CN113667668B - HBV detection based on CRISPR/Cas system - Google Patents
HBV detection based on CRISPR/Cas system Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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Abstract
The invention provides brand-new HBV recognition target regions 1 and 2 and brand-new crRNA. The crRNA of the invention has the sequence shown as SEQ ID NO.3 or SEQ ID NO. 4. The invention also provides CRISPR-based kits comprising any of the crrnas. Under the premise of ensuring detection sensitivity, the detection technology can improve the HBV detection efficiency, reduce the detection cost, does not depend on professional equipment and technicians, and has the advantages of easy popularization and the like.
Description
Technical Field
The invention relates to an HBV detection method.
Background
Hepatitis b is an infectious disease mainly caused by Hepatitis B Virus (HBV) infection, and is one of the recognized global public health problems seriously jeopardizing human health. In a statistics, 68% of infected individuals who die from chronic hepatitis B have been concentrated in the western Pacific region and in developing countries of Africa. These countries have a lag in economic development, possibly resulting in a low HBV detection rate due to a lag in medical level.
CRISPR consists of genes encoding Cas-related proteins and CRISPR arrays, with more and more CRISPR/Cas systems emerging and being used for nucleic acid detection. Cas13a has two different RNA cleavage activities, remaining active after cleavage of its target RNA, exhibiting indiscriminate cleavage activity.
Clinically qPCR detection HBV technology is very mature, and CRISPR/Cas combined qPCR detection HBV is also available, but in areas with lagging medical conditions, the requirement of on-site immediate detection of HBV still cannot be met, and the PCR operation is complex and long.
Disclosure of Invention
The invention overcomes the defects of the prior art, obtains brand-new HBV identification target regions 1 and 2 through repeated screening, designs crRNA, amplification primers and the like, and finally successfully provides an excellent CRISPR/Cas detection system.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a novel crRNA which recognizes the above novel target-sequence of HBV as shown in SEQ ID NO.1 or SEQ ID NO.2, and the specific sequence of which is shown in SEQ ID NO.3 or SEQ ID NO. 4.
In a second aspect, the invention correspondingly provides a CRISPR-based kit comprising the crRNA composition described above, which may contain a Cas13a protein, such as an LwCas13a protein.
Optionally, the CRISPR-based kit further comprises RAA primers Primer3 or Primer2, wherein the upstream and downstream primers in the Primer2 are sequentially shown as SEQ ID NOs.5-6 (NOs represent parallel list of serial numbers in the field), and the upstream and downstream primers in the Primer3 are sequentially shown as SEQ ID NOs.7-8.
Optionally, the kit further comprises a reporting system for reporting the activation status of Cas13a protein, which can amplify the signal of Cas protein activation cleavage, which can be constructed based on colorimetric, fluorescent or lateral flow test strips, which can be hybrid detect test strips.
Optionally, the system uses a fluorescent probe or a Biotin probe, the fluorescent probe structure can be 5'6-FAM-UUUUUU-3' BHO, and the Biotin probe structure can be 5'-/6-FITC/UUUUUU/Biotin/-3'.
Optionally, the kit further comprises a transcriptional enzyme, such as T7 RNA polymerase.
Optionally, the kit further comprises an rnase inhibitor for inhibiting other possible rnases than the proteins described above.
It should be noted that, based on the simple modification of the present patent idea, infringement to the present patent is constituted, for example: the crRNA is transformed into a fusion molecule fused with other nucleic acid fragments, which may be single-stranded RNA or double-stranded DNA, and the "other nucleic acid fragments" may be sequences, such as T7 transcribed sequences, which are attached upstream of the crRNA and recognized and transcribed by an RNA polymerase (e.g., T7 polymerase), and specific fusion examples are attachment of T7 sequences upstream of the crRNA encoding DNA, and the fusion molecule is transcribed once to obtain an RNA product in which the T7 sequence is in tandem with the crRNA, as can be seen in particular embodiments of this patent. Similarly, the RAA primer can be transformed into a fusion molecule, for example, a T7 sequence is connected to the upstream of the primer of SEQ ID NO.5, and an amplified product of the fusion molecule is transcribed once to obtain ssRNA with the T7 sequence connected with region1 in series, and the specific embodiment of the present patent can be seen.
