CN116479171A - Human rhinovirus B-type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12a - Google Patents
Human rhinovirus B-type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12a Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a human rhinovirus B-type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12a, which comprises an RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system; the RT-RPA isothermal amplification system comprises an RT-RPA primer pair designed based on human rhinovirus B nucleic acid, wherein the RT-RPA primer pair comprises a forward primer and a reverse primer; the CRISPR/Cas12a detection system includes crrnas designed based on human rhinovirus type B nucleic acids. The invention adopts CRISPR/Cas12a to detect the human rhinovirus B nucleic acid sequence for the first time, has the advantages of high sensitivity, strong specificity, short time consumption, no dependence on large-scale instruments and the like, and is suitable for meeting the on-site rapid high-sensitivity screening requirement of a basic medical structure on the human rhinovirus B.
Description
Technical Field
The invention relates to a detection system, in particular to a human rhinovirus B type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12 a.
Background
Human Rhinoviruses (HRV) were isolated by tissue culture from price in 1956, and HRV serotypes have been found to be more than 169, one of the most known serotypes of human. Human rhinovirus is a non-enveloped, single-stranded positive-strand RNA virus comprising 4 structural proteins (VP 1-VP 4) and 7 non-structural proteins, the genome being about 7200bp, belonging to the genus Enterovirus of the family Picornaviridae. Human rhinoviruses are considered to be the main causative viruses of respiratory viral infections in children, and can cause more than 50% of common colds, often in spring and autumn. In recent years, human rhinoviruses have also caused lower respiratory tract diseases such as pneumonia, bronchiolitis, asthma attacks, chronic obstructive pulmonary disease, and the like. HRV is one of the major pathogens responsible for acute respiratory infections in infants and children, with clinical symptoms mainly nasal obstruction, sneezing, headache, mild sore throat, fever. The virus has the advantages of rapid variation, low infection dosage, short incubation period after infection, long toxin expelling time, short immune protection time and various transmission ways, so that the human rhinovirus B has high infectivity and rapid transmission capability. Among them, type B is also a more common rhinovirus that causes common cold and other lower respiratory tract related diseases. Currently, there are no specific antiviral drugs and vaccines against human rhinovirus B, and non-pharmaceutical preventive measures are mainly adopted for the prevention and control thereof. Therefore, the method can effectively screen the pathogenic agents of patients in time, has important significance for early clinical diagnosis and guiding treatment, and is very critical to develop a rapid, accurate and portable pathogen detection technology.
The current methods for detecting human rhinovirus B mainly comprise a virus isolation culture method, an immunological method and a nucleic acid detection method. The virus separation culture method is complex in operation, long in time consumption, high in cost and not suitable for on-site rapid diagnosis; immunological methods are the most widespread technique at present, but because of the more serotypes of human rhinovirus B and the lack of common group antigens, antigen detection and serological detection cannot be used on a large scale; the most commonly used molecular diagnosis method for nucleic acid detection is RT-PCR technology, which is a gold standard for current nucleic acid detection, but the method requires expensive equipment and professionals, and generally requires 2-3 hours, so the method does not have the advantage of rapid and portable detection and cannot meet the key conditions of rapid detection on site. Cas proteins can target degradation of foreign target nucleic acids through RNA-guided, wherein the CRISPR-Cas9 protein family has been widely used in numerous fields of gene editing, antiviral agents, and biological imaging, CRISPR-Cas12a (Cpf 1) belongs to the fifth family of Casases for guiding RNA-guided double-stranded DNA cleavage of a single ruvC catalytic domain.
