RPA-CRISPR / Cas12a-based colourless booklice visual detection and identification method and application thereof
By applying RPA-CRISPR/Cas12a technology in colorless lice detection, using specific RPA primer pairs and crRNA to combine Cas12a protein, the difficulty of detecting colorless lice in the prior art is solved, and rapid, accurate and specific detection is achieved, and the detection cost is reduced.
Patent Information
- Application Number
- CN202411948706.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to detect colorless lice quickly, accurately and specifically, and has problems such as high cost, cumbersome operation, high requirements for instruments and equipment, and prone to false positives.
Using a complete set of reagents based on RPA-CRISPR/Cas12a, including specific RPA primer pairs, crRNA and Cas12a proteins, the rapid detection of colorless lice is achieved through the binding of RPA amplification and the CRISPR/Cas12a system.
It realizes fast, accurate and specific detection of colorless book lice, reduces detection costs, simplifies the operation process, does not require special instruments, and is suitable for on-site inspection.
Smart Images
Figure BDA0005214330950000061 
Figure BDA0005214330950000071 
Figure BDA0005214330950000081
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for visually detecting and identifying colorless booklice based on RPA-CRISPR / Cas12a and an application thereof. Background Art
[0002] Booklice, belonging to the order Psocoptera, family Liposcelididae, genus Liposcelis, is a common storage pest worldwide. Booklice are tiny (body length 0.6-1.5mm) and widely distributed worldwide. Currently, 127 species are known, 27 of which have been reported in my country. Most species live indoors and are common pests of stored goods such as grain, Chinese medicinal materials, books, archives and biological specimens. Among them, the colorless booklice Liposcelis decolor is one of the most common worldwide storage booklice and has been distributed in my country.
[0003] While stored book lice feed directly on grain, their corpses, excrement, and shed skins can contaminate the grain, reduce the quality of stored grain, and pose a threat to national food security. When book lice occur in large numbers, the temperature and humidity of grain can rise, causing mold. In addition, book lice can carry and spread pathogenic microorganisms, and as potential allergens, they can cause skin allergies in humans, endangering human health. With the increasing frequency of international trade and passenger exchanges, the number of intercepted batches of stored book lice at my country's entry ports has gradually increased. In recent years, global economic integration, climate change, and biological evolution have further exacerbated biological invasions, and the prevention and control of invasions by foreign stored book lice has received more attention.
[0004] In the protection of stored grain and the prevention and control of invasion of foreign storage pests, the identification of storage pests is particularly important. The accuracy and timeliness of identification affect the formulation and implementation of prevention and control decisions. Traditional identification of book lice is mainly based on morphological characteristics, but due to their small individual size and similar morphological characteristics between species, species identification based on traditional morphology requires certain professional knowledge and skills. With the rapid development of molecular biotechnology, molecular identification technologies based on DNA barcodes, specific primer PCR, gene chips, and LAMP have been developed for book lice storage pests. However, due to the high cost, cumbersome operation, high requirements for instruments and equipment, easy false positives, and difficulty in achieving on-site instant detection, the above methods are relatively difficult to apply. Therefore, it is urgent to establish a method for colorless book lice that is low in cost, simple to operate, and can achieve rapid, accurate, and specific on-site detection. Summary of the invention
[0005] The technical problem to be solved by the invention is how to establish a method for colorless book lice with low cost, simple operation and the ability to achieve on-site rapid, accurate and specific detection.
[0006] In order to solve the above technical problems, the present invention first provides a set of reagents for identifying or assisting in identifying colorless book lice.
[0007] The reagent set provided by the present invention for identifying or assisting in identifying colorless book lice comprises an RPA primer pair;
[0008] The RPA primer pair consists of primer-F and primer-R;
[0009] The primer-F is as follows a1) or a2):
[0010] a1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing;
[0011] a2) a single-stranded DNA molecule having the same function as sequence 1 after one or more nucleotides are substituted and / or deleted and / or added;
[0012] The primer-R is as follows a3) or a4):
[0013] a3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing;
[0014] a4) A single-stranded DNA molecule having the same function as sequence 2 after one or several nucleotides are substituted and / or deleted and / or added.
[0015] In the above kit, the molar ratio of the primer-F to the primer-R may be 1:1.
