reaction system is pink, the to-be-tested fruit fly is not or is not a candidate for B. dorsalis.

[0045] To solve the above technical problems, the application further provides a method for detecting or assisting in detecting whether a to-be-tested sample contains B. dorsalis.

[0046] The method for detecting or assisting in detecting whether a to-be-tested sample contains B. dorsalis provided by the application is as follows T1) or T2):

[0047] The T1) comprises the following steps: extracting nucleic acid of the to-be-tested sample, using the nucleic acid of the to-be-tested sample as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested sample contains B. dorsalis is determined by detecting whether the reaction product presents ladder bands after electrophoresis; if the reaction product presents ladder bands after electrophoresis, the to-be-tested sample contains or is suspected to contain B. dorsalis; if the reaction product does not present ladder bands after electrophoresis, the to-be-tested sample does not contain or is suspected not to contain B. dorsalis.

[0048] The T2) comprises the following steps: extracting nucleic acid of the to-be-tested sample, using the nucleic acid of the to-be-tested sample as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification is completed, whether the to-be-tested sample contains B. dorsalis is determined by observing the color of the reaction system; if the reaction system is yellow, the to-be-tested sample contains or is suspected to contain B. dorsalis; if the reaction system is pink, the to-be-tested sample does not contain or is suspected not to contain B. dorsalis.

[0049] To solve the above technical problems, the application finally provides a method for distinguishing or assisting in distinguishing B. dorsalis from other fruit flies.

[0050] The method for distinguishing or assisting in distinguishing B. dorsalis from other fruit flies provided by the application is as follows U1) or U2):

[0051] The U1) comprises the following steps: extracting nucleic acid of the to-be-tested fruit fly, using the nucleic acid of the to-be-tested fruit fly as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested fruit fly is B. dorsalis is determined by detecting whether the reaction product presents ladder bands after electrophoresis; if the reaction product presents ladder bands after electrophoresis, the to-be-tested fruit fly is or is suspected to be B. dorsalis; if the reaction product does not present ladder bands after electrophoresis, the to-be-tested fruit fly is or is suspected to be other fruit flies.

[0052] The U2) comprises the following steps: extracting nucleic acid of the to-be-tested fruit fly, using the nucleic acid of the to-be-tested fruit fly as a template, and performing loop-mediated isothermal amplification by using the primer set; after the loop-mediated isothermal amplification is completed, whether the to-be-tested fruit fly is B. dorsalis is determined by observing the color of the reaction system; if the reaction system is yellow, the to-be-tested fruit fly is or is suspected to be B. dorsalis; if the reaction system is pink, the to-be-tested fruit fly is or is suspected to be other fruit flies.

[0053] In any of the above-mentioned methods, the loop-mediated isothermal amplification reaction system can specifically be system 1, with a total volume of 25 μL, consisting of 12.5 μL Warm Start LAMP 2x Master Mix, 1 μL DNA template, 9 μL primer mixture, and 2.5 μL sterile ultrapure water.

[0054] In any of the above-mentioned methods, the loop-mediated isothermal amplification reaction system can specifically be system 2, with a total volume of 25 μL, consisting of 12.5 μL Warm Start Colorimetric LAMP 2x Master Mix, 1 μL DNA template, 9 μL primer mixture, and 2.5 μL sterile ultrapure water.

[0055] In any of the above-mentioned methods, the loop-mediated isothermal amplification reaction conditions can be 63-67°C (such as 63-65°C, 65-67°C, 63°C, 65°C, or 67°C) constant temperature for 50 min.

[0056] The primer mixture is a mixture of each primer in the above-mentioned primer set. The final concentration of the primer-F3 and the primer-B3 in system 1 or system 2 can specifically be 0.2 μM, and the final concentration of the primer-FIP and the primer-BIP in system 1 or system 2 can specifically be 1.6 μM.

[0057] In any of the above-mentioned methods, the nucleic acid is genomic DNA.

[0058] In any of the above-mentioned applications or kits or methods, the to-be-tested real fly can specifically be an egg, a larva, a pupa, and / or an adult of the to-be-tested real fly.

[0059] In any of the above-mentioned applications or kits or methods, the to-be-tested sample can include various citrus fruits that can be damaged by the B. minax, such as sweet orange, mandarin, sour orange, red orange, grapefruit, lemon, citron, and bergamot.