CRISPR/Cas12a enzymes recognize the spacer adjacent motif (PAM) rich in Thymine (T) nucleotides, catalyzing their own maturation of guide CRISPRRNA (crRNA) to Rloop, thereby releasing the active site of RuvC for Cas 12. When mature guide RNAs direct CRISPR/Cas12a proteins to cleave double-stranded DNA (dsDNA) in a sequence-specific manner, powerful nonspecific single-stranded DNA (ssDNA) trans-cleavage activity can be induced. Based on the above characteristics of CRISPR/Cas12a, we developed a rapid and accurate detection method for detecting human rhinovirus B detection in clinical specimens. Genomic DNA is extracted from a clinical sample to be examined and subjected to basic reverse transcription isothermal amplification (RT-RPA) under isothermal conditions. The CRISPR/Cas12a-crRNA complex binds to and cleaves target DNA, which activates the trans-cleavage activity of ssDNA, thereby cleaving the ssdnarometer fluorescent reporter, generating a fluorescent signal upon cleavage, which is collected by a fluorescence detector and presented in real time. The novel method provides a powerful platform for rapidly and accurately detecting the human rhinovirus B nucleic acid. At present, no report exists that CRISPR/Cas12a detection technology is applied to human rhinovirus B detection.
Disclosure of Invention
The invention mainly solves the technical problems existing in the prior art, thereby providing a human rhinovirus B-type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12a, and having the advantages of rapid detection, high sensitivity and high specificity.
The technical problems of the invention are mainly solved by the following technical proposal:
a human rhinovirus type-B detection system based on RT-RPA and CRISPR/Cas12a, comprising an independently packaged RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system; the RT-RPA isothermal amplification system comprises an RT-RPA primer pair designed based on human rhinovirus B-type nucleic acid, wherein the RT-RPA primer pair comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2; the CRISPR/Cas12a detection system comprises crRNA designed based on human rhinovirus B-type nucleic acid, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3.
Optionally, the RT-RPA isothermal amplification system also comprises nuclease-free water and buffer solution.
Optionally, the RT-RPA isothermal amplification system also comprises nuclease-free water, a buffer solution and magnesium ions.
Optionally, the RT-RPA isothermal amplification system comprises:
forward primer, concentration 10. Mu.M, volume 2. Mu.L;
reverse primer, concentration 10. Mu.M, volume 2. Mu.L;
2 Xbuffer, 25. Mu.L volume;
magnesium ion, concentration 280-300mM, volume 2-3. Mu.L;
nuclease-free water, 13.5-14.5. Mu.L in volume.
Alternatively, the crRNA comprises a set of single-stranded sequences of oligo DNA comprising the set of single-stranded sequences of oligo DNA required for the preparation of crrna2.1; the oligonucleotide DNA single-stranded sequence group comprises crRNA2.1-F and crRNA2.1-R, the nucleotide sequence of the crRNA2.1-F is shown as SEQ ID NO.4, and the nucleotide sequence of the crRNA2.1-R is shown as SEQ ID NO. 5.
Optionally, the CRISPR/Cas12a detection system further comprises a Cas12a protein, a ssDNA reporter molecule and a buffer solution.
Optionally, the CRISPR/Cas12a detection system further comprises Cas12a protein, ssDNA reporter, buffer and nuclease-free water.
Alternatively, the ssDNA reporter is 5'-F-TTATTATT-Q-3', wherein F is fluorescein and Q is a fluorescence quencher.
Alternatively, F is 6-carboxyfluorescein and Q is fluorescence quencher BHQ1.
Alternatively, the buffer is NEB2.1 buffer.
Optionally, the CRISPR/Cas12a detection system comprises:
NEB2.1 buffer, 5. Mu.L;
crRNA，50-1000nM，0.5-10μL；
ssDNA reporter, 1-2. Mu.M, 2. Mu.L;
cas12a protein, 1-2.5 μΜ,1-2 μΙ_;
no nuclease water, 26-37 mu L.
Optionally, the CRISPR/Cas12a detection system comprises:
NEB2.1 buffer, 5. Mu.L;
crRNA，1000nM，2.5μL；
ssDNA reporter, 1 μΜ,2 μΜ;
cas12a protein, 2.5 μΜ,2 μΙ_;
nuclease-free water, 33.5 μl.
Use of the human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a in the preparation of a product for detecting human rhinovirus type B nucleic acid.
