A CRISPR-Cas12a system and detection method for visual detection of HIV

Abstract

The present invention discloses a CRISPR‑Cas12a system and a detection method for visual detection of HIV, and belongs to the field of virus detection technology. It includes Cas12a protein, crRNA and fluorescent reporter molecules; the sequence of the crRNA is shown in SEQ ID NO.1 or SEQ ID NO.2. The present invention finds a target site for HIV nucleic acid detection based on the CRISPR / Cas12a system, and HIV nucleic acid detection can be achieved by using the CRISPR / Cas12a system for the site, with good detection specificity, high sensitivity, better specificity and compatibility, low detection cost, convenient and fast operation. The detection limit of the present invention can reach 1 copies / μL, realizing single molecule detection of the target.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a CRISPR-Cas12a system and a detection method for visually detecting HIV. Background Art

[0002] The main transmission routes of HIV include sexual contact (including homosexual, heterosexual and bisexual contact), blood and blood products (including shared needles for intravenous drug use, invasive medical procedures, etc.) and mother-to-child transmission (including prenatal, intravenous and postnatal). The general population is susceptible to HIV.

[0003] HIV belongs to the human lentivirus group in the genus Lentivirus of the family Retroviridae. It is a spherical particle with a diameter of about 100 to 120 nm, consisting of two parts: the core and the envelope. The core includes two single-stranded RNA chains, core structural proteins, and enzymes necessary for viral replication, including reverse transcriptase (RT, P51 / P66), integrase (INT, P32), and protease (PI, P10). Outside the core are viral capsid proteins (P24, P17). The outermost layer of the virus is the envelope, which contains outer membrane glycoproteins (gp120) and transmembrane glycoproteins (gp41).

[0004] According to the differences in HIV genes, it is divided into HIV-1 and HIV-2, and the homology of the amino acid sequence between the two subtypes is 40% to 60%. Currently, HIV-1 is the main one prevalent in the world. HIV-1 can be further divided into different subtypes, including the M subtype group (the main subtype group), the O subtype group and the N subtype group, among which the M group has 11 subtypes: A, B, C, D, E, F, G, H, I, J, and K. In addition, several popular recombinant types have been discovered in recent years. The biological characteristics of HIV-2 are similar to those of HIV-1, but its infectivity is lower, and the clinical progression of AIDS caused by it is slower and the symptoms are milder.

[0005] HIV-1 is the main prevalent strain in my country, and eight subtypes have been found, including A, B (European and American B), B' (Thailand B), C, D, E, F and G, as well as different prevalent recombinant types. According to the fourth molecular epidemiological survey, there are four main dominant strains in my country. These four dominant HIV-1 viruses accounted for 89.3% of all HIV-infected people in 2015, namely CRF07_BC (41.3%), CRF01_AE (32.7%), CRF08_BC (11.3%) and B subtype (4.0%). There are differences in the distribution of subtypes in different regions. The prevalent strains in Northwest China and Central China are relatively simple. About 86.7% of the prevalent strains in Northwest China are CRF07_BC subtypes, while the absolute dominant strain in Central China is B' subtype, accounting for about 90.7%. There are three subtypes of HIV-1 strains prevalent in Northeast China, North China, East China, and South China, with CRF01_AE being the main subtype, followed by CRF07_BC and B' subtypes, and CRF08_BC accounting for only 5%. The subtype distribution of HIV-1 strains in Southwest China is more complex, with CRF08_BC and CRF01_AE subtypes being the main subtypes, followed by CRF07_BC subtype, and other subtypes (mostly unique recombinant subtypes, URFs) also accounting for a large proportion.

[0006] The distribution of strains in different transmission populations is also different. CRF01_AE is mainly prevalent in sexually transmitted populations, but it is also prevalent to a certain extent in drug users; CRF07_BC subtype and CRF08_BC subtype are mainly prevalent in drug users, CRF07_BC is also prevalent to a certain extent in heterosexual transmission populations and MSM populations, and CRF08_BC is also prevalent in heterosexual transmission populations in the southwest region; B' subtype is mainly prevalent in people who used to donate blood for compensation.

