阅读说明：本技术 基于CRISPR/Cas系统的HBV检测 (HBV detection based on CRISPR/Cas system ) 是由 段广才 丁荣华 陈帅印 袁明珠 杨海燕 晋乐飞 郗园林 于 2021-08-04 设计创作，主要内容包括：本发明提供了全新的HBV识别靶点region1、region2,还提供了全新的crRNA。本发明的crRNA,其序列可如SEQ ID NO.3或SEQ ID NO.4所示。本发明还提供了基于CRISPR的试剂盒,其包含任一所述crRNA。本专利检测技术在保证检测灵敏度的前提下,可提高HBV检测的效率,降低检测成本,并且不依赖专业设备和技术人员,具有易推广普及等优势。(The invention provides brand-new HBV recognition targets region1 and region2 and also provides brand-new crRNA. The sequence of the crRNA can be shown as SEQ ID NO.3 or SEQ ID NO. 4. The invention also provides CRISPR-based kits comprising any of the crrnas. The detection technology can improve the detection efficiency of HBV 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.)

The crRNA can identify a target spot shown as SEQ ID NO.1 or SEQ ID NO.2 of HBV.

The sequence of the crRNA is shown as SEQ ID NO.3 or SEQ ID NO. 4.

3. A CRISPR-based kit comprising the crRNA of any preceding claim.

4. The kit of claim 3, further comprising a Cas13a protein.

5. The kit of claim 4, wherein the Cas13a protein is an LwCas13a protein.

6. The kit of any preceding claim, further comprising a RAA Primer3 or Primer2, wherein the upstream and downstream primers of Primer2 are shown in sequence as SEQ ID nos.5-6, and the upstream and downstream primers of Primer3 are shown in sequence as SEQ ID nos. 7-8.

7. The kit of the preceding claim, further comprising a reporter system for reporting the activation status of Cas13a protein, the system being constructed based on colorimetric, fluorescent or lateral flow dipstick.

8. Kit according to the preceding claim, characterized in that the system uses a fluorescent probe or a biotin probe.

9. The kit according to the preceding claim, wherein the kit further comprises a transcription enzyme, such as T7 RNA polymerase.

10. A method for detecting HBV comprising the use of a kit according to any preceding claim.

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 harming human health. By a statistical measure, 68% of those who die from chronic hepatitis B are concentrated in the western pacific and developing countries in Africa. These countries have lagged economic development and may have low detectable rates of HBV due to lagged medical levels.

CRISPR consists of genes encoding Cas-associated proteins and CRISPR arrays, and more CRISPR/Cas systems are emerging and used for nucleic acid detection. Cas13a has two distinct RNA cleavage activities, remains active after cleaving its target RNA, showing indiscriminate cleavage activity.

Clinical qPCR detection HBV technology is mature, and HBV detection by combination of CRISPR/Cas and qPCR is also available, but in regions with laggard medical conditions, the requirement of HBV on-site instant detection cannot be met, and PCR operation is complex and long in time.

Disclosure of Invention

The invention overcomes the defects of the prior art, obtains brand new HBV recognition targets region1 and region2 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 purpose, the invention adopts the technical scheme that:

in a first aspect, the present invention provides a novel crRNA, which can recognize the above-mentioned novel target-sequence of HBV as shown in SEQ ID NO.1 or SEQ ID NO.2, and the specific sequence thereof can be shown in SEQ ID NO.3 or SEQ ID NO. 4.

In a second aspect, the invention correspondingly provides CRISPR-based kits comprising the above crRNA compositions, which may contain a Cas13a protein, such as an LwCas13a protein.

Optionally, the CRISPR-based kit further comprises an RAA Primer3 or a Primer2, wherein the upstream Primer and the downstream Primer in the Primer2 are sequentially shown as SEQ ID NOs.5-6 (NOs are shown in the field as a side-by-side list of sequence numbers), and the upstream Primer and the downstream Primer in the Primer3 are sequentially shown as SEQ ID NOs.7-8.

