CN118240979A - CRISPR-Cas12a system for visually detecting HIV and detection method - Google Patents

Abstract

The invention discloses a CRISPR-Cas12a system for visually detecting HIV and a detection method, belonging to the technical field of virus detection. Including Cas12a protein, crRNA, and fluorescent reporter molecules; the sequence of the crRNA is shown as SEQ ID NO.1 or SEQ ID NO. 2. The invention discovers an HIV nucleic acid detection target site based on a CRISPR/Cas12a system, and can realize HIV nucleic acid detection by utilizing the CRISPR/Cas12a system aiming at the site, and has the advantages of good detection specificity, high sensitivity, better specificity and compatibility, low detection cost and convenient and quick operation. The detection limit value of the invention can reach 1 copies/. Mu.L, thereby realizing target single molecule detection.

Description

CRISPR-Cas12a system for visually detecting HIV and detection method

Technical Field

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

Background

The major routes of transmission of HIV include three by sexual contact transmission (including homosexual, anisotropic and bipolar contact), blood and blood products (including intravenous drug administration with a common needle, interventional medical procedures, etc.), and maternal and infant transmission (including prenatal, prenatal and postnatal), with which the population is generally susceptible.

HIV belongs to human lentivirus in lentivirus genus of retrovirus family, is spherical particle with diameter of about 100-120 nm, and consists of core and envelope. The core consists of two single strand RNA strands, a core structural protein and enzymes necessary for viral replication, containing 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, in which are embedded the outer membrane glycoprotein (gp 120) and the transmembrane glycoprotein (gp 41).

According to HIV gene difference, it is divided into HIV-1 type and HIV-2 type, and the homology of amino acid sequence between two subtypes is 40% -60%. The current global popularity is mainly HIV-1.HIV-1 can be further divided into different subtypes, including the M subtype group (main subtype group), the O subtype group and the N subtype group, where the M group has A, B, C, D, E, F, G, H, I, J, K subtypes. In addition, a number of popular recombinant types have been discovered in recent years. HIV-2 has similar biological properties to HIV-1, but is less infectious, causing a slower clinical progression of AIDS and a lighter symptom.

The HIV-1 type is taken as a main epidemic strain in China, and A, B (European and American B), B' (Thailand B), C, D, E, F and G8 subtypes and different flow recombination types are found. According to the fourth molecular epidemiological investigation statistics, the main dominant strains in China are four, and the four dominant epidemic HIV-1 viruses account for 89.3% of all HIV-infected people in 2015, which are CRF07_BC (41.3%), CRF01_AE (32.7%), CRF08_BC (11.3%) and subtype B (4.0%), respectively. There are differences in subtype distribution in different regions. The epidemic strains in northwest and China are single, about 86.7% of the epidemic strains in northwest are CRF07_BC subtype, and the absolute dominant strains in China are B' subtype, which account for about 90.7%. The strains of 3 subtypes are mainly prevalent in northeast, north China, east China and south China, CRF01_AE is mainly, CRF07_BC and B' subtype are secondarily, and CRF08_BC accounts for only 5%. The subtype distribution of HIV-1 strains in southwest areas is complex, the CRF08_BC and CRF01_AE subtypes are mainly used, the CRF07_BC subtype is inferior, and the proportion of other subtypes (mostly unique recombinant subtypes, URFs) is also larger.

The distribution of strains in different transmission populations also differs. Crf01_ae is prevalent mainly in sexually transmitted people, but also in drug-taking people; the CRF07_BC subtype and the CRF08_BC subtype are mainly popular among drug-taking people, the CRF07_BC is popular in a certain proportion of sexually transmitted people and MSM people, and the CRF08_BC is popular in the southwest region of the sexually transmitted people; subtype B' is prevalent mainly in the existing paid blood collection population.

The recombinase-mediated strand displacement nucleic acid amplification technology (RAA technology) is a technology for rapid amplification of nucleic acid at constant temperature, and utilizes a recombinase obtained from bacteria or fungi, which can be tightly combined with primer DNA at normal temperature to form an enzyme and primer aggregate, when the primer searches for a complementary sequence which is completely matched with the primer on the template DNA, the double-stranded structure of the template DNA is opened with the help of a single-stranded DNA binding protein, a new DNA complementary strand is formed under the action of DNA polymerase, and an amplification product grows exponentially.