[0007] Recombinase-mediated strand replacement nucleic acid amplification technology (RAA technology) is a rapid nucleic acid amplification technology at a constant temperature. It uses recombinases obtained from bacteria or fungi. At room temperature, the recombinase can tightly bind to the primer DNA to form a polymer of enzyme and primer. When the primer searches for a complementary sequence that completely matches it on the template DNA, it opens the double-stranded structure of the template DNA with the help of single-stranded DNA binding proteins, and forms a new complementary DNA chain under the action of DNA polymerase, and the amplified product grows exponentially.

[0008] At present, the CRISPR-Cas system represented by SpCas9 has been widely used in gene, epigenome and base editing of eukaryotic cells, and many newly identified Cas effector nucleases with unique properties have also been successfully used in genome engineering tasks. Among them, the Cas12a protein family has several outstanding advantages suitable for genetic engineering tasks. This family is represented by As-(Acidaminococcus sp.) and Lb-(Lachnospiraceae bacterium)Cas12a nucleases. These features include (1) using a single shorter crRNA; (2) recognizing T-rich PAM sequences; (3) cleaving the target DNA at the distal position of the PAM; (4) generating protruding DNA ends after cleavage; and (5) having indiscriminate single-stranded DNase activity after target binding.

[0009] By combining the isothermal amplification of DNA by RAA technology, the nuclease characteristics of Cas12a, and fluorescent probes, isothermal viral DNA detection without complex instrument dependence is achieved. crRNA guides Cas12a to recognize the target viral DNA fragments amplified by RAA, activates the indiscriminate single-stranded DNase activity of Cas12a, cuts the fluorescent probe and the target DNA fragment, and combines blue light or ultraviolet light to achieve visual detection of viral DNA.

[0010] Currently, HIV testing can be divided into two categories: antibody testing and viral testing. Viral testing includes cell culture (virus isolation), p24 antigen testing and viral nucleic acid testing.

[0011] Antibody testing consists of initial screening and confirmation tests. Initial screening tests require high sensitivity to avoid missed detections. The enzyme-linked immunosorbent assay (ELISA) method has a certain degree of sensitivity and is simple and fast to operate, suitable for the detection of a large number of samples. Therefore, it is currently the most commonly used initial screening test method in clinical practice. There are three confirmation test methods internationally, including immunoblotting, strip immunoassay, and immunofluorescence test. Currently, the immunoblotting test is the most commonly used. In addition, in recent years, researchers have also begun to study methods for detecting HIV antibodies in saliva and urine.

[0012] There is a 3-8 week window period for antibody testing. During the window period, viral antibodies cannot be detected, but virus-related antigens or isolated viruses can be detected. Antigens can be detected 2-18 days before seroconversion after an individual is infected. Therefore, there is a great advantage in detecting p24 antigen during the seroconversion period, and it can be used as a method for early auxiliary diagnosis of HIV infection. The US FDA has stipulated that from August 1995, blood donors and blood products must be tested for HIV-1 P24 antigen as a supplement to anti-HIV testing. Virus culture is the most accurate method for detecting HIV infection, and HIV diagnosis is generally carried out by culturing peripheral blood mononuclear cells (PBMC).

[0013] Viral nucleic acid testing usually reflects the viral load by detecting HIV RNA levels. It has high sensitivity and uses real-time fluorescent polymerase chain reaction (PCR) technology to detect viral nucleic acid in the first two weeks of HIV infection. Viral nucleic acid testing methods can be used for early diagnosis of HIV, such as auxiliary diagnosis during the window period, disease course monitoring, guiding treatment plans and efficacy determination, and predicting disease progression. Currently, commonly used testing methods include reverse transcription PCR experiments (RT-PCR), nucleic acid sequence amplification experiments (NASBA), branched DNA hybridization experiments (bDNA), etc.