[0060] In any of the above-mentioned applications or kits or methods, the other real fly can be at least one of the following real flies: B. tsuneonis, B. dorsalis.

[0061] In any of the above-mentioned applications or kits or methods, the B. minax can be B. minax from different geographical populations, including B. minax from Yiliang in Yunnan, Wangcang in Sichuan, Xichong in Sichuan, Chengbu in Hunan, Xupu in Hunan, Shimen in Hunan, Longshan in Hunan, Jiangjin in Chongqing, Fengjie in Chongqing, Wulong in Chongqing, Wanzhou in Chongqing, Zigui in Hubei, and Ankang in Shaanxi.

[0062] The application is based on loop-mediated isothermal amplification technology, and establishes a rapid identification method for Bactrocera minax. Four specific primers are designed according to six regions of a specific segment of a COI gene DNA barcode fragment of Bactrocera minax. Through optimization of a reaction system and a reaction condition, a LAMP molecular identification technology and a visual detection method are established according to the characteristics that a pH indicator changes from pink to yellow due to the pH change of the reaction system in the reaction process, so that sample positive contamination in the detection link caused by opening cover operation is effectively avoided. The loop-mediated isothermal amplification technology for identifying Bactrocera minax can realize rapid identification of eggs, larvae / adult individuals of Bactrocera minax, has the advantages of rapidity, high efficiency, strong specificity, high sensitivity, low cost, simple operation, no need of special instruments, and the like, is suitable for on-site, is easy to popularize at the grassroots level, can provide reliable technical basis for prevention and control of Bactrocera minax, and has important significance for plant quarantine and citrus fruit trade. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 The detection result of the primer set BM1 and BM2 in step one in Example 1.

[0064] Figure 2 The visual detection result in step two in Example 2.

[0065] Figure 3 The specific partial detection result in Example 3.

[0066] Figure 4 The partial detection result of sensitivity in Example 4. DETAILED DESCRIPTION

[0067] The following examples facilitate better understanding of the present application, but are not limited to the present application.

[0068] In the following examples, the experimental methods are all conventional methods unless otherwise specified.

[0069] In the following examples, the experimental materials are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0070] In the following examples, three repeated experiments are set for the quantitative test, and the average value is taken.

[0071] In the following examples, the blood / cell / tissue genomic DNA extraction kit is a product of Tiangen Company, and the product catalog number is DP304. The Warm Start LAMP 2xMaster Mix and the Warm Start Colorimetric LAMP 2xMaster Mix are both products of NEW ENGLAND BioLabs Company, and the product catalog numbers are E1700S and E1800S, respectively.

[0072] The sample number, species name and collection site information of the test fruit fly samples in the following examples are shown in Table 1. All the test fruit fly samples were identified using DNA barcoding identification method before testing.

[0073] Table 1

[0074]

[0075] Example 1, obtaining of primer set and kit for identifying B. dorsalis

[0076] I. Screening of primer set for identifying B. dorsalis

[0077] 1. The COI gene DNA barcode sequence in the mitochondrial genome of B. dorsalis was used as the target gene to design and synthesize primer set BM1 and primer set BM2 for identifying B. dorsalis. The primer sequences of each primer set are shown in Table 2.

[0078] Table 2

[0079]

[0080]

[0081] 2. The genomic DNA of the fruit fly samples was extracted using the blood / cell / tissue genomic DNA extraction kit to obtain the genomic DNA of the fruit fly samples.

[0082] The fruit fly samples were B. aurantii with sample number 3, B. dorsalis with sample number 6 and B. minor with sample number 18 in Table 1.

[0083] 3. The genomic DNA of the fruit fly samples was used as the template to perform loop-mediated isothermal amplification using primer set BM1 or primer set BM2 to obtain the amplification product.

[0084] The reaction system was 25 μL, which was composed of 12.5 μL Warm Start LAMP 2x Master Mix, 1 μL DNA template, primer mixture and sterile ultrapure water. The primer mixture was a mixture of primers in the primer set. In the reaction system, the final concentration of outer primer F3 and outer primer B3 was 0.2 μM, and the final concentration of inner primer FIP and inner primer BIP was 1.6 μM.

[0085] Reaction conditions: 65℃ for 50 min.

[0086] 4. The amplification product was subjected to 2% agarose gel electrophoresis.

[0087] According to the above method, the template was replaced with sterile ultrapure water, and the other steps were unchanged, serving as a negative control.

