HIV1 / 2 total nucleic acid detection kit based on isothermal amplification and CRISPR (clustered regularly interspaced short palindromic repeats) and application thereof

Abstract

The invention relates to an HIV1 / 2 total nucleic acid detection kit based on isothermal amplification and CRISPR (clustered regularly interspaced short palindromic repeats) and application thereof. Comprising an RT-isothermal amplification freeze-drying reagent of an HIV-1-POL total nucleic acid specific target, an RT-isothermal amplification freeze-drying reagent of an HIV-1-LTR total nucleic acid specific target, an RT-isothermal amplification freeze-drying reagent of an HIV-2-POL total nucleic acid specific target, an RT-isothermal amplification freeze-drying reagent of an HIV-2-LTR total nucleic acid specific target, and an RT-isothermal amplification freeze-drying reagent of an HIV-2-LTR total nucleic acid specific target. An RT-isothermal amplification freeze-drying reagent of an internal reference sequence and an RT-isothermal amplification freeze-drying reagent without a primer; and a CRISPR detection freeze-drying reagent for an HIV-1-POL total nucleic acid amplification product, a CRISPR detection freeze-drying reagent for an HIV-1-LTR total nucleic acid amplification product, a CRISPR detection freeze-drying reagent for an HIV-2-POL total nucleic acid amplification product, a CRISPR detection freeze-drying reagent for an HIV-2-LTR total nucleic acid amplification product, a CRISPR detection freeze-drying reagent for an internal reference sequence and a CRISPR detection freeze-drying reagent without containing crRNA. By simplifying operation steps and anti-pollution design, rapid, simple, efficient and accurate detection of HIV (1 + 2 type) total nucleic acid is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of HIV nucleic acid detection, and particularly relates to an HIV 1 / 2 total nucleic acid detection kit based on isothermal amplification+CRISPR and application thereof. BACKGROUND

[0002] The existing HIV detection technologies mainly include immunological detection and nucleic acid detection.

[0003] The immunological detection is mainly directed against HIV antibodies or antigens, and although it is simple to operate and widely used, it has the problem of insufficient detection sensitivity in the "window period" after viral infection, and cannot be used for early detection, and it is also difficult to distinguish active infection from past infection.

[0004] The nucleic acid detection (NAT) directly targets viral nucleic acid, can effectively shorten the window period, and can be used for viral load monitoring and antiviral effect evaluation. At present, the mainstream nucleic acid amplification technology is polymerase chain reaction (PCR). However, the PCR-based technology has the following limitations:

[0005] ①The detection process is time-consuming: the PCR detection process takes more than 2 hours, and the skill requirement for the operator is high.

[0006] ②The detection target is relatively single and difficult to comprehensively evaluate: most existing nucleic acid detection methods are usually designed for HIV-1 type, and lack simultaneous detection of HIV-2 type. In addition, many methods only specifically detect viral RNA (for diagnosis and viral load analysis) or proviral DNA (for latent reservoir evaluation), and the detection scheme that can simultaneously cover RNA and DNA, i.e. "total nucleic acid", is less. This leads to the need for different detection when a comprehensive assessment of the viral infection state (such as new infection identification, latent infection detection) is needed, which increases the complexity and cost of operation. SUMMARY

[0007] In order to solve the problems that it is difficult to cover HIV-1 and HIV-2 and viral RNA and proviral DNA (i.e. total nucleic acid) in one detection system, and the detection of HIV total nucleic acid is time-consuming and complicated in the prior art, the present application provides an HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR and its application, which achieves the detection of HIV-1 and HIV-2 total nucleic acid in one detection system, and has the advantages of short detection time, simple operation, high detection sensitivity and high specificity. The kit comprises RT-isothermal amplification reagents for detecting HIV-1 and HIV-2 total nucleic acid in a sample, CRISPR reagents, and the amplification and detection of HIV-1 and HIV-2 total nucleic acid are realized by isothermal amplification primers and crRNA for the long terminal repeat (LTR) region and the polymerase (Pol) gene (double target) of the highly conserved genome of HIV-1 and HIV-2. The kit also controls the effectiveness of the sample by detecting the internal reference human beta-actin gene. The detection principle is as follows: after 42°C reverse transcription and constant temperature amplification reaction, the amplification product is transferred into the CRISPR detection system and incubated at 37°C. When the target sequence exists in the amplification product, the crRNA guides the Cas protein to specifically recognize the target, activates its side cleavage activity, and causes the report probe to be cut to produce a fluorescence signal; if the target sequence does not exist, the Cas protein remains in an inactive state, and the report probe is not cut. The final fluorescence intensity is related to the amount of amplified target nucleic acid, and qualitative detection can be carried out by real-time fluorescence monitoring.