Optionally, the kit further comprises a reporting system for reporting the activation condition of the Cas13a protein, wherein the system can amplify the signal of Cas protein activation cleavage, and the system can be constructed based on a colorimetry method, a fluorescence method or a lateral flow test strip, and the lateral flow test strip can be a HybriDetect test strip.

Optionally, the system uses a fluorescent probe or Biotin probe, the fluorescent probe can be 5 '6-FAM-UUUUUU-3' BHO, and the Biotin probe can be 5 '-/6-FITC/UUUUU/Biotin/-3'.

Optionally, the kit further comprises a transcription enzyme, such as T7 RNA polymerase.

Optionally, the kit further comprises an rnase inhibitor for inhibiting other possible rnases than the above proteins.

It should be noted that all simple modifications based on the patent idea constitute infringement of the patent, such as: the crRNA is transformed into a fusion molecule fused with other nucleic acid segments, the molecule can be single-stranded RNA or double-stranded DNA, the "other nucleic acid segments" can be sequences which are connected with the upstream of the crRNA and can be recognized and transcribed by RNA polymerase (such as T7 polymerase), such as T7 transcription sequences, a specific fusion example is that T7 sequences are connected with the upstream of the crRNA coding DNA, and the fusion molecule obtains an RNA product of the T7 sequences and the crRNA in tandem through one-time transcription, and specific embodiments of the patent can be seen. Similarly, the RAA primer can also be modified into a fusion molecule, for example, a T7 sequence is connected to the upstream of the primer of SEQ ID NO.5, and the amplification product of the fusion molecule is transcribed once to obtain ssRNA in which the T7 sequence is connected with region1 in series, as shown in the detailed description of the present patent.

Accordingly, the present invention provides a method for detecting HBV using the above-mentioned nucleic acid or a kit form thereof, which can be used for non-diagnostic purposes.

The invention has the beneficial effects that: the research provides a brand new HBV detection target spot and crRNA, the HBV detection technology of the invention 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 in Cas13a detection system.

FIG. 2 is a fluorescence curve of the crRNA1 of the present patent for HBV detection at different DNA concentrations.

FIG. 3 is a fluorescence curve of the crRNA2 of this patent for HBV detection at different DNA concentrations.

Detailed Description

Acronyms in English that may be involved

2 materials and methods

Materials/methods mentioned in the detailed description are conventional in the art, and the acronyms refer to the table above, unless otherwise noted.

2.1 part of Experimental materials notes

2.1.1HBV Standard plasmid Synthesis

Standard plasmids of the HBV P coding region (GenBank: MN683731.1) were recombined with the HBV P sequence using a PUC57 vector plasmid (Shanghai Biosynthesis).

2.1.2 reagent LwCas13a protein as compared with Cas13a protein disclosed in the literature (Cas13a from Leptotrichia wadei)

2.2 Experimental procedures and methods

2.2.1 Collection and sequence alignment of HBV sequences

Collecting all HBV sequences, using Clustal X biological information software to carry out sequence alignment on the collected HBV sequences, using MEGA7.0 software to screen conserved regions with relatively few mutations for visualization of sequence alignment results, and using the screened conserved regions as target regions for subsequent Cas13a detection and design regions of universal isothermal amplification primers.

2.2.2 clinical serum HBV DNA nucleic acid extraction

Clinical serum sample extraction of HBV DNA the experimental procedures were carried out using the HiPure Viral DNA Kits column virus DNA extraction kit (Meiji organism) according to the general procedure.

2.2.3 design and screening of isothermal amplification primers

After the above sequence alignment, we use the conserved region as a candidate target sequence for the subsequent Cas13a detection. Primers designed for recombinase-mediated strand-displacement nucleic acid amplification technology (RAA method) were then performed at conserved regions upstream and downstream of the candidate Cas13a target sequence. Isothermal amplification primers were designed using Primer-BLAST and Oligo7.0 in NCBI according to the standards required in the RAA nucleic acid amplification reagents (basic types) instructions.

