MicroRNA primer group for detecting infection of honeybee by n. ceranae, kit and application thereof

Abstract

The application relates to the field of biotechnology, and particularly relates to a MicroRNA primer group for detecting Nosema infection of Apis mellifera, a kit and application thereof. The primer group comprises the following primers: miR-6941-F, miR-6941-R, U6-F and U6-R; the nucleotide sequence of the miR-6941-F is shown in SEQ ID No. 1; the nucleotide sequence of the miR-6941-R is shown in SEQ ID No. 2; the nucleotide sequence of the U6-F is shown in SEQ ID No. 3; and the nucleotide sequence of the U6-F is shown in SEQ ID No. 4. The present application finds and identifies a specific MicroRNA molecule miR-6941 which is significantly changed after Nosema infection of Apis mellifera worker larvae. The primer group provided by the present application can be used for early diagnosis of Italian bee chalkbrood, and the diagnosis time is earlier than that of traditional methods. The primer group can provide a diagnosis basis for whether the larvae of a bee colony are infected with chalkbrood before the larvae are sick, provide a new perspective and target for elucidating the molecular mechanism of Apis mellifera resistance to chalkbrood, and lay a theoretical foundation for future development of a new bee disease prevention and control technology based on RNA interference.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a microRNA primer set, kit, and application for detecting *Gastrocystis apiae* infection in Italian honeybees. Background Technology

[0002] As one of the superior subspecies of the Western honeybee, the Italian honeybee (Apis mellifera ligustica) is widely used in beekeeping in my country. This bee species possesses advantages such as a docile temperament, strong queen egg-laying ability, concentrated pollination, high wax production, rapid comb building, strong ability to defend and clean the hive, and ease of maintaining large and strong colonies. Ascosphaeraapis is a deadly fungal disease that specifically infects larvae, causing chalkbrood and resulting in a significant decline in colony strength and productivity, causing substantial losses to the beekeeping industry.

[0003] However, the diagnosis of Italian bees infected with Coccidioides apiae currently relies mainly on the culture of fungal pathogens, morphological observation, or conventional molecular markers (such as ITS sequences). These methods suffer from low sensitivity, long processing time, and strong dependence on experimental conditions, making it difficult to meet the needs of early diagnosis in apiaries. There is a lack of early and sensitive molecular diagnostic methods, and research on the role of microRNA in the disease resistance process of Italian bees is also scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a microRNA primer set, kit, and application for detecting Coccidioidomyces apiaceus infection in Italian honeybees.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a microRNA primer set for detecting *Gnaphalium apiae* infection in Italian honeybees. The primer set includes the following primers: miR-6941-F, miR-6941-R, U6-F, and U6-R; The nucleotide sequence of miR-6941-F is shown in SEQ ID No. 1; The nucleotide sequence of miR-6941-R is shown in SEQ ID No. 2; The nucleotide sequence of U6-F is shown in SEQ ID No. 3; The nucleotide sequence of the U6-F is shown in SEQ ID No. 4.

[0006] The present invention also includes a kit for detecting Italian honeybee infection with Coccidioidomyces apiaceus, comprising the primer set said therein.

[0007] The present invention also includes the application of the primer set described herein in the preparation of a reagent for detecting Coccidioidomyces beesiensis.

[0008] Compared with existing technologies, this study discovered and identified a specific microRNA molecule, miR-6941, whose expression changed significantly after *Gnaphalium affine* infection of Italian honeybee worker larvae. The primer set provided in this study can be used for the early diagnosis of chalkbrood in Italian honeybees, with a diagnostic time earlier than traditional methods. It can provide a diagnostic basis for whether bee colony larvae are infected with chalkbrood before the larvae develop symptoms, providing a new perspective and target for elucidating the molecular mechanism of Italian honeybee resistance to chalkbrood, and laying a theoretical foundation for the future development of novel bee disease control technologies based on RNA interference.