[0008] Isothermal amplification technology is a kind of molecular biology method that realizes efficient amplification of nucleic acid target sequences at a constant temperature. Unlike traditional polymerase chain reaction (PCR) which requires cyclic temperature changes, isothermal amplification simplifies equipment requirements and improves detection speed, making it particularly suitable for on-site rapid detection and resource-limited environments. According to the core amplification mechanism, general isothermal amplification technology can be mainly divided into two categories: the first category is dependent on primer displacement to initiate repeated template copying: this method continuously displaces synthesized nucleic acid chains through enzymatic or chemical means, exposing new template sequences for primer binding, thereby achieving exponential amplification. Representative technologies include helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), recombinase-mediated amplification (RAA / RDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), multiple displacement amplification (MDA), and cross primer amplification (CPA). The second category is dependent on the continuous reuse or de novo synthesis of single primer molecules: this method usually involves enzymes with strand displacement activity, allowing a single primer or transcription process to continue, resulting in a large number of copies. Representative technologies include strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification (TMA).

[0009] In the present application, the isothermal amplification reaction is one or more of recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, rolling circle amplification, multiple displacement amplification, cross primer amplification, recombinase-mediated amplification, novel recombinase-dependent amplification, strand displacement amplification, and helicase-dependent amplification.

[0010] Among the many technologies, recombinase-mediated amplification technology represented by recombinase polymerase isothermal amplification (RPA / RAA) is a more preferred solution. The core is to simulate the principle of in vivo DNA homologous recombination, and the synergistic effect of three key enzyme proteins is used: recombinase (in some embodiments, uvsX or RecA derived from bacteria or fungi): combined with primers to form a complex under the action of auxiliary factors, scan double-stranded DNA and mediate the pairing and displacement of primers with homologous template sequences to form a "D-loop" structure; single-stranded DNA binding protein (in some embodiments, gp32 is used): immediately stabilizes the displaced single-stranded DNA to prevent its reannealing; strand displacement DNA polymerase (in some embodiments, Bacillus subtilis DNA polymerase I or Staphylococcus aureus DNA polymerase I, i.e., Bsu or Sau): initiates synthesis at the 3' end of the primer and displaces the downstream DNA strand to achieve strand extension and displacement.

[0011] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology originates from a kind of acquired immune system of prokaryotes (such as bacteria and archaea). This system forms a "memory" by integrating a fragment of exogenous virus or plasmid DNA into the CRISPR sequence of its own genome, and when encountering the same invader again, it uses guide RNA (crRNA) to guide Cas protein to specifically cut exogenous genetic material, thereby achieving defense. In recent years, especially the CRISPR-Cas system represented by Cas9, Cas12, Cas13, etc., has been revolutionarily transformed into a high-specificity molecular diagnostic tool due to its excellent "gene scissors" or "molecular detective" properties.

[0012] In the field of molecular diagnostics, Cas proteins with "side cutting" activity have become the core engine. After completing their specific targeted cutting (cis-cutting), these proteins are activated to have a non-specific, continuous cutting activity that can indiscriminately cut surrounding reporter nucleic acid molecules (such as single-stranded DNA or RNA probes with fluorescent groups and quenching groups), thereby converting invisible nucleic acids into detectable fluorescent, colorimetric, or test strip signals.

[0013] The CRISPR-Cas system has high specificity: derived from its sequence recognition mechanism. crRNA must accurately bind to the target nucleic acid sequence through base complementary pairing principles to effectively activate Cas protein, thereby effectively distinguishing single-base differences.

