Primer group for rapidly and visually identifying Bactrocera mangifera and application of primer group

Abstract

The invention discloses a primer group for rapidly and visually identifying Bactrocera mangifera and application of the primer group. The invention belongs to the technical field of biology, and particularly relates to a primer group for rapidly and visually identifying Bactrocera mangifera and application of the primer group. The invention relates to a composition for identification or auxiliary identification of Bactrocera mangifera, the composition is a composition A or a composition B, the composition A is composed of an upstream primer named as Ccos-1F and a downstream primer named as Ccos-1R, and the composition B is composed of an upstream primer named as Ccos-1F and a downstream primer named as Ccos-1R; the composition B is composed of an upstream primer with the name of Ccos-1F, a downstream primer with the name of B-Ccos-1R, and a fluorescent probe sequence Ccos-Probe1 specific to the to-be-detected bactrocera indica. The MIRA-LFD primer disclosed by the invention is more flexible in design, high in sensitivity (capable of detecting trace DNA) and strong in specificity, is particularly suitable for on-site rapid detection requirements of port quarantine, field monitoring and the like, and provides an efficient technical support for prevention and control of the Bactrocera mangifera.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a primer set for rapid visual identification of Bactrocera mangostana and an application thereof. Background Art

[0002] Mango fruit fly, scientific name Ceratitis cozyra (Walker), English name mango fruit fly, also known as African mango fruit fly, Diptera, Tephritidae, genus Ceratitis The mango fruit fly is a dangerous and extremely destructive pest of fruits and vegetables, and is a quarantine pest for agricultural plants in my country. Adults lay eggs beneath the skin of their host fruit. After hatching, the larvae bore into the fruit, feeding and growing inside, causing the fruit to rot and become inedible, thus losing its economic value. The mango fruit fly is widely distributed in sub-Saharan Africa and to a lesser extent in Europe. It currently has no natural distribution in my country. Although it has no natural distribution in China, due to the influence of global trade, the mango fruit fly has the potential to spread to other regions through imported and exported fruit. Research indicates that the potential distribution of the mango fruit fly in my country primarily includes parts of Yunnan, Hainan, and Sichuan. With climate change and the development of global trade, the distribution range of the mango fruit fly may gradually expand, posing a major challenge to agricultural production and plant quarantine in my country. Plant quarantine is a key measure to prevent the spread of pests and is crucial for preventing the spread of the mango fruit fly. Existing quarantine methods primarily include morphological identification, traditional PCR technology, and gene chip technology. While these methods can accurately identify B. mangostana to a certain extent, they still have many limitations. For example, morphological identification requires a high level of expertise and experience, while PCR technology is complex and time-consuming. Therefore, a simpler, faster detection method suitable for routine quarantine work is urgently needed. Therefore, the development of a multi-enzyme constant-temperature rapid amplification and identification technology for B. mangostana is extremely necessary for those in this field.

[0003] In vitro amplification can be specifically described as a multi-enzyme isothermal rapid amplification reaction. MIRA (Multi-enzyme Isothermal Rapid Amplification) is an isothermal amplification technique that relies on the synergistic action of multiple functional proteins (T4 bacteriophage DNA helicase gp41 protein, Streptomyces coelicolor recA protein, single-stranded binding protein, and Escherichia coli DNA polymerase I, among others). The MIRA system requires two specific primers that recognize the target gene. At 37°C-42°C, the recombinase and primer form a protein / single-stranded nucleotide complex, Rec / ssDNA. With the help of accessory proteins and single-stranded binding protein, the complex invades the double-stranded DNA template, forming a D-loop region at the invasion site and initiating chain scanning. Upon finding the target region complementary to the primer, the Rec / ssDNA complex disintegrates, while DNA polymerase I binds to the 3' end of the primer, initiating chain extension. This process is rapid and efficient, resulting in exponential amplification of the target fragment within 20-40 minutes. It is currently widely used for the efficient detection of human, animal, and plant pathogens.

[0004] It has been further applied to lateral flow dipsticks. MIRA combined with lateral flow dipstick (LFD) technology requires the design of a colloidal gold probe between the upstream and downstream primers. This probe consists of a 46-52 nt sequence complementary to the target fragment, modified with an antigen label (such as FAM) at the 5' end, labeled with a dSpacer (tetrahydrofuran, THF) midway between the 5' and 3' ends, and labeled with a modifying group (such as C3-Spacer) at the 3' end. The downstream primer also requires a modifying group (commonly biotin) at the 5' end. When the target fragment, colloidal gold probe, primer, and endonuclease nfo are present in the system, the nfo enzyme recognizes and cleaves the THF molecule in the probe, leaving a free hydroxyl end in the probe, which becomes an upstream primer. This is then paired with a downstream primer labeled with biotin for amplification, resulting in an amplification product with a FAM group at the 5' end and a biotin at the 3' end. The FAM group on the amplified product binds to the anti-FAM antibody on the colloidal gold particles on the sample pad to form an immune complex. The biotin on the complex is captured by the biotin ligand on the detection line, causing the amplified product to aggregate with the colloidal gold particles, and a red strip appears on the detection line. Summary of the Invention

[0005] The main problem to be solved by the present invention is how to quickly and accurately identify the fruit fly.

[0006] In order to solve the above problems, the present invention provides a composition for identifying or assisting in identifying the mango fruit fly.