[0014] The cell culture method is highly specific for HIV detection and will not produce false positives. It is of great significance for confirming whether individuals with uncertain antigen / antibody tests and newborns of positive mothers are infected with HIV. However, the virus culture method requires a certain number of infected cells to culture and isolate the virus. Therefore, it has poor sensitivity, long operation time, complex operation, must be performed in a specific P3 laboratory, and has high costs (each culture costs about 200 to 500 US dollars). Therefore, it is not suitable for clinical use.

[0015] The p24 antigen test can detect soluble p24 antigen in the blood after the virus begins to replicate, but it is prone to false positives, which may be due to interference from other substances and the influence of complex formation with antibodies. Therefore, a positive result must be confirmed by a neutralization test before the result can be used as an auxiliary diagnosis basis for HIV infection. A negative HIV-1 P24 antigen test only means that there is no reaction in this test and cannot rule out HIV infection.

[0016] Viral nucleic acid detection methods have high sensitivity and are very important for monitoring disease progression, observing antiviral efficacy, and monitoring drug resistance. However, due to the diversity of HIV genes, no set of primers can cover all HIV sequences, which limits the sensitivity of the test. In addition, existing viral nucleic acid detection methods are either expensive in detection instruments and reagents, or complex in operation and require high operator requirements. They are difficult to promote in general laboratories, are not suitable for rapid detection of a large number of patients, and are also not suitable for widespread clinical applications.

[0017] Therefore, improving the sensitivity and specificity of detection, shortening the window period, and making it simple, rapid and cost-effective have become the requirements and directions for the development of HIV detection technology. Many studies are dedicated to finding alternative technologies for virus detection. Summary of the invention

[0018] The object of the present invention is to provide a CRISPR-Cas12a system and a detection method for visually detecting HIV to solve the problems existing in the above-mentioned prior art.

[0019] To achieve the above object, the present invention provides the following solutions:

[0020] One of the technical solutions of the present invention is a CRISPR-Cas12a system for visual detection of HIV, including Cas12a protein, crRNA and fluorescent reporter molecules;

[0021] The sequence of the crRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0022] The second technical solution of the present invention is a method for detecting HIV for non-disease detection or treatment purposes, which uses the CRISPR-Cas12a system to detect the sample to be tested to determine whether it contains HIV.

[0023] Based on the above technical solution, the present invention has the following technical effects:

[0024] The present invention discovers an HIV nucleic acid detection target site based on the CRISPR / Cas12a system. HIV nucleic acid detection can be achieved by using the CRISPR / Cas12a system for the site. The detection has good specificity and high sensitivity, and can have better specificity and compatibility, low detection cost, and convenient and quick operation.

[0025] 1. The detection limit of this method can reach 1 copies / μL, realizing single-molecule detection of the target.

[0026] 2. The degenerate RAA primers and crRNA in this method have high coverage of HIV strains, high conservatism, and strong specificity. After testing, the present invention can cover multiple classic strains of HIV-1 and domestic prevalent strain genotypes.

[0027] 3. The degenerate RAA primers and crRNA in this method have strong specificity for HIV detection and can effectively avoid interference of other viruses on the test results.

[0028] 4. The HIV detection method proposed in the present invention has the advantages of being rapid, accurate, and convenient, and does not rely on expensive detection instruments, which is conducive to popularization in grassroots testing institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 Schematic diagram of HIV virus detection principle based on CRISPR-Cas12a system;

[0031] Figure 2 The schematic diagram of the structure of the synthetic construct clone NL4-3_mutant_LDNA;

[0032] Figure 3 To determine the effectiveness of crRNA16 and crRNA88;

[0033] Figure 4 Determination of the optimal temperature for Cas12a cleavage of RAA products;