[0016] The above-mentioned set of reagents also includes crRNA corresponding to the RPA primer pair; the crRNA is as follows a5) or a6):
[0017] a5) a single-stranded RNA molecule shown in Sequence 3 in the sequence listing;
[0018] a6) A single-stranded RNA molecule having the same function as sequence 3, wherein one or several nucleotides are substituted and / or deleted and / or added.
[0019] The above-mentioned set of reagents may also include Cas12a protein and reporter DNA.
[0020] The reporter DNA is a DNA molecule with a signal reporting function. When the DNA molecule is cut and degraded by the Cas12a protein, a positive signal can be reported and detected.
[0021] In some embodiments, the Cas12a protein is a LbCas12a protein.
[0022] In some embodiments, the reporter DNA is a single-stranded DNA with a FAM fluorescent group connected to the 5' end and a BHQ-1 quenching group connected to the 3' end. The nucleotide sequence of the single-stranded DNA is as follows: TTATT.
[0023] In order to solve the above technical problems, the present invention also provides new uses of the above set of reagents or the above RPA primer pair or the above crRNA.
[0024] The present invention provides the use of the above-mentioned set of reagents or the above-mentioned RPA primer pair or the above-mentioned crRNA in any one of the following b1)-b8):
[0025] b1) identifying or assisting in the identification of colorless book lice or their nucleic acids;
[0026] b2) preparing products for identifying or assisting in the identification of colorless book lice or their nucleic acids;
[0027] b3) detecting or assisting in detecting whether the sample to be tested contains colorless book lice or its nucleic acid;
[0028] b4) preparing products for detecting or assisting in detecting whether a sample to be tested contains colorless book lice or its nucleic acid;
[0029] b5) distinguishing or assisting in distinguishing colorless book lice from other book lice;
[0030] b6) preparing products that distinguish or assist in distinguishing colorless book lice from other book lice;
[0031] b7) To control or assist in the control of colorless book lice;
[0032] b8) preparing products for controlling or assisting in controlling colorless book lice.
[0033] In order to solve the above technical problems, the present invention also provides a kit for identifying or assisting in identifying colorless book lice;
[0034] The function of the kit is any one of the following c1)-c4):
[0035] c1) identifying or assisting in the identification of colorless book lice or their nucleic acids;
[0036] c2) detecting or assisting in detecting whether the sample to be tested contains colorless book lice or its nucleic acid;
[0037] c3) distinguish or assist in distinguishing colorless book lice from other book lice;
[0038] c4) To control or assist in the control of colourless book lice.
[0039] The kit for identifying or assisting in identifying colorless book lice provided by the invention comprises the above-mentioned complete set of reagents.
[0040] Furthermore, the kit also includes other reagents for identifying colorless book lice. The other reagents for identifying colorless book lice are: Primer Free Rehydration buffer, MgOAc, Cas12a, ssDNA fluorescent probe, 10×NEBuffer, and Ribonuclease RNase Inhibitor (RRI).
[0041] Furthermore, the kit also includes a negative control (such as sterile ultrapure water) and a positive control (such as colorless booklice genomic DNA), as well as a blue light and a yellow filter for observing the color development reaction results.
[0042] In order to solve the above technical problems, the present invention also provides a method for identifying or assisting in identifying whether a book louse to be detected is a colorless book louse.
[0043] The method for identifying or assisting in identifying whether the book louse to be tested is a colorless book louse provided by the present invention is as follows R1) or R2):
[0044] The R1) comprises the following steps: extracting nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the above RPA primer pair; after the RPA amplification reaction is completed, determining whether the booklice to be tested is a colorless booklice by detecting the amplification product; if the primer pair can amplify a band with a size of 100-250 bp, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is not a colorless booklice;
[0045] The R2) comprises the following steps: extracting the nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the above RPA primer pair; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the booklice to be tested is a colorless booklice: if the color development reaction system is green, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is not a colorless booklice.
[0046] In order to solve the above technical problems, the present invention also provides a method for detecting or assisting in detecting whether a sample to be tested contains colorless book lice.
[0047] The method for detecting or assisting in detecting whether a sample to be tested contains colorless booklice provided by the present invention is as follows S1) or S2):
[0048] The step S1) comprises the following steps: extracting nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing RPA amplification using the above RPA primer pair; after the RPA amplification reaction is completed, determining whether the sample to be tested contains colorless booklice by detecting the amplification product; if the primer pair can amplify a band with a size of 100-250 bp, the sample to be tested contains colorless booklice, otherwise the sample to be tested does not contain colorless booklice;
[0049] The step S2) comprises the following steps: extracting nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template and using the above RPA primer pair to perform RPA amplification; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the sample to be tested contains colorless booklice: if the color development reaction system is green, the sample to be tested contains colorless booklice, otherwise the sample to be tested does not contain colorless booklice.