At present, CRISPR-Cas systems typified by SpCas9 have been widely used for gene, epigenomic and base editing of eukaryotic cells, and many newly identified Cas effect nucleases with unique properties have also been successfully used for genome engineering tasks. Among these, the Cas12a protein family has several prominent advantages for genetic engineering tasks, which family is represented by As- (Acidaminococcus sp.) and Lb- (Lachnospiraceae bacterium) Cas12a nucleases. These features include (1) the use of a single shorter crRNA; (2) identifying a T-rich PAM sequence; (3) cleaving the target DNA at a PAM distal site; (4) cleavage to produce protruding DNA ends; (5) Has an indiscriminate single-stranded DNase activity after target binding.

By combining the RAA technology with isothermal amplification DNA, cas12a nuclease characteristics and fluorescent probes, isothermal and complex instrument-free virus DNA detection is realized. The crRNA guides the Cas12a to recognize the target virus DNA fragment amplified by the RAA, activates the indifferent single-stranded DNase activity of the Cas12a, cuts the fluorescent probe and the target DNA fragment, and combines blue light or ultraviolet light to realize the visual detection of the virus DNA.

Currently, HIV detection can be largely divided into two major classes, antibody detection and virus detection, including cell culture (virus isolation), p24 antigen detection, and virus nucleic acid detection.

Antibody detection consisted of a primary screening and validation assay. The primary screening test requires high sensitivity to avoid missed detection, wherein the enzyme-linked immunosorbent assay (ELISA) method has certain sensitivity, is simple and quick to operate, and is suitable for detecting a large number of samples, so that the method is a primary screening detection method which is clinically common at present. There are 3 methods of confirmatory testing, including immunoblotting, strip immunoblotting and immunofluorescence, which are currently most commonly used. In addition, in recent years, researchers have also begun to conduct research on methods for detecting HIV antibodies in saliva and urine.

The antibody detection was present for a window period of 3-8 weeks. During the window, the viral antibodies cannot be detected, but viral-associated antigens can be detected or the virus isolated. The antigen can be detected 2-18 days before seroconversion after infection of an individual, so that the detection of the p24 antigen has great advantages in the seroconversion period and can be used as a method for early auxiliary diagnosis of HIV infection. The U.S. FDA has regulated that blood donors and blood products must be tested for HIV-1P24 antigen since month 8 of 1995 as a complement to anti-HIV testing. Virus culture is the most accurate method for detecting HIV infection, and diagnosis of HIV is generally performed by culturing Peripheral Blood Mononuclear Cells (PBMCs).

Viral nucleic acid detection is typically reflected in viral load by detection of HIV RNA levels with high sensitivity using real-time fluorescent Polymerase Chain Reaction (PCR) techniques, which can detect viral nucleic acid in the first two weeks of HIV infection. The virus nucleic acid detection method can be used for early diagnosis of HIV, such as window period auxiliary diagnosis, disease course monitoring, guiding treatment scheme and curative effect measurement, predicting disease course and the like. The commonly used methods of measurement include reverse transcription PCR (RT-PCR), nucleic acid sequence amplification (NASBA), branched DNA hybridization (bDNA) and the like.

The cell culture method has strong specificity for detecting HIV, does not have false positive, and has important significance for confirming whether the newborn infant of individuals and positive mothers with uncertain antigen/antibody detection is infected with HIV. However, the method of virus culture requires the presence of a certain number of infected cells to culture and isolate the virus, and thus has poor sensitivity, long operation time, complicated operation, must be performed in a specific P3 laboratory, and is expensive (about $200.about.500 per culture). Therefore, it is not suitable for clinical use.

The p24 antigen detection is able to detect soluble p24 antigen in the blood after the virus has started replication, but is prone to false positives, possibly due to interference by other substances and the effect of complex formation with antibodies. Therefore, a positive result must be confirmed by a neutralization test, and the result can be used as an auxiliary diagnosis basis for HIV infection. HIV-1P24 antigen detection was negative, indicating no reaction only in this assay and no rejection of HIV infection.

The virus nucleic acid detection method has high sensitivity and is very important for monitoring disease progress, antiviral curative effect observation and drug resistance monitoring. However, due to the diversity of HIV genes, no set of primers can cover all HIV sequences, which limits the sensitivity of detection; in addition, the existing virus nucleic acid detection method is expensive in detection instrument and detection reagent, or complex in operation, has high requirements on operators, is difficult to popularize in a common laboratory, is not suitable for rapid detection of a large number of patients, and is also not suitable for wide clinical application.