A method for detecting human rhinovirus type B nucleic acid for non-disease diagnosis based on the human rhinovirus type B detection system of RT-RPA and CRISPR/Cas12a, comprising the steps of:
s1, adding RNA of a sample to be detected into an RT-RPA isothermal amplification system to carry out RT-RPA isothermal amplification to obtain an RT-RPA amplification product;
s2, adding the RT-RPA amplification product into a CRISPR/Cas12a detection system for reaction;
s3, detecting fluorescence change of the CRISPR/Cas12a detection system in the S2 in real time, and drawing a fluorescence curve; in the drawn fluorescence curve, judging the detection result that the fluorescence value rises in a curve along with the time extension as positive; the result that the fluorescence value is in a horizontal line along with the time extension is judged to be negative; .
Optionally, in the step S1, the isothermal amplification conditions of RT-RPA are: the reaction is carried out for 20-30min at 37-40 ℃.
Alternatively, the RT-RPA isothermal amplification conditions are: the reaction was carried out at 39℃for 20min.
Optionally, in the step S2, the reaction conditions are: the reaction is carried out for 20-30min at 37-40 ℃.
Alternatively, the reaction conditions are: the reaction was carried out at 37℃for 30min.
The human rhinovirus B-type nucleic acid rapid detection system based on RT-RPA and CRISPR/Cas12a adopts the technical scheme:
1. according to the invention, by combining a CRISPR/Cas12a detection system by using an RT-RPA technology, a targeting RNA specific amplification system based on RT-RPA and target DNA prepared by combining a CRISPR/Cas12a-crRNA complex with RT-RPA, the indiscriminate trans-DNA cutting activity of Cas12a protein is activated, the trans-cutting of a ssDNA reporter molecule is performed, and a fluorescent signal is generated after the ssDNA reporter molecule is cut, so that the high-sensitivity and high-specificity rapid detection of human rhinovirus B-type nucleic acid is realized, and the method has the advantages of short time consumption, simplicity in operation, no dependence on large experimental equipment and the like. The detection method of human rhinovirus B type is RT-qPCR, the detection limit is up to 1000 copies/. Mu.L, and the experiment shows that the sensitivity of the detection method established by the kit is higher than that of RT-qPCR, and the detection limit can be up to 0.5 copies/. Mu.L.
2. The method for detecting the human rhinovirus B type nucleic acid by utilizing the human rhinovirus B type detection system non-disease diagnosis based on the RT-RPA and the CRISPR/Cas12a provided by the invention can finish sample nucleic acid extraction and detection within 1h in the whole process under a constant temperature condition. The method has low requirements on equipment, can carry out the reaction by using simple constant temperature equipment, and is favorable for carrying out nucleic acid detection in areas with low economy.
3. The method for detecting the human rhinovirus B type nucleic acid by utilizing the human rhinovirus B type detection system non-disease diagnosis based on RT-RPA and CRISPR/Cas12a can observe results through fluorescence. When fluorescence detection is used, a DNA (ssDNA) reporting system in the CRISPR/Cas12a detection system is a ssDNA reporter molecule, and when the reaction is finished, the result can be observed through a fluorescence value. Is suitable for clinical and basic medical staff to develop quick and timely virus detection.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a result of sensitivity analysis of human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a in experimental example 1 of the present invention.
FIG. 2 is the result of a specific analysis of human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a in experimental example 1 of the present invention.
In the figure: HBoV: human bocavirus, hmPV: human metapneumovirus, HRV-B: human rhinovirus type B, ADV: adenovirus, RSV: human syncytial virus, HRV-Sup>A/C type: human rhinovirus type a and type C nucleic acid mixture, NC: negative control samples.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
In the invention, the non-nuclease water (RNase free water), the buffer solution (Rehydration Buffer) and the magnesium acetate (MgAc) in the RT-RPA isothermal amplification system come from Lesun biological company kit (Lesun, product number: LS-RNA 01); in vitro transcription Kit In vitro TranscriptionT Kit, available from TaKaRa company; the synthesis of RT-RPA primer pair and ssDNA reporter molecule is completed by Shanghai Biotechnology and general biological systems (Anhui) limited company respectively; the nucleic acid rapid extraction kit (magnetic bead method) of the Shuoshi biotechnology company is used for extracting and preparing human rhinovirus B-type clinical sample nucleic acid; the isothermal amplification instrument is Axixin ISO-T8.
pBluescript II SK + carrier, supplied by the division of bioengineering (Shanghai); the carrier bacteria TOP10, supplied by the division of bioengineering (Shanghai) Co.
