A kit for detecting n. apis based on digital PCR and a detection method thereof

Abstract

The application discloses a kit for detecting Ascosphaera apis based on digital PCR and a detection method thereof, and belongs to the technical field of molecular biology detection. The kit comprises a specific primer pair and a probe for the ITS sequence of Ascosphaera apis, a hot-start DNA polymerase, a dNTPs mixture, and a digital PCR reaction buffer. The detection method comprises the following steps: extracting total RNA from a to-be-detected honeybee sample and reversely transcribing the total RNA into cDNA; preparing a digital PCR reaction system; performing microdroplet treatment and PCR amplification; and finally realizing absolute quantification of a target gene through fluorescence detection. The application does not need to rely on a standard curve, has the advantages of high sensitivity, high specificity and simple operation, is suitable for early screening of chalkbrood disease of a bee colony, research on a pathogen transmission mechanism and drug evaluation, and has high practicability.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a kit and detection method for detecting Coccidioidomyces apis based on digital PCR. Background Technology

[0002] Bee cysts ( Ascosphaera apis *Gynocystis aeruginosa* is an obligate parasitic fungus that causes chalkbrood in bees, severely impacting the health of bee larvae and leading to colony weakening or even extinction. Currently, commonly used detection methods for *Gynocystis aeruginosa* include morphological observation, conventional PCR, and quantitative real-time PCR. Morphological observation has low sensitivity and relies on experience; conventional PCR can only detect qualitatively; while quantitative real-time PCR can quantify, it depends on a standard curve and is easily affected by amplification efficiency and matrix interference, resulting in limited accuracy. Therefore, developing an absolute quantitative detection method that does not require a standard curve, has high sensitivity, and strong specificity is of great significance. Summary of the Invention

[0003] The purpose of this invention is to provide a kit and method for detecting Gastrocystis apis based on digital PCR, enabling quantitative detection of Gastrocystis apis without the need for a standard curve.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a kit for detecting Corydalis apiae based on digital PCR, comprising: (a) Primer pair for specifically amplifying the ITS sequence of Coccidioides apis; the forward primer sequence of the primer pair is shown in SEQ ID NO:1 and the reverse primer sequence is shown in SEQ ID NO:2; (b) A probe for the specific detection of the amplification product; (c) Hot-start DNA polymerase; (d) dNTPs mixture; (e) Digital PCR reaction buffer.

[0005] Furthermore, the sequence of the probe is shown in SEQ ID NO:3, and the 5′ end of the probe is labeled with a fluorescent reporter group and the 3′ end is labeled with a quencher group.

[0006] Furthermore, the fluorescent reporter group is FAM, and the quencher group is TAMRA.

[0007] The present invention also includes a method for detecting *Gyrodactylus apis* using any of the kits described herein, comprising the following steps: (1) Sample preparation: Obtain bee samples to be tested, extract total RNA and reverse transcribe it into cDNA as a detection template; (2) Preparation of digital PCR reaction system: Mix the detection template with the primer pair, probe, hot-start DNA polymerase, dNTPs mixture and digital PCR reaction buffer to prepare the reaction system; (3) Microdroplet generation and PCR amplification: The reaction system prepared in step (2) is microdropletized to generate a large number of independent reaction units, and then PCR amplification reaction is performed. (4) Fluorescence detection and data analysis: The fluorescence signal of each reaction unit after PCR amplification is detected, and the absolute copy number of the target gene of Coccidia apis in the sample is calculated based on the number and proportion of positive microdroplets.

[0008] Furthermore, the bee sample to be tested in step (1) is the intestinal tissue of a bee larva.

[0009] Furthermore, the PCR amplification reaction procedure in step (3) is as follows: pre-denaturation at 95℃ for 10 min; followed by 42 cycles of amplification, each cycle including denaturation at 94℃ for 30 s, annealing at 60℃ and extension for 45 s; and finally storage at 4℃.

[0010] Furthermore, the total volume of the reaction system described in step (2) is 30 μL, which includes: 6 μL of Probe dPCRHiTaqMix reagent A, 1.5 μL of reagent B, 5 μL of cDNA template, 1.5 μL each of upstream and downstream primers at a concentration of 10 pmol / μL, 1.5 μL of probe at a concentration of 10 pmol / μL, and the remainder is ddH2O.

