Detection method of apiary bee coccus

Abstract

The invention discloses a detection method of honeybee coccus, and relates to the technical field of biological detection, and the technical key points are as follows: a recombinant prokaryotic expression vector of LbCas12a is constructed and purified, crRNA is designed by taking a conserved region of a 16s gene of the honeybee coccus as a target spot, a trans-cleavage report system based on CRISPR / Cas12a is constructed, and the detection method of the honeybee coccus is realized. The method has the advantages that the method is simple and convenient to operate, the method is combined with RAA (recombinase-aid amplification) to establish the constant-temperature rapid visual detection method for the honeybee cocci, the detection limit value of the method for plasmid templates is 100 copies / mu L, specific test results show that the method has no cross reaction with other bee pathogenic microorganisms, and the detection time is only 80 minutes; according to the invention, a low-cost, simple, rapid and accurate nucleic acid detection technology aiming at the honeybee coccus is established by utilizing a CRISPR / Cas enzyme system and combining a recombinase-mediated isothermal nucleic acid amplification technology platform.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a method for detecting honeycomb Apis mellifera. Background Art

[0002] Bees are social insects whose emergence coincides with the emergence of early angiosperms, first appearing approximately 120-130 million years ago. Through a long evolutionary process, bees have become one of the world's most important economic pollinators. Approximately 80% of the pollination services provided by insects for major crops comes from bees. Furthermore, bees provide humans with nutritious bee products, including honey, propolis, and royal jelly. A sharp decline in bee populations could have serious negative impacts on ecosystem stability, crop yields, and human well-being.

[0003] Honeybee populations have continued to decline in recent years, and the threat of pests and diseases is considered one of the main reasons. Among them, European foulbrood (EFB), caused by the bacterium Melissococcus plutonius, has become a major challenge to global beekeeping health management due to its high infectivity and lethality. Larvae aged 1-3 days are susceptible to infection, and the pathogen is transmitted via royal jelly, resulting in reduced larval food intake, changes in body color, and spots on the body surface, which in turn lead to larval death. The colony's brooding capacity declines, the queen's egg production decreases, and the colony's development stagnates. In severe cases, larval mortality can reach as high as 80%, significantly reducing colony size and honey production. This increases beekeeping costs, and frequent replacement of beehives or queens becomes necessary.

[0004] Rapid and accurate detection of European rot is key to preventing and controlling bee colony diseases. Current detection methods, both domestically and internationally, primarily encompass traditional microbiology techniques, molecular biology, and emerging rapid diagnostic tools. These include staining (such as Gram staining) and microscopy to observe Apis cerevisiae in the larval midgut contents. This method, commonly used in grassroots apiaries, is low-cost but insensitive, and can be easily confused with symbiotic Lactobacilli. Larval samples are cultured on selective media (such as J-medium), and the pathogen is identified through colony morphology and biochemical reactions. Apis cerevisiae is an anaerobic bacterium, making cultivation difficult and time-consuming.

[0005] To this end, the present invention aims to provide a method for detecting Apis mellifera to solve the above problems. Summary of the Invention

[0006] The object of the present invention is to solve the above problems and provide a detection method for Apis mellifera. By constructing a recombinant prokaryotic expression vector of LbCas12a and a recombinant vector of the 16s region of Apis mellifera (i.e., pMD18-T-T7-Mp-16s), utilizing the CRISPR / Cas enzyme system, and combining the recombinase-mediated isothermal nucleic acid amplification technology (RAA) platform, a low-cost, simple, rapid and accurate nucleic acid detection technology for Apis mellifera is established. This method has the potential to be applied to on-site detection, improves the working efficiency of front-line clinical diagnosis, and helps the scientific prevention and control of honey bee European rot.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a method for detecting Apis mellifera, the method comprising the following steps:

[0009] S1. Using the pMV-Mp-16s plasmid as a template, primers were designed to amplify the Mp-16s gene sequence with a T7 promoter. The obtained Mp-16s gene sequence was ligated into pMD18-T and transformed into DH5α competent cells. The cells were cultured on ampicillin plates at 37°C overnight, and single colonies were picked and expanded to obtain T7-Mp-16s.

