CN111187804A - Rapid detection kit and detection method for mycoplasma pneumoniae nucleic acid based on CRISPR/Cas12a - Google Patents
Rapid detection kit and detection method for mycoplasma pneumoniae nucleic acid based on CRISPR/Cas12a Download PDFInfo
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Abstract
The invention discloses a CRISPR/Cas12a kit for rapid detection of mycoplasma pneumoniae nucleic acid and a detection method thereof, wherein the CRISPR/Cas12a kit comprises a CRISPR/Cas12a detection system; the CRISPR/Cas12a detection system comprises: a specific crRNA, CRISPR/Cas12a protein and single-stranded DNA reporter system for mycoplasma pneumoniae; the specific crRNA is any one or more designed and synthesized according to the mycoplasma pneumoniae dsDNA locus; the single-stranded DNA report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip. The method adopts CRISPR/Cas12a to detect the mycoplasma pneumoniae nucleic acid sequence for the first time, and has the advantages of high sensitivity, strong specificity, short time consumption, independence on large-scale experimental equipment and the like.
Description
Technical Field
The invention relates to the field of gene detection of mycoplasma pneumoniae, in particular to a method for rapidly detecting mycoplasma pneumoniae nucleic acid based on CRISPR/Cas12a and a kit thereof, belonging to the technical field of biology.
Background
Mycoplasma Pneumoniae (MP) is a common pathogenic microorganism, mainly causing respiratory infections in humans, especially in children and adolescents. Mycoplasma pneumoniae infection is generally in a dispersed form and widely exists in all parts of the world, the pathogenic symptoms of the mycoplasma pneumoniae infection are general respiratory symptoms such as headache, angina, fever, cough and the like, and the currently widely accepted pathogenic mechanism is that MP directly damages respiratory epithelial cells through adhesion and cytotoxic effects, and severe pneumonia and other system injuries can also be caused through an immune system.
Mycoplasma pneumoniae MP belongs to the class mollicutes, Mycoplasma, and is the smallest microorganism capable of self-replication and capable of surviving in vitro independently of living cells. The genome is circular double-stranded DNA (dsDNA), the genome is about 800000bp in length, and comprises about 700 protein-coding genes and some non-coding DNA sequences. MP is 150nm in diameter and about 1-2 μm in length, has no cell wall, and has intrinsic resistance to antibiotics affecting cell wall synthesis, and drugs inhibiting or affecting protein or nucleic acid synthesis, such as macrolides, capanolones, aminoglycosides, tetracyclines, etc., should be selected clinically for treatment. Since 2000, the evidence that macrolide drug-resistant MP is prevalent worldwide has increased, and the macrolide drug-resistant MP has a certain influence on clinical antibacterial treatment. Severe MP pneumonia is a highly challenging treatment, and requires early diagnosis and treatment, and laboratory etiology diagnosis is the key to diagnosis of MP infection.
The detection method of mycoplasma pneumoniae mainly comprises separation culture, serological detection and nucleic acid detection. The detection of the mycoplasma pneumoniae isolation culture needs longer time, the culture condition is harsh, the sensitivity is lower, and the significance for clinical diagnosis is not large; based on this, MP detection is mainly divided into two main categories: one is serum mycoplasma pneumoniae antibody IgM or IgG detection, and the other is mycoplasma pneumoniae nucleic acid (MP-DNA) detection. The serological detection is simple, convenient and quick, and is widely applied clinically. However, the method can only provide a better detection index for the past infection, and has lower application value for assessing the current infection. The most common molecular diagnostic method for nucleic acid detection is PCR technology, primers are designed aiming at an ATPase gene sequence of mycoplasma pneumoniae, a P1 adhesin gene sequence, a conserved region of 16SrRNA and the like for PCR amplification, real-time quantitative PCR can also be used for detecting the mycoplasma pneumoniae, compared with common PCR, the method can shorten the sample turnover time and reduce the steps of electrophoresis and the like, but the method is expensive, and special equipment and technology are needed to limit the further popularization.