[0011] Furthermore, the target gene of the *Gnaphalium apiaceum* is the ITS1 gene.

[0012] Compared with existing technologies, this invention uses digital PCR technology, which can achieve absolute quantification without the need for a standard curve and has high sensitivity; the primers and probes are designed for the conserved sequences of *Gastromyxobolus apis*, which has high specificity; it is suitable for early screening of chalkbrood in bee colonies, research on pathogen transmission mechanisms and drug evaluation, and has strong practicality. Attached Figure Description

[0013] Figure 1 The image shows the results of digital PCR detection of ITS1 gene copy number in the intestine of larvae on days 4, 5, and 6 after infection with Coccidioides apis. 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.

[0015] In one embodiment, a kit for detecting *Gyrodactylus apis* based on digital PCR comprises: (a) Primer pair for specifically amplifying the ITS sequence of Coccidioides apis; the forward primer sequence of the primer pair is shown in SEQ ID NO:1 and the reverse primer sequence is shown in SEQ ID NO:2; (b) A probe for the specific detection of the amplification product; (c) Hot-start DNA polymerase; (d) dNTPs mixture; (e) Digital PCR reaction buffer.

[0016] This kit contains specific primer pairs targeting the conserved sequences of ITS1, 5.8S rRNA, and ITS2 of *Gyrodactylus beesiensis*. These primers specifically bind to the target gene region of *Gyrodactylus beesiensis*, enabling large-scale replication of the target gene through PCR amplification, thereby improving detection sensitivity. The hot-start DNA polymerase is inactive at room temperature and is only activated under high-temperature conditions, effectively avoiding non-specific amplification of the PCR reaction at low temperatures and improving the specificity and accuracy of the detection reaction for *Gyrodactylus beesiensis*. The dNTPs mixture contains four deoxyribonucleotides: dATP, dCTP, dGTP, and dTTP, providing the raw materials needed for DNA synthesis in the PCR amplification reaction of the target gene of *Gyrodactylus beesiensis*. The digital PCR reaction buffer contains various ions and components to maintain the stability of the reaction system, providing a suitable pH and ionic strength environment for the PCR reaction of *Gyrodactylus beesiensis*, ensuring the smooth progress of the amplification reaction.

[0017] In one embodiment, the probe sequence is shown in SEQ ID NO:3, wherein the 5′ end of the probe is labeled with a fluorescent reporter group and the 3′ end is labeled with a quencher group; during PCR amplification, when the probe binds to the target gene, the 5′-3′ exonuclease activity of the hot-start DNA polymerase will cleave the fluorescent reporter group from the probe, separating it from the quencher group, thereby releasing a fluorescent signal for subsequent detection and analysis.

[0018] In the above embodiments, the fluorescent reporter group is FAM and the quencher group is TAMRA.

[0019] In one embodiment, the kit also includes consumables required for microdroplet generation and detection.

[0020] In the above embodiments, the consumables include a microdroplet generation chip, a microdroplet detection chip, an 8-tube array, and corresponding sealing gaskets.

[0021] In one embodiment, a method for detecting *Gyrodactylus apis* using any of the kits described herein includes the following steps: (1) Sample preparation: Obtain bee samples to be tested, extract total RNA and reverse transcribe it into cDNA as a detection template; (2) Preparation of digital PCR reaction system: Mix the detection template with the primer pair, probe, hot-start DNA polymerase, dNTPs mixture and digital PCR reaction buffer to prepare the reaction system; (3) Microdroplet generation and PCR amplification: The reaction system prepared in step (2) is microdropletized to generate a large number of independent reaction units, and then PCR amplification reaction is performed. (4) Fluorescence detection and data analysis: Detect the fluorescence signal of each reaction unit after PCR amplification, and calculate the absolute copy number of the target gene of Coccidioides bees in the sample based on the number and proportion of positive microdroplets.

[0022] In one embodiment, the bee sample to be tested in step (1) is the intestinal tissue of a bee larva.

[0023] In one embodiment, the PCR amplification reaction procedure in step (3) is as follows: pre-denaturation at 95°C for 10 min; followed by 42 cycles of amplification, each cycle including denaturation at 94°C for 30 s, annealing and extension at 60°C for 45 s; and finally storage at 4°C.

