A rapid detection primer set, kit and identification method for Jackbell's mealybug

Abstract

The present invention discloses a rapid detection primer set, a kit and an identification method for Jackbell's mealybugs. It relates to the field of molecular biology technology. It includes: JKB-RPA-F, JKB-RPA-R and provides related kits and identification methods. The present invention establishes a rapid detection system of rapid crude DNA extraction, RPA (recombinase polymerase isothermal amplification technology) combined with CRISPR-Cas12a. The system can complete the detection of target insects within 1.2 hours under a constant temperature of 37°C. It has the characteristics of short time, high sensitivity and strong specificity, and provides an efficient method for rapid on-site detection of Jackbell's mealybugs. Compared with traditional PCR technology and other rapid detection methods, it is more suitable for on-site detection, and suitable for promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and more particularly to a primer set, a kit and an identification method for rapid detection of Jackbell's mealybugs. Background Art

[0002] Traditional scale insect identification relies primarily on the morphological characteristics of adult female insects. Using morphology to identify species is tedious, time-consuming, and difficult, requiring experience to observe morphology. Furthermore, the morphological characteristics of scale insect eggs and nymphs are less distinct from those of closely related species, making identification challenging. Molecular identification techniques are not limited by insect structure or developmental stage and are accurate and rapid, making them widely used in insect identification. Current molecular detection techniques for Jackbell's mealybugs include conventional PCR and DNA barcoding.

[0003] However, molecular detection methods such as genomic DNA extraction and conventional PCR require large laboratory instruments, relatively complex detection procedures, long reaction times, and result determination requires the use of imaging systems or sequence comparisons, making them unsuitable for rapid on-site or field testing.

[0004] In summary, how to provide an efficient, accurate, simple and visual method for identifying Jackbell's mealybugs is an urgent problem that technicians in this field need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a primer set, a kit, and an identification method for the rapid detection of Jackbell's mealybugs. A rapid detection system combining rapid crude DNA extraction, RPA (recombinase polymerase isothermal amplification technology) and CRISPR-Cas12a has been established. This system can complete the detection of target insects within 1.2 hours at a constant temperature of 37°C. It has the characteristics of short detection time, high sensitivity, and strong specificity, providing an efficient method for the rapid on-site detection of Jackbell's mealybugs. Compared with traditional PCR technology and other rapid detection methods, it is more suitable for on-site detection and suitable for promotion and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A primer set for rapid detection of Jackbell's mealybugs, comprising: JKB-RPA-F and JKB-RPA-R, the specific nucleotide sequences of which are as follows:

[0008] JKB-RPA-F:

[0009] 5'-GCGCGTCGTCGGTTCGATATTTCCGTCGCCGACCA-3', as shown in SEQ ID NO. 1;

[0010] JKB-RPA-R:

[0011] 5'-CGCCGACTAGAGGCCGCCGCGATCACCGCTTCGCA-3', as shown in SEQ ID NO.2.

[0012] The present invention also provides a Jackbell's mealybug rapid detection kit, comprising the above primer set.

[0013] Preferably, crRNA1 or crRNA2 is also included, and the nucleotide sequence of crRNA1 or crRNA2 is as follows:

[0014] crRNA1: 5′-UAAUUUCUACUAAGUGUAGAUGCGAGUGCGCGUUCUUU UCUGGCC-3′, as shown in SEQ ID NO. 3.

[0015] crRNA2: 5′-UAAUUUCUACUAAGUGUAGAUGCACGUCUCCUGCAGAC CGACAC-3′, as shown in SEQ ID NO.4.

[0016] Preferably: it also includes a fluorescent reporter molecule or a lateral flow chromatography test strip reporter molecule;

[0017] The fluorescent reporter molecule is 5′-TTATT-3′, 5′-labeled with 6-FAM and 3′-labeled with BHQ1;

[0018] The reporter molecule of the lateral flow chromatography test strip is 5′-TTATT-3′, 5′ labeled with 6-FAM and 3′ labeled with Biotin.

[0019] Preferably, the method further comprises extraction buffer, test strip 1, washing buffer, rehydration buffer, MgOAc, RNase-free H2O, single tube enzyme dry powder and Cas12a;

[0020] Extraction buffer: Dissolve 2.42 g Tris base, 1.46 g NaCl, and 20 g PVP-40 in 900 mL of water. Add 0.73 g EDTA and 5 mL of 10% SDS. Adjust the pH to 8 with hydrochloric acid and make up to 1 L.