CRISPR-Cas (Clustered regulated short palindromic repeats, CRISPRs) is an adaptive immune system in bacteria, and Cas proteins target degradation of foreign nucleic acids through RNA-guided nucleases. Among them, the CRISPR-Cas9 protein family has been widely applied to numerous fields such as gene editing, antiviral agents, biological imaging, and the like. CRISPR-Cas12a (Cpf1) belongs to Cas enzyme second family and is used to guide RNA to guide double stranded DNA cleavage of a single RuvC catalytic domain. The CRISPR/Cas12a enzymes recognize a spacer adjacent motif (PAM) rich in Thymine (Thymine, T) nucleotides, catalyze their own directed CRISPR RNA (crRNA) maturation, and produce PAM distal dsDNA breaks with staggered 5 'and 3' end incompliance. When the CRISPR/Cas12a protein cleaves double-stranded dna (dsdna) in a sequence-specific manner, a strong non-specific single-stranded dna (ssdna) trans-cleavage activity can be induced. Based on the characteristics of CRISPR/Cas12a, a rapid and accurate detection method is developed for detecting mycoplasma pneumoniae in clinical specimens. Genomic dsDNA is extracted from clinical samples to be tested and Recombinase Polymerase Amplification (RPA) is performed under isothermal conditions. The CRISPR/Cas12a-crRNA complex binds to and cleaves the target MP dsDNA, which activates the trans-cleavage of ssDNA. Fluorescent reporter molecules coupled to ssDNA generate a fluorescent signal upon cleavage. The novel method called DNA endonuclease targeting CRISPR trans-reporter gene provides a powerful platform for rapidly and accurately detecting mycoplasma pneumoniae nucleic acid.
Colloidal gold immunoassay is an efficient technical scheme for clinical rapid detection. When the colloidal gold particle-labeled antibody binds to the corresponding antigen, the colored immunoreactive reagent can be visually detected. The colloidal gold has the characteristics of short detection action time, long-term stable storage and relatively low cost, and the characteristics make the colloidal gold widely applicable to high specificity, high sensitivity, convenience and quickness in clinical detection of the mycoplasma pneumoniae nucleic acid.
Disclosure of Invention
The invention aims to provide a CRISPR/Cas12a kit for quickly detecting mycoplasma pneumoniae nucleic acid, which has high sensitivity, strong specificity and quick visualization and is used for quickly detecting the mycoplasma pneumoniae nucleic acid clinically and a detection method thereof.
In order to achieve the aim, the invention provides a CRISPR/Cas12a kit for quickly detecting mycoplasma pneumoniae nucleic acid, which comprises a CRISPR/Cas12a detection system suitable for mycoplasma pneumoniae.
The CRISPR/Cas12a detection system comprises: a crRNA specific for mycoplasma pneumoniae adhesion gene P1, a CRISPR/Cas12a protein, and a single-stranded dna (ssdna) reporter system.
The specific crRNA is any one or more of crRNA1 to crRNA4 designed aiming at the Mycoplasma pneumoniae P1 gene, and the sequence of the specific crRNA is shown in SEQ NO.4 to SEQ NO. 7.
The single-stranded DNA (ssDNA) report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip; wherein the ssDNA FQ reporter is ssDNA labeled by 6-carboxyfluorescein (6-FAM) and a fluorescence quencher (BHQ1), and the labeling products are as follows: 56 FAM/ATTATTT/3BHQ1/, designated ssDNA FQ reporter/56 FAM/ATTATTT/3BHQ 1/; the ssDNA DBreporter is ssDNA labeled by Digoxin (Digoxin) and Biotin (Biotin), and the labeling products are as follows: the gene is named ssDNA DB reporter/5Dig/ATTATTT/3 Bio/.
Preferably, the CRISPR/Cas12a kit for rapid detection of mycoplasma pneumoniae nucleic acid further comprises an immune colloidal gold test strip; the immune colloidal gold test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC back lining; the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are sequentially adhered to the PVC backing; the conjugate of the mouse anti-digoxin antibody marked by colloidal gold is coated on the combination pad; the cellulose nitrate membrane is respectively coated with a quality control line formed by streptavidin and a detection line formed by rabbit anti-mouse IgG antibody.
Preferably, the preparation method of the specific crRNA comprises: aiming at an adhesion gene P1, searching a targeting sequence containing CRISPR/Cas12a recognition sequence (PAM) TTTN, designing crRNA with the length of 19nt, respectively naming the crRNA as crRNA1 to crRNA10, synthesizing DNA oligo after the design is finished, constructing to a vector pGL3-T7-crRNA, and obtaining the target crRNA through in vitro transcription.
Preferably, the preparation method of the CRISPR/Cas12a protein comprises the following steps: prokaryotic codon optimization is carried out on a CRISPR/Cas12a protein nucleic acid sequence to obtain a sequence, a pET28a expression vector is constructed, low-temperature induction soluble protein expression is carried out, and a target protein is obtained through affinity purification and molecular sieve purification.
The CRISPR/Cas12a kit for quickly detecting mycoplasma pneumoniae nucleic acid provided by the invention can be used for fluorescence detection by an enzyme-labeling instrument and detection by an immune colloidal gold test strip. When the enzyme-linked immunosorbent assay is used for fluorescence detection, a DNA (ssDNA) reporting system in the CRISPR/Cas12a detection system is ssDNA FQ reporter, and when the test strip of immune colloidal gold is used for detection, the DNA (ssDNA) reporting system is ssDNA DB reporter.