[0007] The present invention provides a composition for identifying or assisting in the identification of Bactrocera spp., wherein the composition is composition A or composition B, wherein composition A is composed of an upstream primer named Ccos-1F and a downstream primer named Ccos-1R; The composition B is composed of an upstream primer named Ccos-1F, a downstream primer named B-Ccos-1R, and a fluorescent probe sequence Ccos-Probe1 specific to the fruit fly to be detected; The Ccos-1F is a DNA having a nucleotide sequence of SEQ ID No. 1 in the sequence list; The Ccos-1R is a DNA having a nucleotide sequence of SEQ ID No. 2 in the sequence list; The B-Ccos-1R is a DNA whose nucleotide sequence is SEQ ID No. 3 in the sequence list; The probe sequence Ccos-Probe1 is a DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence list.

[0008] In the above primer combination, the molar ratio of the primer Ccos-1F to the primer Ccos-1R may be 1:1.

[0009] In the above primer combination, the molar ratio of the primer Ccos-1F to the primer B-Ccos-1R can be 1:1.

[0010] The present invention also provides the use of the above-mentioned composition in any of the following: a1) Preparation of products for the identification or differentiation of Bactrocera mangostana; a2) Identification or differentiation of the fruit fly; a3) preparing a product for detecting whether a sample to be tested contains Bactrocera mangostana; a4) Detecting whether the sample to be tested contains the fruit fly Bactrocera spp.

[0011] The present invention also provides a kit for identifying or assisting in identifying Bactrocera mangostana, wherein the kit contains the composition described above and conventional reagents of a DNA constant temperature rapid amplification kit or / and a lateral flow test strip.

[0012] Furthermore, the kit is a constant temperature rapid amplification kit.

[0013] Furthermore, the constant temperature rapid amplification kit can be a DNA constant temperature rapid amplification kit (product of Anpu Future Biotechnology Co., Ltd., product number WLB8201KIT, WLN8203KIT).

[0014] In the above, the detection line of the lateral flow chromatography test paper is coated with anti-biotin antibody.

[0015] In the above description, the quality control line of the lateral flow chromatography test strip is coated with an anti-fluorophore antibody. The fluorophore can be a fluorophore selected from FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LCRED640, LCRED705, Quasar705, or Texas Red. Specifically, in the embodiments of the present application, the fluorophore is specifically a FAM fluorophore.

[0016] Furthermore, the lateral flow chromatography test strips are from HybriDetect colloidal gold test strips (product of Anpu Future Biotechnology Co., Ltd., product number WLFS8201).

[0017] The present invention also provides a method for preparing the kit described above, comprising the steps of separately packaging the upstream primer of Ccos-1F, the downstream primer named Ccos-1R or B-Ccos-1R, and the probe sequence Ccos-Probe1 specific to the fruit fly to be detected.

[0018] The 5' end of the B-Ccos-1R is labeled with biotin.

[0019] Herein, the probe sequence is labeled with a fluorescent group, and the fluorescent group is selected from at least one of FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas Red.

[0020] Herein, the 5' end of the probe sequence Ccos-Probe1 carries a FAM fluorescent group, and the FAM is 5-carboxyfluorescein. The molecular formula of the 5-carboxyfluorescein is C 21 H 12 O7.

[0021] The 3' end of the probe sequence Ccos-Probe1 carries a C3-spacer modification. In the above, the C3-spacer is a short 3-carbon spacer arm modification.

[0022] In the above, the short 3-carbon chain inter-arm modification refers to the connection of a 3-carbon chain to the terminal 3' hydroxyl group of the oligonucleotide.

[0023] The present invention also provides the use of the above-mentioned kit in any of the following: a1) Preparation of products for the identification or differentiation of Bactrocera mangostana; a2) Identification or differentiation of the fruit fly; a3) preparing a product for detecting whether a sample to be tested contains Bactrocera mangostana; a4) Detecting whether the sample to be tested contains the fruit fly Bactrocera spp.

[0024] The present invention also provides a method for detecting Bactrocera spp., comprising using the composition described above to detect whether the sample to be tested contains the nucleic acid of Bactrocera spp.

[0025] The present invention also provides a method for identifying or assisting in identifying whether a sample to be tested is Bactrocera spp., comprising the following steps: N1: Extract genomic DNA from suspected samples of Bactrocera mangostana; N2: Using the genomic DNA of the sample as template DNA, perform multi-enzyme constant temperature rapid amplification using the kit described in claim 3 or 4 to obtain an amplification product, and determine whether the sample to be tested is Bactrocera spp. based on the amplification product. The determination method is as follows: 1) If the amplified product shows a band at 188 bp during gel electrophoresis, the sample to be tested is Bactrocera mangostana; if there is no band at 188 bp, the sample to be tested is not Bactrocera mangostana; 2) subjecting the amplified product to a color reaction using a test strip, and determining whether the sample to be tested is Bactrocera mangostana based on the color of the color reaction; if the detection line of the test strip appears red, the sample to be tested is Bactrocera mangostana; if the detection line of the test strip does not appear red, the sample to be tested is not Bactrocera mangostana.

[0026] The present invention also provides a method for identifying or assisting in identifying whether a sample to be tested contains Bactrocera mangostana, comprising the following steps: M1: Extract genomic DNA of the sample to be tested; M2: Using the genomic DNA of the sample as template DNA, perform multi-enzyme constant temperature rapid amplification using the kit described in claim 3 or 4 to obtain an amplification product, and determine whether the sample to be tested contains Bactrocera mangostana based on the amplification product. The determination method is as follows: 1) If the amplified product shows a band at 188 bp on gel, the sample to be tested contains Bactrocera mangostana; if there is no band at 188 bp, the sample to be tested does not contain Bactrocera mangostana; 2) subjecting the amplified product to a color reaction using a test strip, and determining whether the sample to be tested contains Bactrocera spp. based on the color of the color reaction; if the detection line of the test strip appears red, the sample to be tested contains Bactrocera spp.; if the detection line of the test strip does not appear color, the sample to be tested does not contain Bactrocera spp.