[0021] The preparation method of the test strip 1 is as follows:

[0022] Melt paraffin wax and dye on a hot plate at a mass ratio of 95:0.67. Pour the mixture into a disposable plastic Petri dish. Place filter paper in the wax until two-thirds of the paper is soaked. Place the wax-soaked filter paper on aluminum foil to harden. Draw a pencil line parallel to the edge of the wax on the uncovered portion of the filter paper. Cut the filter paper into test strips using a microtome.

[0023] Wash buffer: Dissolve 1.21 g Tris base in 900 mL water, adjust the pH to 8 with hydrochloric acid, and make up to 1 L with water.

[0024] The present invention also provides a method for rapid detection and identification of Jackbell's mealybugs, using the above primer set or any one of the above kits.

[0025] Preferably, the method comprises the following steps:

[0026] (1) Crude DNA extraction;

[0027] (2) Using the DNA obtained in step (1) as a template, isothermal amplification was performed using JKB-RPA-F and JKB-RPA-R to obtain RPA amplification products;

[0028] (3) using crRNA1 or crRNA2, adding a fluorescent reporter molecule or a lateral flow chromatography test strip reporter molecule and the RPA amplification product of step (2), and performing a cleavage reaction in the CRISPR-Cas12a system to obtain a cleavage product;

[0029] (4) Identify the cleavage products.

[0030] Preferably, the specific operation of step (1) is: adding extraction buffer to the sample to be tested, grinding, then immersing the test strip 1 in the grinding liquid, and then immersing the test strip 1 in the washing buffer to purify the DNA.

[0031] Preferred:

[0032] When crRNA1 is used in step (3) and a fluorescent reporter molecule is added, the specific operation of step (4) is as follows: the cleavage product of step (3) is developed under 365nm ultraviolet light; if the cleavage product shows white light under ultraviolet light, it indicates that the test sample is Jackbell's mealybug; if the cleavage product has no fluorescent brightness under ultraviolet light, it indicates that the test sample is not Jackbell's mealybug;

[0033] When step (3) utilizes crRNA2 and adds a lateral flow chromatography test strip reporter molecule, the specific operation of step (4) is: the cleavage product of step (3) is detected using a lateral flow test strip. If a red strip appears on the test line, it indicates that the test sample is Jackbell's mealybug; if no red strip appears on the test line, it indicates that the test sample is not Jackbell's mealybug.

[0034] The present invention also provides the use of the above primer set, any of the above kits, and any of the above detection methods in agricultural production.

[0035] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a rapid detection primer set, a kit and an identification method for Jackbell's mealybugs, and the technical effects achieved are:

[0036] (1) DNA can be extracted at room temperature without the use of a water bath, resulting in a short extraction time and high efficiency. The extraction buffer and wash buffer do not use toxic or hazardous reagents, do not require sterilization, and can be stored at room temperature (18-25°C) for at least 1 year.

[0037] (2) DNA amplification can be performed at room temperature without the need for large laboratory instruments such as PCR instruments.

[0038] (3) It has strong specificity and the results are visualized, eliminating the need for gel electrophoresis imaging to confirm the bands.

[0039] (4) The rapid crude DNA extraction method provided by the present invention greatly shortens the DNA extraction time, uses RPA technology to amplify the target sequence at a constant temperature, combines CRISPR-Cas12a technology to identify the enzyme-cleaved target sequence, and relies on fluorescent reporter molecules / lateral flow chromatography test strip reporter molecules to visualize the test results. The overall method achieves efficient, accurate, and visual identification compared to traditional methods, and is more suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0041] Figure 1 This is a fluorescence detection diagram of the RPA / CRISPR-Cas12a system provided by the present invention.

[0042] Figure 2 This is a fluorescence colorimetric detection diagram provided by the present invention.

[0043] Figure 3 This is a test strip method detection diagram provided by the present invention.

[0044] exist Figures 1 to 3In the table, No.1 to No.10 correspond to Jackbell's mealybug, Papaya mealybug, Lycoris mealybug, Hibiscus mealybug, New pineapple gray mealybug, Nanyang hip-striped mealybug, Ocean hip-striped mealybug, Citrus thorny mealybug, Citrus mealybug, Oldman mealybug, and W is the negative control. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments 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 are within the scope of protection of the present invention.