[0034] Figure 5 To determine the optimal time for RAA amplification;

[0035] Figure 6 The specificity of crRNA16 and crRNA88 for HIV detection;

[0036] Figure 7 The sensitivity of crRNA16 and crRNA88 in detecting HIV;

[0037] Figure 8 The sensitivity of QPCR method for HIV detection;

[0038] Fig. 9The detection sensitivity of crRNA16 and crRNA88 for three domestic prevalent strains (CRF07_BC, B' and CRF08_BC). DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0045] The embodiment of the present invention provides a CRISPR-Cas12a system for visual detection of HIV, including Cas12a protein, crRNA and fluorescent reporter molecules;

[0046] The sequence of the crRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0047] In some specific embodiments, the fluorescent reporter molecule is a single-stranded DNA oligodeoxynucleotide, and its nucleotide sequence is shown in SEQ ID NO.7; it has a fluorescent FAM at one end and a BHQ at the other end.

[0048] The embodiment of the present invention provides a method for detecting HIV for non-disease detection or treatment purposes, using the CRISPR-Cas12a system to detect a sample to be tested to determine whether it contains HIV.

[0049] In some specific embodiments, before the CRISPR-Cas12a system is used to detect the sample to be tested, a process of amplifying the sample to be tested is also included.

[0050] In some specific embodiments, the amplification is RAA amplification; and the RAA amplification time is 40 min.

[0051] In some specific embodiments, the amplification primers for RAA amplification consist of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0052] In some specific embodiments, the amplification primers for RAA amplification consist of an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6.

[0053] In some specific embodiments, the use of the CRISPR-Cas12a system to detect the sample to be tested specifically comprises: using the CRISPR-Cas12a system to perform an enzyme cleavage reaction on the sample to be tested;

[0054] The temperature of the enzyme cleavage reaction is 45°C.

[0055] In some specific embodiments, the reaction system of the detection method is 25 μL: Cleavage Buffer 2.5 μL, Cas12a protein 0.5 μL, crRNA 0.3 μL, fluorescent reporter molecule 1 μL, sample to be tested 5 μL and H2O15.7 μL.

[0056] In some specific embodiments, the method for determining whether HIV is present is: using a blue light gel cutting instrument to read, with the negative control as a reference, and observing a luminescent group is positive;

[0057] Or dilute the reaction result to 300 μL with nuclease free water, and then use a fluorescence spectrophotometer to detect the fluorescence intensity at 490 nm for excitation light and 520 nm for emission light. When the fluorescence intensity value of the sample to be tested is more than 3 times higher than that of the negative control, it is judged as a positive result.

[0058] Or at any time, the fluorescence intensity is greater than or equal to 600 a.u., which can be judged as a positive result.

[0059] In some specific embodiments, the negative control is obtained by replacing the RAA product used as a template in Table 5 with Nuclease free water, while keeping other components unchanged, that is, a negative control.

[0060] Example 1 Design and screening of the best candidate crRNA that can stably bind to Cas12a and viral RNA

[0061] First, the sequences of the classic HIV strains NL4 and HXB2 and the popular CRF01_AE, CRF07_BC, B', and CRF08_BC strains in my country were obtained from the database HIV Database (https: / / www.hiv.lanl.gov / content / index). The sequences were aligned by Snapgene, and the relatively conserved regions in the sequences of these strains were selected, mainly located in the Gag and Pol regions of HIV. A crRNA library was designed using the online design tool Benchling (https: / / www.benchling.com), with the human genome sequence as the off-target evaluation object, and crRNAs with off-target scores greater than 90 points (the full score is 100, and the lower the score, the higher the off-target possibility) were selected. Subsequently, CRISPR-DT was used to evaluate the effectiveness of the candidate crRNAs, and crRNAs with effectiveness scores greater than 0.9 (the full score is 1) were selected. The higher the score, the better the stability of the crRNA and Cas12a complex, the higher the cutting activity, and the crRNA with the highest docking score was considered to be the potential best crRNA. The designed crRNA was synthesized by Huzhou Hippo Biotechnology Co., Ltd.