Accordingly, the present invention provides a method for detecting HBV using the above-described nucleic acid or kit form thereof, which method can be used for non-diagnostic purposes.
The beneficial effects of the invention are as follows: the research provides a brand new HBV detection target and crRNA, and the HBV detection technology of the patent can improve the HBV detection efficiency and reduce the detection cost on the premise of ensuring the detection sensitivity, does not depend on professional equipment and technicians, and has the advantages of easy popularization and the like.
Drawings
Fig. 1 shows the detection efficiency of crRNA1 and crRNA2 of the present patent in a Cas13a detection system.
FIG. 2 is a fluorescence curve of crRNA1 of the present patent for HBV detection at different DNA concentrations.
FIG. 3 is a fluorescence curve of crRNA2 of the present patent for HBV detection at different DNA concentrations.
Detailed Description
English abbreviations that may be involved
2 materials and methods
Unless otherwise noted, materials/methods referred to in the detailed description are all conventional in the art, and english abbreviations refer to the above tables.
Part 2.1 laboratory Material annotation
2.1.1HBV Standard plasmid Synthesis
The standard plasmid of HBV P coding region (GenBank: MN 683731.1) was synthesized by recombination with HBV P region sequence using the PUC57 vector plasmid (Shanghai Biosynthesis).
2.1.2 reagent LwCas13a protein the same literature discloses Cas13a protein (Cas 13a from Leptotrichia wadei)
2.2 Experimental procedures and methods
Collection of 2.2.1HBV sequences and sequence alignment
All HBV sequences are collected, the collected HBV sequences are subjected to sequence comparison by using Clustal X bioinformatics software, and the conserved regions with relatively few mutation are screened by using MEGA7.0 software for visualizing the sequence comparison result, and the screened conserved regions are used as target regions for subsequent detection of Cas13a and design regions of universal isothermal amplification primers.
2.2.2 clinical serum HBV DNA nucleic acid extraction
Clinical serum samples extraction of HBV DNA the experimental procedure was performed using HiPure Viral DNA Kits post viral DNA extraction kit (meiji organism) according to conventional procedures.
2.2.3 design and screening of isothermal amplification primers
After the sequence alignment described above, we used the conserved region as a candidate target sequence for subsequent Cas13a detection. Primers were then designed for recombinase-mediated strand-exchange nucleic acid amplification technology (RAA method) at conserved regions upstream and downstream of the candidate Cas13a target sequence. Primers for isothermal amplification were designed according to the standard required in the specifications of RAA nucleic acid amplification reagents (base form) using Primer-BLAST and oligo7.0 in NCBI.
RAA primers were designed as shown in Table 2.5. And then carrying out isothermal amplification on 5 pairs of designed primers for 30min at the temperature of RAA 37 ℃ and then carrying out agarose gel electrophoresis experiments. Agarose gel electrophoresis experiments used a 1.5% agarose gel configured. The setting of DYY-6C electrophoresis apparatus is voltage U:120V, time T:30min. The gel after gel electrophoresis was imaged using a Gene Genius Bio gel imaging system. The optimal RAA primers are then screened for subsequent RAA-Cas13a detection based on the electrophoresis band. And screening out a primer with optimal amplification effect according to the strip of the electrophoresis gel, and taking the primer as a primer for RAA amplification in subsequent detection. In addition, since Cas13a system detects RNA, we skillfully add T7 sequence to the RAA upstream primer, which can be transcribed into single stranded RNA (ssRNA) under the action of the subsequent T7 RNA polymerase.
TABLE 2.5 primer sequences for isothermal amplification of RAA in this study (upstream primer preceded by T7 sequence)
2.2.4 isothermal amplification of recombinase-mediated strand-exchange nucleic acids (RAA)
Isothermal amplification of HBV DNA will be performed using a basic RAA nucleic acid amplification kit (hangzhou mass). The amplification system in Table 2.6 was prepared according to the procedure of the reagent specification, and then the prepared mixture was put into a constant temperature metal bath for incubation at 37℃for 30-60min. And placing the amplified HBV product on ice or in a refrigerator at-20 ℃ for subsequent detection of Cas13 a.