When the mycoplasma pneumoniae gene exists in the CRISPR/Cas12a detection system, the endonuclease activity of the CRISPR/Cas12a protein is specifically activated under the mediation of mycoplasma pneumoniae-specific crRNA when the fluorescence detection is carried out by using a microplate reader. The activated CRISPR/Cas12a protein cleaves ssDNA FQ reporter labeled with a fluorophore and a quencher, thereby releasing the activated fluorophore, and a fluorescence reading can be detected using a plate reader. Correspondingly, when no mycoplasma pneumoniae gene sequence exists in the sample to be detected, the fluorescence reading is displayed as a basal value.
When the immune colloidal gold test strip is used for detection, after a sample to be detected is added into the colloidal gold test strip after the CRISPR/Cas12a is cut, a mouse-antibody digoxin antibody marked by colloidal gold is combined with a digoxin-marked ssDNA (single-stranded deoxyribonucleic acid) report system, and a compound moves from a quality control line to a detection line along the direction of liquid flow; the quality control line streptavidin saturation captures a ssDNA report system marked with a biotin label, thereby displaying a strip; when the CRISPR/Cas12a detects the gene of Mycoplasma pneumoniae, the ssDNA reporter system marked with digoxin and biotin is cut off, so that the digoxin-marked ssDNA fragments are captured and developed by the detection line, and when the CRISPR/Cas12a cannot detect the gene sequence of Mycoplasma pneumoniae, the digoxin-marked ssDNA reporter system is not cut off, so that the color captured by the detection line cannot be generated.
The invention also provides a mycoplasma pneumoniae nucleic acid rapid detection method, and the CRISPR/Cas12a kit for rapidly detecting mycoplasma pneumoniae nucleic acid is adopted.
Preferably, the method for rapidly detecting the mycoplasma pneumoniae nucleic acid comprises the following steps:
step a: releasing nucleic acid in a sample to be detected by using a nucleic acid quick release reagent;
step b: amplifying nucleic acid in a sample to be detected by using an isothermal amplification primer: b, adding the product obtained in the step a into an RPA isothermal amplification system by using mycoplasma pneumoniae specific primers SEQ ID NO. 2-SEQ ID NO.3, and reacting at 37 ℃ for 20min for amplification to obtain a specific product;
step c: cleavage of mycoplasma pneumoniae nucleic acid using CRISPR/Cas12a detection system: adding the product obtained in the step b into a CRISPR/Cas12a detection system, and reacting for 30min at 37 ℃;
step d: and detecting the mycoplasma pneumoniae nucleic acid in the sample by using an enzyme-labeling instrument or an immune colloidal gold test strip.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention realizes high sensitivity, high specificity and rapid visual detection of the mycoplasma pneumoniae nucleic acid by using CRISPR/Cas12a specific recognition nucleic acid combined immunoassay chromatography technology. According to research, the adhesion gene P1 of the mycoplasma pneumoniae is a main pathogenic gene of the mycoplasma pneumoniae, the gene has high specificity and high conservation, and the P1 gene is selected as a detection target sequence. According to the characteristics that CRISPR/Cas12a recognizes a specific PAM sequence, 4 pieces of specific crRNA are designed on a P1 gene target sequence. Detection shows that 4 crRNAs can specifically recognize Mycoplasma pneumoniae. The crRNA1 has higher sensitivity for detecting the mycoplasma pneumoniae, so the crRNA1 is selected to be used in the detection of the mycoplasma pneumoniae, and a CRISPR/Cas12a rapid detection mycoplasma pneumoniae nucleic acid system is further established.
(2) The invention relates to a quick detection tool based on CRISPR/Cas12a mycoplasma pneumoniae, which comprises immunochromatography strip detection and can realize convenient and quick result interpretation.
(3) The rapid, high-specificity, high-sensitivity and visual detection of the mycoplasma pneumoniae nucleic acid is realized by using CRISPR/Cas12a to cut a specific sequence and an immunoassay chromatography technology. Meanwhile, based on the design of crRNA with different sequences, the optimal crRNA analysis for detecting the mycoplasma pneumoniae is realized. The method for quickly detecting the mycoplasma pneumoniae nucleic acid, which is established by the invention, provides an accurate, quick, simple and convenient detection method for clinical diagnosis and laboratory research.
(4) The invention discloses a series of CRISPR/Cas12a reaction systems for mycoplasma pneumoniae nucleic acid detection, crRNA and RPA amplification primers, wherein the sequences of the CRISPR/Cas12a reaction systems and the crRNA and RPA amplification primers are shown in SEQ ID No.2 to No.8 in sequence. The CRISPR/Cas12a, crRNA and RPA amplification primer combination can be used for Mycoplasma pneumoniae nucleic acid detection. The method adopts CRISPR/Cas12a to detect the mycoplasma pneumoniae for the first time, and has the advantages of high sensitivity, strong specificity, short time consumption, high flux, independence on large-scale experimental equipment and the like. These advantages make the CRISPR/Cas12 a-based colloidal gold test strip detection method developed by the present invention convenient for rapid detection and diagnosis of mycoplasma pneumoniae nucleic acid in laboratories and clinical medicine.