[0050] In order to solve the above technical problems, the present invention finally provides a method for distinguishing or assisting in distinguishing colorless book lice from other book lice.
[0051] The method for distinguishing or assisting in distinguishing colorless book lice from other book lice provided by the present invention is as follows T1) or T2):
[0052] Said T1) comprises the following steps: extracting nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the above RPA primer pair; after the RPA amplification reaction is completed, judging whether the booklice to be tested is a colorless booklice or other booklice by detecting the amplified product; if the primer pair can amplify a band with a size of 100-250bp, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is other booklice;
[0053] The step T2) comprises the following steps: extracting nucleic acid of the booklice to be detected, using the nucleic acid of the booklice to be detected as a template, and performing RPA amplification using the above RPA primer pair; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the booklice to be detected is a colorless booklice or other booklice: if the color development reaction system is green, the booklice to be detected is a colorless booklice, otherwise the booklice to be detected is other booklice.
[0054] In any of the above methods, the band with a size of 100-250 bp may be a band with a size of 154 bp. The nucleotide sequence of the band with a size of 154 bp is shown in Sequence 4.
[0055] In any of the above methods, the total volume of the RPA amplification reaction system is 50 μL, consisting of 29.5 μL PrimerFree Rehydration buffer, 1 μL DNA template, 2.4 μL primer-F, 2.4 μL primer-R, 2.5 μL MgOAc and 12.2 μL sterile ultrapure water. The final concentrations of the primer-F and the primer-R in the RPA amplification reaction system are both 0.48 μM.
[0056] In any of the above methods, the total volume of the color reaction system is 25 μL, consisting of 2.5 μL 10×NEBuffer, Cas12a, crRNA, ssDNA fluorescent probe, 0.5 μL RNase inhibitor (40U / μL), 2 μL RPA reaction product and 8 μL sterile ultrapure water. The final concentration of Cas12a in the color reaction system is 200 nM; the final concentration of crRNA in the color reaction system is 1 μM; the final concentration of ssDNA fluorescent probe in the color reaction system is 800 nM.
[0057] In any of the above methods, the RPA amplification reaction conditions may be a constant temperature of 37-39° C. for 5-20 min, preferably a constant temperature of 37° C. for 10 min.
[0058] In any of the above methods, the color development reaction condition can be a constant temperature of 37° C. for 5-25 min, preferably a constant temperature of 37° C. for 15 min.
[0059] In any of the above methods, the booklice to be detected may be adult booklice to be detected.
[0060] In any of the above methods, the nucleic acid may be genomic DNA.
[0061] In any of the above-mentioned uses, kits or methods, the other booklice may be species with similar morphology to the colorless booklice, specifically, the entomophilous booklice, the curled booklice, the small-eyed booklice, the gnawing booklice, the dark brown booklice, the red booklice, and the false booklice.
[0062] In any of the above-mentioned uses, kits or methods, the colorless book lice may include colorless book lice from Chongqing, China, colorless book lice from Lijiang, Yunnan, China, and colorless book lice from Yantai, Shandong, China.
[0063] The present invention provides a pair of recombinase polymerase amplification (RPA) primer pairs capable of realizing rapid amplification of colorless booklice nucleic acid under constant temperature conditions and crRNA sequences that specifically recognize colorless booklice target regions, and also optimizes the color development reaction system and reaction conditions, and according to the characteristics that the ssDNA fluorescent probe is cut by Cas12a during the color development reaction, thereby changing from colorless and transparent to green, a colorless booklice visualization detection and identification method based on RPA-CRISPR / Cas12a is established. The colorless booklice visualization detection and identification method established by the present invention based on RPA-CRISPR / Cas12a has the advantages of being fast, efficient, highly specific, highly sensitive, easy to operate, and not requiring special instruments, and is low in cost, suitable for on-site detection, and easy to promote at the grassroots level, which can not only provide a reliable technical basis for the prevention and control of colorless booklice, but also has important significance for grain storage protection, prevention and control of alien biological invasion, and international trade. BRIEF DESCRIPTION OF THE DRAWINGS
[0064] Figure 1 The results of the RPA amplification product detection of primer-F / primer-R. Among them, M is DNA Marker; N is sterile ultrapure water; 1 is colorless book lice; 2 is red book lice; 3 is dark brown book lice; 4 is curled book lice.