[0027] In the above method, the multi-enzyme isothermal amplification reaction temperature is 37-39°C, specifically 37°C.

[0028] In the above method, the multi-enzyme isothermal amplification reaction time is 8-25 min, specifically 10 min.

[0029] In the above method, the primer concentration in the multi-enzyme isothermal amplification reaction system is 0.20 μM-1.00 μM, and can be specifically 0.40 μM.

[0030] In the above method, the probe concentration in the multi-enzyme isothermal amplification reaction system is 0.05 μM-0.20 μM, and can be specifically 0.20 μM.

[0031] In the above method, the sample to be tested is genomic DNA.

[0032] In the above method, the reaction conditions for the isothermal rapid amplification are 37° C. for 8 minutes.

[0033] In a specific embodiment, the reaction system (50 μL) of the multi-enzyme isothermal rapid amplification can be: 29 μL A1 buffer ((Kit No. WLN8201KIT, Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (1-100 ng / μL), 2 μL primer Ccos-1F (final concentration 0.40 μM), 2 μL primer Ccos-1R (final concentration 0.40 μM), 2.5 μL B buffer and 9.5 μL sterile ultrapure water.

[0034] In a specific embodiment, the reaction system (50 μL) of the multi-enzyme isothermal rapid amplification can be: 29 μL A2 buffer (product number WLN8203KIT, product of Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (final concentration of 1-100 ng / μL), 2 μL primer Ccos-1F (final concentration of 0.40 μM), 2 μL B-Ccos-1R (final concentration of 0.40 μM), 1 μL Ccos-Probe1 (final concentration of 0.20 μM), 2.5 μL B buffer and 8.5 μL sterile ultrapure water.

[0035] Herein, the sample to be tested may further contain Mediterranean fruit flies, Keratitis paillidual , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 At least one of the custard apple fruit fly, the citrus fruit fly, the guava fruit fly, and the melon fruit fly.

[0036] As used herein, the term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) that are involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons).

[0037] As used herein, the term "template" refers to any nucleic acid molecule that can be used for amplification according to the present invention. Non-naturally double-stranded RNA or DNA can be made into double-stranded DNA and used as double-stranded DNA. Any double-stranded DNA or preparation containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci of interest contained within the template DNA.

[0038] As used herein, the term "primer" refers to an oligonucleotide that can be used in an amplification method, such as the polymerase chain reaction (PCR), for amplifying a nucleotide sequence based on a polynucleotide sequence that corresponds to a specific genomic sequence. At least one PCR primer used to amplify a polynucleotide sequence is sequence-specific for that sequence.

[0039] As used herein, the term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (commonly referred to as a "target polynucleotide") through complementary base pairing. The probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. The probe can be labeled directly or indirectly.

[0040] As used herein, the term "amplification reaction" refers to a process used to copy a nucleic acid one or more times. In embodiments, the amplification method includes, but is not limited to, polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or overlap extension splicing polymerase chain reaction. In some embodiments, single-molecule nucleic acids are amplified, for example, by digital PCR.

[0041] As used herein, the term "kit" refers to any delivery system used to deliver a substance. In assays, such delivery systems include systems for storing, transporting, or delivering reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., boxes) containing the relevant reagents and / or support materials.

[0042] In this document, the terms "include", "including", "have", "contain", etc. are open terms, which mean including but not limited to.

[0043] As used herein, the term "nucleic acid" refers to a polymer comprising at least two deoxynucleotides or nucleotides in single or double-stranded form. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to those of reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, specific nucleic acid sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as sequences clearly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassolet et al. (1992); Rossolin et al., Mol. Cell. Probes 8:91-98 (1994)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. DNA can be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0044] In this article, the terms "multienzyme isothermal rapid amplification technology" and "multienzyme isothermal amplification" refer to the same technology, namely "Multienzyme Isothermal Rapid Amplification (MIRA)", which is a constant temperature rapid nucleic acid amplification technology that relies on the synergistic action of multiple functional proteins (helicase, recombinase, single-strand binding protein, DNA polymerase, etc.) to achieve rapid nucleic acid amplification at room temperature.

[0045] This invention discloses a primer set and its application for rapid visual identification of Bactrocera mangostoma. Based on the MIRA-LFD (Multi-Enzyme Isothermal Rapid Amplification-Lateral Flow Digest) detection system, it offers advantages such as speed, sensitivity, specificity, and portability. The core of this technology lies in a highly efficient primer set designed for the specific gene sequence of Bactrocera mangostoma. Combined with the Multi-Enzyme Isothermal Amplification (MIRA) technique, this technology enables rapid nucleic acid amplification within 15 minutes at a constant temperature (37°C). The results are then visually interpreted using lateral flow digestion (LFD) strips, eliminating the need for complex instrumentation.

