Kit and method for detecting phenacoccus spongii RPA-CRISPR / Cas

Abstract

The invention discloses an RPA-CRISPR / Cas detection kit and method for phenacoccus solenopsis, the protein of Cas is cas12, and the kit comprises an RPA primer and crRNA (Complementary Ribonucleic Acid), and the RPA-CRISPR / Cas detection method for the phenacoccus solenopsis is characterized in that the protein of Cas is cas12. According to the on-site visual rapid detection kit and the detection method for the phenacoccus solenopsis of the CRISPR / Cas12a, reaction can be carried out under the constant temperature condition of 37-42 DEG C, complex instruments and equipment are not needed, and the CRISPR / Cas12a can recognize and cut a specific nucleic acid sequence, so that the specificity and the sensitivity of detection are further improved on the basis of RPA, the detection sensitivity is extremely high, and the kit and the method are suitable for being used for detecting the phenacoccus solenopsis of the CRISPR / Cas12a. The kit fills the blank of on-site high-sensitivity rapid detection of the phenacoccus solenopsis, and plays an important role in early discovery and invasion prevention and control of the phenacoccus solenopsis.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection and identification, and in particular to an RPA-CRISPR / Cas detection kit and method for Phenacoccus hibiscus. Background Art

[0002] The hibiscus mealybug (Phenacoccussolenopsis) belongs to the genus Phenacoccus, superfamily Coccoidea, family Pseudococcidae, subfamily Phenacoccinae, order Hemiptera, and is an important invasive pest originating from North America.

[0003] The female adults and nymphs of the hibiscus mealybug suck plant sap, mainly harming the tender parts of plants such as hibiscus and cotton, including young branches, leaves, flower buds and petioles. The affected plants grow weak, slow down or stop growing, and become dehydrated and dry, causing the buds, flowers and young bolls of the plants to fall off; it can cause the stems and leaves or even the entire plant to twist and deform, and in severe cases, it can cause the death of the host plant. The honeydew secreted by the hibiscus mealybug easily breeds black mold, which in turn induces sooty mold disease, affects the photosynthesis of the host plant, and inhibits the growth of the host plant or even kills it. At the same time, the honeydew will also summon invasive pests such as red fire ants, posing a threat to the health of humans and animals. The hibiscus mealybug has many host plants, including 207 species of plants in 57 families, 149 genera, and 57 families, such as cotton, tomatoes, corn, morning glory, and Xanthium. Data show that cotton production after being attacked by the hibiscus mealybug may be reduced by more than 40%, and some fields may even have no harvest.

[0004] The hibiscus mealybug has not only caused significant economic losses in China, but its damage has also spread to many countries and regions around the world.

[0005] It is particularly urgent to develop rapid detection technology for this pest so that timely control measures can be taken to reduce its impact on agriculture. Developing rapid detection technology for P. hibiscus that is not based on morphological characteristics, especially efficient identification technology suitable for on-site detection, is the basis for monitoring and early warning of P. hibiscus, and is also the basis for further prevention and control measures. Summary of the invention

[0006] One of the purposes of the present invention is to provide a RPA-CRISPR / Cas detection kit for Phenacoccus sangonis in view of the above problems, wherein the Cas protein is cas12, and the kit comprises an RPA primer and crRNA, wherein the RPA primer is a primer combination Ps1-F / Ps1-R, and the sequence is as follows:

[0007] Ps1-F: 5'-TTCATCATCAGATTTAATATTTCCACGATTAAAC-3',

[0008] Ps1-R: 5'-TGAAGATAGGCCGTTTAAATGAAGAGAG-3',

[0009] The crRNA is Ps-crRNA1, and its sequence is:

[0010] UAAUUUCUACUAAGUGUAGAUUACCCUCCUUUAAUUAAUCA.

[0011] Preferably, the kit further comprises a signal reporter molecule, and the sequence of the signal reporter molecule is: 5'-TTATTATT-3' or 5'-TTTTTTTTTT-3'.

[0012] Preferably, the kit further comprises an RPA amplification reagent, and the RPA amplification reagent comprises: RPA enzyme and MgOAc.

[0013] Preferably, the kit further comprises a CRISPR / Cas detection reagent, which comprises: cas12a protein, an RNAse inhibitor, and DTT, and the cas12a protein is selected from AsCas12a, Lb4Cas12a, Lb5Cas12a, FnCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a and BoCas12a.

[0014] Another object of the present invention is to provide a use of any of the above-mentioned kits in detecting Phenacoccus hibiscus.