[0010] S2, in vitro transcription using T7-Mp-16s as template;

[0011] S3. Use the FastFire qPCR PreMix kit and the 16s gene of the Apis mellifera strain as the template sequence to perform fluorescent quantitative PCR.

[0012] S4, construct crRNA primer and Cas12a reaction system;

[0013] S5, constructing RAA primers and RAA reaction system;

[0014] S6. Perform sensitivity and specificity tests.

[0015] The specific steps of S2 are: using T7-Mp-16s as a template to perform in vitro transcription according to HiScribe TMQuickT7 High Yield RNA Synthesis Kit instructions: The reaction system includes 17μL Nuclease-free water, 10μL NTP Buffer Mix, 1μL T7-Mp-16s, and 2μL T7 RNA Polymerase Mix. After vortex mixing, incubate at 37°C for 4 hours. After the reaction, add 20μL Nuclease-free water and 2μL DNase I, vortex mix, and incubate at 37°C for 15 minutes.

[0016] The crRNA primer in the S4 includes a T7 promoter, a LbCas12a repeat sequence, and a LbCas12a spacer sequence. The 25 μL Cas12a reaction system includes 2.5 μL 10 × NEB buffer 2.1, 1.2 μM crRNA primer, 0.4 μM purified LbCas12a, 0.4 μM ssDNA fluorescent reporter (5'-6-FAM-TTATT-BHQ-3') and 1 μL DNA template or RAA product (Mp-16s amplification product); at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, a fluorescence quantifier was used to incubate for 50 minutes at 37 ° C, and a fluorescence value was taken every 30 seconds for fluorescence kinetic analysis.

[0017] In S5, the upstream and downstream crRNA target sequences were screened based on the Mp-16s gene, and RAA amplification primers were designed. The RAA reaction system was performed according to the instructions of the RAA nucleic acid amplification kit. First, 25 μL of buffer V, 2 μL of RAA amplification primer, and 16.5 μL of purified water were added to the reaction tube. The lyophilized powder in the reaction tube was mixed by hand, and the liquid was collected at the bottom of the tube by brief centrifugation. Then, 2.5 μL of magnesium acetate was added to the inside of each tube cap. 2 μL of template DNA was added below the liquid surface in the tube, and the reaction was carried out at 37°C for 30 minutes.

[0018] Compared with the existing technology, this solution has the following beneficial effects:

[0019] The present invention constructs a recombinant prokaryotic expression vector of LbCas12a and obtains high-purity LbCas12a protein by using Strep affinity chromatography and molecular sieve purification technology. At the same time, crRNA is designed with the conserved region of the 16S gene of Apis mellifera as the target, a trans-cleavage reporter system based on CRISPR / Cas12a is constructed, and a constant-temperature rapid visual detection method for Apis mellifera is established in combination with RAA. The detection limit of this method for plasmid templates is 10 0The results of specificity tests showed that it had no cross-reaction with other bee-derived microorganisms, and the detection time was only 80 minutes. The present invention conducted research on the rapid molecular detection technology of the above three bee-derived microorganisms, and established a low-cost, simple, rapid and accurate nucleic acid detection technology for Apis mellifera by using the CRISPR / Cas enzyme system and combining it with the recombinase-mediated isothermal nucleic acid amplification technology (RAA) platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In the embodiment of the present invention;

[0021] Figure 2 Schematic diagram of crRNA sequence optimization in an embodiment of the present invention, where a is the fluorescence signal intensity and b is the result of naked eye reading under ultraviolet light;

[0022] Figure 3 Schematic diagram of RAA primer optimization in an embodiment of the present invention, wherein a is the fluorescence signal intensity; b is the result read by naked eye under ultraviolet light;