Drawings
Fig. 1 is a schematic diagram of a CRISPR/Cas12 a-based method for rapid detection of mycoplasma pneumoniae nucleic acid.
Figure 2 specific crRNA design for rapid detection of mycoplasma pneumoniae nucleic acids based on CRISPR/Cas12 a.
FIG. 3 CRISPR/Cas12a detection of Mycoplasma pneumoniae fluorescence detection results with different crRNAs.
FIG. 4 CRISPR/Cas12a sensitivity of fluorescence method for detecting Mycoplasma pneumoniae.
FIG. 5 CRISPR/Cas12a fluorescence method for detecting Mycoplasma pneumoniae nucleic acid in clinical samples.
Fig. 6 CRISPR/Cas12a colloidal gold test strip detects mycoplasma pneumoniae nucleic acid in clinical samples.
Fig. 7 is a quantitative graph of mycoplasma pneumoniae nucleic acid in clinical samples detected by CRISPR/Cas12a colloidal gold test strips.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the invention: RPA amplification kit
Basic kit was purchased from twist amp, inc; the crRNA in vitro Transcription cassette MEGA shortscript T7 Transcription Kit and the purification cassette MEGA clear Kit were purchased from Ambion; conventional reagents such as Tris-Base, NaCl, Tris-HCl, MgCl2BSA and glycerol, etc. were purchased from ThermoFisher; nucleic acid and ssDNA probe synthesis was done by Nanjing Kinsley; the present invention uses a rapid nucleic acid release agent from nuozoken to obtain a pretreated nucleic acid.
The general technical schematic diagram of the invention is shown in the attached figure 1, and comprises the following 4 parts: preparing a nucleic acid sample to be detected, pre-amplifying a target gene, designing and preparing a CRISPR/Cas12a detection component, constructing a system, designing a fluorescent and colloidal gold test strip and reading a result.
Example 1: rapid and sensitive detection of mycoplasma pneumoniae nucleic acid fragments
1.1 nucleic acid preparation
In this case, the MP gene fragment refers to the P1 gene fragment of MP in NCBI database, and 399bp SEQ NO.1 of P1 partial gene is synthesized by Nanjing Kingsler company and constructed into a pUC57 vector, which is named as pUC 57-MP-P1.
The sample to be detected is obtained by amplification by using the RPA amplification primers RPA-F1 SEQ NO.2 and RPA-R1 SEQ NO.3 of the invention and referring to the RPA isothermal amplification operation steps. The specific operation is as follows:
a1. mu.L (10ng) sample of pUC57-MP-P1 was subjected to RPA amplification: 25 mu L of 2 × Buffer, 2 mu L of RPA-F1, 2 mu L of RPA-R1 and 2.5 mu L of magnesium acetate are mixed uniformly and reacted at 37 ℃ for 20min to obtain a sample for the next nucleic acid detection.
1.2 design preparation of MP-specific crRNA
As shown in fig. 2, crRNA preparation was performed according to the following protocol, searching for a targeting sequence comprising CRISPR/Cas12a recognition sequence (PAM) TTTN against the P1 region of MP gene, and designing crRNA of 19nt length, which were named crRNA1 to crRNA4, respectively. After the design is finished, the DNA oligo is synthesized by Nanjing Kinshire company and constructed on a vector pGL3-T7, and the target crRNA is obtained through in vitro transcription.
The MP crRNA provided by the invention comprises SEQ ID NO.4 to SEQ ID NO.7, and the specific information is shown in Table 1.
TABLE 1 MP Gene-specific crRNA
The detection adopts a 20 mu L system as shown in the table 2, but is not limited to the system, and comprises the following steps of adjusting the proportion of corresponding components:
TABLE 2 Mycoplasma pneumoniae CRISPR/Cas12a detection system
Wherein the ssDNA reporter is ssDNA FQ reporter or ssDNA DB reporter.
1.3 full-wavelength ELIASA fluorescence detection
In the fluorescence detection of a microplate reader, various components are sequentially added into a target gene detection system by the CRISPR/Cas12 a. The components are mixed evenly and then react for 30min at 37 ℃. Wherein, the concentration of RNase Inhibitors in the reaction system is 40U/muL, the concentration of CRISPR/Cas12a is 200 ng/muL, the concentration of ssDNA FQ reporter is 25 pmol/muL, and the concentration of crRNA is 1 pmol/muL.
