CN110257556A - A kind of kit for detecting nucleic acid of sexually transmitted disease infective pathogen - Google Patents
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Abstract
The invention discloses a kind of kit for detecting nucleic acid of sexually transmitted disease (Sexually transmitted diseases, STD) infective pathogen.The present invention studies to obtain the specific nucleic acid sequence site that can detect STD correlation cause of disease based on CRISPR/Cas12a, the express delivery of STD cause of disease, sensitive, special qualitative detection can be realized for the site, STD etiology nucleic acid detection method and detection kit are constructed based on the technology, both target unimolecule had been able to achieve precisely to detect, it can also realize multidigit point while detect, clinical detection excellent effect, has great importance for the detection and screening of STD cause of disease and application prospect.
Description
Technical field
The invention belongs to technical field of molecular biology.More particularly, to a kind of core of sexually transmitted disease infective pathogen
Acid detection kit.
Background technique
Sexually transmitted disease (Sexually Transmitted Disease, STD) is popular one in worldwide
The most common infectious disease of kind, it is one group using property contact as main mode of transmission that morbidity, prevalence are closely related with life style
Disease.The prevalence of sexually transmitted disease gradually shows Epidemic Scope and expands, suffers from age attenuating and severity in the latest 20 years
The situation of exacerbation, it has also become the severe public health problem that the whole mankind must face jointly.Syphilis, stranguria syndrome, genital herpes, point
8 kinds of sharp condyloma, chancroid, non-gonococcal urethritis, lymphogranuloma venereum and AIDS STD are listed in Chinese keypoint control
Venereal disease.STD can be caused by virus, bacterium and helminth, and common pathogen has several: human immunodeficiency virus (Human
Immunodeficiency Virus, HIV), chlamydia trachomatis (Chlamydia trachomatis, CT), spirochaeta pallida
(Treponema Pallidum, TP), NEISSERIA GONORRHOEAE (Neisseria gonorrhoeae, NG), toxoplasma gondii
(Toxoplasma Gondii, TG).Risk, developing low-cost, accurate, height are propagated to realize the early detection of STD and controlling it
Effect, the diagnostic method for rapidly detecting STD cause of disease are extremely important.
Conventional Pathogen test technology such as (1) is separately cultured technology: pathogen isolation culture, especially for the micro- life of cause of disease
Object is separately cultured with virus, is the goldstandard of the pathogen detection of early stage.But this method is separately cultured that time-consuming, cannot achieve
Testing result is obtained in short time rapidly, it is necessary to which height relies on testing laboratory's hardware and experiment operator condition, and uncomfortable
For not there is the detection of the pathogenic microorganism of maturation culture means and virus at present.(2) immunology detection: with anti-based on antigen-
The immune response of body identifies pathogenesis-related protein, detects from protein level to pathogen.There are detection sensitivities for this method
It is lower, and specificity is affected by environment etc., the window phase of detection is longer, is unable to satisfy diagnosis and treatment demand, is only applicable to primary dcreening operation
And the problems such as cannot function as the foundation made a definite diagnosis in time, can not identifying the different subtype of same class cause of disease.
The speed and accuracy of detection resistant gene can be improved in diagnostic method based on molecule, this is to hospital and community's ring
Infection control in border, prevent, treat it is meaningful.Molecular diagnosis method is mainly polymerase chain reaction (PCR) at present: including
Regular-PCR, ApoE gene, real-time fluorescence quantitative PCR, PCR-Sanger sequencing technologies, PCR- biochip technology
Deng.PCR is to detect from nucleic acid level to pathogen, and entire experiment needs complete for 1~2 hour.The major defect of this method
It is the real-time PCR and other a variety of corollary equipments for carrying out needing to rely on PCR instrument or valuableness when PCR detection, Yi Jizhuan
The PCR Lab of door and professional operator.PCR detection cannot achieve real-time test, the diagnosis of bed side and examine without special laboratory
The scene application of survey condition, thus be unable to satisfy base, user terminal, scene inspection demand.Meanwhile PCR detection may deposit
The false positive and insufficient sensitivity the problems such as.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect and deficiency of existing STD Pathogen test technology, research is obtained
A kind of STD etiology nucleic acid detection site based on CRISPR/Cas12a system utilizes CRISPR/Cas12a system for the site
The detection of nucleic acids for achievable STD cause of disease of uniting, specific good, high sensitivity, false positive are low.