[0065] Figure 2 The results of RPA specificity detection of primer-F / primer-R. Among them, M is DNA Marker; N is sterile ultrapure water; 1-3 are colorless book lice; 4 is entomophilous book lice; 5 is curled book lice; 6 is small-eyed book lice; 7 is gnawing book lice; 8 is dark brown book lice; 9 is red book lice; 10 is false book lice.
[0066] Figure 3 1 is the color development reaction system with the addition of colorless booklice DNA amplification product, and N is the color development reaction system with the addition of sterile ultrapure water.
[0067] Figure 4 The results of specific detection of colorless book lice RPA-CRISPR / Cas12a visualization system. Among them, 1-3 are colorless book lice; 4 is entomophilous book lice; 5 is curled book lice; 6 is small-eyed book lice; 7 is gnawing book lice; 8 is dark brown book lice; 9 is red book lice; 10 is false book lice; N is sterile ultrapure water.
[0068] Figure 5The sensitivity test results of the colorless book lice RPA-CRISPR / Cas12a visual detection and identification system. Among them, M is DNA Marker; N is sterile ultrapure water; the DNA template concentrations of 1-7 are: 100.0ng / μL, 10.0ng / μL, 1.0ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL. DETAILED DESCRIPTION
[0069] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0070] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0071] The quantitative tests in the following examples were all repeated three times, and the results were averaged.
[0072] Sources of reagents or kits in the following examples: Blood / cell / tissue genomic DNA extraction kit is a product of Tiangen. Primer Free Rehydration buffer and MgOAc are both from TwistAmp Basic Kit, and TwistAmp Basic Kit is a product of Twist Dx, a British company. Cas12a (LbCas12a) and 10×NEBuffer are both products of New England Biolabs. RNase inhibitor is a product of Acori Biotech.
[0073] The numbers, species names and collection location information of the booklice samples used in the following examples are shown in Table 1. All the booklice samples used in the tests were identified by morphological and DNA barcode molecular identification methods before testing.
[0074] In the following examples, each numbered sample includes samples from multiple corresponding collection locations. In specific experiments, multiple samples (at least three) are taken from each numbered sample for repeated experiments.
[0075] Table 1
[0076]
[0077] Example 1, RPA primer pairs, crRNA sequences and kits for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0078] 1. Design and validation of RPA primer pairs for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0079] 1. Design of RPA primer pairs for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0080] The present invention uses the NCBI website biological macromolecule sequence alignment search tool (Basic Local Alignment Search Tool, BLAST) to obtain the target region of the colorless book louse through a large number of sequence analyses, and designs and synthesizes the RPA primer pair for the target region. The primer sequences are shown in Table 2.
[0081] Table 2
[0082]
[0083] The amplification effect of each RPA primer pair was verified through preliminary experiments, and it was finally determined that the RPA primer pair with the best amplification effect consisted of primer-F shown in sequence 1 and primer-R shown in sequence 2.
[0084] 2. Validation of the effectiveness of RPA primer pairs for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0085] 1) Using a blood / cell / tissue genomic DNA extraction kit to extract genomic DNA from a booklice sample, thereby obtaining genomic DNA from the booklice sample.
[0086] The book lice samples were the colorless book lice numbered 1, the curled book lice numbered 5, the dark brown book lice numbered 8, and the red book lice numbered 9 in Table 1. The book lice samples were all in the adult stage.
[0087] 2) Using the genomic DNA of the booklice sample as a template, RPA amplification was performed using primer-F / primer-R to obtain RPA amplification products.
[0088] The total volume of the RPA reaction system is 50 μL, consisting of 29.5 μL Primer Free Rehydration buffer, 1 μL DNA template, 2.4 μL Primer-F (10 μM), 2.4 μL Primer-R (10 μM), 2.5 μL MgOAc, and 12.2 μL sterile ultrapure water. The final concentrations of primer-F and primer-R in the reaction system are both 0.48 μM.
[0089] RPA reaction conditions: constant temperature at 37°C for 10 min.
[0090] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.
[0091] 3) The RPA amplification product was subjected to 2% agarose gel electrophoresis.
[0092] The results are as follows Figure 1 As shown in the figure, M is DNA Marker; N is sterile ultrapure water; 1 is colorless book lice; 2 is red book lice; 3 is dark brown book lice; 4 is curled book lice.
