CN113969281B - Modified CrRNA fragment and African swine fever virus kit - Google Patents
Modified CrRNA fragment and African swine fever virus kit Download PDFInfo
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Abstract
The invention discloses a modified CrRNA fragment and an African swine fever virus kit. The modified CrRNA segment is a DNA modified segment added to the 3' end of the CrRNA segment. The modified CrRNA fragment can further improve the reaction rate of a CRISPR/DX detection system, thereby shortening the detection time. The CRISPR/DX detection system adopts unique double-target matching, and two modified CrRNAs aiming at different targets are matched in one reaction system, so that the overall reaction rate can be improved. The kit for CcMFD African swine fever virus can be constructed after the CRISPR/DX detection system is combined with the RPA amplification system. Particularly, the cleavage efficiency of the LBcas12a protein can be improved, and the target specificity can be improved.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a modified CrRNA fragment and an African swine fever virus kit.
Background
African swine fever is a strong and highly-contact animal infectious disease caused by African Swine Fever Virus (ASFV), the death rate of pigs once infected can reach 100%, the world animal health Organization (OIE) classifies the swine infectious disease as an animal epidemic disease which needs to be reported, and China also classifies the African swine fever as a type of animal epidemic disease. The African swine fever virus genome is a double-linked closed linear DNA molecule with the size of about 170-194 kb, contains 150-160 Open Reading Frames (ORF) and totally encodes 54 structural proteins and more than 100 non-structural proteins. The African swine fever virus genome mainly comprises three parts, namely a hairpin loop structure at the tail end, a stable gene region in the middle part and a variable region consisting of a tandem repeat sequence and a multigene family, wherein the change of the copy number of the gene in the variable region is a main factor causing the unequal genome size. 24 genotypes have been identified based on the difference in nucleotides of about 500bp at the end of the gene encoding major capsid p72(B646L), but the genotypes do not reflect the serological properties of the virus, nor the immunological identity of the strain. The genetic evolution analysis of different virus genotypes by utilizing the PCR technology discovers that the epidemic is mainly genotype I in the west Africa region, and more than 20 genotypes are existed in east Africa and south Africa. Gene II epidemic in the southeast part of Africa, in 2007 to Grugia, Russia and eastern Europe; in 2017 the genotype was transmitted to the Russian Kurtz region. The current strain popular in China is genotype II, and belongs to the same branch with the strains popular in Russia and eastern Europe.
Because no effective vaccine against African swine fever virus is developed in the world at present, live pigs in epidemic sites can only be killed and treated harmlessly to prevent disease transmission, and huge economic loss is brought to the breeding industry. Therefore, under the current severe prevention and control situation, the research and development of a simple, rapid and accurate field detection technology for the African swine fever virus is urgent.
Disclosure of Invention
The invention aims to provide a modified CrRNA fragment and an African swine fever virus kit, which are used for solving one or more technical problems in the prior art and providing at least one beneficial selection or creation condition.
The first purpose of the invention is to provide a modified CrRNA fragment. The 3' end of the CrRNA fragment is provided with a DNA modified fragment with the length of 5-10 bp. The CrRNA fragment added with the DNA modified fragment can further improve the reaction rate in a CRISPR/DX detection system, thereby shortening the detection time.
Further, the length of the DNA modified fragment is 7 bp. Specifically, the DNA modified fragment is 5 '-TCCCCCCC-3' or 5 '-TATTATTATT-3'. After the modified fragment is added at the 3' end of the CrRNA fragment, the later-stage cutting efficiency of a CRISPR/DX detection system using the modified CrRNA fragment is improved compared with that of a CrRNA fragment without the modification.
Further, the modified CrRNA fragment is any one shown in table 1:
TABLE 1 CRISPR/DX assay systems with DNA-modified CrRNA
The second purpose of the invention is to provide a detection system, which comprises the CrRNA segment after the DNA modification segment. The detection efficiency of the improved detection system is improved.
Further, the detection system simultaneously uses two segments of the modified CrRNA segment, including a first CrRNA segment and a second CrRNA segment, wherein the sequence of the first CrRNA segment is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the sequence of the second CrRNA segment is shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8. The detection system using the two sections of modified CrRNA segments is paired through double targets, so that the reaction rate can be further accelerated, and the detection efficiency is improved.
Further, the detection system also comprises an upstream primer and a downstream primer. The upstream primer and the downstream primer are shown in Table 2:
TABLE 2 RPA amplification System primers
The third purpose of the invention is to provide a kit. The kit comprises the detection system.