Therefore, to improve the sensitivity and specificity of detection, shorten the window period, and simply, quickly and reduce the cost has become a requirement and direction of development of HIV detection technology, and many researches are being conducted to find alternative technologies for virus detection.

Disclosure of Invention

The invention aims to provide a CRISPR-Cas12a system for visually detecting HIV and a detection method thereof, so as to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

according to one of the technical schemes, the CRISPR-Cas12a system for visually detecting HIV comprises Cas12a protein, crRNA and fluorescent reporter molecules;

the sequence of the crRNA is shown as SEQ ID NO.1 or SEQ ID NO. 2.

According to the second technical scheme, the CRISPR-Cas12a system is utilized to detect a sample to be detected, and whether the sample contains HIV is judged.

Based on the technical scheme, the invention has the following technical effects:

The invention discovers an HIV nucleic acid detection target site based on a CRISPR/Cas12a system, and can realize HIV nucleic acid detection by utilizing the CRISPR/Cas12a system aiming at the site, and has the advantages of good detection specificity, high sensitivity, better specificity and compatibility, low detection cost and convenient and quick operation.

1. The detection limit value of the method can reach 1 copies/. Mu.L, and target single molecule detection is realized.

2. The degenerate RAA primer and crRNA in the method have high coverage rate on HIV strains, high conservation and strong specificity. The invention can cover a plurality of classical strains of HIV-1 and domestic epidemic strain genotypes through tests.

3. The degenerate RAA primer and crRNA in the method have strong specificity for detecting HIV, and can effectively avoid interference of other viruses on detection results.

4. The HIV detection method provided by the invention has the advantages of rapidness, accuracy, convenience and the like, does not depend on expensive detection instruments, and is favorable for popularization in basic-level inspection institutions.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the principle of HIV virus detection based on CRISPR-Cas12a system;

FIG. 2 is a schematic diagram of SYNTHETIC CONSTRUCT CLONE NL 4-3_variant_LDNA structure;

FIG. 3 is a graph showing the determination of the effectiveness of crRNA16 and crRNA 88;

FIG. 4 is a determination of optimal temperature for Cas12a cleavage of RAA product;

FIG. 5 is a graph showing the determination of the optimal time for RAA amplification;

FIG. 6 is the specificity of crRNA16 and crRNA88 for detecting HIV;

FIG. 7 is the sensitivity of crRNA16 and crRNA88 to detect HIV;

FIG. 8 shows the sensitivity of QPCR method for detecting HIV;

FIG. 9 shows the sensitivity of crRNA16 and crRNA88 detection for three domestic epidemic strains (CRF07_BC, B' and CRF08_BC).

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The technical scheme of the invention is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.

The embodiment of the invention provides a CRISPR-Cas12a system for visually detecting HIV, which comprises Cas12a protein, crRNA and fluorescent reporter molecules;

the sequence of the crRNA is shown as SEQ ID NO.1 or SEQ ID NO. 2.

In some specific embodiments, the fluorescent reporter is a single-stranded DNA oligodeoxynucleotide having the nucleotide sequence shown in SEQ ID No. 7; one end of the kit is provided with fluorescein FAM, and the other end is provided with BHQ.

The embodiment of the invention provides a detection method for non-disease detection or treatment destination HIV, which utilizes the CRISPR-Cas12a system to detect a sample to be detected and judge whether the sample contains HIV.

In some specific embodiments, prior to detecting the test sample with the CRISPR-Cas12a system, a process of amplifying the test sample is further included.

In some specific embodiments, the amplification is RAA amplification; the RAA amplification time was 40min.

In some 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.

In some 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.

In some specific embodiments, the detecting a sample to be detected using the CRISPR-Cas12a system is specifically: performing enzyme digestion reaction on a sample to be detected by using a CRISPR-Cas12a system;

The temperature of the cleavage reaction was 45 ℃.

In some specific embodiments, the detection method has a reaction system of 25 μl: cleavage buffer2.5 mu L, cas a protein 0.5 mu L, crRNA 0.3.3 mu L, fluorescent reporter 1 mu L, sample to be tested 5 mu L and H ₂ O15.7 mu L.