The RAA primers were designed as shown in Table 2.5. Then 5 pairs of designed primers are subjected to RAA isothermal amplification for 30min at 37 ℃, and then an agarose gel electrophoresis experiment is carried out. Agarose gel electrophoresis test was performed using a prepared 1.5% agarose gel. Settings for model DYY-6C electrophoresis apparatus are voltage U: 120V, time T: and (3) 30 min. The gel was imaged after gel electrophoresis using a Gene Genius Bio gel imaging system. The optimal RAA primer was then screened for subsequent RAA-Cas13a detection based on the electrophoretic bands. And (3) screening out a primer with the optimal amplification effect according to the band of the electrophoresis gel as a primer for RAA amplification in subsequent detection. In addition, since the Cas13a system detects RNA, we skillfully added T7 sequence to the RAA upstream primer, which was transcribed into single-stranded RNA (ssrna) by the subsequent T7 RNA polymerase.

TABLE 2.5 primer sequences for isothermal amplification of RAA in this study (T7 sequences added before the upstream primer)

2.2.4 recombinase mediated strand displacement isothermal amplification (RAA)

Isothermal amplification of HBV DNA will be performed using the basic RAA nucleic acid amplification kit (well-known in Hangzhou). The amplification system in table 2.6 was prepared according to the instructions of the reagents, and the prepared mixture was incubated in a constant temperature metal bath at 37 ℃ for 30-60 min. The amplified HBV product is placed on ice or in a refrigerator at-20 ℃ for subsequent Cas13a detection.

TABLE 2.6 RAA isothermal amplification sample application System

2.2.5 principles of the design of crRNA

The designed crRNA can be connected with LwCas13a protein and can be complementarily paired with a target sequence of HBV, a T7 sequence is additionally added at the upstream of the designed crRNA sequence, and the fused total sequence is shown in the table in the following section, so that the Cas13a detection experiment is convenient.

2.2.6 Experimental procedure for the Synthesis of crRNA

The optimal RAA primers were selected by agarose gel electrophoresis as described above, and two corresponding crRNAs were designed according to the criteria for the crRNAs (Table 2.7). In view of the fact that direct RNA synthesis is not necessary, we have used In Vitro Transcription (IVT) from a DNA template of crRNA. Therefore, we add T7 sequence at the upstream of the crRNA, which makes the crRNA gene fragment transcribe into single-stranded crRNA under the subsequent action of T7 RNA polymerase.

TABLE 2.7 CRRNA designed in this study and templates for the synthesis of crRNA (in this table, the crRNA is added with T7 sequence upstream)

The specific crRNA synthesis test comprises the steps of 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 conditions are 95 ℃ and 10 min. And (3) closing the PCR instrument after the reaction is finished, and naturally cooling the PCR instrument to room temperature, so that the double-stranded DNA of the crRNA and the T7 which are subjected to complementary pairing is slowly annealed, extended and fused.

2.2.7 transcription of T7 from crRNA after Synthesis

Next, the double-stranded crRNA DNA synthesized in the previous step was subjected to HiScribe using a kit^TMT7 Quick High Yield RNA Synthesis Kit (NEB, E2050S) for T7 transcription, and preparing T7 transcription mixture according to the steps of the instruction 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 the transcription of double-stranded crRNA DNA into single-stranded crRNA.

2.2.8 purification of crRNA after transcription

However, the crRNA transcribed by T7 may contain impurities (the mixture may contain some untranscribed DNA or the reagents in the above steps), so we purify the RNA of the transcribed product of T7 to obtain crRNA with better purity and concentration, and we use the kitThe RNA Cleanup Kit (NEB) was used for crRNA purification.