[0088] The detection results of primer set BM1 and BM2 are shown in the following figures Figure 1 . The left figure is primer set BM1, and the right figure is primer set BM2. M is DNA Marker, 1 is B. minax, 2 is B. aurantii, 3 is B. dorsalis, and N is sterile ultrapure water.

[0089] The results show that only primer set BM1 shows a typical ladder band in identifying B. minax, and the negative control does not show a band. Therefore, only primer set BM1 can be used to identify B. minax.

[0090] II. Preparation of a kit for identifying B. minax

[0091] The kit for identifying B. minax comprises primer set BM1.

[0092] Example 2, application of the kit prepared in Example 1 in identifying whether the to-be-tested fruit fly is or is a candidate for B. minax

[0093] I. Identifying whether the to-be-tested fruit fly is or is a candidate for B. minax by agarose gel electrophoresis

[0094] 1. The genomic DNA of the to-be-tested fruit fly is extracted using a blood / cell / tissue sample genomic DNA extraction kit to obtain the genomic DNA of the to-be-tested fruit fly.

[0095] 2. The genomic DNA of the to-be-tested fruit fly is used as a template, and primer set BM1 is used for loop-mediated isothermal amplification to obtain an amplification product.

[0096] The reaction system is 25 μL, which is composed of 12.5 μL Warm Start LAMP 2x Master Mix, 1 μL DNA template, 9 μL primer mixture, and 2.5 μL sterile ultrapure water. The primer mixture is a mixture of the primers in primer set BM1. In the reaction system, the final concentration of the outer primers F3 and B3 is 0.2 μM, and the final concentration of the inner primers FIP and BIP is 1.6 μM.

[0097] The reaction conditions are 65℃ for 50 min.

[0098] 3. The amplification product is subjected to 2% agarose gel electrophoresis.

[0099] According to the above method, the template is replaced with sterile ultrapure water, and the other steps remain unchanged, serving as a negative control.

[0100] According to the above method, the template is replaced with the genomic DNA of B. minax, and the other steps remain unchanged, serving as a positive control.

[0101] 4. According to the electrophoresis result, the following judgment is made:

[0102] When the positive control presents a typical ladder-like band and the negative control has no band, if the test fruit fly presents a typical ladder-like band, the test fruit fly is or is a candidate for the citrus fruit fly; if the test fruit fly does not present a typical ladder-like band (such as no band), the test fruit fly is not or is not a candidate for the citrus fruit fly.

[0103] When the positive control does not present a typical ladder-like band or the negative control has a band, the identification result of the test fruit fly is invalid.

[0104] II. Visual identification of whether the test fruit fly is or is a candidate for the citrus fruit fly

[0105] 1. The genomic DNA of the test fruit fly is extracted by using a blood / cell / tissue sample genomic DNA extraction kit to obtain the genomic DNA of the test fruit fly.

[0106] 2. The genomic DNA of the test fruit fly is used as a template, and loop-mediated isothermal amplification is performed by using primer set BM1.

[0107] The reaction system is 25 μL, which is composed of 12.5 μL Warm Start Colorimetric LAMP 2x Master Mix, 1 μL DNA template, 9 μL primer mixture, and 2.5 μL sterile ultrapure water. The primer mixture is a mixture of the primers in primer set BM1. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 is 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP is 1.6 μM.

[0108] Reaction conditions: 65℃ for 50 min.

[0109] According to the above method, the template is replaced with sterile ultrapure water, and the other steps are unchanged, which is used as a negative control.

[0110] According to the above method, the template is replaced with the genomic DNA of the citrus fruit fly, and the other steps are unchanged, which is used as a positive control.

[0111] 3. After step 2 is completed, the color of the reaction system is observed with the naked eye, and then the following judgment is made:

[0112] When the reaction system of the positive control is yellow and the reaction system of the negative control is pink, if the reaction system of the test fruit fly is yellow, the test fruit fly is or is a candidate for the citrus fruit fly; if the reaction system of the test fruit fly is pink, the test fruit fly is not or is not a candidate for the citrus fruit fly.

[0113] When the reaction system of the positive control is not yellow or the reaction system of the negative control is not pink, the identification result of the test fruit fly is invalid.