And (4) determining the CRISPR/Cas12a detection system to detect the activity by using fluorescence detection. And (3) measuring the fluorescence of the detection reaction by using a full-wavelength microplate reader, monitoring the fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, and detecting the fluorescence value after the reaction is carried out for 30 min. The detection of the cutting of each crRNA of the mycoplasma pneumoniae is shown in figure 3, and the detection of the mycoplasma pneumoniae can be realized by utilizing a fluorescence method result judgment scheme.
1.4 colloidal gold test strip detection
In the colloidal gold test strip detection, various components are sequentially added into a target gene detection system by the CRISPR/Cas12 a. The components are mixed evenly and then react for 30min at 37 ℃. Wherein, the concentration of RNase Inhibitors in the reaction system is 40U/muL, the concentration of CRISPR/Cas12a is 200 ng/muL, the concentration of ssDNA DB reporter is 25 pnol/muL, and the concentration of crRNA is 1 pmol/muL.
The detection steps of the immune colloidal gold test strip are as follows: 20 μ L of CRISPR/Cas12a cleavage product was mixed with 40 μ L of colloidal gold dipstick buffer (4XSSC, 2% BSA and 0.05% Tween-20, pH 7.0). The test strip was immersed in the mixture and after 3 minutes of reaction, the results were visually determined and recorded by photography.
In this embodiment, the CRISPR/Cas12a fluorescence and colloidal gold are used to realize rapid detection of mycoplasma pneumoniae nucleic acid. And it was found that crRNA1 and crRNA4 have high sensitivity for detection of Mycoplasma pneumoniae P1 gene (FIG. 3).
Example 2: sensitivity of CRISPR/Cas12a in detection of Mycoplasma pneumoniae nucleic acid
In the case of sensitive assay, the pUC57-MP-P1 plasmid was converted to copy number based on molecular weight and 10-fold gradient dilutions were performed to obtain copy number (copy/. mu.l) containing 2 ae 7, 2 ae 6, 2 ae 5, 2 ae 4, 2 ae 3, 2 ae 2, 2 ae 1 and 2e 0 per microliter. Samples were diluted in 1 μ L gradient and subjected to RPA amplification reactions: 25 mu L of 2 × Buffer, 2 mu L of RPA-F1, 2 mu L of RPA-R1 and 2.5 mu L of magnesium acetate are mixed uniformly and reacted at 37 ℃ for 20min to obtain a sample for the next nucleic acid detection.
According to the results obtained in example 1, crRNA has higher sensitivity to the detection of Mycoplasma pneumoniae P1 gene, so that the subsequent detection is carried out by using crRNA 1. The detection adopts a 20 mu L system as shown in the table 3, but is not limited to the system, and comprises the following steps of adjusting the proportion of corresponding components:
TABLE 3 Mycoplasma pneumoniae CRISPR/Cas12a detection system
Wherein the ssDNA reporter is ssDNA FQ reporter or ssDNA DB reporter.
In the embodiment, the detection activity of the CRISPR/Cas12a detection system is determined by fluorescence detection. And (3) measuring the fluorescence of the detection reaction by using a full-wavelength microplate reader, monitoring the fluorescence dynamics, wherein the excitation wavelength is 485nm, the emission wavelength is 520nm, and detecting the fluorescence value after the reaction is carried out for 30 min.
In this case, the CRISPR/Cas12a fluorescence method is used to detect mycoplasma pneumoniae nucleic acid, which can realize high-sensitivity detection of 2e1 copies of mycoplasma pneumoniae (fig. 4).
Example 3: rapid detection of mycoplasma pneumoniae nucleic acid in clinical sample nucleic acid
This example provides a rapid detection of nucleic acids from clinical samples, all samples and manipulations are done in the laboratory, in this case CRISPR/Cas12a detection of DNA obtained from buccal swabs from the sample. This example uses a rapid nucleic acid release agent from Novomedium to obtain pre-treated nucleic acids. The method comprises the following steps: and (3) cleaning the oral swab with PBS and centrifuging, taking 2 mu L of centrifuged supernatant, adding 20 mu L of nucleic acid lysate, standing at normal temperature for 3 minutes, adding 20 mu L of neutralizing solution, mixing uniformly, and carrying out next detection.
By using the RPA amplification primers SEQ NO.2 and SEQ NO.3, 2 mul of each sample to be detected is subjected to RPA pre-amplification by referring to the RPA isothermal amplification operation steps to obtain the sample to be detected. The specific operation is as follows:
25 mu L of 2 × Buffer, 2 mu L of RPA-F, 2 mu L of RPA-R, 2.5 mu L of magnesium acetate and 2 mu L of DNA sample are added with water to be supplemented to 50 mu L, the mixture is uniformly mixed and reacts for 20min at 37 ℃, and the sample is obtained to be used for next nucleic acid detection.