[0014] However, its sensitivity is limited: the CRISPR-Cas system itself is a "one-to-one" signal transducer. One target molecule activates one Cas protein, which in turn cleaves multiple reporter molecules to generate a signal. Although this linear amplification capability is considerable, its intrinsic sensitivity (usually in the pM to nM range) is still insufficient for reliable direct detection when faced with extremely low copy numbers that may exist in clinical samples (such as in early infection or treatment monitoring).

[0015] The technical problem solved by this invention is achieved by the following technical solution:

[0016] The present invention aims to provide an HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR, comprising RT-isothermal amplification lyophilized reagents for HIV-1-POL total nucleic acid specific targets, RT-isothermal amplification lyophilized reagents for HIV-1-LTR total nucleic acid specific targets, RT-isothermal amplification lyophilized reagents for HIV-2-POL total nucleic acid specific targets, RT-isothermal amplification lyophilized reagents for HIV-2-LTR total nucleic acid specific targets, RT-isothermal amplification lyophilized reagents for internal reference sequences, and primer-free reagents. RT-isothermal amplification lyophilized reagents; and CRISPR detection lyophilized reagents for total nucleic acid amplification products of HIV-1-POL, total nucleic acid amplification products of HIV-1-LTR, total nucleic acid amplification products of HIV-2-POL, total nucleic acid amplification products of HIV-2-LTR, CRISPR detection lyophilized reagents for internal reference sequences, and CRISPR detection lyophilized reagents without crRNA.

[0017] Furthermore, the RT-isothermal amplification lyophilized reagent for HIV-1-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO.1-2 and SEQ ID NO.9-10, and the CRISPR detection lyophilized reagent for HIV-1-POL total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO.3, SEQ ID NO.5-6 and SEQ ID NO.11-12;

[0018] The RT-isothermal amplification lyophilized reagent for HIV-1-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 13-14 and SEQ ID NO. 20-21, and the CRISPR detection lyophilized reagent for HIV-1-LTR total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 16-17, SEQ ID NO. 22 and SEQ ID NO. 24.

[0019] The RT-isothermal amplification lyophilized reagent for HIV-2-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 25-26 and SEQ ID NO. 34-35, and the CRISPR detection lyophilized reagent for HIV-2-POL total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 27-28, SEQ ID NO. 30, SEQ ID NO. 32 and SEQ ID NO. 36-38.

[0020] The RT-isothermal amplification lyophilized reagent for HIV-2-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 40-41 and SEQ ID NO. 46-47, and the CRISPR detection lyophilized reagent for HIV-2-LTR total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 42, SEQ ID NO. 44 and SEQ ID NO. 49-51.

[0021] Furthermore, the combination of isothermal amplification primers and CRISPR detection crRNA sequence is as follows: SEQ ID NO. 1-3, SEQ ID NO. 13-14, SEQ ID NO. 16, SEQ ID NO. 25-27 and SEQ ID NO. 40-42.

[0022] Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, with sequences SEQ ID NO.1–2, SEQ ID NO.5, SEQ ID NO.13–14, SEQ ID NO.17, SEQ ID NO.25–26, SEQ ID NO.28, SEQ ID NO.40–41, and SEQ ID NO.44.

[0023] Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, with the sequences being SEQ ID NO.1–2, SEQ ID NO.6, SEQ ID NO.20–22, SEQ ID NO.25–26, SEQ ID NO.30, SEQ ID NO.46–47, and SEQ ID NO.49.

[0024] Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO. 9–11, SEQ ID NO. 20–21, SEQ ID NO. 24, SEQ ID NO. 25–26, SEQ ID NO. 32, SEQ ID NO. 46–47 and SEQ ID NO. 50;

[0025] Alternatively, the isothermal amplification primers and CRISPR detection crRNA sequence combination five, with sequences SEQ ID NO.9–10, SEQ ID NO.12, SEQ ID NO.13–14, SEQ ID NO.16, SEQ ID NO.34–36, SEQ ID NO.46–47 and SEQ ID NO.51;

[0026] Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.1–3, SEQ ID NO.13–14, SEQ ID NO.17, SEQ ID NO.34–35, SEQ ID NO.37, SEQ ID NO.46–47 and SEQ ID NO.49;

[0027] Alternatively, there is combination seven of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.1–2, SEQ ID NO.5, SEQ ID NO.20–22, SEQ ID NO.34–35, SEQ ID NO.38, SEQ ID NO.46–47 and SEQ ID NO.50.