Furthermore, the detection system also comprises reagents required by an RPA amplification system and a CRISPR/DX detection system, such as buffer A, buffer B, CRISPR-Cas12a protease, a fluorescence quenching probe, magnesium chloride, buffer or RNase inhibitor and the like.
Further, the reaction temperature of the CRISPR/DX detection system is 47 ℃. The reaction temperature of the existing CRISPR/DX detection system is generally 37 ℃ or 45 ℃, but the CRISPR/DX detection system provided by the invention can obviously improve the reaction rate under the reaction condition of 47 ℃.
Further, the magnesium ion concentration of the CRISPR/DX detection system is 17.5 mM. The fastest reaction rate can be achieved when the concentration of magnesium ions used by the existing CRISPR/DX detection system is 15 mM. Surprisingly, the CRISPR/DX detection system provided by the invention can obviously improve the reaction rate under the reaction condition that the magnesium ion concentration is 17.5mM, and has a detection effect far superior to that of the detection system using the magnesium ion concentration of 15 mM.
Further, the RPA amplification system adopts a 10 μ l reaction system, which is as follows:
buffer A5.9. mu.l; primer mix 0.8. mu.l (any pair of forward and reverse primers in Table 2); water 0-0.8 μ l; DNA sample 2-2.8. mu.l; buffer B0.5. mu.l; and mixing the RPA amplification system with a 20 mu l CRISPR/DX detection system, reacting at 47 ℃, and detecting the nucleic acid of the African swine fever virus according to the fluorescence intensity. And the lowest limit value of the detectable value of the reaction system is 500 copy/ml.
Furthermore, the detection system has lower requirement on the purity of the nucleic acid sample, and is beneficial to reducing the quality threshold of extracting the nucleic acid of the sample, thereby further shortening the whole detection time. The sample roughly processed by the nucleic acid extraction-free kit (the processing process is 5 minutes) can also be detected by using the detection system provided by the technology of the invention, and an accurate fluorescence signal is obtained.
The invention has the following beneficial effects:
the invention relates to an African swine fever virus rapid detection system based on CRISPR technology. The African swine fever virus is detected by using a specific primer group and a CRISPR technology, so that the detection time is shortened, and the detection can be completed within 20 min. In addition, the specific sequence combination is obtained by screening and is used as a primer group for detection, the detection condition is screened at the same time, the African swine fever virus is detected by the primer combination condition, the primer combination condition has the advantages of high sensitivity and strong specificity, the detection limit can reach 500copies/ml, and the clinical detection requirement is met.
Unexpectedly, the inventors found that the detection efficiency of a CRISPR/DX detection system can be remarkably improved by connecting an oligonucleotide fragment to the 3' end of CrRNA. Particularly, the cleavage efficiency of the LBcas12a protein can be improved, and the target specificity can be improved. The unique double-target matching is realized by matching two CrRNAs aiming at different targets in a reaction system, and combining the reaction system used by the invention, the concentration of magnesium ions is increased to 17.5mM, the reaction temperature is set to 47 ℃, the overall reaction rate can be obviously improved, and the detection time is ensured to be completed within 20 minutes.
The RPA amplification reaction system contained in the invention is reduced to 10 mul, which can reduce the detection cost while ensuring the detection limit value and shortening the detection time.
In order to further shorten the detection time, a nucleic acid surface extraction kit is adopted to process a sample (the total processing process only needs 5 minutes), and the detection system is used for detecting, so that the total time of nucleic acid extraction and detection is reduced to 25 minutes.