The object of the present invention is to provide a kind of STD etiology nucleic acid detection site based on CRISPR/Cas12a system and
GRNA combination.
Another object of the present invention is to provide a kind of CRISPR/Cas12a detection system of STD pathogenic genes.
Still a further object of the present invention is to provide a kind of detection method of STD etiology nucleic acid based on CRISPR/Cas12a system.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The research of the invention finds that a kind of STD etiology nucleic acid based on CRISPR/Cas12a system detects target site, for
The site can carry out the detection of the STD etiology nucleic acid based on CRISPR/Cas12a system, detect specific good, high sensitivity.
Shown in the sequence such as SEQ ID NO.1-16 of the STD etiology nucleic acid detection target site is any.It can specific area
Divide the different types of STD cause of disease and includes the PAM sequence of Cas12a identification.
Application of the target site in terms of as STD etiology nucleic acid detection site simultaneously, and as being based on
Application in terms of the STD etiology nucleic acid detection site of CRISPR/Cas12a system, should all be within protection scope of the present invention.
Based on the studies above achievement, the present invention also provides a kind of Pathogen test sides STD based on CRISPR/Cas12a system
Method is to utilize the above-mentioned target site of CRISPR/Cas12a system detection.
Specifically CRISPR detection of nucleic acids is carried out using the gRNA of Cas12a albumen and the corresponding target site.
The design principle of the gRNA are as follows: when choosing gRNA targeting sequence, targeting sequence 5 ' end should have 5 '-TTTN-
3 ' sequences, and target and do not form stable secondary structure between sequence itself, targeting sequence and remaining sequence.
Scheme may be selected as preferred, the sequence of the gRNA such as SEQ ID NO.17-51 is any or appoints several shown.
The present invention also provides a kind of CRISPR/Cas12a detection system of STD etiology nucleic acid or kits simultaneously, including
Cas12a albumen and gRNA, the sequence of the gRNA correspond to any shown target site of SEQ ID NO.1-16.Preferably, institute
State gRNA sequence such as SEQ ID NO.17-51 it is any or appoint it is several shown in.
In addition, above-mentioned Cas12a albumen is with endonuclease activity and with the Cas12a egg of attached cleavage activity
It is white.Such as LbCas12a, SsCas12a, ScCas12a, FnCas12a, AsCas12a etc..
The sequence of the ScCas12a is as shown in SEQ ID NO.52, the sequence of the SsCas12a such as SEQ ID NO.53
It is shown, sequence reference Addgene pMAL-his-LbCpf1-EC (Plasmid#79008) of the LbCas12a,
Sequence of the sequence of FnCas12a referring to Addgene 6-His-MBP-TEV-FnCpf1 (Plasmid#90094), AsCas12a
Referring to Addgene AsCpf1-2NLS (Plasmid#102565).
The invention has the following advantages:
The research of the invention finds that a kind of STD etiology nucleic acid based on CRISPR/Cas12a system detects target site, for
The site can realize that STD etiology nucleic acid detects using CRISPR/Cas12a system, detect specific good, high sensitivity, can be
25-37 DEG C of realization at room temperature is highly sensitive, high-precision Molecular Detection, has preferably specificity and compatibility, testing cost
It is cheap, easy to operate, quick.Detection limit value can reach A Moer grades (10-18Mole/L), realize target Single Molecule Detection；Simultaneously
It is also able to achieve multidigit point while detecting, clinical detection excellent effect, the detection and screening for STD cause of disease have important meaning
Justice.