[0046] Compared to traditional PCR methods, MIRA-LFD eliminates the need for a thermal cycler, offering simpler operation and faster detection (down to 30 minutes). Compared to other isothermal amplification technologies (such as LAMP), MIRA-LFD offers greater flexibility in primer design, and LFD eliminates the risk of contamination from opening the cap. The system boasts high sensitivity (capable of detecting trace amounts of DNA) and strong specificity (no cross-reactivity with closely related species), making it particularly suitable for rapid on-site testing in applications such as port quarantine and field monitoring, providing effective technical support for the prevention and control of the fruit fly. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The specific detection results of the primer pair Ccos-1F / Ccos-1R in step 2 of Example 1, wherein M: D2000 DNA marker, N: ddH2O, and channels 1-11 are respectively Bactrocera spp. 1, Bactrocera spp. 2, Ceratitis spp. Keratitis paillidual , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 Sugar apple fruit fly, citrus fruit fly, guava fruit fly, and melon fruit fly.

[0048] Figure 2 This is the optimal time detection result of the primer pair Ccos-1F / Ccos-1R in step 2 of Example 1, where M: D2000 DNA marker, N: ddH2O, and channels 1-5 are 5 min, 10 min, 15 min, 20 min, and 25 min, respectively.

[0049] Figure 3 The sensitivity test results of the primer pair Ccos-1F / Ccos-1R in step 2 of Example 1 are shown, where M: D2000 DNA marker, N: ddH2O, and the concentrations of wells 1-6 are 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, and 1.0× 10 -1 ng / μL, 1.0 × 10 -2 ng / μL, 1.0 × 10 -3 ng / μL.

[0050] Figure 4 The specific detection results of Ccos-Probe1 in Example 2. Wherein, N is ddH2O, 1-12 are respectively No. 1 Bactrocera mangostoma, No. 2 Bactrocera mangostoma, Mediterranean fruit fly, Keratitis paillidual , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 Sugar apple fruit fly, citrus fruit fly, guava fruit fly, and melon fruit fly.

[0051] Figure 5 The optimal time detection result of Ccos-Probe1 in Example 2. Wherein, N is ddH2O, and 1-5 are 6 min, 7 min, 8 min, 9 min, and 10 min, respectively.

[0052] Figure 6 The sensitivity test results of Ccos-Probe1 in Example 2 are shown. Wherein, N is ddH2O, and 1-6 are 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.

[0053] Figure 7 This is the process and principle diagram of the combination of multi-enzyme constant temperature rapid amplification and lateral flow chromatography test strips in Example 2. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0055] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0056] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0057] The blood / cell / tissue genomic DNA extraction kit (Cat. No. DP316) used in the following examples is a product of Tiangen Biotechnology Co., Ltd. Buffer A1 was obtained from the DNA Constant Temperature Rapid Amplification Kit (Cat. No. WLB8201KIT, a product of Ampu Future Biotechnology Co., Ltd.) or Buffer A2 was obtained from the DNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip) (Cat. No. WLN8203KIT, a product of Ampu Future Biotechnology Co., Ltd.). Buffer B was obtained from the DNA Constant Temperature Rapid Amplification Kit (Cat. No. WLB8201KI, a product of Ampu Future Biotechnology Co., Ltd.) or the DNA Constant Temperature Rapid Amplification Kit (Colloidal Gold Test Strip) (Cat. No. WLN8203KIT, a product of Ampu Future Biotechnology Co., Ltd.). Lateral flow chromatography test strips were obtained from HybriDetect colloidal gold test strips (Cat. No. WLFS8201, a product of Ampu Future Biotechnology Co., Ltd.).

[0058] The numbers, species names, and collection locations of the test fruit fly DNA samples in the following examples are shown in Table 1. The samples were stored at -20°C for future use. The test fruit fly samples of the genus Bactrocera for this study were kindly provided by Dr. Marc DeMeyer and Dr. Massimiliano Virgilio of the Royal Museum of Central Africa. The test insect samples in Table 1 are described in: Zhang Yue (2021). Molecular phylogeny of important economic fruit flies and traceability of the invasion of the citrus fruit fly, a doctoral dissertation at China Agricultural University. The public may obtain this biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.

[0059] Using DNA barcoding technology for species identification: 1. Use blood / cell / tissue genomic DNA extraction kit (DP316, Tiangen Company) to extract DNA of various fruit fly samples in Table 1, and obtain genomic DNA of fruit fly samples (1 Bactrocera mangostoma, 2 Bactrocera mangostoma, 3 Mediterranean fruit fly, 4 Keratitis paillidual , No. 5 Natal fruit fly, No. 6 five-spotted fruit fly 、 No. 7 Ceratitis complaint, No. 8 sugar apple fruit fly, No. 9 citrus fruit fly, No. 10 guava fruit fly, No. 11 melon fruit fly).

[0060] 2. PCR amplification of DNA was performed using the universal primer pair LCO1490 / HCO2198. The forward primer was LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and the rear primer was HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'). The PCR system used was a 25 μL system consisting of 12.5 μL of 2× Taq PCR MasterMix (KT201, Tiangen Biochemical Technology (Beijing) Co., Ltd.), 9.5 μL of ddH2O, 1.0 μL each of the forward and rear primers, and 1.0 μL of genomic DNA template. Amplification conditions were: 95°C denaturation for 3 min, followed by denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 1 min. 34 cycles were performed, with extension at 72°C for 10 min. PCR products were stored at 4°C. If the electrophoresis results show that a bright band can be amplified, it means that the DNA sample can be used for subsequent sequencing identification.

[0061] 3. The corresponding PCR products with bright bands shown in the electrophoresis results were sent to Beijing Liuhe BGI Biotechnology Co., Ltd. for double-end first-generation sequencing. The sequencing results obtained were the mtDNA of each fruit fly sample. COI The obtained sequences were analyzed using the BLAST function on the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website. COI The sequences were compared in the core nt database. The similarity was more than 99%, and the phylogenetic tree was constructed for the first 100 results. COI Sequences on the same branch were considered to be of the same species. The results are shown in Table 1.