[0093] The results showed that Primer-F / Primer-R only amplified a clear and bright band in colorless booklice (band size was 154 bp, nucleotide sequence was shown in Sequence 4), the amplification product size of Primer-F / Primer-R was between 100-250 bp, and Primer-F / Primer-R did not produce bands in non-colorless booklice and negative controls. It can be seen that the Primer-F / Primer-R of the present invention can effectively amplify the target fragment of colorless booklice.
[0094] 3. Specificity verification of RPA primer pairs for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0095] According to the method in step 2, the specificity of Primer-F / Primer-R was further verified using the tested booklice 1-10 as experimental materials.
[0096] The book louse 1 to be tested is a colorless book louse numbered 1.
[0097] The book louse 2 to be tested is the colorless book louse numbered 2.
[0098] The book louse 3 to be tested is the colorless book louse numbered 3.
[0099] The book louse 4 to be tested is the entomophilous book louse numbered 4.
[0100] The book louse 5 to be tested is the book louse No. 5.
[0101] The book louse 6 to be tested is the small-eyed book louse numbered 6.
[0102] The book louse 7 to be tested is the gnawing book louse numbered 7.
[0103] The book louse 8 to be tested is the dark brown book louse numbered 8.
[0104] The book louse 9 to be tested is the red book louse numbered 9.
[0105] The book louse 10 to be tested is the false book louse numbered 10.
[0106] The book lice samples to be tested were all in the adult stage.
[0107] The results are as follows Figure 2 As shown in the figure, M is DNA marker; N is sterile ultrapure water; 1-3 are colorless book lice; 4 is entomophilous book lice; 5 is curled book lice; 6 is small-eyed book lice; 7 is gnawing book lice; 8 is dark brown book lice; 9 is red book lice; 10 is false book lice.
[0108] The results showed that when the booklice to be tested were colorless booklice, Primer-F / Primer-R could amplify a clear, bright band; when the booklice to be tested were non-colorless booklice or negative controls, Primer-F / Primer-R did not produce any bands. This shows that Primer-F / Primer-R has good specificity in identifying colorless booklice or distinguishing different booklice species.
[0109] 2. Design and validation of crRNA for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0110] 1. Design of crRNA for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0111] The corresponding crRNA was designed and synthesized using the RPA amplification product sequence of primer-F / primer-R in colorless book lice as the target. The crRNA sequence is shown in Table 3.
[0112] Table 3
[0113]
[0114] The performance of each crRNA sequence was verified through preliminary experiments, and it was finally determined that the crRNA sequence with the best performance was the crRNA sequence shown in Sequence 3.
[0115] 2. Verification of the effectiveness of detecting crRNA of colorless booklice based on RPA-CRISPR / Cas12a
[0116] 1) Using a blood / cell / tissue sample genomic DNA extraction kit to extract genomic DNA of a booklice sample to be tested, to obtain genomic DNA of the booklice to be tested. The booklice sample to be tested is the colorless booklice numbered 1 in Table 1, and the insect stage of the booklice sample to be tested is an adult.
[0117] 2) Using the genomic DNA of the booklice to be tested as a template, RPA amplification is performed using primer-F / primer R to obtain an RPA amplification product.
[0118] The total volume of the RPA reaction system is 50 μL, consisting of 29.5 μL Primer Free Rehydration buffer, 1 μL DNA template, 2.4 μL Primer-F (10 μM), 2.4 μL Primer-R (10 μM), 2.5 μL MgOAc, and 12.2 μL sterile ultrapure water. The final concentrations of primer-F and primer-R in the reaction system are both 0.48 μM.
[0119] RPA reaction conditions: constant temperature at 37°C for 10 min.
[0120] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.
[0121] According to the above method, the template was replaced with the genomic DNA of the colorless booklice, and the other steps remained unchanged as a positive control.
[0122] 3) adding the RPA amplification product obtained in step 2) into a color development reaction system, and observing the color of the color development reaction system with the naked eye after the reaction.
[0123] The total volume of the color reaction system is 25 μL, consisting of 2.5 μL 10×NEBuffer, Cas12a, crRNA, ssDNA fluorescent probe (ssDNA fluorescent probe is synthesized by Qingke Biotechnology Co., Ltd., the sequence is as follows: FAM-TTATT-BHQ-1), 0.5 μL RNase inhibitor (40U / μL), 2 μL RPA amplification product and 8 μL sterile ultrapure water. The final concentration of Cas12a in the color reaction system is 200nM, the final concentration of crRNA in the color reaction system is 1 μM, and the final concentration of ssDNA fluorescent probe in the color reaction system is 800nM.