Detailed description of the invention
Fig. 1 is in embodiment 2 to the gRNA detection effect of the different target sites of human immunodeficiency virus (HIV).
Fig. 2 is in embodiment 2 to the gRNA detection effect of the different target sites of chlamydia trachomatis (CT).
Fig. 3 is in embodiment 2 to the gRNA detection effect of the different target sites of microspironema pallidum (TP).
Fig. 4 is in embodiment 2 to the gRNA detection effect of the different target sites of toxoplasma gondii (TG).
Fig. 5 is in embodiment 2 to the gRNA detection effect of the different target sites of NEISSERIA GONORRHOEAE (NG).
Fig. 6 is in embodiment 4 to the detection effect of the clinical sample of 10 NEISSERIA GONORRHOEAE positives.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.To those skilled in the art, other any without departing from Spirit Essence and original of the invention
Changes, modifications, substitutions, combinations, simplifications made by reason is lower, should be equivalent substitute mode, are included in protection of the invention
Within the scope of.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Unless otherwise indicated, the present invention uses immunology, biochemistry, chemistry, molecular biology, microbiology, thin
Born of the same parents' biology, genomics and recombinant DNA etc. are the conventional technical ability of this field.Referring to Pehanorm Brooker (Sambrook), not in
Odd (Fritsch) and the Germania base of a fruit this (Maniatis), " molecular cloning: laboratory manual " (MOLECULAR CLONING:A
LABORATORY MANUAL), the 2nd editor (1989)；" Current Protocols laboratory manual " (CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY) (F.M. Austria Su Beier (F.M.Ausubel) et al. editor, (1987))；" Enzymology method "
(METHODS IN ENZYMOLOGY) series (Academic Press Inc): " PCR2: practical approach " (PCR 2:A PRACTICAL
APPROACH) (M.J. McPpherson (M.J.MacPherson), B.D. Hei Musi (B.D.Hames) and Taylor G.R.
(G.R.Taylor) edit (1995)), Ha Luo (Harlow) and draw in (Lane) edit (1988) " antibody: laboratory manual "
(ANTIBODIES, A LABORATORY MANUAL), and " animal cell culture " (ANIMAL CELL CULTURE) (R.I.
Fu Leixieni (R.I.Freshney) edits (1987)).
The discovery of STD etiology nucleic acid detection site of the embodiment 1 based on CRISPR/Cas12a system
We obtain STD pathogenic genes group sequence, are compared by bioinformatic analysis, find each plant type of STD cause of disease
Specificity identification region.The specific operation method is as follows, in the whole genome sequence of this cause of disease of NCBI nucleic acid sequence library lookup, obtains
Existing all whole genome sequences of this cause of disease are simultaneously filtered out according to sequence integrity degree with reference to genome；To said gene group sequence
Column carry out homology analysis, find the cause of disease and guard between different genes group sequence but relative to human genome (hg19) specificity
Target gene sequence.As unit of 20bp base, the sequence containing " TTTN " is searched in above-mentioned sequence and exports related sequence
Arrange the alternate data library as gRNA targeting sequence.The STD common infection cause of disease that we screen in this patent has: human immunity
Defective virus (Human Immunodeficiency Virus, HIV), chlamydia trachomatis (Chlamydia trachomatis,
CT), NEISSERIA GONORRHOEAE (Neisseria gonorrhoeae, NG), spirochaeta pallida (Treponema Pallidum, TP), just
Ground toxoplasma (Toxoplasma Gondii).By assessing the different type difference of related cause of disease target spot, each alternative gRNA sequence
Specificity between different strains, the parameters in series such as G/C content, base homogeneity, the sequence conservation of alternative gRNA sequence, and
By largely testing, designs gRNA for different zones and construct CRISPR/Cas12a system and studied, finally confirmed
To the special identification target of STD cause of disease, sequence is as shown in SEQ ID NO.1-16.With sequence shown in SEQ ID NO.17-51
GRNA is detected for the STD etiology nucleic acid based on CRISPR/Cas12a system, gained detection scheme all has good detection effect
(table 1).