[0062] Table 1. Types and origins of test insect samples covered by this patent

[0063] Example 1: Primer pair specificity experiment, optimal time test, sensitivity test and kit acquisition for identifying Bactrocera mangostana 1. Design of primer pairs for identifying Bactrocera mangostana 1. The present invention designed and synthesized three front and back primers for identifying Bactrocera mangostana: Ccos-1F, Ccos-2F, Ccos-3F, Ccos-1R, Ccos-2R, Ccos-3R. The sequences of each primer are shown in Table 2.

[0064] Table 2. Primer sequence information

[0065] 2. Specificity Verification of Primer Pairs for Identifying Bactrocera mangostana 1. Take the above fruit fly samples (No. 1 mango fruit fly, No. 2 mango fruit fly, Mediterranean fruit fly, Keratitis paillidual , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 Using genomic DNA from Bactrocera annona, Bactrocera dorsalis, Bactrocera guava, and Bactrocera cucurbitae as templates, multi-enzyme isothermal amplification was performed using the primer combination Ccos-1F / Ccos-1R to obtain MIRA amplification products. The specific method is as follows: The reaction system was 50 μL, consisting of 29 μL A1 buffer ((Kit No. WLN8201KIT, Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (100 ng / μL), 2 μL primer Ccos-1F (final concentration 0.40 μM), 2 μL primer Ccos-1R (final concentration 0.40 μM), 2.5 μL B buffer, and 9.5 μL sterile ultrapure water. The reaction conditions were: constant temperature at 37°C for 10 min. 2. The amplified product in step 2 was subjected to 1% agarose gel electrophoresis and detected under ultraviolet light.

[0066] Test results are shown in Figure 1 The primer combination Ccos-1F / Ccos-1R showed a clear, bright band at 188 bp (nucleotide sequence is SEQ ID No. 5) when identifying Bactrocera mangostana; when the fruit fly species to be tested is not Bactrocera mangostana, there is no band (i.e., no clear, bright band at around 188 bp).

[0067] Therefore, the primer combination Ccos-1F / Ccos-1R was selected as the optimal identification primer for subsequent research. The nucleotide sequence of primer Ccos-1F is SEQ ID No. 1 in the sequence listing, and the nucleotide sequence of primer Ccos-1R is SEQ ID No. 2 in the sequence listing.

[0068] 3. Optimization of reaction time for multi-enzyme constant temperature amplification for identification of Bactrocera mangostana 1. Take the genomic DNA of Bactrocera mangostana No. 1 used in step 2 above.

[0069] 2. Using the genomic DNA of the fruit fly sample (Bactrocera mangosteenii No. 1) as a template, perform multi-enzyme isothermal amplification using the primer combination Ccos-1F / Ccos-1R to obtain the MIRA amplification product. The specific method is as follows: The experiment was divided into 5 min, 10 min, 15 min, 20 min, 25 min and negative control group (ddH2O).

[0070] 5-minute group: The reaction system was 50 μL, consisting of 29 μL A1 buffer (product in the kit (Cat. No. WLN8201KIT), a product of Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (100 ng / μL), 2 μL primer Ccos-1F (final concentration 0.40 μM), 2 μL primer Ccos-1R (final concentration 0.40 μM), 2.5 μL B buffer and 9.5 μL sterile ultrapure water. The reaction conditions were: constant temperature at 37°C for 5 min. The only difference between the 10 min, 15 min, 20 min, 25 min and 5 min groups is that the reaction time of 5 min is replaced by 10 min, 15 min, 20 min, and 25 min, and the other operations are the same as those of the 5 min group.

[0071] Negative control group (ddH2O): The reaction template was replaced with ddH2O, the reaction time was controlled at 25 min, and the operation was the same as that of other treatment groups.

[0072] 3. The amplified product in step 2 was subjected to 1% agarose gel electrophoresis and detected under ultraviolet light. Test results are shown in Figure 2 When the reaction time was longer than 10 minutes, a clear, bright band appeared at 188 bp. When the reaction time was shorter than 10 minutes, no band was observed (i.e., no clear, bright band appeared at around 188 bp). Furthermore, no band was observed in the negative control. Therefore, the optimal MIRA reaction time for the primer pair Ccos-1F / Ccos-1R for identifying Bactrocera mangostana is greater than 10 minutes. To meet the requirement for rapid identification, 10 minutes was subsequently selected as the final reaction time.

[0073] 4. Validation of the sensitivity of primer pairs for identifying Bactrocera mangostana 1. Take the genomic DNA of Bactrocera mangostana No. 1 used in step 1 above.

[0074] 2. The genomic DNA obtained in step 1 was diluted with sterile water to obtain 100.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 10.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 1.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 1.0×10 -1 ng / μL Bactrocera spp. genomic DNA dilution solution, 1.0×10 -2 ng / μL Bactrocera spp. genomic DNA dilution solution, 1.0×10 -3ng / μL dilution of Bactrocera spp. genomic DNA.

[0075] The experimental groups were as follows: 100.0 ng / μL Bactrocera genomic DNA diluent group, 10.0 ng / μL Bactrocera genomic DNA diluent group, 1.0 ng / μL Bactrocera genomic DNA diluent group, 1.0×10 -1 ng / μL Bactrocera mangostana genomic DNA dilution set, 1.0×10 -2 ng / μL Bactrocera mangostana genomic DNA dilution set, 1.0×10 -3 ng / μL Bactrocera mangostana genomic DNA dilution liquid group and negative control group.