[0028] Furthermore, the RT-isothermal amplification lyophilized reagent for HIV-1-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO.52-53 and SEQ ID NO.58-59, and the CRISPR detection lyophilized reagent for HIV-1-POL total nucleic acid amplification products includes Cas13a-crRNA, with sequences SEQ ID NO.54-55 and SEQ ID NO.60-61;

[0029] The RT-isothermal amplification lyophilized reagent for HIV-1-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 63-64 and SEQ ID NO. 69-70, and the CRISPR detection lyophilized reagent for HIV-1-LTR total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 65-66 and SEQ ID NO. 71-72;

[0030] The RT-isothermal amplification lyophilized reagent for HIV-2-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 74-75 and SEQ ID NO. 80-81, and the CRISPR detection lyophilized reagent for HIV-2-POL total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 76, SEQ ID NO. 78 and SEQ ID NO. 82-83;

[0031] The RT-isothermal amplification lyophilized reagent for HIV-2-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 85-86 and SEQ ID NO. 91-92, and the CRISPR detection lyophilized reagent for HIV-2-LTR total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 88-89 and SEQ ID NO. 93-94.

[0032] Furthermore, it includes eight combinations of isothermal amplification primers and CRISPR detection crRNA sequences, with sequences SEQ ID NO. 52–54, SEQ ID NO. 63–65, SEQ ID NO. 74–76, SEQ ID NO. 85–86 and SEQ ID NO. 88;

[0033] Alternatively, isothermal amplification primers and CRISPR detection crRNA sequence combination nine, the sequences being SEQ ID NO.52~53, SEQ ID NO.55, SEQ ID NO.63~64, SEQ ID NO.66, SEQ ID NO.74~75, SEQ ID NO.78, SEQ ID NO.85~86 and SEQ ID NO.89;

[0034] Alternatively, a combination of isothermal amplification primers and CRISPR detection crRNA sequences could be used, with sequences defined as SEQ ID NO. 58–60, SEQ ID NO. 69–71, SEQ ID NO. 80–82, and SEQ ID NO. 91–93.

[0035] Alternatively, there is combination eleven of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.58-59, SEQ ID NO.61, SEQ ID NO.69-70, SEQ ID NO.72, SEQ ID NO.80-81, SEQ ID NO.83, SEQ ID NO.91-92 and SEQ ID NO.94.

[0036] Furthermore, it also includes isothermal amplification internal reference primers and CRISPR detection internal reference crRNA, with sequences SEQ ID NO. 96–103.

[0037] Furthermore, the RT-isothermal amplification lyophilized reagents for HIV-1-POL total nucleic acid specific targets, HIV-1-LTR total nucleic acid specific targets, HIV-2-POL total nucleic acid specific targets, HIV-2-LTR total nucleic acid specific targets, internal control sequence RT-isothermal amplification lyophilized reagents, and primer-free RT-isothermal amplification lyophilized reagents all contain: trehalose 8-12%, dextran 1-4%, Tris-HCl buffer 40-60 mM, recombinase 150-250 nM, single-stranded DNA binding protein 400-600 nM, DNA polymerase 90-110 nM, dNTPs 0.1-2 mM, ATP 1-6 mM, creatine phosphate 30-40 mM, and creatine kinase 5-15 mM. nM, RT enzyme 0.05U / μL-0.2U / μL, specific primer 0.1-0.5μM, RNase inhibitor 0.2U / μL-0.6U / μL. All concentrations are after reconstitution.

[0038] Furthermore, the following CRISPR lyophilized reagents are available for detecting HIV-1-POL total nucleic acid amplification products, HIV-1-LTR total nucleic acid amplification products, HIV-2-POL total nucleic acid amplification products, HIV-2-LTR total nucleic acid amplification products, CRISPR lyophilized reagents for internal control sequences, and CRISPR lyophilized reagents without crRNA. All reagents contain: trehalose 10-15%, dextran 4-8%, PEG 8000 0.5-5%, HEPES 20-50 mM, KCl 50-100 mM, DTT 1-7 mM, specific crRNA 10-150 nM, fluorescent probe 50-120 nM, and MgSO4 7-17 mM. All concentrations are after reconstitution.