The target gene referred in 1 this patent of table and corresponding gRNA target sequence
Embodiment 2 detects case based on the STD etiology nucleic acid of CRISPR/Cas12a system
1, CRISPR/Cas12a gene cloning and protein expression
Using the Cas12a protein gene for being originated from Lachnospiraceae bacterium, codon optimization makes base
Because being more suitable for expressing in mammalian cells.Cas12a protein gene cloning after optimization enters with 6-His histidine tag
PET28a plasmid, facilitates protein purification to express.The conversion of Cas12a Protein reconstitution expression vector, expression bacterium use BL21star
(DE3)。
Specific protein expression condition are as follows: in culture bacterium solution OD6000.5mMIPTG is added when=0.6 to cultivate 4 hours.Collect bacterium
Body carries out protein purification.Purification condition are as follows: thallus is resuspended in lysate, and (50mM Tris, pH8.0,300mM NaCl, 5% is sweet
Oil, 20mM imidazoles), carry out ultrasonication (70% amplitude, 2s On/4s Off, 3 minutes, Sonics 750w Ultrasound Instrument), centrifugation
Supernatant is separated, with ni-sepharose purification, is eluted with the lysate of the imidazoles containing 250mM, elution fraction is concentrated, with Superdex 200,
10/300 gel chromatographic columns of Tricorn are purified.SDS-PAGE detection and gel column purification, acquisition after purification
Cas12a albumen puts -80 DEG C of preservations.
2, target DNA is prepared
Target nucleotide can pass through PCR amplification, recombinase polymeric enzymatic amplification (RPA), NASBA isothermal duplication or ring mediation etc.
Temperature amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HDA) and nickase amplified reaction (NEAR) mode
Expand target DNA.
Recombinase polymeric enzymatic amplification RPA (Recombinase Polymerase Amplification): NCBI is used
Primer blast designs RPA primer, and amplified fragments size is 80-120nt, and the denaturation temperature of primer can be 54-67 DEG C, Opt
=60, length 30-35nt, Opt=32, G/C content is 40-60% in primer, according to implementation sequence synthetic DNA primer.
Template sequence is (to derive from HIV genome sequence) shown in SEQ ID NO.1 in embodiment 1.
Wherein RPA primer includes:
FP:AAGGAGTAGTGGAGTCTATGAATAAGGAA
RP:TATATCGTTATTGTCCTGTATTACCACTGC
It refers to respectivelyBasic andIt is anti-that BasicRT (TwistDx) kit carries out RPA
It answers, unlike, before template segments addition, the MgAc of 280mM, i.e. magnesium acetate is first added.It is reacted 30 minutes at 37 DEG C.
Using acquisition target DNA after MinElute gel extraction kit (Qiagen) kits.
3, gRNA is prepared
GRNA primer sequence design principle: when choosing targeting sequence, targeting sequence 5 ' end should have 5 '-TTTN-3 ' sequence；
And stable secondary structure is not formed between targeting sequence itself, targeting sequence and remaining sequence.Http can be passed through: //
Www.rgenome.net/cas-designer/ online software Computer Aided Design.
GRNA primer construction:
5 '-targeting sequence-" ATCTACACTTAGTAGAAATTA "-CCCTATAGTGAGTCGTATTACA-3 '
Wherein " ATCTACACTTAGTAGAAATTA " sequence can be replaced " ATCTACAACAGTAGAAATTA " or
" ATCTACAACAGTAGAAATTA " or " ATCTACAACAGTAGAAATTA " or " GCATGAGAACCATGCATTTC " or
" ACCTAATTACTAGGTAATTT " or " ATCTACAAAAGTAGAAATCC " or " ATCTACAATAGTAGAAATTA " or
" ATCTACAAAGTAGAAATTAT " or " ATCTACAAACAGTAGAAATT ".