[0124] Color development reaction conditions: constant temperature at 37°C for 15 min.
[0125] 4) Under blue light, observe with the naked eye through a yellow filter, and then make the following judgment: if the reaction system with crRNA added is green and the reaction system of the negative control is colorless and transparent, crRNA can be used for visual identification of colorless book lice; if the reaction system with crRNA added is colorless and transparent, crRNA cannot be used for visual identification of colorless book lice. When the reaction system of the negative control is green, the test result is invalid.
[0126] The results are as follows Figure 3 As shown in FIG. 1, 1 is colorless book lice, and N is sterile ultrapure water.
[0127] The results show that when the sample is colorless booklice and Primer-F / Primer-R and its corresponding crRNA are added, the RPA-CRISPR / Cas12a visualization system appears green, and when the sample is a negative control, the RPA-CRISPR / Cas12a visualization system is colorless and transparent. It can be seen that the Primer-F / Primer-R and its corresponding crRNA designed by the present invention can successfully detect colorless booklice.
[0128] 3. Kit for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0129] The kit for detecting colorless booklice based on RPA-CRISPR / Cas12a of the present invention includes the primer-F / primer-R designed in the above step one and the reagents required for PRA amplification, the crRNA designed in step two and the reagents required for CRISPR / Cas12a detection.
[0130] Example 2: Specificity analysis experiment for detecting colorless booklice based on RPA-CRISPR / Cas12a
[0131] The book louse 1 to be tested is a colorless book louse numbered 1.
[0132] The book louse 2 to be tested is the colorless book louse numbered 2.
[0133] The book louse 3 to be tested is the colorless book louse numbered 3.
[0134] The book louse 4 to be tested is the entomophilous book louse numbered 4.
[0135] The book louse 5 to be tested is the book louse No. 5.
[0136] The book louse 6 to be tested is the small-eyed book louse numbered 6.
[0137] The book louse 7 to be tested is the gnawing book louse numbered 7.
[0138] The book louse 8 to be tested is the dark brown book louse numbered 8.
[0139] The book louse 9 to be tested is the red book louse numbered 9.
[0140] The book louse 10 to be tested is the false book louse numbered 10.
[0141] The book lice samples to be tested were all in the adult stage.
[0142] Each book louse to be tested performs the following steps:
[0143] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the booklice to be tested.
[0144] 2. Using the genomic DNA of the booklice to be tested as a template, use Primer-F / Primer-R to perform RPA amplification to obtain the RPA amplification product.
[0145] The total volume of the RPA reaction system is 50 μL, consisting of 29.5 μL Primer Free Rehydration buffer, 1 μL DNA template, 2.4 μL Primer-F (10 μM), 2.4 μL Primer-R (10 μM), 2.5 μL MgOAc, and 12.2 μL sterile ultrapure water. The final concentrations of primer-F and primer-R in the reaction system are both 0.48 μM.
[0146] RPA reaction conditions: constant temperature at 37°C for 10 min.
[0147] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.
[0148] According to the above method, the template was replaced with the genomic DNA of the colorless booklice, and the other steps remained unchanged as a positive control.
[0149] 3. Add the RPA amplification product obtained in step 2 to the color development reaction system, and observe the color of the color development reaction system with the naked eye after the reaction.
[0150] The total volume of the color reaction system is 25 μL, consisting of 2.5 μL 10×NEBuffer, Cas12a, crRNA, ssDNA fluorescent probe (ssDNA fluorescent probe is synthesized by Qingke Biotechnology Co., Ltd., the sequence is as follows: FAM-TTATT-BHQ-1), 0.5 μL RNase inhibitor (40U / μL), 2 μL RPA amplification product and 8 μL sterile ultrapure water. The final concentration of Cas12a in the color reaction system is 200nM, the final concentration of crRNA in the color reaction system is 1 μM, and the final concentration of ssDNA fluorescent probe in the color reaction system is 800nM.
[0151] Color development reaction conditions: constant temperature at 37°C for 15 min.