[0046] The embodiment of the invention discloses a primer set, a kit and an identification method for rapid detection of Jackbell's mealybugs.

[0047] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0048] Technical Principle: Test strips made of cellulose filter paper rapidly bind nucleic acids and retain them for a short period of time. Washing steps remove some contaminants. The nucleic acids are then eluted directly into an RPA amplification solution. After specific amplification by RPA, CRISPR-Cas12a is used to verify the specific target sequence. Detection is then performed using both fluorescence and lateral flow strips, visualizing the results.

[0049] Example 1

[0050] A method for rapid identification of Jackbell's mealybugs comprises the following steps:

[0051] (1) DNA crude extraction: Place the rapid crude extraction sample in a centrifuge tube at room temperature and add extraction buffer. The mass volume ratio of the rapid crude extraction sample to the extraction buffer is 1:99 g / mL. Use a grinding rod to break the sample to release DNA. Then immerse the test strip 1 until the nucleic acid binding area is completely soaked for 5 seconds to capture the nucleic acid. Then immerse the test strip in the washing buffer 5 times. Wipe the test strip on the edge of the washing tube to ensure that there are no large drops of washing buffer to remove some impurities and purify the DNA.

[0052] Extraction buffer: Dissolve 2.42 g Tris base, 1.46 g NaCl, and 20 g PVP-40 in 900 mL of water. Add 0.73 g EDTA and 5 mL of 10% SDS. Adjust the pH to 8 with hydrochloric acid and make up to 1 L.

[0053] Wash buffer: Dissolve 1.21 g Tris base in 900 mL of water, adjust the pH to 8 with hydrochloric acid, and dilute to 1 L with water.

[0054] The preparation method of the test strip 1 is:

[0055] Melt paraffin wax and blue candle dye at a mass ratio of 95:0.67 on a hot plate and pour into a disposable plastic Petri dish. Place cellulose filter paper in the wax, allowing the wax to soak two-thirds of the filter paper. Place the wax-soaked filter paper on aluminum foil to harden for 1 minute. Draw a pencil line parallel to the wax edge on the unwaxed filter paper, 6 mm from the wax edge. This will serve as the length of the nucleic acid-binding zone on the test strip. (Wear gloves, work on a clean surface, and use pencils, rulers, and scissors that are free of DNA and RNA contamination.) Cut the filter paper into 2 mm wide strips using a microtome.

[0056] (2) Using the DNA obtained in step (1) as a template, isothermal amplification was performed using RPA primers (including forward primer JKB-RPA-F, sequence 5'-GCGCGTCGTCGGTTCGATATTTCCGTCGCCGACCA-3', SEQ ID NO. 1; reverse primer JKB-RPA-R, sequence 5'-CGCCGACTAGAGGCCGCC GCGATCACCGCTTCGCA-3', SEQ ID NO. 2) to obtain an RPA amplification product.

[0057] The RPA amplification system and conditions are:

[0058] Buffer (rehydration buffer) 29.5μL, 10μM JKB-RPA-F 2.4μL, 10μM JKB-RPA-R 2.4μL, 280mM MgOAc 2.5μL, RNase-free H2O 13.2μL, mix well and add single tube enzyme dry powder ( The test strip with DNA captured in step (1) was washed 15 times in the above mixture and reacted at 37°C for 25 minutes.

[0059] (3) Using crRNA1 (5'-UAAUUUCUACUAAGUGUAGAUGCGAGUGCGC GUUCUUUUCUGGCC-3', SEQ ID NO.3), a fluorescent reporter molecule (sequence: 5'-TT ATT-3', 5' labeled with 6-FAM, 3' labeled with BHQ1, i.e., 5'(6-FAM)-TTATT-3'(BHQ1), and the RPA amplification product of step (2) was added to perform a cleavage reaction in the CRISPR-Cas12a system to obtain a cleavage product.

[0060] The system and conditions of the cracking reaction are:

[0061] 0.3 μL of 20 μM crRNA, 2.5 μL of 1 μM Cas12a, 2 μL of 10×NEBuffer, 1.5 μL of 4 μM fluorescent reporter molecule, 2 μL of RPA amplification product obtained in step (2), 11.7 μL of H2O, and reaction at 37°C for 30 minutes.