Referring to T7RNApolymerase kit (Thermo) kit specification, by the DNA fragmentation with T7 promoter, T7
Primer, the mixing of T7 polymerase, 37 DEG C of overnight incubations；RNeasy mini kit (Qiagen) is used again, obtains the gRNAs of purifying.
T7 primer sequence: TGTAATACGACTCACTATAGGG
T7gRNA primer sequence:
" targeting sequence " -5 '-ATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTACA-3 '
Targeting sequence includes: SEQ ID NO.17-51
4, the validation verification of substance CRISPR/Cas12a Pathogen test system
Detection architecture includes: 2 μ l RPA products, the LbCas12a of 45nM purifying, 22.5nM gRNA, and 100nM exists
The reporter dna chain of LbCas12a capable of emitting fluorescence when cutting, i.e., non-specific single stranded DNA fluorescence probe (DNAseAlert QC
System, Thermo Scientific), 0.5 μ l RNase inhibitor (Promega) and nuclease detect buffer (20mM
Tris,60mM NaCl,10mM MgCl 2,pH 7.3).Reaction system is placed in fluorescence analyser (BioTek), 37 DEG C (unless
Be otherwise noted) under react 30min, take terminal fluorescent value carry out result interpretation.
Analysis CRISPR/Cas12a reacts fluorescence data: the fluorescence data in order to calculate removal background facilitates different condition
Between comparison, the initial fluorescence of sample is removed.Background fluorescence (no target nucleotide or without gRNA under conditions of) can be from sample
Middle removal, to obtain the data of background correction fluorescence.After removing sample background fluorescence, more than or equal to negative control sample
3 times of fluorescent value are defined as the positive, and 3 times less than the fluorescent value of negative control sample are defined as feminine gender.
Testing result is as shown in Figs. 1-5, the results showed that the Cas12a albumen and designed gRNA can recognize cutting target
Mark point simultaneously generates fluorescence signal, illustrate design gRNA sequence can the relevant cause of disease target sequence of specific recognition, can be used for
The qualitative detection of related cause of disease.
5, the specificity verification of substance CRISPR/Cas12a detection architecture
3 groups of experiments are set, takes the RPA product of 3 kinds of different target genes, selects a kind of corresponding gRNA and two respectively
The uncorrelated gRNA of kind is combined reaction, verifies the specificity of gRNA sequence.Concrete operations: from 16 kinds of target sequences in table 1
3 kinds of target genes (SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3) of middle selection take this 3 kinds different target genes
RPA product respectively carries out 3 kinds of different gRNA (SEQ ID NO.17, SEQ ID NO.21 and SEQ ID NO.23) above-mentioned
CRISPR/Cas12a cleavage reaction, aqua sterilisa does blank control, and the sample of no target nucleic acid is negative control, and other conditions are not
Become.After removing sample background fluorescence, 3 times more than or equal to the fluorescent value of negative control sample are defined as the positive.
As the result is shown: SEQ ID NO.1 corresponding gRNA (SEQ ID NO.17) reaction is the positive, with its non-corresponding
GRNA (SEQ ID NO.21 and SEQ ID NO.23) reaction, testing result unstressed configuration value generate, for feminine gender.SEQ ID
NO.2 corresponding gRNA (SEQ ID NO.21) reaction is the positive, gRNA (SEQ ID NO.17 and SEQ with its non-corresponding
ID NO.23) reaction, testing result unstressed configuration value generates, for feminine gender.SEQ ID NO.3 corresponding gRNA (SEQ ID
NO.23) reaction for the positive, reacted with the gRNA (SEQ ID NO.17 and SEQ ID NO.21) of its non-corresponding, testing result without
Fluorescent value generates, for feminine gender.The above results prove that the target sequence of three kinds of cause of diseases and gRNA sequence are special each other, corresponding
GRNA has good specificity.Concrete outcome is as shown in table 2.