[0152] 4. Under the irradiation of blue light, observe with the naked eye through a yellow filter, and then make the following judgment: when the reaction system of the positive control is green and the reaction system of the negative control is colorless and transparent, if the reaction system of the book louse to be tested is green, then the book louse to be tested is or the candidate is a colorless book louse; if the reaction system of the book louse to be tested is colorless and transparent, then the book louse to be tested is not or the candidate is not a colorless book louse. When the reaction system of the negative control is green, the identification result of the book louse to be tested is invalid.
[0153] The results are as follows Figure 4Among them, 1-3 are colorless book lice; 4 is entomophilous book lice; 5 is curled book lice; 6 is small-eyed book lice; 7 is gnawing book lice; 8 is dark brown book lice; 9 is red book lice; 10 is false book lice; N is sterile ultrapure water.
[0154] The results showed that the RPA-CRISPR / Cas12a visualization system was green only when the sample was colorless booklice, and when the sample was non-colorless booklice and negative control, all RPA-CRISPR / Cas12a visualization systems were colorless and transparent. It can be seen that the colorless booklice visualization detection and identification method established by the present invention based on RPA-CRISPR / Cas12a has good specificity.
[0155] Example 3: Sensitivity analysis experiment of detecting colorless booklice based on RPA-CRISPR / Cas12a
[0156] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the colorless booklice (adult) numbered 1 in Table 1.
[0157] 2. Take the genomic DNA of colorless book lice and dilute it with sterile water to obtain the concentrations of colorless book lice genomic DNA as 100.0 ng / μL, 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 - 3 ng / μL, 1.0×10 -4 ng / μL dilution.
[0158] 3. Using the dilution obtained in step 2 as a template, respectively, perform RPA amplification using primer-F / primer-R to obtain an RPA amplification product.
[0159] The RPA reaction system is 50 μL, consisting of 29.5 μL Primer Free Rehydration buffer, 1 μL DNA template, 2.4 μL Primer-F (10 μM), 2.4 μL Primer-R (10 μM), 2.5 μL MgOAc, and 12.2 μL sterile ultrapure water. The final concentrations of primer-F and primer-R in the reaction system are both 0.48 μM.
[0160] RPA reaction conditions: constant temperature at 37°C for 10 min.
[0161] According to the above method, the template was replaced with sterile ultrapure water, and the other steps remained unchanged as a negative control.
[0162] 4. Add the RPA amplification product obtained in step 3 to the color development reaction system, and observe the color of the color development reaction system with the naked eye after the reaction.
[0163] The color reaction system is 25 μL, consisting of 2.5 μL 10×NEBuffer, Cas12a, crRNA, ssDNA fluorescent probe (ssDNA fluorescent probe is synthesized by Qingke Biotechnology Co., Ltd., the sequence is as follows: FAM-TTATT-BHQ-1), 0.5 μL RNase inhibitor (40U / μL), 2 μL RPA amplification product and 8 μL sterile ultrapure water. The final concentration of Cas12a in the color reaction system is 200nM, the final concentration of crRNA in the color reaction system is 1μM, and the final concentration of ssDNA fluorescent probe in the color reaction system is 800nM.
[0164] Reaction conditions: 37°C for 15 min.
[0165] 5. Under blue light, observe with the naked eye through a yellow filter, and then make the following judgments: If the visualization system appears green, it indicates that the corresponding DNA template content in the reaction system can be detected; if the visualization system does not appear green, it indicates that the corresponding DNA template content in the reaction system cannot be detected; if the negative control appears green, the experimental results are invalid.
[0166] The results are as follows Figure 5 As shown. Wherein, M is DNA Marker; N is sterile ultrapure water; the DNA template concentrations of 1-7 are: 100.0ng / μL, 10.0ng / μL, 1.0ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng / μL.
[0167] The results show that when the DNA template concentration is higher than or equal to 1.0 ng / μL, the RPA-CRISPR / Cas12a visualization system is green, indicating that the sensitivity of the colorless booklice visualization detection and identification method based on RPA-CRISPR / Cas12a of the present invention can reach 1.0 ng / μL. It can be seen that the colorless booklice visualization detection and identification method based on RPA-CRISPR / Cas12a of the present invention has a high sensitivity.
[0168] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. A set of reagents for identifying or assisting in identifying colorless book lice, the set of reagents comprising an RPA primer pair; The RPA primer pair consists of primer-F and primer-R; The primer-F is as follows a1) or a2): a1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; a2) a single-stranded DNA molecule having the same function as sequence 1 after one or more nucleotides are substituted and / or deleted and / or added; The primer-R is as follows a3) or a4): a3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; a4) A single-stranded DNA molecule having the same function as sequence 2 after one or several nucleotides are substituted and / or deleted and / or added.