TABLE 2.6RAA isothermal amplification sample addition system
2.2.2 principle of design of 5crRNA
The crRNA designed by the patent can be connected with LwCas13a protein and can be complementarily paired with a HBV target sequence, so that the detection experiment of Cas13a is convenient, a T7 sequence is additionally added at the upstream of the designed crRNA sequence, and the total sequence after fusion is shown in the table of the following section.
2.2.6 test procedure for crRNA synthesis
The optimal RAA primers were selected according to agarose gel electrophoresis as described above, and two corresponding crRNAs were designed according to the criteria for crRNAs as described above (Table 2.7). Considering the unnecessary direct synthesis of RNA, we used transcription from the DNA template of crRNA by In Vitro Transcription (IVT). So we add a T7 sequence upstream of the crRNA, which allows the crRNA gene fragment to be transcribed into single stranded crRNA by the subsequent T7 RNA polymerase.
TABLE 2.7 templates for crRNA and synthetic crRNA designed in this study (crRNA in this table refers to both upstream added T7 sequences)
The specific crRNA synthesis test steps are as follows, firstly, preparing a mixed solution of a T7 Primer and a crRNA template according to a crRNA synthesis system, and then placing the mixed solution in a PCR instrument for high-temperature fusion, wherein the reaction condition is 95 ℃ for 10min. After the reaction is finished, the PCR instrument is closed, the temperature of the PCR instrument is naturally reduced to the room temperature, and thus, the double-stranded DNA of complementary pairing of the crRNA and the T7 is formed by slow annealing, extension and fusion.
2.2.7T 7 transcription of crRNA after synthesis
Next, the double-stranded crRNA DNA synthesized in the previous step was used in the kit HiScribe TM T7 Quick High Yield RNA Synthesis Kit (NEB, E2050S) T7 transcription was performed, and a T7 transcription mixture was prepared according to the procedure of the specification and the required system. Then, the prepared mixed solution is placed into a constant temperature incubator at 37 ℃ for incubation for 8-10 hours. This allows transcription of double-stranded crRNA DNA into single strandscrRNA。
2.2.8 purification of crRNA after transcription
However, the above-mentioned crRNA transcribed by T7 may contain impurities (the mixture may contain some untranslated DNA or reagents present in the above steps), so we use a kit to purify the product transcribed by T7 to obtain crRNA with better purity and concentrationRNA clearup Kit (NEB) was used for crRNA purification.
2.2.9 evaluation of crRNA detection efficiency
The purified crrnas described above were diluted to 300ng/μl and 30ng/μl using rnase-free water gradient for subsequent Cas13a detection. Prior to performing Cas13a detection, we need to evaluate the detection efficiency of crrnas: the HBV standard plasmid (10 concentration) was first primed with RAA primer 8 cobies/. Mu.L) and then adding the RAA amplification product to the crRNA-Cas13a cocktail. The configuration of the mixed liquor of crRNA-Cas13a was performed according to the CRISPR/Cas13a detection system of table 2.10, and then the mixed liquor was placed into a Fast 7500 fluorescence quantitative PCR machine for Cas13a fluorescence detection and fluorescence collection. And judging whether the crRNA is the optimal crRNA according to the fluctuation of fluorescence expression quantity (RFU) after crRNA-Cas13a detection and the early and late arrival of the plateau of the fluorescence curve. The results are shown in FIG. 1. .
2.2.10 construction of CRISPR/Cas13a System for HBV detection
The HBV Cas13a detection comprises a previous RAA isothermal amplification target sequence and a CRISPR/Cas13a detection test, and the specific RAA-Cas13a detection steps are as follows: the HBV DNA was first amplified isothermally according to the RAA amplification assay procedure and conditions described above, and then the amplified RAA product was added to the crRNA-Cas13a cocktail configured according to the CRISPR/Cas13a detection system of table 2.10. Visualization of Cas13a detection results was then presented using both fluorescence reading and lateral flow dipstick detection.
For Cas13a fluorescence readout, we used the ABI Fast 7500qPCR system (Applied Biosystems) to collect fluorescence from the nonspecific cleavage fluorescent probe (FAM-uuuuu-BHO) following Cas13a protein activation. Also we can use a portable fluorescence collector.