The combination of 2. substance CRISPR/Cas12a detection architecture specificity verification of table and experimental result
| Reaction combination | Template | gRNA | As a result |
| Combination 1 | SEQ ID NO.1 | SEQ ID NO.17 (target sequence is SEQ ID NO.1) | It is positive |
| Combination 2 | SEQ ID NO.1 | SEQ ID NO.21 (target sequence is SEQ ID NO.2) | It is negative |
| Combination 3 | SEQ ID NO.1 | SEQ ID NO.23 (target sequence is SEQ ID NO.3) | It is negative |
| Combination 4 | SEQ ID NO.2 | SEQ ID NO.17 (target sequence is SEQ ID NO.1) | It is negative |
| Combination 5 | SEQ ID NO.2 | SEQ ID NO.21 (target sequence is SEQ ID NO.2) | It is positive |
| Combination 6 | SEQ ID NO.2 | SEQ ID NO.23 (target sequence is SEQ ID NO.3) | It is negative |
| Combination 7 | SEQ ID NO.3 | SEQ ID NO.17 (target sequence is SEQ ID NO.1) | It is negative |
| Combination 8 | SEQ ID NO.3 | SEQ ID NO.21 (target sequence is SEQ ID NO.2) | It is negative |
| Combination 9 | SEQ ID NO.3 | SEQ ID NO.23 (target sequence is SEQ ID NO.3) | It is positive |
Multiple STD etiology nucleic acid detection method of the embodiment 3 based on CRISPR/Cas12a system
Detection while in order to realize to a variety of pathogens, we have developed the Multiple detection bodies for being directed to above-mentioned cause of disease target
System.Since human immunodeficiency virus (HIV) is RNA virus in STD cause of disease, just can be carried out after needing reverse transcription or RT-RPA
CRISPR/Cas12a detection, so we are only to wherein 4 kinds of STD cause of diseases (chlamydia trachomatis, NEISSERIA GONORRHOEAE, pale spirals
Body, toxoplasma gondii) carry out Multiple detection system development.Identification region and corresponding gRNA sequence to this 4 kinds of pathogenic genes groups
Analyzed, according to the similitude of sequence, G/C content, base homogeneity, whether there is or not formed second level hairpin structure, it is same reaction whether there is or not
The parameters such as cross reaction optimize reaction system and gRNA combination.
1, concrete operations: after preparing gRNA according to the method for step 3 in embodiment 2,4 kinds of gRNA is taken to mix by equal proportion
(SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.28 and SEQ ID NO.33), then according to step 4 in embodiment 2
Method prepare CRISPR/Cas12a detection architecture, detect the specificity of multiple gRNA method.Template in detection reaction is then divided
Not Wei selected gRNA correspond to the RPA product of target gene, they are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5,
SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.Aqua sterilisa does blank control in experiment, and the sample of no target nucleic acid is
Negative control, other conditions are constant.It is glimmering more than or equal to negative control sample after removing sample background fluorescence when interpretation of result
3 times of light value are defined as the positive.
The experimental results showed that RPA product (SEQ ID NO.3, SEQ ID NO.4, the SEQ ID of specific target gene
NO.5 and SEQ ID NO.6) multiple gRNA reaction system is added after, have specificity fluorescent generation, result is the positive；It is non-specific
Property target gene RPA product (SEQ ID NO.7 and SEQ ID NO.8) multiple gRNA reaction system is added after, it is not glimmering
Light generates, and result is feminine gender；Illustrate that the multiple CRISPR/Cas12a detection architecture that experiment is established has good specificity.