2.2.9 evaluation of the detection efficiency of crRNA

The purified crRNA was diluted to 300 ng/. mu.L and 30 ng/. mu.L using a RNase-free water gradient for subsequent detection of Cas13 a. Before proceeding with Cas13a detection, we need to assess the detection efficiency of crRNA: the HBV standard plasmid (concentration 10) was first paired with RAA primer⁸copies/. mu.L) and then adding the RAA amplification product to the crRNA-Cas13a mixture. The preparation of the mixed solution of crRNA-Cas13a was performed according to the CRISPR/Cas13a detection system shown in Table 2.10, and then the mixed solution was placed in 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 the fluorescence expression quantity (RFU) after the detection of the crRNA-Cas13a and the early and late arrival of the plateau phase 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: first, HBV DNA is isothermally amplified according to the RAA amplification test procedure and conditions described above, and then the amplified RAA product is added to a crRNA-Cas13a mixed solution configured according to the CRISPR/Cas13a detection system in Table 2.10. Visualization of the Cas13a test results was then presented using both fluorescence readings and lateral flow test strip tests.

For the Cas13a fluorescence readout, we used the ABI Fast 7500qPCR system (Applied Biosystems) to collect the fluorescence emitted by the nonspecific shear fluorescent probe (FAM-UUUUUU-BHO) after Cas13a protein activation. Also we can do this using a portable fluorescence collector.

The lateral flow dipstick method was carried out using a HybriDetect dipstick (Milenia Biotec, Germany) with a Biotin probe of 5 '-/6-FITC/UUUUUU/Biotin/-3'. The lateral flow test strip detects the probe because one end is fluorescein and the other end is biotin. Streptavidin at the first line (Control line) of the lateral flow strip will bind to the organisms on the probes and will then bind to the cleaved biotin ends and capture all intact uncleaved probes. Gold Nanoparticle (NPs) -labeled anti-FITC antibodies will produce a deep purple band visible to the naked eye upon binding to FITC. If the FITC-Biotin RNA probe is not cleaved (negative detection), a dark purple band is left on the first line. When the FITC-Biotin RNA probe is cleaved (test positive), the gold particle-labeled antibody will follow the flow to the second line (Testing line) due to the presence of the target and the attached activity. The second line of the strip is labeled with a secondary antibody that captures all antibodies, forming a dark purple band, the color of the second band indicating the presence of the pathogen of interest and the extent to which the probe is cleaved. In addition, the lateral flow dipstick method requires incubation at 37 ℃ for 30-60min in a metal bath before performing the test.

TABLE 2.10 CRISPR/Cas13a assay sample application System

2.2.11 RAA-Cas13a system sensitivity analysis for detecting HBV DNA

And (3) carrying out gradient sensitivity analysis on the newly established RAA-Cas13a system, and evaluating the lower detection limit of the RAA-Cas13 a. First, a series of diluted HBV standard plasmids are used to perform RAA isothermal amplification, and then the amplified products are used to perform fluorescence method and lateral flow test strip detection of Cas13 a. The procedure was followed as described previously for the experimental procedure. And then determining the lower limit of the RAA-Cas13a detection according to the fluorescence curve and the test strip detection result.

2.2.12 RAA-Cas13a specific assay for detecting HBV DNA

To ensure that only HBV can be detected and no other pathogens can be detected affecting the assay result. First, we perform bioinformatics analysis, and perform BLAST analysis on the target sequence in crRNA to see if there are similar sequences in other pathogens except HBV subtypes. If the sequences are similar and have fewer different bases, the target sequence of HBV-Cas13a is redesigned, and finally, a specific target sequence is obtained. In addition, other hepatitis viruses including Hepatitis A Virus (HAV), Hepatitis C Virus (HCV), Hepatitis D Virus (HDV) and Hepatitis E Virus (HEV) are clinically and frequently tested for differential diagnosis of hepatitis B. HBV and other hepatitis identification detection are carried out simultaneously by using a sequence alignment method and an HBV-Cas13a fluorescence detection method.