[0076] 3. Use the dilution obtained in step 2 as a template for PCR reaction. The reaction system is 50 μL, consisting of 29 μL A1 buffer (product in the kit (Cat. No. WLN8201KIT), a product of Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (DNA dilution template of each concentration of Bactrocera mangostana genomic DNA in step 2), 2 μL primer Ccos-1F (final concentration 0.40 μM), 2 μL primer Ccos-1R (final concentration 0.40 μM), 2.5 μL B buffer and 9.5 μL sterile ultrapure water.

[0077] Reaction conditions: constant temperature at 37°C for 10 min. Negative control group: The only difference from the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent group is that the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent was replaced with sterile ultrapure water. Other operations were the same as those of the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent group.

[0078] 4. The amplified product in step 3 was subjected to 1% agarose gel electrophoresis and detected under ultraviolet light.

[0079] Test results are shown in Figure 3 When the genomic DNA concentration of Bactrocera mangostana was above 1.0 ng / μL, a clear, bright band appeared at 188 bp, and no band appeared when the concentration was below 1.0 ng / μL and in the negative control. Therefore, the primer pair Ccos-1F / Ccos-1R had a high sensitivity in identifying Bactrocera mangostana.

[0080] Example 2: Preparation of a kit for identifying Bactrocera spp. based on a multi-enzyme constant temperature-lateral flow chromatography test strip method 1. Visual identification specificity test of Bactrocera mangostana 1. Using the MIRA amplified product sequence of Bactrocera mangostana as the target, a probe was designed and synthesized. The probe sequence is detailed in Table 3. Ccos-Probe1, which showed specific amplification results, was selected as the probe for identifying Bactrocera mangostana.

[0081] Table 3. Probe sequence information in the kit

[0082] In Table 3, FAM is 5-carboxyfluorescein, a derivative of fluorescein, in which a carboxyl group (-COOH) is connected to the 5-carbon atom of fluorescein. Its chemical formula is Fam-CO-OCH3, and its molecular formula is C 21 H 12 O7, FAM- indicates that it forms an amide bond through a condensation reaction with the amino (-NH2) group on the DNA molecule and then connects to the nucleic acid.

[0083] [THF] is tetrahydrofuran, whose structural formula is and whose chemical formula is C4H8O, indicating that the oxygen atom of tetrahydrofuran forms a covalent bond with the phosphorus atom on the phosphate group at the 5' end of the oligonucleotide; and the lone pair of electrons on the oxygen atom of tetrahydrofuran will form a hydrogen bond with the hydrogen atom on the hydroxyl group at the 3' end of the oligonucleotide.

[0084] C3-Spacer is a short 3-carbon chain spacer modification, which here refers to the connection of a 3-carbon chain (-CH2-CH2-CH2-) to the terminal 3' hydroxyl group of the oligonucleotide.

[0085] 2. Various fruit flies (No. 1 mango fruit fly, No. 2 mango fruit fly, Mediterranean fruit fly, Ceratitis pale , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 Using genomic DNA from the fruit flies (Bactrocera annona, Bactrocera dorsalis, Bactrocera guava, and Bactrocera cucurbitae) as templates, a multi-enzyme thermostat-lateral flow chromatography (MFCD) amplification was performed using probes, primers Ccos-1F, and Ccos-1R labeled with biotin at the 5' end (abbreviated as B-Ccos-1R, nucleotide sequence: 5'-Biotin-TGAATATGCACTGGAGTTACTAAAGGATTA-3', SEQ ID No. 3 in the sequence listing). MIRA-LFD amplification products were obtained. Biotin is a molecule whose carboxyl group is covalently linked to the amino group of guanine deoxyribonucleotides.

[0086] The specific operations are as follows: The reaction system was 50 μL, consisting of 29 μL A2 buffer (Anpu Future Biotechnology Co., Ltd., catalog number WLN8203KIT), 5 μL DNA template (final concentration of 100 ng / μL), 2 μL primer Ccos-1F (final concentration of 0.40 μM), 2 μL B-Ccos-1R (final concentration of 0.40 μM), 1 μL Ccos-Probe1 (final concentration of 0.20 μM), 2.5 μL Bbuffer, and 8.5 μL sterile ultrapure water, and was kept constant at 37°C for 10 min.

[0087] The above reaction system was used to isolate the fruit flies No. 1, No. 2, Mediterranean fruit flies, Keratitis paillidual , Natal fruit fly, five-spotted fruit fly 、Ceratitis querita、 The genomic genes of the annona fruit fly, the citrus fruit fly, the guava fruit fly, and the cucurbit fruit fly and sterile ultrapure water were used as amplification templates for amplification.

[0088] 3. Dilute the amplified product obtained in step 2 10 times and add 50 μL to the sample pad of the lateral flow chromatography test paper (product of Anpu Future Biotechnology Co., Ltd., product number WLFS8201) for 5 minutes. After the reaction, observe with the naked eye whether the test line of the lateral flow chromatography test paper is colored (the reaction process and principle are as follows Figure 7 Then make the following judgment: 1) If the lateral flow chromatography test strip test line of the positive control (amplification products of genomic DNA of Bactrocera mangostana No. 1 and Bactrocera mangostana No. 2) is red and the lateral flow chromatography test strip test line of the negative control is white, then the probe can be used for visual detection of Bactrocera mangostana; 2) If the test line of the positive control lateral flow chromatography test strip is not red or the test line of the negative control lateral flow chromatography test strip is red, the probe cannot be used for visual detection of Bactrocera mangostana.