[0062] (4) The cleavage product of step (3) is developed under 365 nm ultraviolet light. If the cleavage product shows white light under ultraviolet light, it indicates that the test sample is Jackbell's mealybug. If the cleavage product has no fluorescent brightness under ultraviolet light, it indicates that the test sample is not Jackbell's mealybug.

[0063] Example 2

[0064] The difference from Example 1 is that

[0065] In step (3), crRNA1 is replaced with crRNA2 (5'-UAAUUUCUACUAAGUGUAGAUGCACGUCUCCUGCAGACCGACAC-3', SEQ ID NO. 4.) and the fluorescent reporter molecule is replaced with a lateral flow chromatography test strip reporter molecule (sequence: 5'-TTATT-3', 5' labeled 6-FAM, 3' labeled Biotin, i.e. 5'(6-FAM)-TTATT-3'(Biotin)).

[0066] Step (4) is: the lysis product of step (3) is tested with a lateral flow test strip ( Cas12 / 13 special nucleic acid detection test strips, Cat.No.JY0301) were used for testing. If a red stripe appeared on the test line, it indicated that the test sample was Jackbell's mealybug. If no red stripe appeared on the test line, it indicated that the test sample was not Jackbell's mealybug.

[0067] Technical effect verification:

[0068] The designed RPA-specific primers JKB-RPA-F and JKB-RPA-R were used to amplify Jackbell's mealybugs and other mealybugs (No. 1 to 10). The obtained RPA amplification products were cleaved in the CRISPR-Cas12a system and detected by CRISPR-Cas12a fluorescence using a real-time fluorescence quantitative instrument (ABIQuantStudio 6Pro). The results are as follows: Figure 1 shown.

[0069] The results showed that only the sample of Jackbell's mealybug had a fluorescent signal, while the other mealybugs had no obvious fluorescent signal, showing a negative result ( Figure 1 ).

[0070] The method of Example 1 was used to perform a color reaction on the test samples of Jackbell's mealybug and other mealybugs (No. 1-10) under 365nm ultraviolet conditions. The results are as follows: Figure 2 shown.

[0071] The results showed that only the sample of Jackbell's mealybug was milky white, while the other samples were transparent ( Figure 2 ).

[0072] The method of Example 2 was used to test Jackbell's mealybugs and other mealybugs (No. 1 to 10) using test strips. The results are as follows: Figure 3 shown.

[0073] The results showed that only Jackbell's mealybug had a positive detection band (T), while other mealybugs and blank controls had no detection band ( Figure 3 ).

[0074] The results of experiments 1 to 3 showed that the RPA primers and crRNA designed in the present invention are specific for detecting Jackbell's mealybugs.

[0075] Comparative Experiment 1

[0076] The differences between DNA extraction using a commercially available kit and crude DNA extraction using the present invention are temperature, time, and equipment. The differences and advantages are shown in Table 1:

[0077] Table 1 Different DNA extraction methods

[0078]

[0079] Results show that the method of this invention offers the advantage of being able to extract DNA outdoors at room temperature. Compared to traditional extraction methods, it significantly shortens the extraction time, requiring only 5 minutes. Furthermore, it reduces equipment requirements, eliminating the need for laboratory equipment such as water baths, centrifuges, and vortexers. This makes it more suitable for rapid DNA extraction in the field or on-site compared to traditional techniques.

[0080] Comparative Experiment 2

[0081] The differences between RPA / CRISPR-Cas12a and conventional PCR in identifying Jackbell's mealybugs lie in temperature, time, instrumentation, and result presentation. The differences and advantages are shown in Table 2:

[0082] Table 2 Different PCR methods

[0083]

[0084] The results showed that the advantage of the RPA / CRISPR-Cas12a method is that it does not require specialized thermal cycling equipment. A pair of primers can be used to exponentially amplify nucleic acids at 37°C. Combining it with the CRISPR-Cas12a detection system can greatly improve the amplification specificity and sensitivity. The detection results are concise, clear and visual, and gel electrophoresis is not required. It is simpler, faster and easier to operate.

[0085] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0086] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for rapid detection of Jackbell's mealybugs, characterized in that: include: The specific nucleotide sequences of JKB-RPA-F and JKB-RPA-R are as follows: JKB-RPA-F: 5'-GCGCGTCGTCGGTTCGATATTTCCGTCGCCGACCA-3', as shown in SEQ ID NO.1; JKB-RPA-R: 5'-CGCCGACTAGAGGCCGCCGCGATCACCGCTTCGCA-3', as shown in SEQ ID NO.