Example 1
Preparation of human rhinovirus B-type standard DNA, RT-RPA primer pair and crRNA
The embodiment of the invention discovers that a segment of gene fragment (the cDNA sequence of which is shown as SEQ ID NO. 6) selected from human rhinovirus B-type conserved gene VP-2 is named as human rhinovirus B-type standard DNA through a large number of experiments, and the human rhinovirus B-type can be detected with high sensitivity and high specificity according to the RT-RPA primer pair and crRNA specifically designed by the human rhinovirus B-type standard DNA.
Nucleotide sequence of human rhinovirus type B standard DNA:
5’-CCGCCAATCAACTACGTAACAGCTAGTAGCATTTTGTTGTTGACTGGACGTTCGATCAGGTGGTTTACCCCCCCACTAGTTTGGTCGATGAGGCTGGAAATTCCCCACGGGTGACCGTGTTCCAGCCTGCGTGGCGGCCAACCCAGCTCATGCTGGGACGCCCTTTCAATGACATGGTGTGAAGACTCGCATGTGCTTGATTGTGAATCCTCCGGCCCCTGAATGCGGCTAACCCTAACCCCGGAGCCTTGCATCACAATCCAGTGATGTTAGGGTCGTAATGAGTAATTCTGGGATGGGACCGACTACTTTGGGTGTCCGTGTTTCTCATTTTTCTTTGAATTGTCTTATGGTCACAGCATATAGTAATATATACTGTGATCATGGGCGCTCAAGTCTCAACACAAAAGAGC-3’(SEQ ID NO.6)。
the designed RT-RPA primer pair for amplifying human rhinovirus B type standard RNA comprises a forward primer (RT-RPA-F) and a reverse primer (RT-RPA-R), and is as follows:
RT-RPA-F：5’-ATGTGCTTGRTTGWGANTCCTCCGGCCCCTGAATG-3’(SEQ ID NO.1)；
RT-RPA-R：5’-GTWGANACYTGHGCDCCCATGRTCACAGTATAT-3’(SEQ ID NO.2)。
searching for a targeting sequence comprising a CRISPR/Cas12a recognition sequence TTTN (N represents base A, T, C or G), based on a crRNA designed to target human rhinovirus type B standard DNA, the nucleotide sequence of the crRNA being shown as SEQ ID No.3, as follows:
5’-UAAUUUCUACUAAGUGUAGAUGAAACACGGACACCCAAAGU-3’(SEQ ID NO.3)。
example 2
Preparation of crRNA
The crRNA sequence is prepared by adopting an in vitro transcription mode, and is used for preparing an oligonucleotide single-stranded sequence group required by crRNA, wherein the crRNA2.1 is prepared by adopting the oligonucleotide single-stranded sequence group required by crRNA2.1-F and crRNA2.1-R. The set of oligo DNA single-stranded sequences comprises: t7 promoter sequence (1-26 nt) which is used for recognition and binding of T7RNA polymerase during in vitro transcription of crRNA; cas protein-bound anchor scaffold sequences (27-46 nt) that create a stable hairpin structure that aids in the formation of Cas12 a/crRNA/target sequence complexes; and a guide sequence (28-66 nt) complementary to the targeting sequence, wherein the fragment promotes the formation of a Cas12 a/crRNA/target sequence trisome complex through specific recognition and complementary pairing with the targeting sequence, further activates the trans ssDNA shearing activity of Cas12a, and realizes the accurate detection of human rhinovirus B.