[0062] Table 1 Screening of crRNA

[0063]

[0064] After testing, the crRNA designed by the present invention can target and bind to NL4, HXB2, NDK in the classical HIV strains and CRF01_AE, CRF07_BC and B', CRF08_BC, which are prevalent in China.

[0065] Example 2 Establishment of HIV virus detection system based on CRISPR-Cas12a system

[0066] The detection principle of the CRISPR-Cas12a detection system is as follows Figure 1 shown.

[0067] 1. Preparation of DNA samples.

[0068] The sequence from the HIV classical strain Synthetic construct clone NL4-3_mutant_L, the structure diagram is shown in Figure 2 The plasmid was transformed into Stb13 bacteria, and the plasmid was purified using TIANprep Mini Plasmid Kit. The band was verified by enzyme digestion and gel running. The plasmid concentration was 1715.9 ng / μL and OD260 / 280 was 1.80 measured by Nanodrop. The plasmid was stored at -20°C.

[0069] Use the formula: copies / μL = 6.02×10 23 ×(ng / μL)×10 -9 / DNA Length×330, the DNA copy number is 1.5×10 13 copies / μL.

[0070] 2. Design of RAA amplification primers

[0071] The design requirements of RAA technology primers are extremely strict. The replacement or addition or subtraction of individual bases will have a significant impact on the experimental results. Only after experimental verification and testing can the available primers be screened. The design principles are: primer length is 25-35nt, amplicon size is 100-200bp, and Tm value is between 54℃-67℃. The primer sequences are shown in Table 2 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0072] Table 2 RAA amplification primers

[0073]

[0074]

[0075] 3. Design of signal reporter molecules

[0076] Because Cas12a has collateral cleavage activity, after identifying the target DNA, it continues to cut other non-target DNAs, so a short single-stranded DNA oligodeoxynucleotide can be designed as a fluorescent reporter molecule, with a fluorescein (such as FAM) at one end and BHQ at the other end, as shown in Table 3.

[0077] Table 3 Reporter molecule sequences

[0078]

[0079] 4. Obtaining RAA amplification products

[0080] Using the DNA sample in step 1 as a template, perform RAA amplification using the primers designed in step 2 to obtain RAA amplification products. Configure the RAA amplification reaction system as shown in Table 4, add 1.0 μL of the DNA sample obtained in step 1 to the detection unit tube, cover the tube, mix and centrifuge (Note: Whether "sufficient mixing" in this step will determine the repeatability of the test results); place the reaction unit tube in a 37°C constant temperature water bath and incubate for 1 hour.

[0081] Table 4 RAA amplification basic system

[0082]

[0083] 5. Preparation of CRISPR-Cas12a detection system

[0084] Take 5 μL of the RAA amplification product obtained in step 3 as a template, and prepare the CRISPR-Cas12a detection system according to Table 5. Cleavage Buffer is prepared in the laboratory, LbCas12a protein is purchased from Bio-Lifesci (http: / / www.bio-lifesci.com / ), and the RAA product used as a template in Table 5 is replaced with Nuclease free water, and the other components are kept unchanged, which is a negative control.

[0085] Table 5 Cas12a detection reaction system

[0086]

[0087] 6. Reading of test results

[0088] When crRNA is complementary to the target DNA, the collateral cleavage activity of Cas12a is activated, the ss-DNA connecting FAM and the quenching group is broken, and the two are separated, and observable fluorescence can be emitted under blue light or ultraviolet light. The results of this technology can be read using a blue light gel cutting instrument, with the negative control as a reference, and the luminescent group observed is positive, or the reaction result can be diluted to 300 μL with nuclease free water, and then the fluorescence intensity is detected at 490 nm of excitation light and 520 nm of emission light using a fluorescence spectrophotometer (Shimadzu Spectro fluorophotometer RF-6000). When the fluorescence intensity value of the experimental group is more than 3 times higher than the fluorescence intensity value of the negative control, it is judged as a positive result, or at any time, the fluorescence intensity is greater than or equal to 600 a.u. (3 times the highest value that the fluorescence intensity of the negative control can reach) It can be judged as a positive result, such as Figure 3 shown.