[0114] The inventors of the present application identified whether the to-be-tested fruit flies were B. dorsalis by the method of Step 2. The to-be-tested fruit flies were B. aurantii No. 3, B. dorsalis No. 5, B. dorsalis No. 6, B. dorsalis No. 10, B. dorsalis No. 13, B. dorsalis No. 16, B. dorsalis No. 17, and B. minor No. 18 in Table 1, respectively.

[0115] Some results are shown in Table 2. Figure 2 (1-8 are B. dorsalis No. 6, B. dorsalis No. 10, B. dorsalis No. 13, B. dorsalis No. 5, B. dorsalis No. 16, B. dorsalis No. 17, B. aurantii No. 3, and B. minor No. 18, respectively, and N is a negative control). The results show that the reaction systems of B. dorsalis of different geographical populations are yellow, and the reaction systems of B. aurantii and B. minor are pink.

[0116] Example 3, specificity experiment

[0117] The to-be-tested fruit fly 1 was B. dorsalis No. 5.

[0118] The to-be-tested fruit fly 2 was B. dorsalis No. 6.

[0119] The to-be-tested fruit fly 3 was B. dorsalis No. 7.

[0120] The to-be-tested fruit fly 4 was B. dorsalis No. 8.

[0121] The to-be-tested fruit fly 5 was B. dorsalis No. 9.

[0122] The to-be-tested fruit fly 6 was B. dorsalis No. 10.

[0123] The to-be-tested fruit fly 7 was B. dorsalis No. 11.

[0124] The to-be-tested fruit fly 8 was B. dorsalis No. 12.

[0125] The to-be-tested fruit fly 9 was B. dorsalis No. 13.

[0126] The to-be-tested fruit fly 10 was B. dorsalis No. 14.

[0127] The to-be-tested fruit fly 11 was B. dorsalis No. 15.

[0128] The to-be-tested fruit fly 12 was B. dorsalis No. 16.

[0129] The to-be-tested fruit fly 13 was B. dorsalis No. 17.

[0130] The to-be-tested fruit fly 14 was B. aurantii No. 1.

[0131] The to-be-tested fruit fly 15 is the citrus fruit fly numbered 2.

[0132] The to-be-tested fruit fly 16 is the citrus fruit fly numbered 3.

[0133] The to-be-tested fruit fly 17 is the citrus fruit fly numbered 4.

[0134] The to-be-tested fruit fly 18 is the orange fruit fly numbered 18.

[0135] Each to-be-tested fruit fly is subjected to the following steps:

[0136] 1. The genomic DNA of the to-be-tested fruit fly is extracted by using a blood / cell / tissue sample genomic DNA extraction kit.

[0137] 2. The genomic DNA of the to-be-tested fruit fly extracted in step 1 is used as a template, and a loop-mediated isothermal amplification is performed by using primer set BM1 to obtain an amplification product.

[0138] The reaction system is 25 μL, which is composed of 12.5 μL Warm Start LAMP 2x Master Mix, 1 μL DNA template, 9 μL primer mixture and 2.5 μL sterile ultrapure water. The primer mixture is a mixture of each primer in primer set BM1. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 is 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP is 1.6 μM.

[0139] The reaction condition is 65℃ for 50 min.

[0140] 3. The amplification product is subjected to 2% agarose gel electrophoresis.

[0141] According to the above method, the template is replaced with sterile ultrapure water, and the other steps are unchanged, serving as a negative control.

[0142] According to the above method, the template is replaced with the genomic DNA of the citrus fruit fly, and the other steps are unchanged, serving as a positive control.

[0143] Some of the detection results are shown in Figure 3 (1-18 are to-be-tested fruit flies 1-18, and N is a negative control). The results show that when the to-be-tested fruit fly is the citrus fruit fly, it shows a typical ladder-like band; when the to-be-tested fruit fly is not the citrus fruit fly, there is no band (i.e., no typical ladder-like band); and the negative control also has no band.

[0144] It can be seen that the primer set BM1 has good specificity for identifying the citrus fruit fly.

[0145] Example 4, Sensitivity Experiment

[0146] 1. Extract the genomic DNA of Bactrocera minax using the blood / cell / tissue sample genomic DNA extraction kit.

[0147] 2. Take the genomic DNA of Bactrocera minax, and perform gradient dilution with sterile water to obtain dilutions with concentrations of 10.0 ng / μL, 1.0 ng / μL, 1.0 x 10 -1 ng / μL, 1.0 x 10 -2 ng / μL, 1.0 x 10 -3 ng / μL, and 1.0 x 10 -4 ng / μL of genomic DNA of Bactrocera minax.