[0023] Figure 4 Schematic diagram of the optimization conditions of Mp-RT-RAA-CRISPR in an embodiment of the present invention, wherein a is ssDNA; b is crRNA; c is Cas12a;

[0024] Figure 5 Schematic diagram of the RT-RAA-Cas12a reaction sensitivity test of Apis mellifera in an embodiment of the present invention, wherein a is the plasmid sensitivity fluorescence signal intensity; b is the plasmid sensitivity result read by naked eye under ultraviolet light; c is the sensitivity of fluorescence quantitative PCR;

[0025] Figure 6 Schematic diagram of a specific test in an embodiment of the present invention, wherein a is the fluorescence signal intensity of the specific test; b is the result of the specific test read by naked eyes under ultraviolet light;

[0026] Figure 7 1 is a schematic diagram of the simulated clinical sample detection of Apis mellifera RT-RAA-Cas12a in an embodiment of the present invention, wherein a is the fluorescence signal intensity; b is the naked eye reading under ultraviolet light; and c is the result of fluorescence quantitative PCR. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0028] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0029] Example:

[0030] 1. Materials and Methods

[0031] 1.1 Plasmids, strains, and genomes

[0032] Apis mellifera, Escherichia coli, Staphylococcus aureus, Paenibacillus, Enterococcus faecalis, and Hafnia alvei were isolated and preserved in our laboratory; the 16S gene sequence was synthesized by Beijing Qingke Biological Co., Ltd.

[0033] 1.2 Main Reagents

[0034] Table 1 Main reagents

[0035]

[0036]

[0037] 1.3 Main instruments

[0038] Table 2 Main instruments used

[0039]

[0040] 1.4 Construction of recombinant plasmids and in vitro transcription of the 16s gene of Apis mellifera

[0041] 1.4.1 Construction of pMD18-T-T7-Mp-16s recombinant plasmid

[0042] Primers were designed using the pMV-Mp-16s plasmid as a template to amplify the full-length sequence of the Mp-16s gene with a T7 promoter (Table 3). The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The obtained target fragment was ligated to pMD18-T, transformed into DH5α competent medium, cultured overnight at 37°C on ampicillin plate medium, and monoclonal colonies were picked and expanded to obtain pMD18-T-T7-Mp-16s (i.e., the recombinant prokaryotic expression vector of LbCas12a) and sent to Changchun Kumei for sequencing.

[0043] Upstream and downstream primer sequences:

[0044] T7-Mp-16s-F:

[0045] 5'-TAATACGACTCACTATAGCTGAGACACGGCCCAGACTCCTACGGG-3'

[0046] T7-Mp-16s-R: 5'-TTGCTGCAGCACTGAAGGGCGGAAACCC-3'

[0047] Table 3 PCR reaction program of M.p-16s gene

[0048]

[0049]

[0050] 1.4.2 M.p-16s gene in vitro transcription

[0051] The full-length T7-Mp-16s obtained by PCR was used as a template for in vitro transcription. TM According to the Quick T7 High Yield RNA Synthesis Kit instructions, the reaction system includes 17 μL Nuclease-free water, 10 μL NTP Buffer Mix, 1 μL template DNA, and 2 μL T7 RNA Polymerase Mix. Vortex and incubate at 37°C for 4 hours. After the reaction, add 20 μL Nuclease-free water and 2 μL DNase I (RNase-free), vortex and incubate at 37°C for 15 minutes to digest any remaining DNA.