In some embodiments, the method of determining whether HIV is present is: reading by using a blue light gel cutting instrument, and taking negative control as a reference, wherein the observed luminescence group is positive;

Or adding nucleotide FREE WATER into the reaction result to dilute the reaction result to 300 mu L, then detecting fluorescence intensity at the position of 490nm of excitation light and 520nm of emission light by using a fluorescence spectrophotometer, and judging the sample to be tested as a positive result when the fluorescence intensity value of the sample to be tested is more than 3 times higher than that of a negative control fluorescence intensity value;

Or the fluorescence intensity is larger than or equal to 600a.u. at any time, and then positive results can be judged.

In some embodiments, the negative control is a negative control in which the RAA product as a template in table 5 is replaced with nucleic FREE WATER, while the other components are kept unchanged.

Example 1 design and screening of optimal candidate crrnas that may stably bind Cas12a and viral RNAs

Firstly, the HIV classical strain NL4 and HXB2 sequences and the CRF01_AE, CRF07_BC and B', CRF08_BC strain sequences popular in China are obtained in a Database HIV Database (https:// www.hiv.lanl.gov/content/index), and the sequence alignment is carried out by Snapgene, so that relatively conserved regions in the strain sequences are selected and are mainly located in Gag and Pol regions of HIV. A crRNA library was designed using an on-line design tool Benchling (https:// www.benchling.com) and human genomic sequences were used as targets for off-target evaluation, and crRNAs with off-target scores greater than 90 minutes (100 full score, with lower score and higher off-target probability) were selected. The candidate crrnas were then assessed for effectiveness using CRISPR-DT, with crrnas having an effectiveness score greater than 0.9 (split 1) being selected, the higher the score, the better the stability of the crRNA and Cas12a complex, the higher the cleavage activity, and the highest docking score of crRNA being considered as the potentially optimal crRNA. The designed crRNA was synthesized via the hippocampus biotechnology limited in lake.

TABLE 1 screening of the crRNA obtained

Through inspection, crRNA designed by the invention can be targeted to combine with NL4, HXB2 and NDK in HIV classical strains and Chinese epidemic strains CRF01_AE, CRF07_BC and B', CRF08_BC.

Example 2 establishment of HIV Virus detection System based on CRISPR-Cas12a System

The detection principle of the CRISPR-Cas12a detection system is shown in figure 1.

1. Preparation of DNA samples.

The sequence of HIV classical strain SYNTHETIC CONSTRUCT CLONE NL-3_mutant_L is shown in figure 2, plasmid is transformed into Stb13 strain, TIANPREP MINI PLASMIDKIT is used for purifying plasmid, enzyme digestion and gel running are used for verifying that the strip is correct, the concentration of the plasmid is 1715.9 ng/. Mu.L, OD260/280 is 1.80, and the plasmid is stored at-20 ℃.

Using the formula: the calculated DNA copy number was 1.5X10 ¹³ copies/. Mu.L, copies/. Mu.L=6.02X10 ²³×(ng/μL)×10^-9/DNA LENGTH X330.

2. Design of RAA amplification primer

The RAA technology primer design requirement is extremely strict, the substitution or increase and decrease of individual bases can have an important influence on experimental results, and the available primers can be obtained through screening after experimental verification and detection. The design principle is as follows: the primer length is 25-35nt, the amplicon size is 100-200bp, and the Tm value is 54-67 ℃. The primer sequences are shown in Table 2 and were synthesized by the company Highway, inc. of Biotechnology.

TABLE 2RAA amplification primers

3. Design signal reporter

Since Cas12a has the attendant cleavage (collateral cleavage) activity, after recognition of the target DNA, the cleavage of other non-target DNA is continued, a short single-stranded DNA oligodeoxynucleotide can be designed as a fluorescent reporter with fluorescein on one end (e.g., FAM) and BHQ on the other end, as shown in table 3.

TABLE 3 reporter sequence

4. Obtaining RAA amplification products

And (3) taking the DNA sample in the step (1) as a template, and adopting the primer designed in the step (2) to carry out RAA amplification to obtain a RAA amplification product. The RAA amplification reaction system was configured as shown in Table 4, 1.0. Mu.L of the DNA sample obtained by the treatment in step 1 was added to the detection unit tube, the tube cap was covered, and the mixture was homogenized and centrifuged to pay attention to: the step of fully mixing will determine the repeatability of the test result; the reaction unit tube was placed in a 37 ℃ thermostat water bath and incubated for 1h.