2.2.13 clinical specimen 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. Nucleic acid extraction from sample sera was performed using the HiPure Viral DNA Kits column virus DNA extraction kit (magenta) following the above described protocol. The extracted nucleic acid and the remaining serum sample were placed in a-80 ℃ freezer for use. In order to avoid interference of subjective factors on the detection results, clinical serum samples were subjected to the RAA-Cas13a detection and the qPCR detection, respectively, in a blind assay format. Detection of RAA-Cas13a follows the previously described steps. The qPCR Assay was performed using a commercial HBV detection Kit (HBV Nucleic Acid Assay Kit, Sansure Biotech) according to the procedures and standards of the specification. And the effect of the RAA-Cas13 assay was evaluated using qPCR assay as gold standard. In addition, sensitivity (sensitivity), specificity (specificity), Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Receiver Operating Characteristic (ROC) curves will be used to evaluate the efficiency of RAA-Cas13a detection. For the definition of detecting HBV positive samples based on the RAA-Cas13a fluorescence method, we set a signal-to-noise ratio parameter (ratio of fluorescence value of sample to negative control, S/N). The signal-to-noise ratio S/N >3 of the fluorescence value at the 30min time point of Cas13a reaction was taken and defined as 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 data are expressed by adding or subtracting standard errors by means of average numbers. Multiple sets of independent sample data comparisons use analysis of variance. The Dunnett-t test was used to compare samples from the test group with the control group. Data analysis and mapping used Adobe Illustrator CS 6 software, SPSS21.0(IBM, Chicago, IL, USA) and GraphPad Prism 8.3.0(GraphPad software, San Diego, CA, USA). In this study, P < 0.05 was statistically significant, P < 0.05, P < 0.01, P < 0.001, and ns (not statistical significant) indicated no statistical significance.

2.2.15 RAA-Cas13a test protocol the extracted HBV DNA is RAA-amplified isothermally to give more target HBV DNA sequence, and we add T7 RNA polymerase to the Cas13a detection system to integrate the T7 transcription with the Cas13a detection: the RAA amplification product (DNA) is directly added into a Cas13a detection system, and the T7 is transcribed into ssRNA, and the Cas13a detection can be carried out at the same time. The visualization of the detection result adopts two modes of fluorescence reading and lateral flow test strip. The fluorescence reading can be used for observing the change of the fluorescence value of the fluorescent probe FAM-BHQ after being cut in real time, but a fluorescence collecting instrument is required. The lateral flow test strip method is characterized in that a visual detection result is carried out on a test strip display strip after a Biotin probe FITC-Biotin is cut.

3, results:

3.1.1 establishment of an HBV detection System based on RAA-Cas13a

After aligning 7720 collected HBV P regions by using Clustal X software, 5 conservative regions with less variation are selected as candidate Cas13a target regions for detection. Corresponding RAA primers were then designed for each of these 5 conserved regions. After agarose gel electrophoresis test, only primer2 (conserved region1) and primer3 (conserved region2) are found to have better amplification effect and less primer dimer. Thus, the corresponding crrnas (crRNA1 and crRNA2) were designed only for the conserved regions region1 and region 2.

Next, in order to enable the Cas13a system to detect HBV DNA more quickly and sensitively, we perform Cas13a fluorescence detection on crRNA1 and crRNA2, and screen out crRNA with better detection efficiency according to the fluorescence reading and the time of reaching the plateau phase of the fluorescence curve for the subsequent clinical detection of HBV by the Cas13a system, and the results are shown in fig. 1. 3.1.2 analysis of the lower detection limit based on the RAA-Cas13a HBV detection System