[0052] 1.4.3 TaqMan Real-time Fluorescence Quantitative PCR of Apis cerevisiae

[0053] According to the fluorescence quantitative PCR reaction program in Table 4, the FastFire qPCR Pre Mix kit was used, the MP-16s gene of the Apis mellifera strain was used as the template sequence, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0054] Melissococcus pluton fluorescence quantitative PCR upstream and downstream primers and probes:

[0055] Mp-F: 5'-TGTTGTTAGAGAAGAATAGGGGAA-3'

[0056] Mp-R: 5'-CGTGGCTTTCTGGTTAGA-3'

[0057] Probe: 5'-FAM-CGTGGCTTTCTGGTTAGA-BHQ1-3'

[0058] Table 4M.p-16s fluorescence quantitative PCR

[0059]

[0060] 1.4.4crRNA design and Cas12a reaction system

[0061] 1.4.4.1 crRNA Design and Synthesis

[0062] The Mp-16s gene sequence was obtained from the NCBI database, and the alignment analysis was performed using MegAlign software. 22 primers were designed for the conserved region, of which 3 crRNA primers were located in the published fluorescence quantitative sequence (Table 5). The crRNA primers included a T7 promoter, a LbCas12a repeat sequence (different types and sources of Cas protein binding sequences are different) and a spacer sequence (recognition target sequence). All primers were synthesized by Changchun Kumei Company. The two synthesized upstream and downstream primers were mixed in equal amounts, reacted at 95°C for 5 minutes, and then annealed at room temperature to form double-stranded DNA. The double-stranded DNA was incubated at 37°C overnight and transcribed into crRNA. The transcribed crRNA was purified according to the instructions of the RNAClean&Concentrator kit and stored in aliquots at -80°C.

[0063] Table 5 crRNA targeting Mp-16s gene

[0064]

[0065] 1.4.4.2 Cas12a reaction system

[0066] The entire 25 μL reaction system includes 2.5 μL 10×NEB buffer 2.1, 1.2 μM crRNA, 0.4 μM purified LbCas12a, 0.4 μM ssDNA fluorescent reporter (5'-6-FAM-TTATT-BHQ-3', synthesized by Shanghai Shenggong Bioengineering Technology Co., Ltd.) and 1 μL DNA template or RAA product. At an excitation wavelength of 485 nm and an emission wavelength of 520 nm, a fluorescence quantifier was used to incubate at 37 ° C for 50 minutes, and the fluorescence value was taken every 30 seconds for fluorescence kinetic analysis. After the run program is completed, the reaction tube can be observed for fluorescence by naked eye under ultraviolet light.

[0067] 1.4.5 RAA Primer Design and RAA Reaction System

[0068] The AA reaction has the best amplification efficiency for fragments of 100-200bp, and the primer length is generally 30-38nt. If the primer is too short, the recombination rate will be reduced, thereby affecting the amplification speed and detection sensitivity. Based on the optimal crRNA target sequence screened by the Mp-16s gene, RAA amplification primers were designed upstream and downstream. Degenerate bases were used for some single-base mutation sites. A total of 4 upstream primers and 4 downstream primers were designed. By combining them, 16 different primer pairs can be obtained (Table 6). The RAA reaction was performed according to the instructions of the RAA nucleic acid amplification kit. First, 25μL of buffer V, 2μL of F / R, and 16.5μL of purified water were added to the reaction tube. The lyophilized powder in the reaction tube was mixed by hand, and the liquid was collected at the bottom of the tube by brief centrifugation. Then 2.5μL of magnesium acetate was added to the inside of each tube cap. 2μL of template DNA was added below the liquid surface in the tube and reacted at 37°C for 30 minutes.

[0069] Mp-16s upstream primer:

[0070] RAA-Mp-16s-RAA-F1:

[0071] 5'-GGGAGGCAGCAGTAGGGAATCTTCGGCAATG-3';

[0072] Mp-16s-RAA-F2:

[0073] 5'-GAGGCAGCAGTAGGGAATCTTCGGCAATGGA-3';

[0074] Mp-16s-RAA-F3:

[0075] 5'-CTTCGGCAATGGACGAAAGTCTGACCGAGCAA-3';

[0076] Mp-16s-RAA-F4:

[0077] 5'-GCCGCGTGAGTGAAGAAGGTTTTCGGATCGT-3';

[0078] RAA-Mp-16s downstream primers:

[0079] Mp-16s-RAA-R1:

[0080] 5'-AAACCGCCTGCGCTCGCTTTACGCCCAATAA-3';

[0081] Mp-16s-RAA-R2:

[0082] 5'-CTCAAGTCTTCCAGTTTCCAATGACCCTCCC-3';

[0083] Mp-16s-RAA-R3:

[0084] 5'-CACTCAAGTCTTCCAGTTTCCAATGACCTC-3';

[0085] Mp-16s-RAA-R4:

[0086] 5'-GGACAACGCTTGCCACCTACGTATTACCGCGG-3'.