[0089] Example 3Cas12a optimal cutting temperature determination

[0090] After the crRNA designed in Example 1 was synthesized by the company, 1 μM of the standard sample was used as the test object, RNasefree water was used as the negative control, and the Cas12a cleavage reaction system was configured according to Table 5. After the addition of the sample was completed, the PCR tube containing the prepared reaction system was placed in a fluorescent quantitative PCR instrument, and the channel excitation light wavelength was set to 490 nm, the emission light wavelength was 520 nm, the temperature was 31.4 ° C, 34.5 ° C, 39.1 ° C, 45 ° C, and 49.7 ° C. The fluorescence intensity value was read every 0.5 min, 80 times, a total of 40 min, and 3 groups were repeated at different times.

[0091] The results are as follows Figure 4 As shown, when the temperature is 45°C, Cas12a cleaves the RAA product fastest and produces the strongest fluorescence signal, so 45°C is taken as the subsequent experimental condition.

[0092] Example 4 Screening of crRNA and determination of RAA amplification time

[0093] After the crRNA designed in Example 1 was synthesized by the company, a standard sample with a concentration of 10 copies / μL was used as the test object, RNase free water was used as the negative control, and the system was configured according to the RAA amplification basic system in Table 5. After the sample was added, the PCR tube containing the prepared reaction system was placed in a water bath, and the RAA amplification time gradient was set to 10min, 20min, 30min, 40min, 50min, and 60min. The RAA amplification product was added to the enzyme-free PCR tube according to the system in the table below. After the sample was added, the PCR tube containing the prepared reaction system was placed in a fluorescent quantitative PCR instrument, and the channel excitation light wavelength was set to 490nm, the emission light wavelength was 520nm, and the temperature was 45°C. The fluorescence intensity value was read every 0.5min, and 80 times were read for a total of 40min. Three groups were repeated at different times.

[0094] The experimental results are as follows Figure 5 As shown: In the legend, 10c-60min means that the initial sample with a concentration of 10 copies / tube was amplified by RAA reaction for 60min, and so on. When the RAA amplification time is 40min, the subsequent cleavage effect of the two crRNAs and the time cost are the most cost-effective.

[0095] Example 5 Specificity Detection

[0096] 1. Extract DNA of HSV-1, adeno-associated virus (AAV), adenovirus (AdV) and cDNA of Murine Leukemia Virus (MLV). Detect them together with clinical samples according to the established detection method to compare whether this method can cross-detect other coronaviruses.

[0097] 2. After RAA amplification, 5 μL of the amplified product was taken to detect each viral nucleic acid based on the CRISPR / Cas12a system according to the method of Example 2, and the amplified product with water as the template was set as a negative control.

[0098] The results are as follows Figure 6 As shown: the fluorescence values ​​of MLV, HSV, AAV, and AdV are extremely low, while the fluorescence value of HIV samples is above 3000, and is significantly different from MLV, HSV, AAV, and AdV. The above results show that the method for detecting HIV constructed by the present invention has strong specificity and has no cross reaction with other viruses.

[0099] Example 6 Sensitivity Detection

[0100] The standard sample is diluted in series to detect the sensitivity of the method of the present invention. The specific steps are as follows:

[0101] 1. Dilute the standard sample in step 1 of Example 1 to 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 1copies / μL, 0.5copies / μL.

[0102] 2. Perform RAA amplification according to the method of Example 2 to obtain RAA amplification products.