First, we evaluated the newly established HBV RAA-Cas13a detection system using the lower detection limit to determine how sensitive the newly established detection system is. A series of standard gradient diluted HBV DNA plasmids are subjected to RAA isothermal amplification for 30min at 37 ℃, and then the detection is carried out by using a Cas13a fluorescence method according to the steps, and the results are shown in FIGS. 2 and 3. As a result of a fluorescence curve detected by the crRNA1-Cas13a, the detection limit of a Cas13a detection system corresponding to the crRNA1(region1) can be up to 1 copy/. mu.L. In addition, the statistical difference can be obtained from the negative control within 10 min; the Cas13a detection system corresponding to crRNA2(region2) has a lower detection limit of 10 copies/. mu.L after reaction for 10 min. In addition, the detection of crRNA1-Cas13a system has shorter plateau reaching period and faster reaction ending.

In addition, the lateral flow test strip is also used for detecting the crRNA1-Cas13a system and the crRNA2-Cas13a system, the test strip detection result is basically the same as that of the Cas13a detection system corresponding to crRNA1, the detection limit is up to 1 copy/muL, and the detection result shows that the Cas13a system corresponding to crRNA2 is detected, and the detection limit is up to 10 copies/muL. Therefore, subsequent assessment of HBV DNA clinical samples selected a combination of crRNA1-Cas13 a.

3.1.3 specificity assays based on the RAA-Cas13a HBV detection System

First, we performed BLAST analysis of the target sequence in crRNA1, and the BLAST results revealed that no similar sequences were found except for HBV subtypes. Next, we performed differential detection of HBV and other hepatitis viruses (HAV, HCV, HDV and HEV) using the newly established RAA-Cas13a detection system. The results of the MEGA7.0 sequence alignment show that the HBV sequences have a large base difference from the HAV, HCV, HDV and HEV sequences. Furthermore, we also experimentally verified the specificity of the RAA-Cas13a system. HAV, HBV, HCV, HDV and HEV were detected using the RAA-Cas13a method, respectively. The fluorescence detection result of RAA-Cas13a shows that the fluorescence signals of other hepatitis viruses have no fluctuation basically except the fluorescence curve of the HBV sample, and the fluorescence value difference between HBV and other hepatitis viruses is larger after the reaction of the Cas13a system for 30 min. Thus, it can be shown that the RAA-Cas13a system is specific for HBV detection.

3.1.4 clinical validation of the RAA-Cas13 a-based HBV detection System

To evaluate the effectiveness of the newly established RAA-Cas13a test system in clinical sample testing, we tested 74 clinical serum samples using qPCR and the RAA-Cas13 a-based HBV system, respectively. And 74 clinical samples were divided into Positive group (Positive HBV samples) and Negative group (Negative HBV samples) with qPCR assay result most commonly used in clinic at present as gold standard (table 3.2). The RAA-Cas13a detection of HBV is presented by using two modes of a fluorescence method and a lateral flow test strip method. After 30min RAA isothermal amplification, Cas13a fluorescence detection reaction was performed for 30min according to the above experimental conditions. The results in table 3.1 show that the RAA-Cas13a fluorescence method only requires 60min, with a detection sensitivity of 93.8%, specificity of 100%, positive predictive value (PPA) of 100%, and negative predictive value (NPA) of 95.5%. In addition, the area AUC under the ROC curve is 0.9375 (95% CI,0.8536-1), which is close to 1, and thus, the fluorescence detection of Cas13a is true and reliable.

Next, 74 sera were subjected to the RAA-Cas13a lateral flow strip test again, with an extension of Cas13a reaction incubation time to 40min and an additional addition of RAA product to the Cas13a test system. Finally, Cas13a lateral flow strip assay was performed using the adjusted reaction conditions, and the results in table 3.1 show that the RAA-Cas13a lateral flow strip assay had 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 RAA-Cas13a comparison with qPCR detection results

Note: CI is Confidence intervals

TABLE 3.274 clinical specimens qPCR test results

Note: qPCR quantitative detection was performed using a commercial HBV detection kit (Sansure Biotech).

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