[0089] Test results are shown in Figure 4 :The Ccos-Probe1 test line on the lateral flow chromatography test strip of Bactrocera mangostana only turned red; the test lines on the lateral flow chromatography test strips of non-Bactrocera mangostana and the negative control turned white.

[0090] 2. Test on the optimal time for visual identification of Bactrocera mangostana 1. Obtain the genomic DNA of Bactrocera spp. No. 1 used in Example 1 above.

[0091] 2. Using the genomic DNA of the fruit fly sample (Bactrocera mangosteenii No. 1) as a template, perform multi-enzyme thermostat-lateral flow chromatography strip amplification using probe Ccos-Probe1 and primers Ccos-1F and B-Ccos-1R to obtain the MIRA-LFD amplification product. The specific method is as follows: The experiment was divided into 6 min, 7 min, 8 min, 9 min, and 10 min.

[0092] 6-minute group: The reaction system was 50 μL, consisting of 29 μL A2 buffer (product number WLN8203KIT, Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (final concentration of 100 ng / μL), 2 μL primer Ccos-1F (final concentration of 0.40 μM), 2 μL B-Ccos-1R (final concentration of 0.40 μM), 1 μL Ccos-Probe1 (final concentration of 0.20 μM), 2.5 μL Bbuffer and 8.5 μL sterile ultrapure water, and was kept constant at 37°C for 6 min.

[0093] 7 min group, 8 min group, 9 min group, and 10 min group: The only difference from the 6 min group is that the reaction time of 6 min is replaced by 7 min, 8 min group, 9 min group, and 10 min group. Other operations are the same as those of the 6 min group.

[0094] Negative control group: the reaction template was replaced with ddH2O, the reaction time was controlled at 25 min, and the operation was the same as that of other treatment groups.

[0095] 3. Dilute the amplified product obtained in step 2 10-fold and add 50 μL to the sample pad of the lateral flow chromatography test strip (product of Anpu Future Biotechnology Co., Ltd., product number WLFS8201) for 5 minutes. After the reaction, observe the test line of the lateral flow chromatography test strip with the naked eye to see if it is colored, and then make the following judgments: 1) If the lateral flow chromatography test strip test line of the positive control (amplified product of the genomic DNA of Bactrocera mangostana No. 1) is red and the lateral flow chromatography test strip test line of the negative control is white, then the visual detection of Bactrocera mangostana can be effectively performed at that time; 2) When the test line of the positive control lateral flow chromatography test strip is not red, the visual detection of Bactrocera mangostana cannot be effectively performed at that time.

[0096] Test results are shown in Figure 5 : The test line of the lateral flow chromatography test strip is red when the reaction time is more than 8 minutes. The test line of the lateral flow chromatography test strip is white when the reaction time is less than 8 minutes and the negative control.

[0097] In summary, the optimal MIRA-LFD reaction time for identifying Bactrocera mangostana using probe Ccos-Probe1, primers Ccos-1F, and B-Ccos-1R was 8 min.

[0098] 3. Visual identification sensitivity test of Bactrocera mangostana 1. Obtain the genomic DNA of Bactrocera spp. No. 1 used in Example 1 above.

[0099] 2. The genomic DNA obtained in step 1 was diluted with sterile water to obtain 100.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 10.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 1.0 ng / μL genomic DNA dilution solution of Bactrocera mangostana, 1.0×10 -1 ng / μL Bactrocera spp. genomic DNA dilution solution, 1.0×10 -2 ng / μL Bactrocera spp. genomic DNA dilution solution, 1.0×10 -3 ng / μL dilution of Bactrocera spp. genomic DNA.

[0100] 3. Using the dilution obtained in step 2 as a template, perform multi-enzyme constant temperature-lateral flow chromatography test strip amplification using probe Ccos-Probe1 and primers Ccos-1F and B-Ccos-1R to obtain the MIRA-LFD amplification product. The specific method is as follows: The experiment was divided into 100.0 ng / μL Bactrocera genomic DNA dilution group, 10.0 ng / μL Bactrocera genomic DNA dilution group, 1.0 ng / μL Bactrocera genomic DNA dilution group, 1.0×10 -1 ng / μL Bactrocera mangostana genomic DNA dilution set, 1.0×10 -2 ng / μL Bactrocera mangostana genomic DNA dilution set, 1.0×10 -3 ng / μL Bactrocera mangostana genomic DNA dilution liquid group and negative control group.

[0101] 100.0 ng / μL Bactrocera genomic DNA dilution set: The reaction system was 50 μL, consisting of 29 μL A2 buffer (product number WLN8203KIT, Anpu Future Biotechnology Co., Ltd.), 5 μL DNA template (final concentration of 100 ng / μL), 2 μL primer Ccos-1F (final concentration of 0.40 μM), 2 μL B-Ccos-1R (final concentration of 0.40 μM), 1 μL Ccos-Probe1 (final concentration of 0.20 μM), 2.5 μL Bbuffer and 8.5 μL sterile ultrapure water, and was kept constant at 37°C for 8 min.

[0102] 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 Bactrocera genomic DNA diluent group: The difference from the 100.0 ng / μL Bactrocera genomic DNA diluent group is that the 100.0 ng / μL Bactrocera genomic DNA diluent is replaced with 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 Bactrocera mangostana genomic DNA dilution solution, and other operations were the same as those of the 100.0 ng / μL Bactrocera mangostana genomic DNA dilution solution group.