[0103] 3. After RAA amplification, take 5 μL of amplified product and test the standard sample based on the CRISPR\Cas12a system according to the method in Example 2, and set the amplified product with water as the template as a negative control. Put the PCR tube with the prepared reaction system into the fluorescent quantitative PCR instrument, set the channel excitation light wavelength to 490 nm, the emission light wavelength to 520 nm, 37 ° C, read the value every 2 min, read 40 times for a total of 80 minutes, and detect the change of fluorescence intensity in the system.

[0104] The experimental results are as follows Figure 7 As shown: As the reaction time of the cleavage reaction of the two crRNAs increases, the fluorescence values ​​of samples with different dilutions increase, especially when diluted to 0.5 and 1 copies / μL, which is more obvious and positively correlated with the reaction time.

[0105] Comparison of Example 7 with the detection limit of qPCR method

[0106] 1. In vitro synthesis of primers and probes for QPCR detection of HIV virus provided by the Department of Respiratory Viruses, Viral Diseases Division, Centers for Disease Control and Prevention, USA. The primer and probe sequences are shown in Table 6, the reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.

[0107] Table 6 RT-qPCR primers and probes

[0108]

[0109] Table 7 TaqPath TM 1-Step RT-qPCR Master Mix

[0110]

[0111] Table 8 Fluorescence quantitative PCR amplification program

[0112]

[0113] The test samples were serially diluted and 10 6 , 10 5 , 10 4, 10 3 , 10 2 , 10 1 , 1 copies / μL concentration was used for detection. According to the reaction system configuration in Table 7, the mixture was mixed and centrifuged, and then placed in a fluorescence quantitative QPCR instrument, and the detection was performed according to the reaction procedure in Table 8.

[0114] The experimental results are as follows Figure 8 As shown: As the sample concentration decreases, the Ct value increases. When the sample concentration is 10 copies / μL, there is an amplification curve and the average Ct value is 36.86. When diluted to 1 copies / μL, there is no amplification curve and Ct value, that is, 10 copies / μL is the lowest detection concentration of fluorescent quantitative PCR.

[0115] Compared with fluorescent quantitative PCR, the detection method established by the present invention has a sensitivity one order of magnitude higher (ie, 10 times higher).

[0116] 2. Detection sensitivity for domestic strains

[0117] Fig. 9 In the examples, CRF07_BC, B' and CRF08_BC strain plasmid standards were tested by CRISPR method, and the concentration gradient of the samples to be amplified was 1, 10, 100 copies / μL, and each sample was repeated three times.

[0118] The results are as follows Fig. 9 As shown in the figure: When the sample concentration is 100 copies / μL and 10 copies / μL, the two crRNAs all show positive reactions to the detection of the three domestic epidemic strains; when the sample concentration is 1 copy / μL, crRNA16 shows positive reactions to the three domestic strains, while crRNA88 has no positive reaction. That is, 1 copy / μL is the minimum detection concentration of crRNA16 for the three domestic strains, and the minimum detection concentration of crRNA88 for the three domestic strains is 10 copies / μL.

[0119] In summary, the present invention provides a method for HIV nucleic acid detection with wide strain coverage, high sensitivity, high specificity, fast and visualization. The present invention provides a technology based on CRISPR / Cas12a for the first time to quickly and accurately detect HIV, which can be used as a detection method for early monitoring and diagnosis of HIV. Its biggest advantage is its high sensitivity, which can detect a variety of classic strains of HIV and prevalent strains in China. At present, the best detection method for HIV is recognized as fluorescent quantitative PCR. Through experimental comparison, the detection method we established is at least 10 times higher than that of fluorescent quantitative PCR (the current minimum detection concentration of fluorescent quantitative PCR is 10 copies / μL of virus particles, while the minimum detection concentration based on CRISPR / Cas12a technology is 1 copy / μL of virus particles).