[0024] In one embodiment, the total volume of the reaction system in step (2) is 30 μL, which includes: 6 μL of Probe dPCRHiTaqMix reagent A, 1.5 μL of reagent B, 5 μL of cDNA template, 1.5 μL each of upstream and downstream primers at a concentration of 10 pmol / μL, 1.5 μL of probe at a concentration of 10 pmol / μL, and the remainder is ddH2O.

[0025] In one embodiment, the target gene of the bee globosum is the ITS1 gene. Example 1:

[0026] To detect *Gynotrophomonas apiae*, follow these steps: I. Sample Preparation Artificial feed for bee larvae was prepared according to the following ratio: 63% royal jelly, 30% sterile water, 6% honey, and 1% yeast extract. Strong and healthy Italian bee colonies (without chalkbrood symptoms and negative PCR test results) were used as experimental colonies. Empty combs were marked, and the queen was limited to laying eggs for 12 hours. Five days later, combs were extracted from the experimental colony, and 2-day-old larvae were transferred using a transfer needle to 48-well cell culture plates containing the pre-prepared artificial feed. The culture plates were placed in a constant temperature and humidity incubator at 35±0.5℃ and 90% relative humidity (RH).

[0027] Formulate feed containing spores of *Gastromyxobolus apis*, with a final spore concentration of 1 × 10⁻⁶. 7 50 μL of the above spore-containing feed was placed in the wells of a 48-well cell culture plate for inoculation of 3-day-old larvae (n=32). The total amount of spores ingested by each larva was approximately 5 × 10⁶ spores / mL. 5 Each day thereafter, the feed was changed to normal artificial feed (feed without spores).

[0028] Using clean scissors and forceps in a laminar flow hood, the intestines of larvae infected with *Coccidioidomyces beeshuriensis* on day 4 (4 dpi), day 5 (5 dpi), and day 6 (6 dpi) were dissected. The fat bodies adhering to the intestines were carefully removed, and the samples were frozen in liquid nitrogen and then quickly transferred to a -80°C freezer for storage. (1) ≤10 mg of fresh or -80°C frozen tissue samples were transferred to a 1.5 mL centrifuge tube (RNase-free) or a grinding tube containing 300 μL of Buffer QLS lysis buffer. The samples were ground for 3-5 min with a grinding rod or tissue homogenizer until no obvious tissue precipitate was observed. If the homogenate was less than 300 μL, it was made up to 300 μL with Buffer QLS lysis buffer and vortexed to mix. The samples were fully lysed. (2) Centrifuged at 12,000 rpm and 4°C for 5 min. (3) The supernatant was carefully aspirated into a new 1.5 mL centrifuge tube (RNase-free).

[0029] The extracted total RNA (supernatant) was reverse transcribed into cDNA using the Hifair reverse transcription kit. ® The AdvanceFast1st Strand cDNA Synthesis Kit (Yisheng, Shanghai) (catalog number 11149ES60) was used to synthesize cDNA. The mixture shown in Table 1 was prepared in an RNase-free centrifuge tube. The mixture was gently pipetted and incubated at 42°C for 2 minutes to obtain the reaction solution. The reaction solution was then subjected to reverse transcription to obtain cDNA. The reverse transcription reaction system and procedure are shown in Tables 2 and 3.

[0030] Table 1 RNA template denaturation system

[0031] Table 2 Reverse transcription reaction system

[0032] Table 3 Reverse transcription reaction procedure

[0033] II. Preparation of Digital PCR Reaction System The forward primers, reverse primers, and specific probes were synthesized by Qingke (Beijing). The sequences of the primer pairs and probes are as follows: Forward primer: 5′-ATTGGCCCCCCTGTTATTTCC-3′ (SEQ ID NO:1) Reverse primer: 5′-CGATCGCCCAACACAACAG-3′ (SEQ ID NO:2) Probe: 5′-FAM-CGAGCGTCATTGCAACCCCTAAGC-TAMRA-3′ (SEQ ID NO:3) The PCR reaction system was prepared using the Xinyi (Beijing) Probe dPCR HiTaqMix according to the components and dosages in Table 4.