[0009] The reagent kit based on this innovative biomarker enables rapid and visual detection, expanding the approach to bee disease diagnosis and applying host molecular biomarkers to disease monitoring, providing a new technical tool for bee colony health assessment and control. Attached Figure Description

[0010] Figure 1 A: Agarose gel electrophoresis of PCR amplification products, lane 1: DNA Marker, lane 2: PCR product of miR-6941; B: Sanger sequencing peak diagram of amplified fragments.

[0011] Figure 2 The relative expression level of miR-6941 in the intestine of 4-day-old Italian honeybee worker larvae infected with Coccidioides apiae was determined.

[0012] Figure 3 The incidence of chalkbrood in Italian honeybee worker larvae after inoculation with Coccidioides apiae.

[0013] Figure 4 Italian honeybee larvae under naked-eye observation. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. Example 1:

[0015] 1. (1) Select a strong and healthy Italian honeybee colony (artificially raised), quickly transfer the comb to the laboratory, and use a clean transfer needle to transfer 2-day-old larvae into a 48-well plate (1 larva / well) containing 50 µL of feed (63% royal jelly, 30% sterilized water, 6% honey, and 1% yeast extract). Then place the plate in a constant temperature and humidity chamber at 35±0.5℃ and 90% relative humidity (RH) for rearing; (2) When the larvae are reared to 3 days old, prepare a feed containing honeybee spores (final concentration of 1×10⁻⁶).6 The control group was fed a diet without *Gastrocystis bee* (50 µL / larva), while the treatment group was fed a diet containing *Gastrocystis bee* spores (final concentration 1×10⁻⁶ spores / mL). 6 Feed (50 µL / head) of larvae per mL. After the feed is consumed, feed without spores of Coccidioides apiae is fed; (3) Intestinal tissue of 4-day-old control group and treatment group larvae is dissected in a clean bench. Each 3 larvae's intestines constitute a biological replicate, and 3 biological replicates are set up for each age.

[0016] 2. Based on the microRNA molecule (named miR-6941, whose DNA copy sequence obtained by sequencing is shown in SEQ ID No. 6) detected and identified in the intestinal tissue of bee larvae in this study, loop primers and upstream and downstream primers were designed and synthesized using miRNA Design software. Total RNA was extracted from intestinal samples, and residual genomic DNA was removed by digestion with 5×gDNADigester. After treatment at 55°C for 5 min, the samples were heat-shocked at 85°C for 5 s. The total RNA was reverse transcribed using the loop primer (miR-6941-loop, sequence shown in Table 5 or SEQ ID No. 5). The resulting cDNA was used as a template for PCR amplification. The reaction system and procedure for PCR amplification are shown in Tables 1 and 2, where the upstream primer in Table 1 is miR-6941-F from Table 5, and the downstream primer in Table 1 is miR-6941-R from Table 5. The amplified products were detected by 1.5% agarose gel electrophoresis and observed and photographed using a gel imaging analysis system. The target fragment was recovered by gel extraction and TA cloning. The bacterial culture with positive PCR results was sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing.

[0017] Agarose gel electrophoresis results as follows Figure 1 As shown in Figure A, the miR-6941-specific fragment amplified by the circular primers is the expected size (less than 100 bp). Sanger sequencing results are as follows... Figure 1 As shown in Figure B, the target fragment sequence is completely consistent with the next-generation sequencing results, and the target fragment contains the BS site. These results confirm the existence of the BS site in miR-6941.

[0018] Table 1 PCR reaction system

[0019] The PCR Mix used in Table 1 is Hieff® PCR Master Mix (With Dye), catalog number: 10102ES03; it contains Hieff® TaqDNA Polymerase, dNTP mixture, MgCl2 and an optimized buffer system.