[0120] For weakly positive and suspected positive samples with extremely low virus content, efficient detection can be achieved. It only needs to increase the reaction time appropriately to determine whether HIV nucleic acid is positive or negative. The current defect of fluorescence quantitative PCR is that it is impossible to determine the positive or negative nature of HIV nucleic acid for samples with extremely low virus content, that is, Ct values ​​of 30-37. Even if the reaction time is extended, the result is still suspicious, which brings uncertainty to the early monitoring and prevention of HIV. The detection method established by the present invention can increase the fluorescence value for suspected viral nucleic acid positive samples by simply increasing the reaction time, and then determine the positive or negative nature of the sample, greatly improving the early monitoring of HIV on the front line.

[0121] The present invention screened the best crRNA, which further improved the sensitivity of the method established by the present invention. The core of the CRISPR / Cas12a detection method lies in crRNA, so crRNA is directly related to sensitivity and accuracy. Among the 25 crRNAs, crRNA16 and crRNA8 have the highest fluorescence values ​​detected by CRISPR / Cas12a, and cover a variety of HIV classic strains and domestic prevalent strains, indicating that these two have the best effect, the highest sensitivity, and the widest detection range.

[0122] The detection method provided by the present invention is low-cost and does not require expensive instruments and equipment. Currently, fluorescent quantitative PCR instruments are expensive, with ordinary domestically produced ones costing around RMB 150,000. The detection method proposed by the present invention only requires a water bath or a constant temperature incubator and a fluorescence detector, with the total cost of the instruments and equipment being less than RMB 10,000. Moreover, it can be used for large-scale clinical testing and initial screening, without the need for a professional and standardized laboratory.

[0123] The detection method provided by the present invention has a short detection time. The current fluorescence quantitative PCR reaction time is 1.5-2h, and the method established by the present invention is: constant temperature amplification for 40min, and then fluorescence detection with Cas12a for 40min, a total of 80min. Therefore, the detection method established by the present invention is more conducive to the rapid detection of HIV, which is very important for HIV prevention and control.

[0124] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for detecting HIV for non-disease detection or treatment purposes, characterized in that: Use the CRISPR-Cas12a system to test samples to determine whether they contain HIV; The CRISPR-Cas12a system includes Cas12a protein, crRNA and fluorescent reporter molecules; The sequence of the crRNA that stably binds to the viral RNA is shown in SEQ ID NO.1; The use of the CRISPR-Cas12a system to detect the sample to be tested specifically comprises: using the CRISPR-Cas12a system to perform an enzyme cleavage reaction on the sample to be tested; The temperature of the enzyme digestion reaction is 45°C; The reaction system of the enzyme cleavage reaction is 25µL: Cleavage Buffer 2.5µL, Cas12a protein 0.5µL, crRNA 0.3µL, fluorescent reporter molecule 1µL, sample to be tested 5µL and H2O 15.7µL; The fluorescent reporter molecule is a single-stranded DNA oligodeoxynucleotide, the nucleotide sequence of which is shown in SEQ ID NO.7; one end of the molecule carries the fluorescent dye FAM, and the other end carries BHQ; Before using the CRISPR-Cas12a system to detect the sample to be tested, a process of amplifying the sample to be tested is also included; The amplification is RAA amplification; the RAA amplification time is 40 minutes; The amplification primers for RAA amplification consist of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.

4.

2. The detection method according to claim 1, characterized in that: The method for determining whether HIV is present is as follows: using a blue light gel cutting instrument to read, with the negative control as a reference, and observing a luminescent group is positive; Or dilute the reaction result to 300µL with nuclease free water, and then use a fluorescence spectrophotometer to detect the fluorescence intensity at 490nm for excitation light and 520nm for emission light. When the fluorescence intensity value of the sample to be tested is more than 3 times higher than that of the negative control, it is judged as a positive result. Or at any time, the fluorescence intensity is greater than or equal to 600 a.u., which can be judged as a positive result.

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