[0015] The last object of the present invention is to provide an RPA-CRISPR / Cas detection method for Phenacoccus hibiscus, which is detected using any of the above-mentioned kits, comprising the following steps:

[0016] S1. Extract DNA from the sample to be tested;

[0017] S2.RPA amplification: preparing an RPA reaction system, amplifying the DNA of the sample to be tested obtained by the above extraction by the RPA method, and obtaining an amplified product;

[0018] S3.CRISPR / Cas system reaction detection: Take the above amplified product, add signal reporter molecules, cas12a protein and crRNA, perform CRISPR reaction detection, read the detection signal, and obtain.

[0019] The reaction system of RPA amplification includes rehydration buffer, ddH2O, upstream and downstream primers, RPA enzyme lyophilized powder, template DNA, and MgOAc; the reaction conditions of RPA amplification are: 37-42°C for 10-30 minutes;

[0020] The reaction system of the CRISPR / Cas system reaction includes: NE Buffer, LbaCas12a, RNAseInhibitor, DTT, signal reporter molecule and crRNA; the reaction conditions of the CRISPR / Cas system are: 37-42° C. for 5-30 min.

[0021] The components of the RPA amplification reaction system are prepared in the following proportions: 28.5-30.5 μL rehydration buffer, 10.5-12 μL ddH2O, 1-3 μL of 10 μM upstream and downstream primers, lyophilized RPA enzyme powder, 1-3 μL template DNA, and 1.5-3.5 μL MgOAc with a concentration of 280 μmol / L.

[0022] The ratio of each component of the reaction system of the CRISPR / Cas system reaction is prepared according to the following ratio: DEPC-H2O 10-12μL, NEBuffer 1.5-2.5μL, 5μM LbaCas12a0.8-1.2μL, 40U / μL RNAseInhibitor 0.4-0.6μL, 0.1M DTT 0.4-0.6μL, 10μM signal reporter molecule 1.5-2.5μL and 10μM crRNA 0.8-1.2μL.

[0023] Preferably, the ratio of each component of the RPA amplification reaction system is prepared according to the following ratio: 29.5 μL rehydration buffer, 11.2 μL ddH2O, 2.4 μL of 10 μM upstream and downstream primers, RPA enzyme lyophilized powder, 2 μL template DNA, 2.5 μL MgOAc with a concentration of 280 μmol / L; the reaction conditions are incubation at 39°C for 20 min;

[0024] The ratio of each component of the reaction system of the CRISPR / Cas system reaction is prepared according to the following ratio: DEPC-H2O 11μL, NEBuffer 2μL, 5μM LbaCas12a 1μL, 40U / μL RNAse Inhibitor 0.5μL, 0.1MDTT 0.5μL, 10μM signal reporter molecule 2μL and 10μM crRNA 1μL; the reaction conditions are incubation at 39°C for 30min.

[0025] The detection signal is read by using a real-time quantitative PCR instrument to read the fluorescence signal or by using a CRISPR / Cas test strip to determine the result.

[0026] The beneficial effects of the present invention are:

[0027] The present invention is aimed at Phenacoccus hibiscus, and develops a fast visual detection kit and a detection method for Phenacoccus hibiscus on-site based on recombinase polymerase amplification (RPA) technology combined with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated enzyme systems (Cas proteins) - CRISPR / Cas12a. The kit can react under a constant temperature of 37-42°C without complicated instruments and equipment. CRISPR / Cas12a can recognize and cut specific nucleic acid sequences, thereby further increasing the specificity and sensitivity of the detection on the basis of RPA. The kit has extremely high detection sensitivity, fills the gap of high-sensitivity and rapid detection of Phenacoccus hibiscus on-site, and plays an important role in the early discovery and invasion prevention and control of Phenacoccus hibiscus. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of specificity verification of primer pair Ps1-F / Ps1-R (A) and primer pair Ps2-F / Ps2-R (B): In Figures A and B, lanes 1-8 from left to right correspond to: D2000 Marker, Phenacoccus solenopsis, Phenacoccus madeirensis, Phenacoccus solani, Paracoccus marginatus, Planococcus citri, Planococcus ficus, and Planococcus lilacius.

[0029] Figure 2This is the specificity verification result of the RPA-CRISPR / Cas12a test strip for hibiscus mealybug: the samples in the figure are in order from left to right: Phenacoccussolenopsis, Phenacoccusmadeirensis, Phenacoccussolani, Paracoccusmarginatus, Planococcuscitri, Planococcusficus, and Planococcuslilacius.