Sets of artificially synthesized oligo DNA single-stranded sequences, as shown in crRNA2.1-F and crRNA2.1-R:
CrRNA2.1-F:
5’-GAAATTAATACGACTCACTATAGGGTAATTTCTACTAAGTGTAGATGGACACCCAAAGTAGTCGGT-3’(SEQ ID NO.4)；
CrRNA2.1-R:
5’-ACCGACTACTTTGGGTGTCCATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC-3’(SEQ ID NO.5)。
the oligo DNA single-stranded sequence set designed above was directly synthesized by Jin Weizhi company, annealed and passed through IVT kit (purchased from TaKaRa company) to obtain mature crRNA (shown as SEQ ID NO. 3).
Example 3
Human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a
The embodiment provides a human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a, which comprises an independently packaged RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system;
the RT-RPA isothermal amplification system comprises:
forward primer (shown as SEQ ID NO. 1) at a concentration of 10. Mu.M and a volume of 2. Mu.L;
reverse primer (shown as SEQ ID NO. 2), concentration 10. Mu.M, volume 2. Mu.L;
2 Xbuffer, 25. Mu.L volume;
magnesium ion (magnesium acetate), concentration 300mM, volume 3. Mu.L;
nuclease-free water, 13 μl in volume.
CRISPR/Cas12a detection system:
NEB2.1 buffer, 5. Mu.L;
crRNA (shown as SEQ ID NO. 3), 1000nM, 2.5. Mu.L;
ssDNA reporter, 1 μΜ,2 μΜ;
cas12a protein, 2.5 μΜ,2 μΙ_;
nuclease-free water, 33.5 μl.
The ssDNA reporter is 5'-F-TTATTATT-Q-3', wherein F is fluorescein, Q is fluorescence quencher, F is 6-carboxyfluorescein, and Q is fluorescence quencher BHQ1.
Example 4
Human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a
The embodiment provides a human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a, which comprises an independently packaged RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system;
the RT-RPA isothermal amplification system comprises:
forward primer (shown as SEQ ID NO. 1) at a concentration of 10. Mu.M and a volume of 2. Mu.L;
reverse primer (shown as SEQ ID NO. 2), concentration 10. Mu.M, volume 2. Mu.L;
2 Xbuffer, 25. Mu.L volume;
magnesium ion (magnesium acetate), concentration 300mM, volume 3. Mu.L;
nuclease-free water, 13 μl in volume.
CRISPR/Cas12a detection system:
NEB2.1 buffer, 5. Mu.L;
crRNA (shown as SEQ ID NO. 3), 1000nM, 2.5. Mu.L;
ssDNA reporter, 2 μΜ,2 μΙ_;
cas12a protein, 2.5 μΜ,2 μΙ_;
nuclease-free water, 33.5 μl.
The ssDNA reporter is 5'-F-TTATTATT-Q-3', wherein F is fluorescein, Q is fluorescence quencher, F is 6-carboxyfluorescein, and Q is fluorescence quencher BHQ1.
Example 5
Human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a
The embodiment provides a human rhinovirus B-type detection system based on RT-RPA and CRISPR/Cas12a, which comprises an independently packaged RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system;
the RT-RPA isothermal amplification system comprises:
forward primer (shown as SEQ ID NO. 1) at a concentration of 10. Mu.M and a volume of 2. Mu.L;
reverse primer (shown as SEQ ID NO. 2), concentration 10. Mu.M, volume 2. Mu.L;
2 Xbuffer, 25. Mu.L volume;
magnesium ion (magnesium acetate), concentration 300mM, volume 3. Mu.L;
nuclease-free water, 13 μl in volume.
CRISPR/Cas12a detection system:
NEB2.1 buffer, 5. Mu.L;
crRNA (shown as SEQ ID NO. 3), 1000. Mu.M, 2.5. Mu.L;
ssDNA reporter, 1 μΜ,2 μΜ;
cas12a protein, 2.5 μΜ,2 μΙ_;
nuclease-free water, 33.5 μl.