In the embodiment, the detection activity of the CRISPR/Cas12a detection system is determined by fluorescence detection. In a CRISPR/Cas12a detection system, 2 mu L of Buffer, 1 mu L of RNase Inhibitors, 1 mu L of CRISPR/Cas12a, 1 mu L of ssDNA FQ reporter, 5 mu L of RPA sample, 1 mu L of crRNA and 9 mu L H are added in sequence2And O. The components are mixed evenly and reacted for 30min at 37 ℃. The fluorescence kinetics was monitored by measuring the fluorescence of the assay reaction using a full-wavelength microplate reader, with an excitation wavelength of 485nm and an emission wavelength of 520nm, and the fluorescence values were measured at 30min of the reaction, as shown in FIG. 5, with #1, #2, #3, #6 and #8 being positive for Mycoplasma pneumoniae and #4, #5 and #7 being negative.
The colloidal gold method is utilized to detect CRISPR/Cas12a, and 2 muL of Buffer, 1 muL of RnaseInhibitors, 1 muL of CRISPR/Cas12a, 1 muL of ssDNA DB reporter, 5 muL of RPA sample, 1 muL of crRNA and 9 mu L H are added in sequence2And O. The components are mixed evenly and reacted for 30min at 37 ℃. The CRISPR/Cas12a cleavage product was diluted 1:2 into colloidal gold assay buffer, followed by insertion of the colloidal gold test strip of the invention, and incubated for 3 minutes at room temperature. And judging the test result of the test strip, photographing and storing, and performing gray level analysis on the test result of the colloidal gold test strip. The test results of the test strip are shown in FIG. 6, wherein #1, #2, #3, #6 and #8 are positive for Mycoplasma pneumoniae and #4, #5 and #7 are negative. FIG. 7 is the data of the detection of the gray scale scan of the strip in FIG. 6, and the positive determination result is consistent with the fluorescence method. The result shows that the CRISPR/Cas12a fluorescence method and the colloidal gold test strip can realize sensitive, rapid and accurate detection of the mycoplasma pneumoniae nucleic acid in clinical oral swabs.
Sequence listing
<110> institute of science and technology of the national institute of health and wellness
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ggtggcttcg tgaccgaaag cggcgagaag atcaaaggcc tgaacgagta cattaacctg 780
tataaccaga agaccaaaca aaagctgccg aaatttaagc cgctgtataa gcaggtgctg 840
agcgatcgtg aaagcctgag cttctacggc gagggctata ccagcgacga ggaagttctg 900
gaagtgtttc gtaacaccct gaacaaaaac agcgagatct tcagcagcat taagaaactg 960
gaaaagctgt tcaaaaactt tgacgagtac agcagcgcgg gtatctttgt taagaacggc 1020
ccggcgatca gcaccattag caaagatatc ttcggtgaat ggaacgtgat tcgtgacaag 1080
tggaacgcgg agtatgacga tatccacctg aagaaaaagg cggtggttac cgaaaagtac 1140
gaggacgatc gtcgtaaaag cttcaaaaag attggcagct ttagcctgga acagctgcaa 1200
gagtacgcgg acgcggatct gagcgtggtt gaaaaactga aggagatcat tatccagaag 1260
gttgatgaaa tctacaaagt gtatggtagc agcgagaagc tgttcgacgc ggattttgtt 1320
ctggagaaga gcctgaaaaa gaacgacgcg gtggttgcga tcatgaagga cctgctggat 1380
agcgtgaaaa gcttcgaaaa ctacattaag gcgttctttg gtgaaggcaa agagaccaac 1440
cgtgacgaga gcttctatgg cgattttgtt ctggcgtacg acatcctgct gaaggtggac 1500
cacatctacg atgcgattcg taactatgtt acccaaaaac cgtacagcaa ggataagttc 1560
aagctgtact tccagaaccc gcaattcatg ggtggctggg acaaggataa agagaccgac 1620
tatcgtgcga ccatcctgcg ttacggtagc aagtactatc tggcgattat ggataaaaag 1680
tacgcgaaat gcctgcagaa gatcgacaaa gacgatgtta acggtaacta cgaaaagatc 1740
aactacaagc tgctgccggg cccgaacaag atgctgccga aagtgttctt tagcaaaaag 1800
tggatggcgt actataaccc gagcgaggac atccaaaaga tctacaagaa cggtaccttc 1860
aaaaagggcg atatgtttaa cctgaacgac tgccacaagc tgatcgactt ctttaaagat 1920
agcattagcc gttatccgaa gtggagcaac gcgtacgatt tcaactttag cgagaccgaa 1980
aagtataaag acatcgcggg tttttaccgt gaggttgagg aacagggcta taaagtgagc 2040