2. The reagent set according to claim 1, characterized in that: The molar ratio of the primer-F to the primer-R is 1:
1.
3. The reagent set according to claim 1 or 2, characterized in that: The set of reagents also includes crRNA corresponding to the RPA primer pair; The crRNA is as follows a5) or a6): a5) a single-stranded RNA molecule shown in Sequence 3 in the sequence listing; a6) A single-stranded RNA molecule having the same function as sequence 3 after one or several nucleotides are substituted and / or deleted and / or added.
4. A reagent kit according to any one of claims 1 to 3, characterized in that: The kit also includes Cas12a protein and reporter DNA.
5. Use of the kit of reagents described in any one of claims 1 to 4, or the RPA primer pair described in claims 1 to 4, or the crRNA described in claims 1 to 4 in any one of the following b1) to b8): b1) identifying or assisting in the identification of colorless book lice or their nucleic acids; b2) preparing products for identifying or assisting in the identification of colorless book lice or their nucleic acids; b3) detecting or assisting in detecting whether the sample to be tested contains colorless book lice or its nucleic acid; b4) preparing products for detecting or assisting in detecting whether a sample to be tested contains colorless book lice or its nucleic acid; b5) distinguishing or assisting in distinguishing colorless book lice from other book lice; b6) preparing products that distinguish or assist in distinguishing colorless book lice from other book lice; b7) To control or assist in the control of colorless book lice; b8) preparing products for controlling or assisting in controlling colorless book lice.
6. A kit for identifying or assisting in the identification of colorless book lice, comprising a set of reagents as claimed in any one of claims 1 to 4; the function of the kit is any one of the following c1) to c4): c1) identifying or assisting in the identification of colorless book lice or their nucleic acids; c2) detecting or assisting in detecting whether the sample to be tested contains colorless book lice or its nucleic acid; c3) distinguish or assist in distinguishing colorless book lice from other book lice; c4) To control or assist in the control of colourless book lice.
7. A method for identifying or assisting in identifying whether a book louse to be tested is a colorless book louse, which is as follows R1) or R2): The R1) comprises the following steps: extracting nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, determining whether the booklice to be tested is a colorless booklice by detecting the amplification product; if the primer pair can amplify a band with a size of 100-250 bp, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is not a colorless booklice; The R2) comprises the following steps: extracting the nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the booklice to be tested is a colorless booklice: if the color development reaction system is green, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is not a colorless booklice.
8. A method for detecting or assisting in detecting whether a sample to be tested contains colorless book lice, comprising the following S1) or S2): Said S1) comprises the following steps: extracting nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, judging whether the sample to be tested contains colorless booklice by detecting the amplification product; if the primer pair can amplify a band with a size of 100-250bp, the sample to be tested contains colorless booklice, otherwise the sample to be tested does not contain colorless booklice; Said S2) comprises the following steps: extracting nucleic acid of the sample to be tested, using the nucleic acid of the sample to be tested as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system with the naked eye through a yellow filter under the irradiation of a blue light to determine whether the sample to be tested contains colorless booklice: if the color development reaction system is green, the sample to be tested contains colorless booklice, otherwise the sample to be tested does not contain colorless booklice.
9. A method for distinguishing or assisting in distinguishing colorless book lice from other book lice, comprising the following T1) or T2): Said T1) comprises the following steps: extracting nucleic acid of the booklice to be tested, using the nucleic acid of the booklice to be tested as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, determining whether the booklice to be tested is a colorless booklice or other booklice by detecting the amplified product; if the primer pair can amplify a band with a size of 100-250 bp, the booklice to be tested is a colorless booklice, otherwise the booklice to be tested is other booklice; Said T2) comprises the following steps: extracting nucleic acid of the booklice to be detected, using the nucleic acid of the booklice to be detected as a template, and performing RPA amplification using the RPA primer pair described in claims 1-4; after the RPA amplification reaction is completed, adding the amplification product to a color development reaction system, and observing the color of the color development reaction system through a yellow filter under the irradiation of a blue light to determine whether the booklice to be detected is a colorless booklice or other booklice: if the color development reaction system is green, the booklice to be detected is a colorless booklice, otherwise the booklice to be detected is other booklice.
10. The method according to any one of claims 7 to 9, characterized in that: The band with a size of 100-250 bp is a band with a size of 154 bp.