[0103] Negative control group: The only difference from the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent group was that the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent was replaced with sterile ultrapure water. Other operations were the same as those of the 100.0 ng / μL Bactrocera mangostana genomic DNA diluent group.

[0104] 4. Dilute the MIRA amplification product obtained in step 3 10-fold and add 50 μL to the sample pad of the lateral flow chromatography test strip for 5 minutes. After the reaction, observe the test line of the lateral flow chromatography test strip with the naked eye to see if it is colored, and then make the following judgment: If the test line of the lateral flow chromatography test strip is red, it indicates that the corresponding genome content in the reaction system can be detected; if the test line of the lateral flow chromatography test strip is white, it indicates that the corresponding genome content in the reaction system cannot be detected.

[0105] Test results are shown in Figure 6The sensitivity of multi-enzyme constant temperature-lateral flow chromatography test strip amplification for identification of Bactrocera mangostana was 1.0×10 -1 ng / μL.

[0106] In summary, the primer pairs of probe Ccos-Probe1, primer Ccos-1F, and primer B-Ccos-1R were used for multi-enzyme constant temperature-lateral flow chromatography test strip amplification to identify the mango fruit fly with high sensitivity.

[0107] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A composition for identifying or assisting in the identification of Bactrocera spp., the composition being composition A or composition B, wherein composition A is composed of an upstream primer named Ccos-1F and a downstream primer named Ccos-1R; The composition B is composed of an upstream primer named Ccos-1F, a downstream primer named B-Ccos-1R, and a fluorescent probe sequence Ccos-Probe1 specific to the fruit fly to be detected; The Ccos-1F is a DNA having a nucleotide sequence of SEQ ID No. 1 in the sequence list; The Ccos-1R is a DNA having a nucleotide sequence of SEQ ID No. 2 in the sequence list; The B-Ccos-1R is a DNA whose nucleotide sequence is SEQ ID No. 3 in the sequence list; The probe sequence Ccos-Probe1 is a DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence list.

2. Use of the composition according to claim 1 in any of the following: a1) Preparation of products for the identification or differentiation of Bactrocera mangostana; a2) Identification or differentiation of the fruit fly; a3) preparing a product for detecting whether a sample to be tested contains Bactrocera mangostana; a4) Detecting whether the sample to be tested contains the fruit fly Bactrocera spp.

3. A kit for identifying or assisting in identifying Bactrocera mangostana, characterized in that: The kit contains the composition according to claim 1 and conventional reagents of a DNA constant temperature rapid amplification kit or / and a lateral flow test strip.

4. The kit according to claim 3, wherein: The kit is a constant temperature amplification kit.

5. A method for preparing the kit according to claim 3 or 4, comprising the steps of separately packaging the upstream primer of Ccos-1F, the downstream primer named Ccos-1R or B-Ccos-1R, and the probe sequence Ccos-Probe1 specific for the fruit fly to be detected.

6. Use of the kit according to claim 3 or 4 in any of the following: a1) Preparation of products for the identification or differentiation of Bactrocera mangostana; a2) Identification or differentiation of the fruit fly; a3) preparing a product for detecting whether a sample to be tested contains Bactrocera mangostana; a4) Detecting whether the sample to be tested contains the fruit fly Bactrocera spp.

7. A method for detecting Bactrocera spp., comprising using the composition of claim 1 to detect whether a sample to be tested contains nucleic acid of Bactrocera spp.

8. A method for identifying or assisting in identifying whether a sample is Bactrocera mangostana, comprising the following steps: N1: Extract genomic DNA from suspected samples of Bactrocera mangostana; N2: Using the genomic DNA of the sample as template DNA, perform multi-enzyme constant temperature rapid amplification using the kit described in claim 3 or 4 to obtain an amplification product, and determine whether the sample to be tested is Bactrocera spp. based on the amplification product. The determination method is as follows: 1) If the amplified product shows a band at 188 bp during gel electrophoresis, the sample to be tested is Bactrocera mangostana; if there is no band at 188 bp, the sample to be tested is not Bactrocera mangostana; 2) subjecting the amplified product to a color reaction using a test strip, and determining whether the sample to be tested is Bactrocera mangostana based on the color of the color reaction; if the detection line of the test strip appears red, the sample to be tested is Bactrocera mangostana; if the detection line of the test strip does not appear red, the sample to be tested is not Bactrocera mangostana.

9. A method for identifying or assisting in identifying whether a sample contains Bactrocera mangostana, comprising the following steps: M1: Extract genomic DNA of the sample to be tested; M2: Using the genomic DNA of the sample as template DNA, perform multi-enzyme constant temperature rapid amplification using the kit described in claim 3 or 4 to obtain an amplification product, and determine whether the sample to be tested contains Bactrocera mangostana based on the amplification product. The determination method is as follows: 1) If the amplified product shows a band at 188 bp on gel, the sample to be tested contains Bactrocera mangostana; if there is no band at 188 bp, the sample to be tested does not contain Bactrocera mangostana; 2) subjecting the amplified product to a color reaction using a test strip, and determining whether the sample to be tested contains Bactrocera spp. based on the color of the color reaction; if the detection line of the test strip appears red, the sample to be tested contains Bactrocera spp.; if the detection line of the test strip does not appear color, the sample to be tested does not contain Bactrocera spp.

10. The method according to claim 8 or 9, characterized in that The reaction condition of the isothermal rapid amplification is 37° C. for 8 min.

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