[0087] Table 6 Combinations of different RAA primers

[0088]

[0089] 1.4.6 Sensitivity and specificity tests

[0090] 1.4.6.1 Sensitivity test

[0091] pMD18-TM.p-16s was diluted ten-fold to prepare 10 8 -10 -2 The plasmid standard was prepared by 10-fold serial dilution of the Mp-16s-DNA prepared by in vitro transcription. 8 -10 -2 The Mp-RT-RAA-Cas12a assay was used to verify the detection limits of the two templates. A fluorescence quantification method was used to detect the DNA standards, and the sensitivity of the Mp-RT-RAA-Cas12a assay established in this study was compared with that of the fluorescence quantification method.

[0092] 1.4.6.2 Specificity test

[0093] The Mp-RT-RAA-Cas12a method and fluorescence quantitative PCR method were used to detect genomic DNA extracted from Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Enterobacter faecalis (E. faecalis), Paenibacillus larvae (P. larvae), Hafnia alvei (H. alvei), and Melissa pluton (M. pluton) and Mp-16s-DNA prepared by in vitro transcription to determine whether this method has cross-reaction with other pathogenic microorganisms.

[0094] 1.4.7 Simulated clinical sample testing

[0095] To evaluate the detection performance of the established Mp-RT-RAA-CRISPR method in real samples, DNA transcribed in vitro from the Mp-16s gene was added to genomic DNA extracted from larvae to prepare a total of 24 clinical simulation samples. These 24 clinical simulation samples were detected using the Mp-RT-RAA-CRISPR method and real-time fluorescence quantitative PCR.

[0096] 2. Results

[0097] 2.1 Screening of the best crRNA for the 16s gene of Apis mellifera

[0098] The design and screening of specific crRNAs is a key process in establishing CRISPR / Cas12a detection. To achieve optimal reaction sensitivity and accuracy, we designed 22 crRNAs targeting the 16s gene region of Apis mellifera. After software analysis, we selected three optimal crRNAs (all located within the detection area of ​​the fluorescence quantitative method) for the CRISPR / Cas12a system reaction. Judging by the fluorescence signal intensity, all three crRNAs showed varying degrees of activity in this reaction system, reaching a peak at the 10th cycle ( Figure 2 a), the results of visual observation under UV light are consistent with the fluorescence signal value ( Figure 2 b), but the fluorescence values ​​of the negative groups of crRNA8 and crRNA10 continued to increase over time. Therefore, crRNA11 was ultimately determined to be the optimal choice for the CRISPR / Cas12a detection platform for Apis mellifera, and it was able to enter the plateau phase after 10 cycles (5 minutes).

[0099] 2.2 Screening of the best RAA primers for the 16s gene of Apis mellifera

[0100] Based on the optimization screening results of crRNA, 16 sets of specific RAA primers were designed upstream and downstream of the crRNA11 target for isothermal amplification reaction, and the products after RAA amplification were used as the detection substrate of CRISPR / Cas12a system. The RAA template concentration was 10 9 When the number of copies / μL was 1, the amplification efficiency of the RAA7 primer group was higher ( Figure 3 ab), the above results show that primer RAA7 (F2 / R3) has good amplification efficiency, so this primer was selected for the establishment of subsequent detection methods.