2, it is based on the above experimental result, in order to realize to above-mentioned 4 kinds of STD cause of diseases while carry out qualitative detection, we are to this
The target gene and gRNA sequence that patent screening goes out are combined and optimize, and every kind of cause of disease selects 1-2 target site and corresponding
GRNA is combined.According to G/C content, without the principles such as hairpin structure, no cross reaction, optimum organization mode.In the present embodiment
In, we pick two kinds of gRNA assembled schemes by calculating simulation and experimental verification.Scheme one, 7 kind of gRNA etc. are than mixing, and 7
Kind gRNA is respectively as follows: SEQ ID NO.23 (target sequence is SEQ ID NO.3), SEQ ID NO.27 (target sequence SEQ
ID NO.4), SEQ ID NO.32 (target sequence is SEQ ID NO.6), (target sequence is SEQ ID to SEQ ID NO.40
NO.10), SEQ ID NO.43 (target sequence is SEQ ID NO.11), (target sequence is SEQ ID to SEQ ID NO.44
NO.12), SEQ ID NO.49 (target sequence is SEQ ID NO.15)；Scheme two, 7 kind of gRNA etc. divide than mixing, 7 kinds of gRNA
Not are as follows: SEQ ID NO.23 (target sequence is SEQ ID NO.3), SEQ ID NO.31 (target sequence is SEQ ID NO.6),
SEQ ID NO.37 (target sequence is SEQ ID NO.9), SEQ ID NO.40 (target sequence is SEQ ID NO.10), SEQ
ID NO.41 (target sequence is SEQ ID NO.11), SEQ ID NO.47 (target sequence is SEQ ID NO.14) SEQ ID
NO.50 (target sequence is SEQ ID NO.16).
In order to verify both schemes, we use chlamydia trachomatis genome, spirochaeta pallida genome, stranguria syndrome respectively
Neisseria genome, toxoplasma gondii genome verify the specificity of two kinds of gRNA combination as template.According in embodiment 2
The method of step 4 prepares CRISPR/Cas12a detection architecture, detects the specificity of multiple gRNA method.The experimental results showed that four
After the multiple gRNA reaction system of said combination is added in the genomic nucleic acids of kind cause of disease, there is specificity fluorescent generation, result is
Positive (table 3).Illustrate that the multiple CRISPR/Cas12a detection architecture established for 4 kinds of STD cause of diseases has good specificity.
The combination of the multiple CRISPR/Cas12a detection architecture specificity verification of table 3. and experimental result
In the present embodiment, for we present only pathogenic genes group sample of nucleic acid to two kinds of gRNA assembled schemes, use
The gRNA sequence listed in this patent carries out other forms combination, all should be within the protection scope of this patent.Art technology
Personnel are it is understood that can be using Cas12a base conventional in the alternative replacement embodiment of the present invention of this field routine
The clone of cause, the building of recombinant expression carrier, the expression of Cas12a albumen and purifying, the expansion of target nucleotide/target gene segment
Increase and etc. one of or it is a variety of, to obtain similar or equivalent effect.
Embodiment 4 is detected based on the clinical sample of CRISPR/Cas12a system
We have collected 10 Cervical scrapes samples, are the NEISSERIA GONORRHOEAE positive through quantitative fluorescent PCR verifying.To this batch
After sample carries out nucleic acid extraction, the detection of CRISPR/Cas12a technology is carried out using the present invention program.
Specific method:
1, nucleic acid is handled: cleaning swab repeatedly using 1ml physiological saline, eluate extracts nucleic acid (oral cavity using column formulation
Swab genome extraction kit), nucleic acid product is dissolved with 30 μ l aqua sterilisas.
2, CRISPR/Cas12a gene cloning and protein expression, gRNA prepare (SEQ ID NO.43), Pathogen test system
And fluorescence detection method refers to above-described embodiment 2, wherein positive control is the target gene plasmid (SEQ ID NO.11) of synthesis.
3, experimental result has fluorescence generation as shown in fig. 6,10 clinical samples detect, and for the positive, illustrates CRISPR/
Cas12a system can successfully detect NEISSERIA GONORRHOEAE clinical sample.