TABLE 4RAA amplification base System

5. Preparation of CRISPR-Cas12a detection system

Taking 5 mu L of the RAA amplification product obtained in the step 3 as a template, preparing a CRISPR-Cas12a detection system according to Table 5. Wherein the clear Buffer laboratory is self-contained, lbCas a protein is purchased from Bolaisi organism (http:// www.bio-Lifesci. Com /), the RAA product used as a template in Table 5 is replaced by Nuclear FREE WATER, and other components are kept unchanged, namely the negative control.

TABLE 5Cas12a detection reaction System

6. Reading of test results

When crRNA is complementarily paired with target DNA, the collateral cleavage (collateral cleavage) activity of Cas12a is activated, and the ss-DNA linking the FAM and the quenching group breaks apart, where the two can fluoresce observable under blue or ultraviolet light. The technical result can be read by a blue light gel cutting instrument, a negative control is used as a reference, a positive luminescence group is observed, or the reaction result can be diluted to 300 mu L by adding nucleotide FREE WATER, then fluorescence intensity is detected at the position of 490nm of exciting light and 520nm of emitting light by a fluorescence spectrophotometer (Shimadzu Spectro fluorophotometer RF-6000), and when the fluorescence intensity value of the experimental group is more than 3 times higher than that of the negative control, the positive result is judged, or the fluorescence intensity is more than or equal to 600a.u. (3 times of the highest value which can be reached by the negative control fluorescence intensity) at any time, as shown in fig. 3.

Example 3Cas12a optimal cleavage temperature determination

After crRNA designed in example 1 was synthesized by company, a Cas12a cleavage reaction system was prepared according to table 5 with a standard sample of 1 μm as a test substance and RNASE FREE WATER as a negative control, and after the sample addition was completed, PCR tubes containing the prepared reaction system were placed in a fluorescent quantitative PCR instrument, the channel excitation light wavelength was set to 490nm, the emission light wavelength was 520nm, the temperature was 31.4 ℃,34.5 ℃,39.1 ℃,45 ℃,49.7 ℃ and a fluorescence intensity value was read every 0.5min for 80 times for 40min in total, and 3 groups were repeated at different times.

As a result, as shown in fig. 4, cas12a reacted most rapidly to cleave the RAA product when the temperature was 45 ℃, and the generated fluorescent signal was strongest, so 45 ℃ was taken as the subsequent experimental condition.

Example 4CrRNA screening and RAA amplification time determination

After crRNA designed in example 1 is synthesized by a company, a standard sample with the concentration of 10 copies/. Mu.L is used as an object to be detected, RNASE FREE WATER is used as a negative control, the system configuration is carried out according to the RAA amplification basic system in the table 5, after the sample addition is completed, a PCR tube containing the prepared reaction system is placed into a water bath kettle, and the RAA amplification time gradient is set to 10min,20min,30min,40min,50min and 60min. Adding RAA amplification products into enzyme-free PCR tubes according to the following table system, after sample addition, putting the PCR tubes containing the prepared reaction system into a fluorescent quantitative PCR instrument, setting the excitation wavelength of a channel to be 490nm, the emission wavelength to be 520nm, reading the fluorescence intensity value every 0.5min, reading 80 times for 40min, and repeating 3 groups at different times.

The experimental results are shown in fig. 5: in the legend 10c-60min represents the initial sample at a concentration of 10copies/tube amplified by RAA for 60min and so on. When the RAA amplification time is 40min, the cost performance of the subsequent cutting effect and time cost of the two crRNAs is highest.

EXAMPLE 5 specific assay

1. HSV-1, adeno-associated virus (AAV), DNA of adenovirus (adenovirus, adV) and cDNA of murine leukemia virus (Murine Leukemia Virus, MLV) were extracted. And (3) detecting the coronavirus and the clinical sample according to an established detection method, and comparing whether the method can cross detect other coronaviruses.

2. After RAA amplification, 5. Mu.L of the amplified product was used to detect each viral nucleic acid based on the CRISPR/Cas12a system as in example 2, while setting the amplified product with water as template as negative control.