[0030] Figure 3 These are the test results of the RPA-CRISPR / Cas12a detection systems established for Ps1-F / Ps1-R / Ps-crRNA1 (the test strip on the right in the picture) and Ps1-F / Ps1-R / Ps-crRNA2 (the test strip on the left in the picture).

[0031] Figure 4 This is the sensitivity test result of RPA primer reaction of Phenacoccus hibiscus: lanes 1-10 from left to right correspond to: D2000 Marker, Phenacoccus hibiscus DNA (103.7ng / μL), Phenacoccus hibiscus DNA (10.37ng / μL), Phenacoccus hibiscus DNA (1.037ng / μL), Phenacoccus hibiscus DNA (0.1ng / μL), Phenacoccus hibiscus DNA (0.01ng / μL), Phenacoccus hibiscus DNA (1pg / μL), Phenacoccus hibiscus DNA (0.1pg / μL), Phenacoccus hibiscus DNA (0.01pg / μL), Phenacoccus hibiscus DNA (1fg / μL).

[0032] Figure 5 The results of the sensitivity test of the RPA-CRISPR / Cas12a test strips for Phenacoccus hibiscus: The samples in the figure are, from left to right, 10 dilutions of the template DNA of Phenacoccus hibiscus in a 10-fold gradient. 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 8 concentrations.

[0033] Figure 2 , Figure 3 , Figure 5 In the test strip, the upper band on the test strip is the test band, and the lower band is the quality control band. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Example 1

[0037] 1. Experimental Materials

[0038] In this study, Phenacoccus hibiscus was used as the target species, and its closely related species and other economically important mealybug species were selected as control species (see Table 1). The COI gene sequences of the control species in Table 1 were searched in NCBI for the design of specific primers and specificity verification experiments.

[0039] Table 1

[0040]

[0041]

[0042] Among them, the hibiscus mealybug Phenacoccus solenopsis, the midi mealybug Phenacoccus madeirensis, the amaryllis mealybug Phenacoccus solani, the papaya mealybug Paracoccusmarginatus, the citrus mealybug Planococcus citri, Planococcusficus and the South China mealybug Planococcus lilacius used to extract DNA for verification experiments were samples collected by our laboratory. All mealybug samples were identified and confirmed by morphological methods and DNA barcode molecular identification methods before testing.

[0043] 2. DNA Extraction

[0044] DNA was extracted from the test mealybugs using a blood / cell / tissue genomic DNA extraction kit (DP304) (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the DNA was accurately quantified using a Nanodrop ultra-micro spectrophotometer (Thermo Fisher Scientific).

[0045] 3. Design of RPA primers and crRNA specific to Phenacoccus hibiscus

[0046] In order to ensure the specificity of primers and crRNA and the availability of Phenacoccus hibiscus, the COI gene (cytochrome C oxidase subunit I) sequences of all species in Table 1 were downloaded from GenBank. The gene sequences listed in Table 1 are different haplotypes of COI gene homologous sequences of various species. The different haplotype sequences of all species were used for the design of RPA primers and crRNA specific for Phenacoccus hibiscus. According to the SNP sites found by all sequence alignments and the sequence of GenBank sequence number KP981098 of Phenacoccus hibiscus in Table 1, RPA specific primers and crRNA were artificially designed, and the primers and crRNA were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0047] After multiple sequence alignment analysis, two pairs of P. hibiscus-specific RPA primers and two crRNAs were designed (Table 2).

[0048] Table 2

[0049]

[0050] *Note: In Table 2, the underlined target sequence binding region in the crRNA sequence and the bold marked part can bind to the cas12a protein.

[0051] IV. Establishment and specificity verification of RPA-CRISPR / Cas12a detection system

[0052] 1. Establishment of RPA-CRISPR / Cas12a detection system

[0053] Adopt TwistDX (UK) Basic RPA kit (Cat. No.: TABAS03KIT) was used for RPA reaction. The reaction system was referred to the kit manual. The specific ratio was as follows: 29.5 μL Rehydration buffer, 11.2 μL ddH2O, and 2.4 μL of upstream and downstream primers (10 μM) were added to the RPA enzyme lyophilized powder. After the dry powder was dissolved, 2 μL template DNA was added, and finally 2.5 μL MgOAc (280 μmol / L) was added. After mixing, the mixture was placed in a metal bath and incubated at 39°C for 20 min. The RPA reaction product was mixed with 50 μL Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1), centrifuged at 12,000 rpm for 5 min, and 5 μL of the supernatant was taken for electrophoresis to detect the RPA product.