[0101] 2.3Mp-RT-RAA-CRISPR Condition Optimization

[0102] The components added to the reaction system were optimized using crRNA11 and RAA7, including crRNA concentration, Cas12a protein concentration, and ssDNA concentration. The product amplified by RAA was used as the detection substrate for the CRISPR / Cas12a system. The RAA template was 10 9 When the ssDNA concentration was 100 copies / μL, the effect of the ssDNA concentration on the fluorescence value was small, and the minimum concentration of 0.6 μM was selected. As the concentrations of crRNA and Cas12a protein increased, the fluorescence value also gradually increased. The crRNA concentration of 1 μM and the Cas12a protein concentration of 1.2 μM were selected for the establishment of subsequent detection methods.

[0103] 2.4 Sensitivity and specificity test of the Mp-RT-RAA-CRISPR method

[0104] 2.4.1 M.p-RT-RAA-CRISPR reaction sensitivity test

[0105] When the pMD18-TM.p-16s plasmid was diluted tenfold as a template, the minimum detection limit of the Mp-RAA-CRISPR method was 1 copy / μL ( Figure 5 a), the results of visual observation under UV light are consistent with the results of fluorescence signal intensity ( Figure 5 b). The recommended sensitivity of fluorescent quantitative PCR is 10 copies / μL ( Figure 5 c). Compared with the results of fluorescence quantitative PCR, the Mp-RT-RAA-CRISPR detection method has good sensitivity.

[0106] 2.4.2 M.p-RT-RAA-CRISPR reaction specificity test

[0107] Using genomic DNA of different pathogenic microorganisms as templates, such as Figure 6 As shown in a and b, the target DNA collected a strong fluorescence signal value, which was significantly different from that of S. aureus, E. coli, E. faecalis, P. larvae, H. alvei and the negative control (Negative Control, NC). In addition, the negative and positive results could be clearly distinguished by naked eye observation under ultraviolet light, indicating that the method has good specificity.

[0108] 2.5 Clinical simulation sample testing

[0109] All 24 clinical simulation samples were prepared and tested using the Mp-RT-RAA-Cas12a method and real-time PCR. Figure 7c The results showed that the real-time detection results of S1-6 and S13-18 tissue samples were positive, and the fluorescence signal intensity analysis of the Mp-RT-RAA-Cas12a detection platform was consistent with the real-time PCR results ( Figure 7 a), and the results can be directly determined by naked eye observation under ultraviolet light ( Figure 7 b). In summary, the Mp-RT-RAA-Cas12a method can effectively detect positive simulated samples in complex backgrounds and can be used as a reserve technology for early on-site detection of Apis mellifera in beehives.

[0110] 3. Discussion

[0111] The CRISPR / Cas system is not only a powerful tool for gene editing and regulation but also a significant advancement in the development of rapid nucleic acid detection. Its advantages are further enhanced when combined with isothermal amplification technology. RAA (Racine Assay) from Jiangsu Qitian Gene Biotechnology Co., Ltd. and RPA (Reactive Protein Assay) from TwistDx (UK) utilize similar amplification principles, demonstrating consistent specificity and sensitivity. RAA is less expensive than RPA, and therefore, combined with Cas12a-mediated nucleic acid detection platforms, it holds broad application prospects.

[0112] The prevention and control of Apis apiacea plays a key role in the stable and sustainable development of the honey bee industry. Therefore, accurate, rapid, and cost-effective diagnosis is crucial for the prevention and control of Apis apiacea. The development of real-time quantitative RT-PCR has improved the molecular diagnosis of animal diseases and has become a routine method for animal disease diagnosis in laboratories. However, quantitative PCR requires samples to be sent to specialized laboratories for testing using expensive equipment, and the sample transportation process is time-consuming. Compared with quantitative PCR, the Mp-RT-RAA-Cas12a method described in this study has similar detection sensitivity and strong specificity, shortens operation and reaction time, and is not limited to laboratory settings and expensive equipment, allowing for rapid determination in the field. In the testing of simulated samples, all positive samples were detected without false positive results, demonstrating that this method has practical detection capabilities in complex environments and can guide areas affected by Apis apiacea to implement measures more quickly, which is crucial for the prevention and control of Apis apiacea.