The ssDNA reporter is 5'-F-TTATTATT-Q-3', wherein F is fluorescein, Q is fluorescence quencher, F is 6-carboxyfluorescein, and Q is fluorescence quencher BHQ1.
Example 6
Method for detecting human rhinovirus B-type nucleic acid
This example provides a method for detecting human rhinovirus type B nucleic acid using the kit of example 3, comprising the steps of:
(1) Extraction of human rhinovirus B-type genome RNA
200 mu L of clinical tissue fluid is taken, and RNA of a sample to be detected is extracted according to a rapid nucleic acid extraction kit (magnetic bead method) (SDKF 60101) and a full-automatic nucleic acid extraction instrument (SSNP-3000A) provided by Jiangsu Shuo Shi biological company, and specific operation is referred to a kit product instruction book.
(2) Targeted nucleic acid RT-RPA amplification
And (3) adding the RNA (3-5 microliters) of the sample to be detected in the step (1) into an RT-RPA isothermal amplification system, and reacting at 39 ℃ for 20min.
(3) CRISPR/Cas12a system detection
Adding the RT-RPA amplification product (5 mu L) obtained in the step (2) into a CRISPR/Cas12a detection system, uniformly mixing, then placing the mixture at 37 ℃ for reaction for 30min, measuring fluorescence of the detection reaction by using an isothermal amplification instrument ISO-T8, monitoring fluorescence dynamics, wherein the excitation wavelength is 495nm, the emission wavelength is 520nm, the detection is carried out once every 20s, the detection is continued for 30min, and the fluorescence value detected for 15min is taken as a reaction value.
Example 7
Method for detecting human rhinovirus B-type nucleic acid
The present example differs from example 6 in the method of detecting human rhinovirus type B nucleic acid using the kit of example 4; in the step (2), RT-RPA isothermal amplification conditions are as follows: reacting at 39 ℃ for 20min; in the step (3), the mixture is evenly mixed and then is placed at 37 ℃ for reaction for 30min.
Comparative example 1A method for detecting human rhinovirus type B nucleic acid
This comparative example differs from example 6 in that the forward primer of the RT-RPA primer pair is 5'-ATGTGCTTGRTTGWGANTCCTCCGGCCCCTGAATG-3' (SEQ ID NO. 1) and the reverse primer 5'-GTWGANACYTGHGCDCCCATGRTCACAGTATAT-3' (SEQ ID NO. 2), the nucleotide sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUUCCUCCGGCCCCUGAAUGCG-3' (SEQ ID NO. 6) and the target sequence of human rhinovirus type B is TCCTCCGGCCCCTGAATGCG.
Comparative example 2A method for detecting human rhinovirus type B nucleic acid
This comparative example differs from example 6 in that the forward primer pair of RT-RPA primers is 5'-ATGTGCTTGRTTGWGANTCCTCCGGCCCCTGAATG-3' (SEQ ID NO. 1) and the reverse primer 5'-GTWGANACYTGHGCDCCCATGRTCACAGTATAT-3' (SEQ ID NO. 2), the nucleotide sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUACUUUGGGUGUCCGUGUUUC-3' (SEQ ID NO. 7) and the target sequence for human rhinovirus type B is ACTTTGGGTGTCCGTGTTTC.
Experimental example 1 sensitivity and specificity detection
Preparation of Standard template RNA
Human rhinovirus type B conserved nucleic acid sequence (SEQ ID NO. 6) was synthesized by Shanghai Biotechnology and constructed into pBluescriptIISK + The EcoRI site of the vector, designated pBlu-HRV-V, was then transformed into vector strain TOP 10; after culturing for 8 hours at 37℃in LB medium supplemented with 200. Mu.g/mL (final concentration) of ampicillin, plasmid extraction was performed according to the instructions of the plasmid miniprep kit (TIANPREPARIIPLASIMIDkit, available from TIANGEN company under the catalogue number DP 103) and the linearized plasmid was transcribed using the IVT in vitro transcription kit (Takara, daLiang.) to obtain human rhinovirus B standard RNA.