Drawings
FIG. 1 is a graph showing the results of measuring the length of the screening guide sequence in example 1;
FIG. 2 is a graph showing the results of examination of DNA modification sequences screened in example 2;
FIG. 3 is a graph showing the improvement effect of the DNA modification sequence on different guide sequence lengths in example 2;
FIG. 4 is a bar graph of optimized magnesium ion concentration for the CRISPR/DX assay system of example 3;
FIG. 5 is a bar graph of the optimized reaction temperature of the CRISPR/DX detection system of example 3;
FIG. 6 is a bar graph of optimized target pairing and paired concentrations for the CRISPR/DX assay system of example 3;
FIG. 7 is the results of the CcMFD "one pot" assay for samples at concentrations of 140 copy/. mu.l, 14 copy/. mu.l and 1.4 copy/. mu.l, respectively, in example 4;
FIG. 8 is the results of the CcMFD "one-pot" assay at a concentration of 0.5 copy/. mu.l in example 4;
FIG. 9 shows the results of CcMFD "one-pot" assay using samples AT concentrations of 140 copy/. mu.l and 1.4 copy/. mu.l, respectively, after replacing CrRNA (T5-20-AT and T8-23-AT) in example 4;
FIG. 10 is a graph showing the results of the CcMFD "one-pot" assay of blood samples in example 4;
FIG. 11 is a graph of the detection of 5 blood samples containing ASFV with CcMFD using the hands-free reagent in example 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 construction of CrRNA for detection of ASFV
A T5 site and a T8 site of a P72 gene of ASFV are taken as detection targets, guide sequences with the lengths of 23bp, 20bp and 17bp are respectively designed, and the guide sequences and an anchoring sequence aiming at Cas12a protein are combined into CrRNA, so that better cutting efficiency is obtained and off-target rate is reduced. The constructed CrRNA is shown in table 3:
TABLE 3 CrRNA sequences for testing
A CRISPR/DX assay system (20 μ l) was constructed for the detection of ASFV comprising: LBcas12a (1. mu.M) 1. mu.l; CrRNA (1. mu.M) 2. mu.l; probe (10. mu.M) 1. mu.l; RNase inhibitor 1. mu.l; buffer 22 μ l; plasmid 1. mu.l (7X 10)9copy/μl) ； water 10μl； MgCl 22. mu.l (17.5 mM); the reaction temperature was 47 ℃ and the reaction time was 20 minutes. The sequence of the fluorescent reporter molecule (probe) is 5 '-FAM-CCCCCC-BHQ 1-3'.
CrRNA sequences with different lengths are synthesized according to Table 3 to form different CRISPR/DX detection systems, and the detection results are shown in figure 1, wherein the black bars represent guide sequences aiming at the T5 site, and the white bars represent guide sequences aiming at the T8 site. No good reaction rate was obtained with CrRNA having a guide sequence length of 17bp at any site. As can be seen from the figure, the T5 site is optimally selected by using CrRNA with the guide sequence length of 20bp, and the corresponding CrRNA sequence is named as T5-20 (SEQ ID NO. 18); the T8 site was optimally selected using CrRNA with a guide sequence length of 23bp, and the corresponding CrRNA sequence was named T8-23 (SEQ ID NO. 22).
Example 2 CrRNA-DNA Structure optimization design
(2-1) selecting a T5 site of a P72 gene of ASFV, and verifying the influence of adding a DNA modified fragment at the 3' end of CrRNA on the cutting efficiency in a CRISPR/DX detection system. Constructing a detection system: LBcas12a (1. mu.M) 1. mu.l; CrRNA (1. mu.M) 2. mu.l; probe (10. mu.M) 1. mu.l; RNase inhibitor 1. mu.l; buffer 22 μ l; plasmid 0.5. mu.l (7X 10)9copy/ul) ； water 10.5μl； MgCl 22. mu.l (17.5 mM); the reaction temperature was 47 ℃ and the reaction time was 20 minutes. The CrRNA is based on an RNA sequence of a target T5 locus, T5-20 is used as No.1 CrRNA, and the sequences from No.2 CrRNA to No.8 CrRNA are respectively modified with 5 '-TATTATTATT-3', 5 '-AAATAAA-3', 5 '-TTTATTT-3', 5 '-TCCCGCC-3', 5 '-TGGGGGG-3', 5 '-TGGGCGG at the 3' end of T5-20G-3 ' or 5 ' -TCCCCCCC-3 '.
The results of the detection of the respective detection system samples are shown in FIG. 2, and in the same positive plasmid detection, higher Relative Fluorescence Units (RFU) were detected in the detection system samples using CrRNA No.2 and CrRNA No.8, compared with the basic T5-20.
(2-2) after preliminarily judging that the DNA modified fragments of 5 '-TATTATT-3' and 5 '-TCCCCCCC-3' can improve the cutting efficiency of CrRNA, in order to further verify whether the CrRNA has universality, T5-20 and T8-23 are used as seed sequences, and the DNA modified fragments are respectively added to verify the cutting efficiency. The test was carried out using the same detection system as in the aforementioned (2-1). T5-20 is named T5-20-AT (SEQ ID NO. 1) after adding 5 '-TATTATT-3' DNA modification segment; t5-20 was named T5-20-6C (SEQ ID NO. 3) after adding 5 '-TCCCCCCC-3' DNA modification fragment; t8-23 is named T8-23-AT (SEQ ID NO. 6) after adding 5 '-TATTATT-3' DNA modification segment; t8-23 was named T8-23-6C (SEQ ID NO. 8) after adding a 5 '-TCCCCCCC-3' DNA modification fragment.