[0113] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for detecting Apis mellifera in a honeycomb, characterized by: The method comprises the following steps: S1. Using the pMV-Mp-16s plasmid as a template, primers were designed to amplify the Mp-16s gene sequence with a T7 promoter. The obtained Mp-16s gene sequence was ligated into pMD18-T and transformed into DH5α competent cells. The cells were cultured on ampicillin plates at 37°C overnight, and single colonies were picked and expanded to obtain T7-Mp-16s. S2, in vitro transcription using T7-Mp-16s as template; S3. Use the FastFire qPCR PreMix kit and the MP-16s gene of the Apis mellifera strain as the template sequence to perform fluorescent quantitative PCR. S4, construct crRNA primer and Cas12a reaction system; S5, constructing RAA primers and RAA reaction system; S6. Perform sensitivity and specificity tests.

2. The method for detecting Apis mellifera as claimed in claim 1, wherein: The sequences of the crRNA and RAA primers are: crRNA11-F: TAATACGACTCACTATAGGtaatttctactaagtgtagatTGGTTAGATACCGTCACGAG; crRNA11-R: CTCGTGACGGTATCTAACCAatctacacttagtagaaattaCCTATAGTGAGTCGTATTA; Mp-16s-RAA-F2: 5'-GAGGCAGCAGTAGGGAATCTTCGGCAATGGA-3'; Mp-16s-RAA-R3: 5'-CACTCAAGTCTTCCAGTTTCCAATGACCTC-3'.

3. The method for detecting Apis mellifera as claimed in claim 1, wherein: The specific steps of S2 are: using T7-Mp-16s as a template to perform in vitro transcription according to HiScribe TM Quick T7 High Yield RNA Synthesis Kit instructions: The reaction system includes 17μL Nuclease-free water, 10μL NTP Buffer Mix, 1μL T7-Mp-16s, and 2μL T7 RNA Polymerase Mix. After vortex mixing, incubate at 37°C for 4 hours. After the reaction, add 20μL Nuclease-free water and 2μL DNase I, vortex mix, and incubate at 37°C for 15 minutes.

4. The method for detecting Apis mellifera as claimed in claim 1, wherein: The crRNA primer in the S4 comprises a T7 promoter, an LbCas12a repeat sequence and an LbCas12a spacer sequence.

5. The method for detecting Apis mellifera as claimed in claim 1, wherein: In the S4, the 25 μL Cas12a reaction system includes 2.5 μL 10×NEB buffer 2.1, 1.2 μM crRNA primer, 0.4 μM purified LbCas12a, 0.4 μM ssDNA fluorescent reporter molecule and 1 μL Mp-16s amplification product; at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, a fluorescence quantifier is used to incubate at 37 ° C for 50 minutes, and the fluorescence value is taken every 30 seconds for fluorescence kinetic analysis.

6. The method for detecting Apis mellifera as claimed in claim 1, wherein: The sequence of the ssDNA fluorescent reporter molecule is: 5'-6-FAM-TTATT-BHQ-3'.

7. The method for detecting Apis mellifera as claimed in claim 1, wherein: In S5, the upstream and downstream of the crRNA target sequence are screened according to the Mp-16s gene, and RAA amplification primers are designed.

8. The method for detecting Apis mellifera as claimed in claim 1, wherein: In S5, the RAA reaction system was used according to the instructions of the RAA nucleic acid amplification kit. First, 25 μL of buffer V, 2 μL of RAA amplification primer, and 16.5 μL of purified water were added to the reaction tube. The lyophilized powder in the reaction tube was mixed by hand, and the liquid was collected to the bottom of the tube by brief centrifugation. Then, 2.5 μL of magnesium acetate was added to the inside of each tube cap, and 2 μL of template DNA was added below the liquid surface in the tube. The reaction was carried out at 37°C for 30 minutes.

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