The concentration of the extracted human rhinovirus B-standard RNA was measured by an ultra-micro spectrophotometer (B-500 Biophotometer, shanghai Meta analysis instruments Co., ltd.).
1. Sensitivity detection
The standard template RNA of human rhinovirus B type is subjected to gradient dilution to prepare the RNA with the concentration of 1.0X10 3 COPIES/. Mu.l to 5.0X10 -2 A series of concentrations of standard template RNA, copies/. Mu.l, was used as template RNA (sample to be tested). Negative control: template RNA was replaced with sterilized double distilled water. Then, the sample was subjected to the procedure of example 6 (or comparative example 1 or comparative example 2) to give the result that the template concentration was 5.00X 10 as shown in FIG. 1 -1 When the fluorescence curve is still increased in the period of copies/. Mu.l, the sensitivity of the method for detecting the B type human rhinovirus can reach 5.00 multiplied by 10 -1 copies/μl。
While the results of comparative example 1 show that this bar of crRNA is not able to effectively activate Cas12a protein cleavage activity.
The results of comparative example 2 show that the crRNA sequence is not able to bind specifically to Cas12a protein as well as to the target, and thus is not able to activate Cas12a protein cleavage activity.
2. Specific detection
RNA of human rhinovirus B type, RNA of human metapneumovirus, RNA of human syncytial virus, adeno-associated diseaseToxic RNA, RNA mixture of human rhinovirus type A and C, genomic DNA of human bocavirus as template DNA (concentration 1.00X 10) 2 copies/. Mu.l). Then, the detection system of human rhinovirus type B was carried out according to example 6 (or comparative example 1 or comparative example 2), and the results are shown in FIG. 2, and the detection system of human rhinovirus type B has no cross reaction with other pathogens and has better specificity.
While the results of comparative example 1 show that the crRNA used has poor specific binding efficiency to the targeting molecule and Cas12a protein, resulting in the inability to activate the cleavage activity of Cas12a protein.
The results of comparative example 2 show that the crRNA used has low specific binding efficiency to the targeting molecule and Cas12a protein, and cannot effectively activate the indifferent cleavage activity of Cas12a protein.
Experimental example 2 nucleic acid detection of human rhinovirus type B clinical samples
In order to further evaluate the effects of the RT-RPA and CRISPR/Cas12 a-based human rhinovirus B-type detection system and detection method established in the invention, 24 suspected HRV clinical samples were subjected to nucleic acid rapid detection by using a nucleic acid rapid extraction kit (magnetic bead method) (SDKF 60101) and a full-automatic nucleic acid extractor (SSNP-3000A) provided by Jiangsu Shuo Shi biological company, respectively, according to example 6 and human rhinovirus B-type RT-qPCR.
Selecting 24 clinical samples for analysis, wherein 18 of the clinical samples are human rhinovirus B-type positive samples, and 6 of the clinical samples are human rhinovirus B-type negative samples; the detection results of the clinical samples are shown in table 1, 16 detection results in 18 positives are consistent with the fluorescent quantitative RT-qPCR detection, and 6 detection results of the negative samples are consistent with the RT-qPCR detection, namely, the consistency rates of the sample detection results and the RT-qPCR detection results of the detection method established by the invention are respectively 91.67%. (Table 1).
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any changes or substitutions that do not undergo the inventive effort should be construed as falling within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Claims (10)
1. Human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a, characterized in that: the detection system comprises an RT-RPA isothermal amplification system and a CRISPR/Cas12a detection system;
the RT-RPA isothermal amplification system comprises an RT-RPA primer pair designed based on human rhinovirus B nucleic acid, wherein the RT-RPA primer pair comprises a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2;
the CRISPR/Cas12a detection system comprises crRNA designed based on human rhinovirus B-type nucleic acid, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3.
2. The human rhinovirus type B detection system of claim 1 based on RT-RPA and CRISPR/Cas12a, characterized by: the RT-RPA isothermal amplification system also comprises nuclease-free water, a buffer solution and magnesium ions.