ttcgaaagcg cgagcaagaa agaggtggat aaactggtgg aggaaggtaa actgtacatg 2100
ttccaaatct acaacaagga cttcagcgat aagagccacg gcaccccgaa cctgcacacc 2160
atgtacttca agctgctgtt tgacgaaaac aaccatggtc agatccgtct gagcggtggc 2220
gcggagctgt tcatgcgtcg tgcgagcctg aagaaagagg agctggttgt gcacccggcg 2280
aacagcccga ttgcgaacaa aaacccggat aacccgaaaa agaccaccac cctgagctac 2340
gacgtgtata aggataaacg ttttagcgaa gaccaatacg agctgcacat tccgatcgcg 2400
attaacaagt gcccgaaaaa catcttcaag attaacaccg aagttcgtgt gctgctgaaa 2460
cacgacgata acccgtatgt tatcggtatt gaccgtggcg agcgtaacct gctgtacatc 2520
gtggttgtgg acggtaaagg caacattgtg gaacagtata gcctgaacga gattatcaac 2580
aactttaacg gtatccgtat taagaccgat taccacagcc tgctggacaa aaaggagaag 2640
gaacgtttcg aggcgcgtca gaactggacc agcatcgaaa acattaagga gctgaaagcg 2700
ggctatatca gccaagttgt gcacaagatt tgcgaactgg ttgagaaata cgatgcggtg 2760
atcgcgctgg aggacctgaa cagcggtttt aagaacagcc gtgttaaggt ggaaaagcag 2820
gtttaccaaa agttcgagaa gatgctgatc gataagctga actacatggt ggacaaaaag 2880
agcaacccgt gcgcgaccgg tggcgcgctg aaaggttatc agattaccaa caagttcgaa 2940
agctttaaaa gcatgagcac ccaaaacggc ttcatctttt acattccggc gtggctgacc 3000
agcaaaatcg atccgagcac cggttttgtt aacctgctga agaccaaata taccagcatt 3060
gcggatagca aaaagttcat cagcagcttt gaccgtatta tgtacgtgcc ggaggaagac 3120
ctgttcgagt ttgcgctgga ctataagaac ttcagccgta ccgacgcgga ctacatcaaa 3180
aagtggaaac tgtacagcta tggtaaccgt atccgtattt tccgtaaccc gaaaaagaac 3240
aacgtttttg actgggagga agtgtgcctg accagcgcgt ataaggaact gttcaacaaa 3300
tacggtatca actatcagca aggcgatatt cgtgcgctgc tgtgcgagca gagcgacaag 3360
gcgttctaca gcagctttat ggcgctgatg agcctgatgc tgcaaatgcg taacagcatc 3420
accggtcgta ccgatgttga ttttctgatc agcccggtga aaaacagcga cggcattttc 3480
tacgatagcc gtaactatga agcgcaggag aacgcgattc tgccgaagaa cgcggacgcg 3540
aacggtgcgt ataacatcgc gcgtaaagtt ctgtgggcga ttggccagtt caaaaaggcg3600
gaggacgaaa agctggataa ggtgaaaatc gcgattagca acaaagaatg gctggagtac 3660
gcgcaaacca gcgttaagca cgagaacctg tacttccaat cccaccacca ccaccaccac 3720
caccaccacc accaccacca ctga 3744
Claims (6)
1. A CRISPR/Cas12a kit for rapid detection of mycoplasma pneumoniae nucleic acid is characterized by comprising a CRISPR/Cas12a detection system suitable for rapid detection of mycoplasma pneumoniae;
the CRISPR/Cas12a detection system comprises: a specific crRNA, CRISPR/Cas12a protein and ssDNA reporter system for mycoplasma pneumoniae P1 gene;
the specific crRNA is any one or more of crRNA1 to crRNA4, and the sequence of the specific crRNA is SEQ NO.4 to SEQ NO. 7;
the ssDNA report system comprises a ssDNA FQ reporter used for fluorescence detection of a microplate reader and/or a ssDNA DB reporter used for detection of an immune colloidal gold test strip; wherein the ssDNA FQ reporter is ssDNA labeled by 6-carboxyfluorescein and a fluorescence quencher, and the labeling products are as follows: 56 FAM/ATTATTT/3BHQ1/, designated ssDNA FQreporter/56 FAM/ATTATTT/3BHQ 1/; the ssDNA DB reporter is ssDNA labeled by digoxin and biotin, and the labeling products are as follows: the gene is named ssDNA DB reporter/5Dig/ATTATTT/3 Bio/.
2. The CRISPR/Cas12a kit for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, further comprising an immune colloidal gold test strip;
the immune colloidal gold test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC back lining; the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are sequentially adhered to the PVC backing; the conjugate of the mouse anti-digoxin antibody marked by colloidal gold is coated on the combination pad; the cellulose nitrate membrane is respectively coated with a quality control line formed by streptavidin and a detection line formed by rabbit anti-mouse IgG antibody.