[0054] The CRISPR / Cas12a detection system was developed by New England Biolabs (UK). LbaCas12a (Cpf1) nuclease and supporting reagents. Cas12 / 13-specific nucleic acid detection test strips (Beijing Baoying Tonghui Biotechnology Co., Ltd., China) were used for visual detection.

[0055] CRISPR / Cas12a detection system includes 11 μL DEPC-H2O, 2 μL NEBuffer, 1 μL LbaCas12a (Cpf1) (5 μM), 0.5 μL RNAse Inhibitor (RNAse inhibitor) (40U / μL), 0.5 μL DTT (0.1M), 2 μL signal reporter molecules (10 μM), 1 μL crRNA (10 μM), 2 μL RPA reaction products, mixed and put into a metal bath, 39 ° C incubation 30min. After the reaction is completed, 80 μL deionized water is added, and after mixing, the test strip sample end is immersed in the reaction solution and the band is observed. Wherein, the sequence of the signal reporter molecule is 5'-FAM-TTATTATT-Biotin-3' or 5'-FAM-TTTTTTTTTT-Biotin-3' (SEQ ID NO.7), and it is experimentally verified that these two signal reporter molecules can be applied to the present embodiment.

[0056] 2. Specificity detection

[0057] The established RPA reaction system was used to detect the test mealybugs and verify the reaction specificity. The results are as follows Figure 1 As shown in the figure, the results showed that only the target species Phenacoccus hibiscus had an amplified band when the primer pair Ps1-F / Ps1-R was used for RPA reaction ( Figure 1 A), and no amplified bands were found in other control species, indicating that the designed RPA-specific primer pair Ps1-F / Ps1-R for Psophora hibiscus had good specificity for detecting this species. The primer pair Ps2-F / Ps2-R was used for RPA reaction, and the amplified product was detected by electrophoresis. It was found that the primer pair had amplified bands for both Psophora hibiscus and Psophora bisporus ( Figure 1 B), indicating that the primer pair is not specific to Ps. hibiscus. Therefore, the primer pair Ps1-F / Ps1-R was selected for subsequent experiments.

[0058] The RPA-CRISPR / Cas12a detection system established using primer pair Ps1-F / Ps1-R and Ps-crRNA1 was used to detect the test mealybugs to verify the reaction specificity. The results are as follows Figure 2 As shown, the results showed that only the target species Phenacoccus hibiscus was detected with the target band, while no bands were detected in other control species, indicating that the specific primers and crRNA of Phenacoccus hibiscus had good specificity for the detection of this species.

[0059] 3. Sensitivity detection

[0060] (1) Comparison of different RPA-CRISPR / Cas12a detection systems

[0061] The RPA-CRISPR / Cas12a detection system established by Ps1-F / Ps1-R / Ps-crRNA1 and Ps1-F / Ps1-R / Ps-crRNA2 was used for detection. The template DNA concentration of Ps. hibiscus was 103.7 ng / μL. The test strip results showed that the CRISPR / Cas12a cutting reaction efficiency of Ps1-F / Ps1-R / Ps-crRNA2 was extremely low, and the detection band was weak ( Figure 3 Middle left), while the CRISPR / Cas12a cleavage reaction of Ps1-F / Ps1-R / Ps-crRNA1 is highly efficient and the detection bands are clear ( Figure 3 Therefore, the RPA-CRISPR / Cas12a detection system established by Ps1-F / Ps1-R / Ps-crRNA1 was subsequently used for sensitivity testing.

[0062] (2) Sensitivity of the RPA-CRISPR / Cas12a detection system established by Ps1-F / Ps1-R and Ps-crRNA1

[0063] The template DNA of Phenacoccus hibiscus (template concentration was 103.7 ng / μL) was diluted 10-fold, 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 A total of 9 concentrations were used to determine the detection sensitivity using the established RPA reaction system and RPA-CRISPR / Cas12a detection system. Each concentration gradient was repeated 3 times.

[0064] The sensitivity test results of the established Phenacoccus hibiscus RPA reaction system showed that the template DNA concentration was 103.7×10 -7 ng / μL, the target band of about 200bp can still be detected. The detection sensitivity of Ps1-F / Ps1-R can reach 0.01pg / μL ( Figure 4 ).

[0065] The sensitivity test results of the established RPA-CRISPR / Cas12a detection system showed that the template DNA concentration was 103.7×10 -7ng / μL, there is still a relatively clear detection band on the test strip. The detection sensitivity of Ps1-F / Ps1-R and Ps-crRNA1 can reach 0.01pg / μL ( Figure 5 ).