The lateral flow test strip method was performed using a HybriDetect test strip (Milenia Biotec, germany) with a Biotin probe of 5 '-/6-FITC/UUUU/Biotin/-3'. The probe for lateral flow test strip detection is fluorescein at one end and biotin at the other end. Streptavidin in the first line (Control line) position of the lateral flow strip will bind to the organisms on the probe and will then bind to the sheared biotin end and capture all intact uncleaved probe. Gold Nanoparticle (NPs) labeled anti-FITC antibodies will bind to FITC resulting in a macroscopic dark purple band. If the FITC-Biotin RNA probe is not sheared (negative assay), a dark purple band is left on the first line. When the FITC-Biotin RNA probe is cleaved (positive detection), the gold particle-labeled antibody will flow along the second line (Testing line) due to the presence of the target and the attached activity. The secondary antibody is labeled on the second line of the test strip, which captures all antibodies, thereby forming a dark purple band, the color of the second band indicating the presence of the target pathogen and the extent to which the probe is sheared. In addition, the lateral flow strip method requires incubation in a metal bath at 37℃for 30-60min before performing the test.
TABLE 2.10CRISPR/Cas13a detection sample loading System
Sensitivity analysis of 2.2.11RAA-Cas13a system for detecting HBV DNA
Gradient sensitivity analysis was performed on the newly established RAA-Cas13a system to evaluate the lower detection limit of RAA-Cas13 a. RAA isothermal amplification was first performed using a series of diluted HBV standard plasmids, followed by fluorescence and lateral flow dipstick detection of Cas13a using the amplified products. Operating according to the experimental procedure described previously. And then determining the detection lower limit of the RAA-Cas13a according to the fluorescence curve and the detection result of the test strip.
Specific assay for 2.2.12RAA-Cas13a detection of HBV DNA
To ensure that only HBV can be detected and that no other pathogen can be detected. First we performed bioinformatics analysis and BLAST analysis of target sequences in crRNA to see if the test results were similar to sequences of other pathogens except for HBV subtypes. If the sequence similarity difference base is less, the target sequence of HBV-Cas13a is redesigned, and finally the specific target sequence is obtained. In addition, other hepatitis viruses, which are frequently differentially diagnosed clinically with hepatitis B, include Hepatitis A Virus (HAV), hepatitis C Virus (HCV), hepatitis D Virus (HDV), and Hepatitis E Virus (HEV). HBV and other hepatitis identification assays are performed simultaneously using sequence alignment and HBV-Cas13a fluorescence assay.
2.2.13 clinical sample inclusion criteria and clinical validation
Inclusion criteria for clinical samples: all serum samples were sent to a hospital clinical laboratory for HBV screening within 3 months. Sample serum extraction of nucleic acids was performed using a HiPure Viral DNA Kits post viral DNA extraction kit (Magen) according to the test procedure described above. The extracted nucleic acid and the remaining serum sample are placed in a-80 ℃ refrigerator for standby. In order to avoid the interference of subjective factors on the detection result, the clinical serum sample is subjected to RAA-Cas13a detection and qPCR detection respectively in a blind test mode. Detection of RAA-Cas13a follows the previously described procedure. qPCR assays were performed using a commercial HBV detection kit (HBV Nucleic Acid Assay Kit, sansure Biotech) according to the procedures and standards of the instructions. And the RAA-Cas13 detection effect was evaluated with qPCR detection as a gold standard. In addition, sensitivity (sensitivity), specificity (PPV), positive predictive value (positive predictive value), negative predictive value (negative predictive value, NPV) and Receiver Operating Characteristics (ROC) curves will be used to evaluate the efficiency of RAA-Cas13a detection. For the definition of detection of HBV positive samples based on RAA-Cas13a fluorescence, we set signal-to-noise ratio (S/N) parameters (ratio of fluorescence value of sample to negative control). And taking the signal to noise ratio S/N >3 of the fluorescence value of the 30min time point of the Cas13a reaction as the HBV positive sample.