The results are shown in FIG. 6: MLV, HSV, AAV, adV fluorescence values were very low, whereas HIV samples had fluorescence values above 3000, and were very different from MLV, HSV, AAV, adV. The results show that the method for detecting HIV constructed by the invention has strong specificity and has no cross reaction with other viruses.

Example 6 sensitivity detection

The standard sample is subjected to a series of dilutions to detect the sensitivity of the method of the invention. The method comprises the following specific steps:

1. the standard sample in step 1 of example 1 was diluted to 10 ³copies/μL、10²copies/μL、10¹ copies/. Mu.L, 1 copies/. Mu.L, 0.5 copies/. Mu.L in a gradient.

2. RAA amplification was performed as in example 2 to obtain a RAA amplification product.

3. After RAA amplification, 5. Mu.L of the amplified product was taken and standard samples were tested based on the CRISPR\Cas12a system according to the method of example 2, while setting the amplified product with water as template as negative control. The PCR tube with the prepared reaction system is put into a fluorescent quantitative PCR instrument, the excitation wavelength of a channel is set to 490nm, the emission wavelength is set to 520nm, the value is read every 2min, the total number of the reading is 40 times for 80 min, and the change of the fluorescence intensity in the system is detected.

The experimental results are shown in fig. 7: the cleavage reaction of the two crRNAs increases with the reaction time, and the fluorescence value of the samples at different dilutions increases, especially to 0.5 and 1 copies/. Mu.L, and is more obvious and positively correlated with the reaction time.

Example 7 alignment of detection limits with qPCR method

1. In vitro synthesis of primers and probes for QPCR detection of HIV viruses provided by the respiratory virus disease prevention control center of the american virus disease family, 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.

TABLE 6 RT-qPCR primers and probes

TABLE 7 TaqPath ^TM 1-Step RT-QPCR MASTER Mix

TABLE 8 fluorescent quantitative PCR amplification procedure

The test sample was subjected to gradient dilution, and a concentration of 10 ⁶,10⁵,10⁴,10³,10²,10¹, 1 copies/. Mu.L was taken for detection. According to the reaction system configuration shown in Table 7, the mixture was centrifuged, and the mixture was placed in a fluorescent quantitative QPCR instrument, and was subjected to detection according to the reaction procedure shown in Table 8.

The experimental results are shown in fig. 8: as the sample concentration decreases, the Ct value becomes larger, and when the sample concentration is 10 copies/. Mu.L, an amplification curve exists, the average Ct value is 36.86, and when the sample concentration is diluted to 1 copies/. Mu.L, no amplification curve and Ct value exist, namely, 10 copies/. Mu.L is the lowest detection concentration of fluorescent quantitative PCR.

Compared with fluorescent quantitative PCR, the detection method established by the invention has the sensitivity which is higher by an order of magnitude (namely 10 times higher).

2. Detection sensitivity for domestic strains

FIG. 9 shows CRF07_BC and B', CRF08_BC strain plasmid standard tested by CRISPR method, and the concentration gradient of the sample to be amplified is 1, 10, 100 copies/. Mu.L, three replicates per sample.

The results are shown in FIG. 9: at sample concentrations of 100 copies/. Mu.L and 10 copies/. Mu.L, positive reactions appear in the detection of three domestic epidemic strains by two crRNAs; whereas crRNA16 showed positive reactions to all three domestic strains at a sample concentration of 1 copies/. Mu.l, crRNA88 showed no positive reaction. I.e., 1 copies/. Mu.L is the lowest detected concentration of crRNA16 for three domestic strains, while the lowest detected concentration of crRNA88 for three domestic strains is 10 copies/. Mu.L.

In summary, the invention provides a method for detecting HIV nucleic acid, which has wide strain coverage, high sensitivity, high specificity, rapidness and visualization, and the first study of the invention provides a technology for rapidly and accurately detecting HIV based on CRISPR/Cas12a, which can be used as a detection method for early monitoring and diagnosing HIV. The method has the greatest advantages of high sensitivity and capability of realizing detection of various classical strains and epidemic strains of HIV. The currently accepted best detection method for HIV is fluorescent quantitative PCR, which we establish by experimental alignment is at least 10-fold higher than fluorescent quantitative PCR (currently the lowest detection concentration for fluorescent quantitative PCR is 10copies/μl of virion, whereas the lowest detection concentration based on CRISPR/Cas12a technology is 1copy/μl of virion).