3. The human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a of claim 2, characterized by: the RT-RPA isothermal amplification system comprises:
forward primer, concentration 10. Mu.M, volume 2. Mu.L;
reverse primer, concentration 10. Mu.M, volume 2. Mu.L;
2 Xbuffer, 25. Mu.L volume;
magnesium ion, concentration 280-300mM, volume 2-3. Mu.L;
nuclease-free water, 13.5-14.5. Mu.L in volume.
4. A human rhinovirus type B detection system based on RT-RPA and CRISPR/Cas12a according to any one of claims 1-3, characterized in that: the crRNA comprises an oligo-DNA single-stranded sequence group, wherein the oligo-DNA single-stranded sequence group comprises an oligo-DNA single-stranded sequence group required for preparing crRNA 2.1; the oligonucleotide DNA single-stranded sequence group comprises crRNA2.1-F and crRNA2.1-R, the nucleotide sequence of the crRNA2.1-F is shown as SEQ ID NO.4, and the nucleotide sequence of the crRNA2.1-R is shown as SEQ ID NO. 5;
the CRISPR/Cas12a detection system also comprises Cas12a protein, ssDNA reporter molecules, buffer solution and nuclease-free water;
the ssDNA reporter is 5'-F-TTATTATT-Q-3', wherein F is fluorescein and Q is fluorescence quencher;
f is 6-carboxyfluorescein, Q is fluorescence quencher BHQ1;
the buffer solution is NEB2.1 buffer solution.
5. The human rhinovirus type B detection system of claim 4 based on RT-RPA and CRISPR/Cas12a, characterized by: the CRISPR/Cas12a detection system further comprises:
NEB2.1 buffer, 5. Mu.L;
crRNA，10-1000nM，1-10μL；
ssDNA reporter, 1-2. Mu.M, 2-4. Mu.L;
cas12a protein, 1-2.5 μΜ,1-4 μΙ_;
nuclease-free water, 26-37 μl;
the CRISPR/Cas12a detection system comprises:
NEB2.1 buffer, 5. Mu.L;
crRNA，1000nM，2.5μL；
ssDNA reporter, 1 μΜ,2 μΜ;
cas12a protein, 2.5 μΜ,2 μΙ_;
nuclease-free water, 33.5 μl.
6. Use of the RT-RPA and CRISPR/Cas12a based human rhinovirus type B detection system of any one of claims 1 to 5 for the preparation of a product for detecting human rhinovirus type B nucleic acid.
7. A method of detecting human rhinovirus type B nucleic acid for non-disease diagnosis based on RT-RPA and CRISPR/Cas12a based human rhinovirus type B detection system of any one of claims 1 to 5, characterized by comprising the steps of:
s1, adding RNA of a sample to be detected into an RT-RPA isothermal amplification system to carry out RT-RPA isothermal amplification to obtain an RT-RPA amplification product;
s2, adding the RT-RPA amplification product into a CRISPR/Cas12a detection system for reaction;
s3, detecting fluorescence change of the CRISPR/Cas12a detection system in the S2 in real time, and drawing a fluorescence curve; in the drawn fluorescence curve, judging the detection result that the fluorescence value rises in a curve along with the time extension as positive; and judging the result that the fluorescence value is in a horizontal line along with the time extension as negative.
8. The method for detecting human rhinovirus type B nucleic acid of claim 7 wherein in step S1, RT-RPA isothermal amplification conditions are: reacting for 20-30min at 37-40 ℃;
the RT-RPA isothermal amplification conditions are as follows: the reaction was carried out at 39℃for 20min.
9. The method for detecting human rhinovirus type B nucleic acid of claim 7 or 8, wherein the reaction condition is 37-40℃and the reaction time is 20-30min in the step S2.
10. The method of detecting human rhinovirus type B nucleic acid of claim 9 wherein the reaction conditions are: the reaction was carried out at 37℃for 30min.
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