2.2.14 data analysis
For the RAA-Cas13 detection method of HBV, three parallel experiments are carried out to avoid test errors, and the data are expressed by mean plus-minus standard errors. Multiple sets of independent sample data were compared using analysis of variance. The Dunnett-t test was used for comparison of samples from the test group with the control group. Data analysis and mapping used Adobe Illustrator CS software, SPSS21.0 (IBM, chicago, IL, USA) and GraphPad Prism 8.3.0 (GraphPad software, san Diego, CA, USA). In the study, P < 0.05 is statistically significant, P < 0.05, P < 0.01, P < 0.001, ns (not statistically significant) indicates no statistical significance. 2.2.15RAA-Cas13a assay procedure extracted HBV DNA was first RAA isothermally amplified to more target HBV DNA sequences, we added T7 RNA polymerase to Cas13a assay system so that T7 transcription could be integrated with Cas13a assay: directly adding RAA amplification product (DNA) into a Cas13a detection system, and simultaneously, T7 is transcribed into ssRNA, and simultaneously, cas13a detection can be carried out. The visualization of the detection result adopts two modes of fluorescence reading and lateral flow test paper. Fluorescence readings allow real-time observation of the change in fluorescence value of the fluorescent probe FAM-BHQ after cleavage, but require a fluorescence collection instrument. The lateral flow test strip method is to visually inspect the detection result according to the strip of the test strip after the Biotin probe FITC-Biotin is cut.
3 results:
3.1.1 establishment of RAA-Cas13 a-based HBV detection System
After alignment of the collected 7720 HBV P-region sequences using Clustal X software, 5 conserved regions with less variation were screened as candidate Cas13a detection targets. The corresponding RAA primers were then designed for each of these 5 conserved regions. As a result of agarose gel electrophoresis, it was found that only primer2 (region 1) was found, and primer3 (region 2) had a better effect and less primer dimer. Thus, the corresponding crrnas (crRNA 1 and crRNA 2) were designed only for the conserved regions region1 and region 2.
Next, in order to make Cas13a system capable of detecting HBV DNA more rapidly and sensitively, we performed Cas13a fluorescence detection on crRNA1 and crRNA2, and according to the fluorescence reading and the time of reaching the plateau of the fluorescence curve, crRNA with better detection efficiency can be screened out for subsequent clinical detection of HBV by Cas13a system, and the result is shown in fig. 1.
3.1.2 analysis of lower detection limits based on RAA-Cas13a HBV detection System
First, we use the lower detection limit to evaluate the newly established HBV RAA-Cas13a detection system to determine how sensitive the newly established detection system is. We used a series of standard gradient diluted HBV DNA plasmids, which were first subjected to RAA isothermal amplification at 37℃for 30min, and then detected by Cas13a fluorescence according to the procedure described above, with the results shown in FIGS. 2 and 3. Fluorescence curve results of crRNA1-Cas13a detection find that the lower detection limit of the Cas13a detection system corresponding to crRNA1 (region 1) can reach 1copy/μl. Moreover, the negative control can be statistically different from the negative control within 10 min; the lower detection limit of the Cas13a detection system corresponding to crRNA2 (region 2) is 10 copies/. Mu.L after 10min of reaction. In addition, the crRNA1-Cas13a system detection reaches the platform phase shorter and the reaction ends faster.
In addition, the detection of lateral flow test strips is carried out on the crRNA1-Cas13a and the crRNA2-Cas13a systems, and the test strip detection result shows that the detection lower limit of the Cas13a detection system corresponding to the crRNA1 can reach 1 copy/mu L, and the detection lower limit of the Cas13a detection system corresponding to the crRNA2 reaches 10 copies/mu L. Thus, the crRNA1-Cas13a combination was selected for subsequent evaluation of HBV DNA clinical samples.
3.1.3 specific analysis based on RAA-Cas13a HBV detection System
First, we performed BLAST analysis of the target sequence in crRNA1, and BLAST results found no other similar sequences except for HBV subtypes. Next, we used the newly established RAA-Cas13a detection system for differential detection of HBV and other hepatitis viruses (HAV, HCV, HDV and HEV). The MEGA7.0 sequence alignment shows that the base difference between HBV sequence and HAV, HCV, HDV and HEV sequences is large. Furthermore, we also experimentally validated the specificity of the RAA-Cas13a system. HAV, HBV, HCV, HDV and HEV were detected separately using the RAA-Cas13a method. The RAA-Cas13a fluorescence detection result shows that the fluorescence signals of other hepatitis viruses basically have no fluctuation except for the larger fluctuation of the fluorescence curve of HBV samples, and the fluorescence value difference between HBV and other hepatitis viruses is larger after 30min of reaction of the Cas13a system. Thus, it can be demonstrated that the RAA-Cas13a system is specific for HBV detection.