In addition above sequence shown in SEQ ID NO.52 and SEQ ID NO.53 is as follows:
The sequence of SEQ ID NO.52:(ScCas12a)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATC
CCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAA
GAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCC
TGGAGGAATACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAG
AACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAA
GAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCA
CCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCAC
GAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCC
TTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATT
TCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAG
ACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCC
AAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACG
ACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACA
AACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATCTATTTCCGCTCCGG
CACCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATG
CCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGAC
TTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGT
GATCGTCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCG
CCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATC
AAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAA
GGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGC
GGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGC
TGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGAAAACCTGTACTATCTGGGCATCATGGA
CAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTCTTGCTACGAGAAGATGGTGTATA
AGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCC
GCCAAGCTGCTGGAGAACTACGAAAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACCAGCT
GATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCA
CCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCATCGCCGAT
TCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTC
TAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATA
AGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAAT
GAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAG
ATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATC
AGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTG
TACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAA
GGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGA
CAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCC
ATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTT
TGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGA
ACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCC
GCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCA
GGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACT
TCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCC
TGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGA
TGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGG
CCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTAC
ATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGC
CGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGA
AGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCC
ACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGA
CGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC
The sequence of SEQ ID NO.53:(SsCas12a)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATC
CCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAA
GAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCC
TGGAGAAGTACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAG
AACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAA
GAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCA
CCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCAC
GAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCC
TTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATT
TCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAG
ACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCC
AAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACG
ACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACA
AACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATGTATTTCCGCTCCGG
CGCCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATG
CCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGAC
TTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGT
GATCGCCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCG
CCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATC
AAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAA
GGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGC
GGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGC
TGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGATAACCTGTACTATCTGGGCATCATGGA
CAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTTCTGCTACGAGAAGATGGTGTATA
AGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCC
GCCAAGCTGCTGGAGAACTACGCCAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACAAGCT
GATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCA
CCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCGTGGCCGAT
TCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTC
TAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATA
AGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAAT
GAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAG
ATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATC
AGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTG
TACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAA
GGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGA
CAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCC
ATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTT
TGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGA
ACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCC
GCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCA
GGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACT
TCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCC
TGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGA
TGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGG
CCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTAC
ATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGC
CGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGA
AGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCC
ACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGA
CGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC
Claims (9)
1. a kind of STD the Methods of Detection of Pathogens based on CRISPR/Cas12a system, which is characterized in that utilize CRISPR/Cas12a
System detection target site, the nucleotide sequence of the target site such as SEQ ID NO.1-16 is any or appoints several shown.
2. method according to claim 1, which is characterized in that carry out the inspection of CRISPR nucleic acid using Cas12a albumen and gRNA
It surveys, the gRNA is designed using the target site as target sequence, design principle are as follows: when choosing gRNA targeting sequence, target
There should be 5 '-TTTN-3 ' sequence to the end of sequence 5 ', and target and do not form stabilization between sequence itself, targeting sequence and remaining sequence
Secondary structure.
3. method according to claim 2, which is characterized in that the sequence of the gRNA such as SEQ ID NO.17-51 it is any or
Appoint several shown.
4. a kind of CRISPR/Cas12a detection system of STD etiology nucleic acid, which is characterized in that including Cas12a albumen and gRNA,
The gRNA is designed using any shown target site of SEQ ID NO.1-16 as target sequence.
5. detection system according to claim 4, which is characterized in that the sequence of the gRNA such as SEQ ID NO.17-51 appoints
One or appoint it is several shown in.
6. a kind of serial target site based on CRISPR/Cas12a technology detection STD cause of disease, which is characterized in that its sequence is such as
Shown in SEQ ID NO.1-16 is any.
7. application of the target site described in claim 6 in terms of as STD etiology nucleic acid detection site.
8. target site described in claim 6 is as the STD etiology nucleic acid detection site side based on CRISPR/Cas12a system
The application in face.
9. a kind of gRNA combination based on CRISPR/Cas12a detection STD cause of disease, which is characterized in that the gRNA, which is combined, includes
SEQ ID NO.17-51 is any or appoints several shown gRNA.
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