[0148] 3. Use the dilutions obtained in step 2 as templates, and perform loop-mediated isothermal amplification using primer set BM1 to obtain amplification products.

[0149] The reaction system is 25 μL, which is composed of 12.5 μL Warm Start LAMP 2x Master Mix, 1 μL DNA dilution (1 μL of the dilution contains 10.0 ng, 1.0 ng, 1.0 x 10 -1 ng, 1.0 x 10 -2 ng, 1.0 x 10 -3 ng, or 1.0 x 10 -4 ng of genomic DNA of Bactrocera minax), 9 μL primer mixture, and 2.5 μL sterile ultrapure water. The primer mixture is a mixture of the primers in primer set BM1. In the reaction system, the final concentration of outer primer F3 and outer primer B3 is 0.2 μM, and the final concentration of inner primer FIP and inner primer BIP is 1.6 μM.

[0150] Reaction conditions: 65℃ for 50 min.

[0151] 4. Perform 2% agarose gel electrophoresis on the amplification products.

[0152] According to the above method, replace the template with sterile ultrapure water, and the other steps remain unchanged, as a negative control.

[0153] According to the above method, replace the template with genomic DNA of Bactrocera minax, and the other steps remain unchanged, as a positive control.

[0154] If typical ladder-like bands are present, it indicates that the corresponding genomic content in the reaction system can be detected. If typical ladder-like bands are not present (such as no bands), it indicates that the corresponding genomic content in the reaction system cannot be detected.

[0155] Some of the test results are shown in Table 1. Figure 4(1-6 DNA template concentration in turn: 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, 1.0×10 -4 ng / μL, N is negative control). The results show that the sensitivity of primer group BM1 for identifying B. dorsalis is 1.0 ng / μL.

[0156] Therefore, it can be seen that the primer group BM1 has high sensitivity for identifying B. dorsalis.

[0157] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application. SEQUENCE LISTING <110> China Agricultural University <120> LAMP primer group for identifying B. dorsalis and application thereof <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 ttcggggctt gagcaggg 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 ggcccctagt attaggggt 19 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <400> 3 tccgattaag gctcctgggt ggaacttccc ttagaatcc 39 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <400> 4 cgtaatcgtt acagcccacc taatcagttt ccgaatcct 39

Claims

1. A primer set for identifying or assisting in the identification of the citrus fruit fly, consisting of primer-F3, primer-B3, primer-FIP, and primer-BIP; The primer-F3 is the single-stranded DNA molecule shown in sequence 1 of the sequence listing; The primer-B3 is the single-stranded DNA molecule shown in sequence 2 of the sequence listing; The primer-FIP is a single-stranded DNA molecule as shown in sequence 3 of the sequence listing; The primer-BIP is a single-stranded DNA molecule as shown in sequence 4 of the sequence listing; The molar ratio of primer-F3, primer-B3, primer-FIP, and primer-BIP is 1:1:8:

8.

2. The use of the primer set according to claim 1 in any one of the following b1)-b4): b1) Identification or auxiliary identification of the citrus fruit fly; b2) Identify or assist in identifying whether the fruit fly to be tested is the citrus fruit fly; b3) Detect or assist in the detection of whether the sample to be tested contains the citrus fruit fly; b4) To distinguish or assist in distinguishing the citrus fruit fly from other fruit flies.

3. A kit containing the primer set of claim 1; The kit has the function of any one of the following c1)-c4): c1) Identification or auxiliary identification of the citrus fruit fly; c2) Identify or assist in identifying whether the fruit fly to be tested is the citrus fruit fly; c3) Detect or assist in the detection of whether the sample to be tested contains the citrus fruit fly; c4) To distinguish or assist in distinguishing the citrus fruit fly from other fruit flies.

4. The preparation method of the kit according to claim 3 is as follows: d1) or d2). d1) Each primer in the primer set described in claim 1 is packaged separately; d2) Mix the primers in the primer set according to claim 1 in a certain proportion, and mix primer-F3, primer-B3, primer-FIP and primer-BIP in the primer set in a molar ratio of 1:1:8:

8.