3.1.4 clinical verification of RAA-Cas13a HBV detection System
To assess the effectiveness of the newly established RAA-Cas13a detection system in clinical sample detection, we detected 74 clinical serum samples using qPCR and RAA-Cas13a based HBV systems, respectively. And 74 clinical samples were divided into positive (Positive HBV samples) and negative (Negative HBV samples) groups using the qPCR assay results currently most clinically used as gold standard (table 3.2). The detection of HBV by RAA-Cas13a is presented using both fluorescence and lateral flow test strip methods. Following 30min RAA isothermal amplification, cas13a fluorescence detection reactions were performed for 30min according to the above assay conditions. Table 3.1 shows that RAA-Cas13a fluorescence method only requires 60min, with a detection sensitivity of 93.8%, a specificity of 100%, a positive predictive value (PPA) of 100% and a negative predictive value (NPA) of 95.5%. In addition, the area under ROC curve AUC was 0.9375 (95% CI, 0.8536-1), approaching 1, indicating that Cas13a fluorescence detection is more reliable.
Next, RAA-Cas13a lateral flow dipstick detection was performed again on 74 sera, with extension of Cas13a reaction incubation time to 40min and additional addition of RAA products into Cas13a detection system. Finally, cas13a lateral flow test strip detection was performed using the adjusted reaction conditions, and the results in table 3.1 show that the RAA-Cas13a lateral flow test strip method has a detection sensitivity of 90.6%, a specificity of 100%, a positive predictive value (PPA) of 100% and a negative predictive value (NPA) of 91.3% (table 3.1).
TABLE 3.1 comparison of RAA-Cas13a with qPCR detection results
Note that: CI is Confidence intervals, confidence interval
TABLE 3.2 qPCR detection results for 74 clinical samples
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Note that: qPCR quantitative detection was performed using a commercial HBV detection kit (Sansure Biotech).
Sequence listing
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Claims (1)
1. The kit for detecting HBV based on the CRISPR/Cas system is characterized by comprising crRNA shown in SEQ ID NO.3, lwCas13a protein and RAA Primer2, wherein the upstream and downstream Primer sequences of the Primer2 are sequentially shown in SEQ ID NOs.5-6, the kit further comprises a reporting system for reporting the activation condition of the LwCas13a protein, the reporting system is constructed based on a fluorescence method or a lateral flow test strip, the reporting system uses a fluorescent probe or a biotin probe, and the kit further comprises a transcriptase T7 RNA polymerase.
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| CN110184389A (en) * | 2019-06-04 | 2019-08-30 | 中国人民解放军疾病预防控制中心 | Application of the PCR-CRISPR system of crRNA targeting in detection HBV DNA |
| CN110343785A (en) * | 2019-08-05 | 2019-10-18 | 首都医科大学附属北京佑安医院 | Based on PCR-CRISPR-cas13a detects the kit of hepatitis b virus covalence closed cyclic DNA |
| CN111621598A (en) * | 2020-06-12 | 2020-09-04 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper by adopting wire-free method and application thereof in CRISPR nucleic acid detection |
| CN112375847A (en) * | 2020-11-20 | 2021-02-19 | 上海交通大学 | Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system |
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| CN110184389A (en) * | 2019-06-04 | 2019-08-30 | 中国人民解放军疾病预防控制中心 | Application of the PCR-CRISPR system of crRNA targeting in detection HBV DNA |
| CN110343785A (en) * | 2019-08-05 | 2019-10-18 | 首都医科大学附属北京佑安医院 | Based on PCR-CRISPR-cas13a detects the kit of hepatitis b virus covalence closed cyclic DNA |
| CN111621598A (en) * | 2020-06-12 | 2020-09-04 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper by adopting wire-free method and application thereof in CRISPR nucleic acid detection |
| CN112375847A (en) * | 2020-11-20 | 2021-02-19 | 上海交通大学 | Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system |
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| S. Wang et al.."Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system".《Clinical Microbiology and Infection》.2020,第27卷第443-450页和补充实验材料. * |
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