The results are shown in FIG. 3, in which the black bars represent the sequences for the T5 site and the white bars represent the sequences for the T8 site. The CRISPR/DX detection system using T5-20-AT and T5-20-6C has higher RFU than that using T5-20, and the same effect appears in the detection systems of T8-23-AT and T8-23-6C. Therefore, it was determined that the addition of a specific DNA modification fragment to the 3' -end of CrRNA can actually improve the efficiency of CrRNA cleavage.
Example 3 Condition optimization of CRISPR/DX assay System
The reaction rate of the CRISPR/DX detection system is also related to a plurality of reaction conditions, including magnesium ion concentration, reaction temperature, target pairing and other factors, so that higher reaction rate can be obtained by adjusting the conditions in multiple aspects. Based on the CRISPR/DX detection system of (2-1) in example 2, the used CrRNA is T8-23, and the magnesium ion concentration, the reaction temperature and the selection of the target point are respectively optimized.
(3-1) optimization of magnesium ion concentration.
Magnesium chloride solutions with the concentrations of 17.5mM, 16.4mM, 15mM, 13mM and 10mM are respectively prepared and used for the CRISPR/DX detection system to detect the same ASFV positive plasmid, and the reaction temperature is determined as 37 ℃ commonly used for the CRISPR/DX detection system. As shown in FIG. 4, the RFU data of the assay system with a magnesium ion concentration of 17.5mM was optimized.
(3-2) optimization of reaction temperature.
The test was carried out using the same detection system as in the aforementioned (3-1). The reaction temperature of each detection system was 49 ℃, 47 ℃, 45 ℃ and 37 ℃. As shown in FIG. 5, the RFU data of the detection system with the reaction temperature of 47 ℃ is optimal.
(3-3) influence of double targets on reaction speed.
In order to verify and detect the difference of the cutting efficiency of a single target and a double target and the influence of the CrRNA concentration on the cutting efficiency, 4 groups of CrRNA samples are respectively prepared: 2 μ l of T5-23 (SEQ ID NO. 19) (labeled "T5" in FIG. 6); 2 μ l of T8-23 (labeled "T8" in FIG. 6); 2 μ l each of T5-23 and T8-23 (labeled "T5/T8 (2)" in FIG. 6); 1 μ l each of T5-23 and T8-23 (labeled "T5/T8 (1)" in FIG. 6).
Constructing a detection system: LBcas12a (1. mu.M) 1. mu.l; probe (10. mu.M) 1. mu.l; RNase inhibitor 1. mu.l; buffer 22 μ l; plasmid 0.5. mu.l (7X 10)9copy/ul) ； water 10.5μl； MgCl 22. mu.l (17.5 mM); the reaction temperature was 47 ℃ and the reaction time was 20 minutes.
The results are shown in FIG. 6, and the assay system labeled "T5/T8 (1)" measures 3.5X 104Above RFU, much higher than the rest of the group. Therefore, the CRISPR/DX detection system is proved to adopt double targets to effectively improve the detection speed.
Example 4 CcMFD (CRISPR/DX-crRNA Mutant Fluorescence Detect) detection of ASFV
The RPA amplification system (Ampu future RPA amplification kit (DNA)) is combined with the CRISPR/DX detection system obtained after optimization in the embodiment 3 to form CcMFD, and the detection can be completed within 20 minutes. The minimum detection concentration can reach 0.5copy/ul, which takes 17.5 minutes. When the nucleic acid concentration of the sample is 140copy/ul, only about 7 minutes is required to detect a signal.
(4-1) RPA amplification System (10. mu.l) comprising: buffer A5.9. mu.l; primer mix 0.8. mu.l (SEQ ID NO. 9/SEQ ID NO. 13); water 0-0.8 μ l; DNA sample 2-2.8. mu.l; buffer B0.5. mu.l; the reaction is carried out at room temperature.
CRISPR/DX assay system (20 μ l) comprising: LBcas12a (1. mu.M) 1. mu.l; CrRNA (1. mu.M) 1. mu.l + 1. mu.l (T5-20-6C and T8-23-6C); probe (10. mu.M) 1. mu.l; MgCl 22. mu.l (17.5 mM); 0.5 mu l of RNase inhibitor; buffer 22 μ l; water 11.5 μ l; the reaction temperature was 47 ℃.