3. The CRISPR/Cas12a kit for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, wherein the preparation method of the specific crRNA comprises the following steps: aiming at a mycoplasma pneumoniae P1 gene, a targeting sequence containing a CRISPR/Cas12a recognition sequence TTTN is searched, crRNA with the length of 19nt is designed, after the design is finished, oligo is synthesized and constructed to a vector pGL3-T7-crRNA, and the target crRNA is obtained through in vitro transcription.
4. The CRISPR/Cas12a kit for rapid detection of Mycoplasma pneumoniae nucleic acid according to claim 1, wherein the CRISPR/Cas12a protein is prepared by a method comprising the following steps: prokaryotic codon optimization is carried out on a CRISPR/Cas12a protein nucleic acid sequence to obtain a sequence SEQ NO.8, a pET28a expression vector is constructed, low-temperature induction soluble protein expression is carried out, and a target protein is obtained through affinity purification and molecular sieve purification.
5. A rapid detection method for Mycoplasma pneumoniae nucleic acid, which is characterized in that the CRISPR/Cas12a kit for rapid detection of Mycoplasma pneumoniae nucleic acid according to any one of claims 1-4 is adopted.
6. The method for rapidly detecting a Mycoplasma pneumoniae nucleic acid according to claim 5, wherein the method for rapidly detecting a Mycoplasma pneumoniae nucleic acid comprises the following steps:
step a: releasing nucleic acid in a sample to be detected by using a nucleic acid quick release reagent;
step b: amplifying nucleic acid in a sample to be detected by using an isothermal amplification primer: b, adding the product obtained in the step a into an RPA isothermal amplification system by using specific primers SEQ NO.2 and SEQ NO.3, and reacting at 37 ℃ for 20min for amplification to obtain a specific product;
step c: detection of mycoplasma pneumoniae nucleic acid using CRISPR/Cas12a detection system: adding the product obtained in the step b into a CRISPR/Cas12a detection system, and reacting for 30min at 37 ℃;
step d: and detecting the mycoplasma pneumoniae nucleic acid in the sample by using a fluorescence detection method or an immune colloidal gold test strip.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018048194A1 (en) * | 2016-09-07 | 2018-03-15 | 울산대학교 산학협력단 | Composition and method for improving sensitivity and specificity of detection of nucleic acids using dcas9 protein and grna binding to target nucleic acid sequence |
| CN109750116A (en) * | 2019-03-12 | 2019-05-14 | 西安医学院 | PCR- internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection |
| US20190203280A1 (en) * | 2016-09-07 | 2019-07-04 | University Of Ulsan Foundation For Industry Cooperation | Composition and method for improving sensitivity and specificity of detection of nucleic acids using dcas9 protein and grna binding to target nucleic acid sequence |
| CN110551846A (en) * | 2019-08-19 | 2019-12-10 | 上海科技大学 | cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof |
| WO2019233385A1 (en) * | 2018-06-03 | 2019-12-12 | 上海吐露港生物科技有限公司 | Use of high-temperature-resistant cas protein, and method and reagent kit for detecting target nucleic acid molecule |
| CN110804669A (en) * | 2019-11-28 | 2020-02-18 | 广州微远基因科技有限公司 | CRISPR (clustered regularly interspaced short palindromic repeats) detection primer group for mycoplasma pneumoniae and application thereof |
-
2020
- 2020-02-20 CN CN202010104672.2A patent/CN111187804A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018048194A1 (en) * | 2016-09-07 | 2018-03-15 | 울산대학교 산학협력단 | Composition and method for improving sensitivity and specificity of detection of nucleic acids using dcas9 protein and grna binding to target nucleic acid sequence |
| US20190203280A1 (en) * | 2016-09-07 | 2019-07-04 | University Of Ulsan Foundation For Industry Cooperation | Composition and method for improving sensitivity and specificity of detection of nucleic acids using dcas9 protein and grna binding to target nucleic acid sequence |
| WO2019233385A1 (en) * | 2018-06-03 | 2019-12-12 | 上海吐露港生物科技有限公司 | Use of high-temperature-resistant cas protein, and method and reagent kit for detecting target nucleic acid molecule |
| CN109750116A (en) * | 2019-03-12 | 2019-05-14 | 西安医学院 | PCR- internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection |
| CN110551846A (en) * | 2019-08-19 | 2019-12-10 | 上海科技大学 | cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof |
| CN110804669A (en) * | 2019-11-28 | 2020-02-18 | 广州微远基因科技有限公司 | CRISPR (clustered regularly interspaced short palindromic repeats) detection primer group for mycoplasma pneumoniae and application thereof |
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