2.

2. A rapid detection kit for Jackbell's mealybugs, characterized in that: Comprising the primer set according to claim 1.

3. The kit according to claim 2, wherein Also included is crRNA1 or crRNA2, the nucleotide sequence of the crRNA1 or crRNA2 is as follows: crRNA1: 5′-UAAUUUCUACUAAGUGUAGAUGCGAGUGCGCGUUCUUUUCUGGCC-3′, as shown in SEQ ID NO. 3; crRNA2: 5′-UAAUUUCUACUAAGUGUAGAUGCACGUCUCCUGCAGACCGACAC-3′, as shown in SEQ ID NO.

4.

4. The kit according to claim 3, wherein Also included are fluorescent reporter molecules or lateral flow test strip reporter molecules; The fluorescent reporter molecule is 5′-TTATT-3′, labeled with 6-FAM at the 5′ end and BHQ1 at the 3′ end; The reporter molecule of the lateral flow chromatography test strip is 5′-TTATT-3′, the 5′ end of which is labeled with 6-FAM and the 3′ end of which is labeled with Biotin.

5. The kit according to claim 4, wherein Also includes extraction buffer, test strip 1, wash buffer, rehydration buffer, MgOAc, RNase-free H2O, single tube enzyme dry powder and Cas12a; The extraction buffer solution per 1 L is as follows: 2.42 g Tris base, 1.46 g NaCl and 20 g PVP-40 are dissolved in 900 mL of water, 0.73 g EDTA and 5 mL of 10% SDS are added, the pH value is adjusted to 8 with hydrochloric acid, and the volume is made up to 1 L; The preparation method of the test strip 1 is as follows: Melt paraffin wax and dye at a mass ratio of 95:0.67 on a hot plate and pour into a disposable plastic Petri dish. Place filter paper in the wax, allowing the wax to soak two-thirds of the filter paper. Then, place the wax-soaked filter paper on aluminum foil to harden. Draw a pencil line parallel to the wax edge on the uncoated filter paper. Cut the filter paper into test strips using a microtome. The washing buffer solution per 1 L is as follows: 1.21 g Tris base is dissolved in 900 mL of water, the pH value is adjusted to 8 with hydrochloric acid, and the volume is made up to 1 L with water.

6. A method for rapid detection of Jackbell's mealybugs, characterized in that: The DNA of the sample to be tested is detected using the primer set according to claim 1 or the kit according to any one of claims 2 to 5.

7. The method according to claim 6, wherein The steps include: (1) Crude extraction of DNA from the sample to be tested; (2) Using the DNA obtained in step (1) as a template, isothermal amplification was performed using JKB-RPA-F and JKB-RPA-R to obtain RPA amplification products; (3) Using crRNA1 or crRNA2, adding a fluorescent reporter molecule or a lateral flow chromatography test strip reporter molecule and the RPA amplification product of step (2), performing a cleavage reaction in the CRISPR-Cas12a system to obtain a cleavage product; (4) Identify the cleavage products.

8. The method according to claim 7, wherein The specific operation of step (1) is: adding extraction buffer to the sample to be tested, grinding, then immersing the test strip into the grinding solution, and then immersing the test strip into the washing buffer to purify the DNA.

9. The detection method according to claim 8, wherein: When crRNA1 is used in step (3) and a fluorescent reporter molecule is added, the specific operation of step (4) is as follows: the cleavage product of step (3) is developed under 365nm ultraviolet light. If the cleavage product shows white light under ultraviolet light, it indicates that the test sample is Jackbell's mealybug. If the cleavage product has no fluorescence brightness under ultraviolet light, it indicates that the test sample is not Jackbell's mealybug. When crRNA2 is used in step (3) and a lateral flow chromatography test strip reporter molecule is added, the specific operation of step (4) is: the cleavage product of step (3) is detected using a lateral flow chromatography test strip. If a red strip appears on the test line, it indicates that the test sample is Jackbell's mealybug; if no red strip appears on the test line, it indicates that the test sample is not Jackbell's mealybug.

10. Use of the primer set according to claim 1, the kit according to any one of claims 2 to 5, or the detection method according to any one of claims 6 to 9 in the rapid detection of Jackbell's mealybugs.

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