[0034] Table 4 Digital PCR Reaction System

[0035] III. Microdroplet Generation and PCR Amplification Add the reaction system to the droplet generation device (standard operating procedure): Start the sample preparation instrument and perform a self-test. After the self-test is complete, open the instrument cover, first place the 8-tube array into the corresponding position on the instrument, then insert the microdroplet generation chip into the matching chip generation mechanical clamp, press down the clamp cover to fix the chip, add 30 μL of the sample to be tested (PCR reaction system prepared in step two) into the water well, and add 180 μL of microdroplet generation oil into the oil well. Then cover the water well and oil well of the chip with the microdroplet generation chip sealing gasket. Place the clamp containing the microdroplet generation chip into the corresponding position on the instrument. Then press down the handle to fix it and close the instrument cover. Operate the instrument to generate microdroplets. After the microdroplets are generated, remove the clamp, and close the 8-tube array containing microdroplets with the 8-tube array cap, ready for use. Place it in the PCR amplification instrument for amplification, the amplification program is shown in Table 5.

[0036] Table 5 Reaction Procedure

[0037] IV. Fluorescence Detection and Data Analysis Place the 8-tube array containing microdroplets that have undergone PCR amplification into the biochip analyzer. Insert the microdroplet detection chip into the matching fixture. Add 430 μL and 500 μL of microdroplet detection oil to oil wells 1 and 2, respectively. Cover the microdroplet detection chip with the sealing gasket. Place the fixture with the microdroplet detection chip into the biochip analyzer and read the data using the analyzer's FAM channel.

[0038] Test results as follows Figure 1 As shown, on day 4 after infection with *Gnaphalium apiaceum*, the copy number was approximately 1566.6 copies; on day 6, the copy number increased to 52142.3 copies. The number of target molecules can be directly counted without relying on any calibrators or external standards.

[0039] 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 kit for detecting *Gyrodactylus apis* based on digital PCR, characterized in that: include: (a) Primer pair for specifically amplifying the ITS sequence of Coccidioides apis; the forward primer sequence of the primer pair is shown in SEQ ID NO:1 and the reverse primer sequence is shown in SEQ ID NO:2; (b) A probe for the specific detection of the amplification product; (c) Hot-start DNA polymerase; (d) dNTPs mixture; (e) Digital PCR reaction buffer.

2. The kit for detecting *Gyrodactylus apis* based on digital PCR according to claim 1, characterized in that: The sequence of the probe is shown in SEQ ID NO:

3. The 5′ end of the probe is labeled with a fluorescent reporter group, and the 3′ end is labeled with a quencher group.

3. The kit for detecting *Gyrodactylus apis* based on digital PCR according to claim 1, characterized in that: The fluorescent reporter group is FAM, and the quencher group is TAMRA.

4. A method for detecting *Gnaphalium apiaceum* using a kit as described in any one of claims 1-3, characterized in that: Includes the following steps: Sample preparation: Obtain bee samples to be tested, extract total RNA and reverse transcribe it into cDNA as a detection template; Digital PCR reaction system preparation: Mix the detection template with the primer pair, probe, hot-start DNA polymerase, dNTPs mixture and digital PCR reaction buffer to prepare the reaction system; Microdroplet generation and PCR amplification: The reaction system prepared in step (2) was microdropletized to generate a large number of independent reaction units, and then PCR amplification was performed. Fluorescence detection and data analysis: The fluorescence signal of each reaction unit after PCR amplification was detected, and the absolute copy number of the target gene of *Gastrocystis apis* in the sample was calculated based on the number and proportion of positive microdroplets.

5. The method according to claim 4, characterized in that: The bee sample to be tested in step (1) is the intestinal tissue of bee larvae.

6. The method according to claim 4, characterized in that: The PCR amplification reaction procedure in step (3) is as follows: pre-denaturation at 95℃ for 10 min; followed by 42 cycles of amplification, each cycle including denaturation at 94℃ for 30 s, annealing and extension at 60℃ for 45 s; and finally storage at 4℃.

7. The method according to claim 4, characterized in that: The total volume of the reaction system described in step (2) is 30 μL, which includes: 6 μL of Probe dPCR HiTaqMix reagent A, 1.5 μL of reagent B, 5 μL of cDNA template, 1.5 μL each of upstream and downstream primers at a concentration of 10 pmol / μL, 1.5 μL of probe at a concentration of 10 pmol / μL, and the remainder is ddH2O.

8. The method according to claim 4, characterized in that: The target gene of the bee globozobacter is the ITS1 gene.

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