[0020] Table 2 PCR reaction procedure

[0021] 3. Total RNA was reverse transcribed using a loop primer (miR-6941-loop, sequence see Table 5 or SEQ ID No. 5) to obtain miR-6941 cDNA, which was then used as a template for RT-qPCR. Simultaneously, one sample of total RNA was reverse transcribed using an equimolar mixture of OligodT primers and Random 6 Mers Primer primers; the resulting cDNA was used as an internal control. U RT-qPCR was performed using the template from GenBank accession number 6 (LOC725641). Real-time quantitative PCR was performed on an automated PCR analysis system according to the reaction system in Table 3, following the procedure in Table 4. This included two parts: ① Detecting the relative expression level of miR-6941 using the reaction system in Table 3 on an automated PCR analysis system according to the procedure in Table 4, where the upstream primer in Table 3 is miR-6941-F from Table 5, and the downstream primer is miR-6941-R from Table 5; ② Simultaneously, detecting the internal reference gene using the reaction system in Table 3 on an automated PCR analysis system according to the procedure in Table 4. U The expression level of 6 was used to correct for inter-sample variations (such as RNA quality, reverse transcription efficiency, etc.), where the upstream primer in Table 3 is U6-F from Table 5 and the downstream primer is U6-R from Table 5. Ct values ​​ranged from 15 to 30, determined by 2- ΔΔCt Relative expression levels were calculated using a method similar to Sudent's expression level. GraphPad Prism 8 software was used for data analysis and plotting. Experimental data are expressed as Mean ± SD (Sudent's expression level). t Test, ns: P >0.05, *: P <0.05, **: P <0.01, ***: P <0.001,****: P <0.0001).

[0022] The results are as follows Figure 2 As shown, compared with the control group, the expression level of miR-6941 in the intestine of 4-day-old larvae in the treatment group (i.e., the infection group in the figure) was significantly downregulated by 50%, and the statistical difference was significant (P<0.05). The experiment was repeated 3 times.

[0023] When using this invention to detect whether bee larvae are infected with Coccidioides apis, the expression level of miR-6941 in the experimental group (the bee larvae being tested) is more than 50% lower than that in the control group (artificially raised, uninfected bee larvae), which is considered a positive infection.

[0024] Table 3 Reaction System

[0025] The qPCR Mix used in Table 3 is Yeasen; catalog number: HRF0041.

[0026] Table 4 Reaction Procedure

[0027] Table 5 Primer Information Example 2:

[0028] Statistics on the incidence of chalkbrood Three-day-old Italian honeybee worker larvae were inoculated with *Coprinus beescens*, with 24 larvae constituting one biological replicate, for a total of three biological replicates. Feed was changed every 24 hours for seven consecutive days. Before each feed change, the disease incidence rate of the Italian honeybee worker larvae was measured, defined as the appearance of white mycelium on their body surface. The formula for calculating the chalkbrood disease incidence rate was: (Number of larval corpses with white *Coprinus beescens* mycelium on their body surface in each biological replicate / 24) × 100%.

[0029] like Figure 4 The image shows the changes in Italian honeybee larvae before and after disease, as observed by the naked eye. The statistical results are as follows: Figure 3 As shown, the larvae show signs of disease at 4 dpi (i.e., 7-day-old larvae), but with this invention, infection can be detected in the intestines at 1 dpi (4-day-old larvae).

[0030] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A microRNA primer set for detecting *Gnaphalium apiae* infection in Italian honeybees, characterized in that: The primer set includes the following primers: miR-6941-F, miR-6941-R, U6-F and U6-R; The nucleotide sequence of miR-6941-F is shown in SEQ ID No. 1; The nucleotide sequence of miR-6941-R is shown in SEQ ID No. 2; The nucleotide sequence of U6-F is shown in SEQ ID No. 3; The nucleotide sequence of the U6-F is shown in SEQ ID No.

4.

2. A kit for detecting *Gastrocystis apiae* infection in Italian honeybees, comprising the primer set of claim 1.

3. The application of the primer set as described in claim 1 in the preparation of a reagent for detecting Coccidioides beesiensis.

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