FIG. 7 shows the results of the CcMFD "one-pot" assay for standards at concentrations of 140 copy/. mu.l, 14 copy/. mu.l, and 1.4 copy/. mu.l, respectively. FIG. 8 is the result of CcMFD "one-pot" assay for a standard at a concentration of 0.5 copy/. mu.l. Mu.l of the RPA amplification system was mixed with 20. mu.l of the CRISPR/DX detection system and incubated at 47 ℃ for 20 minutes. As is clear from FIG. 8, the lower limit of detection of the present detection technique can be achieved with a sample concentration of 0.5 copy/. mu.l.
(4-2) ASFV clinical blood sample measurement was performed using the CcMFD kit same as that in (4-1) above. The detection results are shown in fig. 10, wherein T1 and T7 are different samples, respectively, and it is known that T1 is a weak positive sample of ASFV and T7 is a strong positive sample of ASFV. As a result, as shown in FIG. 10, the single sample and the mixed sample both obtained accurate measurement results within 20 minutes.
(4-3) the same CcMFD kit as in (4-1) above was used and the effect was detected by replacing CrRNA with T5-20-AT and T8-23-AT. As shown in FIG. 9, when the concentration of the standard substance was 140copy/ul, a positive result was detected; when the concentration of the standard was 1.4 copy/ul, a weak positive was indicated.
Example 5 detection of ASFV by Using hands-free reagent in combination with CcMFD
The degree of match between the CcMFD technique described in example 4 and the hands-free nucleic acid technique was examined using a nucleic acid hands-free kit (Jifan Bionucleic acid face extraction System).
And (3) adding 100 mul of sample lysate B1 into 20 mul of blood sample containing ASFV, shaking, mixing uniformly, standing at room temperature for 5 minutes, adding 100 mul of sample lysate B2, shaking, mixing uniformly, and centrifuging at 10000rpm for 2 minutes. 100 μ l of the supernatant was placed in a new centrifuge tube as a test sample. The results are shown in FIG. 11, and a positive signal was obtained within 20 minutes for each of the 5 positive serum samples.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Shantou university
<120> modified CrRNA fragment and African swine fever virus kit
<130> 2021
<160> 22
<170> PatentIn version 3.5
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uaauuucuac uaaguguaga uguuguccca gucauauc 38
<210> 21
<211> 41
<212> RNA
<213> Artificial sequence
<400> 21
uaauuucuac uaaguguaga uguuguccca gucauauccg u 41
<210> 22
<211> 44
<212> RNA
<213> Artificial sequence
<400> 22
uaauuucuac uaaguguaga uguuguccca gucauauccg uugc 44
Claims (10)
1. A modified CrRNA fragment is characterized in that the sequence of the modified CrRNA fragment is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO. 8.
2. A modified CrRNA fragment is characterized in that the sequence of the modified CrRNA fragment is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5 or SEQ ID NO. 6.
3. An assay product comprising a CrRNA fragment, wherein the CrRNA fragment is the modified CrRNA fragment of any one of claims 1 to 2.
4. The assay product of claim 3, wherein the modified CrRNA fragment comprises a first CrRNA fragment having a sequence set forth in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, or SEQ ID No.4, and a second CrRNA fragment having a sequence set forth in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, or SEQ ID No. 8.
5. The assay product of claim 4, wherein the reaction temperature of the assay product is 47 ℃.
6. The assay product of claim 4, wherein the assay product has a magnesium ion concentration of 17.5 mM.
7. The assay product of any one of claims 3 to 6, further comprising an upstream primer and a downstream primer, wherein the upstream primer has a nucleotide sequence as set forth in any one of SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12, and the downstream primer has a nucleotide sequence as set forth in any one of SEQ ID No.13, SEQ ID No.14, SEQ ID No.15 or SEQ ID No. 16.
8. A kit comprising a modified CrRNA fragment according to any one of claims 1 to 2 or a detection product according to any one of claims 3 to 7.
9. The kit of claim 8, further comprising at least one of a CRISPR-Cas12a protease, a fluorescence quenching probe, magnesium chloride, buffer, or an rnase inhibitor.
10. A modified CrRNA fragment according to any one of claims 1 to 2, a test product according to any one of claims 3 to 7 or a kit according to any one of claims 8 to 9 for the detection of African swine fever virus (African swine fever Virus) (b) for non-disease diagnostic purposesAfrican swine fever virus) The use of (1).
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