5. A method for identifying or assisting in the identification of whether a test fly is a citrus fruit fly, comprising the following S1) or S2): S1) includes the following steps: extracting nucleic acid from the fruit fly to be tested, using the fruit fly nucleic acid as a template, and performing loop-mediated isothermal amplification using the primer set described in claim 1; after the loop-mediated isothermal amplification reaction is completed, determining whether the fruit fly to be tested is a citrus fruit fly by detecting whether a ladder-like band appears after electrophoresis of the reaction product: if a ladder-like band appears after electrophoresis of the reaction product, then the fruit fly to be tested is or is a candidate for a citrus fruit fly; if no ladder-like band appears after electrophoresis of the reaction product, then the fruit fly to be tested is or is not a candidate for a citrus fruit fly. S2) includes the following steps: extracting nucleic acid from the fruit fly to be tested, using the fruit fly nucleic acid as a template, and performing loop-mediated isothermal amplification using the primer set described in claim 1; after completing the loop-mediated isothermal amplification, determining whether the fruit fly to be tested is a citrus fruit fly by observing the color of the reaction system: if the reaction system is yellow, the fruit fly to be tested is or is a candidate for a citrus fruit fly; if the reaction system is pink, the fruit fly to be tested is not or is not a candidate for a citrus fruit fly. The final concentrations of primer-F3 and primer-B3 in the reaction system are both 0.2 μM, and the final concentrations of primer-FIP and primer-BIP in the reaction system are both 1.6 μM.

6. A method for detecting or assisting in the detection of whether a sample contains the citrus fruit fly, comprising the following T1) or T2): The T1 step includes the following steps: extracting nucleic acid from the sample to be tested; using the nucleic acid of the sample to be tested as a template, performing loop-mediated isothermal amplification using the primer set described in claim 1; after the loop-mediated isothermal amplification reaction is completed, determining whether the sample to be tested contains *Bactrocera dorsalis* by detecting whether a ladder-like band appears after electrophoresis of the reaction product: if a ladder-like band appears after electrophoresis of the reaction product, the sample to be tested contains or is a candidate to contain *Bactrocera dorsalis*; if no ladder-like band appears after electrophoresis of the reaction product, the sample to be tested does not contain or is a candidate to not contain *Bactrocera dorsalis*. The T2 step includes the following steps: extracting nucleic acid from the sample to be tested; using the nucleic acid of the sample to be tested as a template, performing loop-mediated isothermal amplification using the primer set described in claim 1; after completing the loop-mediated isothermal amplification, determining whether the sample to be tested contains *Bactrocera dorsalis* by observing the color of the reaction system: if the reaction system is yellow, the sample to be tested contains or is a candidate for containing *Bactrocera dorsalis*; if the reaction system is pink, the sample to be tested does not contain or is a candidate for not containing *Bactrocera dorsalis*. The final concentrations of primer-F3 and primer-B3 in the reaction system are both 0.2 μM, and the final concentrations of primer-FIP and primer-BIP in the reaction system are both 1.6 μM.

7. A method for distinguishing or assisting in distinguishing the citrus fruit fly from other fruit flies, comprising the following U1) or U2): The U1) includes the following steps: extracting nucleic acid from the fruit fly to be tested, using the nucleic acid of the fruit fly to be tested as a template, and performing loop-mediated isothermal amplification using the primer set described in claim 1; after the loop-mediated isothermal amplification reaction is completed, determining whether the fruit fly to be tested is a citrus fruit fly by detecting whether a ladder-like band appears after electrophoresis of the reaction product: if a ladder-like band appears after electrophoresis of the reaction product, then the fruit fly to be tested is or is a candidate for a citrus fruit fly; if no ladder-like band appears after electrophoresis of the reaction product, then the fruit fly to be tested is or is a candidate for another fruit fly; The U2 method includes the following steps: extracting nucleic acid from the fruit fly to be tested, using the fruit fly nucleic acid as a template, and performing loop-mediated isothermal amplification using the primer set described in claim 1; after completing the loop-mediated isothermal amplification, determining whether the fruit fly to be tested is a citrus fruit fly by observing the color of the reaction system: if the reaction system is yellow, the fruit fly to be tested is or is a candidate for a citrus fruit fly; if the reaction system is pink, the fruit fly to be tested is or is a candidate for another fruit fly. The final concentrations of primer-F3 and primer-B3 in the reaction system are both 0.2 μM, and the final concentrations of primer-FIP and primer-BIP in the reaction system are both 1.6 μM.

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