Aiming at weak positive and suspected positive samples with extremely low virus content, high-efficiency detection is realized, and the positive or negative of HIV nucleic acid can be determined only by properly increasing the reaction time. The defects of the current fluorescent quantitative PCR are as follows: for samples with extremely low virus content, that is, ct value of 30-37, the negative and positive HIV nucleic acid can not be determined, even if the reaction time is prolonged, the result is suspicious, and uncertainty is brought to early monitoring, prevention and control of HIV. The detection method established by the invention can increase the fluorescence value for the suspected virus nucleic acid positive sample by only increasing the reaction time, thereby determining the negative positive of the sample and greatly improving the early detection of first-line HIV.

The crRNA with the best effect is screened, so that the sensitivity of the method established by the invention is further improved. The CRISPR/Cas12a detection method has the core of crRNA, so the crRNA is directly related to sensitivity and accuracy. Among 25 crrnas, crRNA16 and crRNA8 have the highest fluorescence values and cover various HIV classical strains and domestic epidemic strains, which means that the 2 crrnas have the best effects, highest sensitivity and the widest detection range.

The detection method provided by the invention has low cost and does not need expensive instruments and equipment. The current fluorescent quantitative PCR instrument is expensive, the common domestic price is about 15 ten thousand, the detection method proposed by the invention only needs a water bath kettle or a constant temperature incubator and a fluorescent detector, the cost of all instruments is less than 1 ten thousand, and the detection method is used for large-scale clinical detection primary screening without a laboratory with professional specifications.

The detection method provided by the invention has short detection time. The current fluorescent quantitative PCR reaction time is 1.5-2h, and the method established by the invention comprises the following steps: amplifying at constant temperature for 40min, and detecting fluorescence by using Cas12a for 40min, wherein the total time is 80min. Therefore, the detection method established by the invention is more beneficial to rapidly detecting HIV and is very important for HIV prevention and control.

It is to be understood that the above examples of the present invention are provided for clarity of illustration only and are not limiting of the embodiments of the present invention. Other variations or modifications of the above description will be apparent to persons of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. A CRISPR-Cas12a system for visually detecting HIV, comprising a Cas12a protein, a crRNA, and a fluorescent reporter;

the sequence of the crRNA is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. The CRISPR-Cas12a system according to claim 1, wherein said fluorescent reporter is a single-stranded DNA oligodeoxynucleotide having the nucleotide sequence shown in SEQ ID No. 7; one end of the kit is provided with fluorescein FAM, and the other end is provided with BHQ.

3. A method for detecting HIV at a non-disease detection or treatment destination, wherein the CRISPR-Cas12a system according to claim 1 or 2 is used to detect a sample to be tested to determine whether HIV is contained.

4. The method of claim 3, further comprising the step of amplifying the test sample prior to detecting the test sample using the CRISPR-Cas12a system of claim 1 or 2.

5. The method of claim 4, wherein the amplification is RAA amplification; the RAA amplification time was 40min.

6. The method according to claim 5, wherein the RAA amplification primer consists of an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.

7. The method according to claim 5, wherein the RAA amplification primer consists of an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO. 6.

8. The detection method according to claim 3, wherein the detection of the sample to be detected using the CRISPR-Cas12a system according to claim 1 or 2 is specifically: performing enzyme digestion reaction on a sample to be tested by using the CRISPR-Cas12a system as claimed in claim 1 or 2;

The temperature of the cleavage reaction was 45 ℃.

9. The method according to claim 3, wherein the reaction system of the method is 25. Mu.L: cleavage Buffer 2.5 mu L, cas a protein 0.5 mu L, crRNA 0.3.3 mu L, fluorescent reporter 1 mu L, sample to be tested 5 mu L and H ₂ O15.7 mu L.

10. The method according to claim 3, wherein the method for determining whether the HIV is contained is: reading by using a blue light gel cutting instrument, and taking negative control as a reference, wherein the observed luminescence group is positive;

Or adding nucleotide FREE WATER into the reaction result to dilute the reaction result to 300 mu L, then detecting fluorescence intensity at the position of 490nm of excitation light and 520nm of emission light by using a fluorescence spectrophotometer, and judging the sample to be tested as a positive result when the fluorescence intensity value of the sample to be tested is more than 3 times higher than that of a negative control fluorescence intensity value;

Or the fluorescence intensity is larger than or equal to 600a.u. at any time, and then positive results can be judged.