Claims

1. A RPA-CRISPR / Cas detection kit for Phenacoccus hibiscus, characterized in that: The Cas protein is cas12, and the kit includes RPA primers and crRNA. The RPA primers are primer combinations Ps1-F / Ps1-R, and the sequences are as follows: Ps1-F: 5'-TTCATCATCAGATTTAATATTTCCACGATTAAAC-3', Ps1-R: 5'-TGAAGATAGGCCGTTTAAATGAAGAGAG-3', The crRNA is Ps-crRNA1, and its sequence is: UAAUUUCUACUAAGUGUAGAUUACCCUCCUUUAAUUAAUCA.

2. The kit according to claim 1, characterized in that: The kit also includes a signal reporter molecule, and the sequence of the signal reporter molecule is: 5'-TTATTATT-3' or 5'-TTTTTTTTTT-3'.

3. The kit according to claim 1, characterized in that: The kit also includes an RPA amplification reagent, which includes: RPA enzyme and MgOAc.

4. The kit according to claim 1, characterized in that: The kit also includes a CRISPR / Cas detection reagent, which includes: cas12a protein, RNAse inhibitor, DTT, The cas12a protein is selected from AsCas12a, Lb4Cas12a, Lb5Cas12a, FnCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a and BoCas12a.

5. Use of the kit according to any one of claims 1 to 4 in detecting Phenacoccus hibiscus.

6. A RPA-CRISPR / Cas detection method for Phenacoccus hibiscus, characterized in that: Detection using the kit according to any one of claims 1 to 4 comprises the following steps: S1. Extract DNA from the sample to be tested; S2.RPA amplification: preparing an RPA reaction system, amplifying the DNA of the sample to be tested obtained by the above extraction by the RPA method, and obtaining an amplified product; S3. CRISPR / Cas system reaction detection: Take the above amplified product, add signal reporter molecules, cas12a protein and crRNA, perform CRISPR reaction detection, read the detection signal, and obtain.

7. The detection method according to claim 6, characterized in that: The reaction system of RPA amplification includes rehydration buffer, ddH2O, upstream and downstream primers, RPA enzyme lyophilized powder, template DNA, and MgOAc; the reaction conditions of RPA amplification are: 37-42°C for 10-30 minutes; The reaction system of the CRISPR / Cas system reaction includes: NE Buffer, LbaCas12a, RNAse Inhibitor, DTT, signal reporter molecule and crRNA; the reaction conditions of the CRISPR / Cas system are: 37-42° C. for 5-30 min.

8. The detection method according to claim 7, characterized in that: The components of the RPA amplification reaction system are prepared in the following proportions: 28.5-30.5 μL rehydration buffer, 10.5-12 μL ddH2O, 1-3 μL of 10 μM upstream and downstream primers, RPA enzyme lyophilized powder, 1-3 μL template DNA, and 1.5-3.5 μL MgOAc with a concentration of 280 μmol / L. The ratio of each component of the reaction system of the CRISPR / Cas system reaction is prepared according to the following proportions: DEPC-H2O 10-12 μL, NEBuffer 1.5-2.5 μL, 5 μM LbaCas12a 0.8-1.2 μL, 40 U / μL RNAse Inhibitor 0.4-0.6 μL, 0.1 M DTT 0.4-0.6 μL, 10 μM signal reporter molecule 1.5-2.5 μL and 10 μM crRNA 0.8-1.2 μL.

9. The detection method according to claim 8, characterized in that: The components of the RPA amplification reaction system were prepared in the following proportions: 29.5 μL rehydration buffer, 11.2 μL ddH2O, 2.4 μL of 10 μM upstream and downstream primers, lyophilized RPA enzyme powder, 2 μL template DNA, and 2.5 μL MgOAc with a concentration of 280 μmol / L; the reaction conditions were incubation at 39°C for 20 min; The ratio of each component of the reaction system of the CRISPR / Cas system reaction is prepared according to the following proportions: DEPC-H2O11μL, NEBuffer 2μL, 5μM LbaCas12a 1μL, 40U / μL RNAse Inhibitor 0.5μL, 0.1M DTT 0.5μL, 10μM signal reporter molecule 2μL and 10μM crRNA 1μL; the reaction conditions are incubation at 39°C for 30min.

10. The detection method according to claim 6, characterized in that: The detection signal is read by using a real-time quantitative PCR instrument to read the fluorescence signal or by using a CRISPR / Cas test strip to determine the result.

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