[0039] In addition to the above-mentioned components, the Cas12a-CRISPR lyophilized reagent also contains: Cas12a 50-200 nM. All concentrations are after reconstitution.

[0040] In addition to the above-mentioned components, the Cas13a-CRISPR lyophilized reagent also contains: NTP Mix 30-50 nM, T7 enzyme 0.02 U / μL-0.05 U / μL, and Cas13a 50-200 nM. All concentrations are after reconstitution.

[0041] Furthermore, it also includes magnesium ion lyophilization reagents: MgCl2 5-20 mM, trehalose 10-30% and dextran 5-15%.

[0042] An application of an HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR includes the following steps:

[0043] S1, RT-isothermal amplification: The extracted total nucleic acid was added to the PCR reaction container for isothermal amplification. The eight-well array was pre-filled with magnesium ion lyophilization reagent and pre-filled with RT-isothermal amplification lyophilization reagents for HIV-1-POL total nucleic acid specific targets, HIV-1-LTR total nucleic acid specific targets, HIV-2-POL total nucleic acid specific targets, HIV-2-LTR total nucleic acid specific targets, internal control sequence, and primer-free RT-isothermal amplification lyophilization reagent.

[0044] S2, CRISPR detection: The isothermal amplification products are transferred to another PCR reaction container for CRISPR reaction. The wells are pre-filled with CRISPR detection lyophilized reagents for HIV-1-POL total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-1-LTR total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-2-POL total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-2-LTR total nucleic acid amplification products, CRISPR detection lyophilized reagents for internal control sequences, and CRISPR detection lyophilized reagents without crRNA.

[0045] S3. Signal detection: Use a fluorescence PCR instrument to detect the fluorescence signal of each detection well.

[0046] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0047] 1. This invention provides an HIV (type 1+2) total nucleic acid detection kit based on RPA-CRISPR, which combines the advantages of isothermal amplification and CRISPR to achieve high sensitivity, high specificity, and speed. It can simultaneously cover the detection of total nucleic acid of HIV-1 and HIV-2 in one detection system, with short detection time and simple operation.

[0048] 2. This invention has high detection efficiency and enables rapid detection: By optimizing the steps and reagents, the total detection time is controlled within 50 minutes, which is significantly shorter than the traditional PCR method (more than 2 hours), making it suitable for rapid screening.

[0049] 3. The operation process of this invention is simplified and human error is reduced: the pre-set lyophilized reagents and standardized well plate design reduce the steps of reagent preparation and pipetting. Operators only need to perform sample extraction and liquid transfer, reducing manual operation steps and lowering the risk of operation error and nucleic acid aerosol contamination.

[0050] 4. This invention offers a broad detection range, enabling comprehensive assessment of HIV infection status. By optimizing isothermal amplification primer sequences and CRISPR detection crRNA sequences, establishing parallel amplification and detection channels, and optimizing reagent formulations, this invention achieves, for the first time, simultaneous detection and typing of total nucleic acids (RNA and DNA) for HIV-1 and HIV-2 in a single detection system. This overcomes the limitations of existing methods that can only detect a single type or category of nucleic acid, providing a more comprehensive detection method for early diagnosis, identification of new infections, and assessment of latent infections.

[0051] 5. This invention has low equipment dependence and wide applicability: It is based on detection using common instruments (such as fluorescence PCR instruments), which reduces dependence on special equipment and improves the applicability of the method. Both amplification and CRISPR detection are isothermal processes, which do not require complex thermal cyclers. Signal detection uses common fluorescence PCR instruments, and the method is easy to promote in grassroots laboratories.

[0052] 6. This invention boasts high specificity and sensitivity, delivering reliable results. By employing a CRISPR-Cas system for secondary specific identification of isothermal amplification products, this method achieves highly specific detection of the target sequence, accurately distinguishing between HIV-1 and HIV-2. The optimized lyophilized reagent formulation ensures the activity and stability of each reaction component, thereby guaranteeing the detection sensitivity of the entire integrated system. The detection limits are as follows: HIV-1 RNA up to 20 IU / mL, HIV-1 DNA up to 200 copies / mL, HIV-2 RNA up to 20 IU / mL, and HIV-2 DNA up to 200 copies / mL, far exceeding clinical testing standards.

[0053] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Detailed Implementation

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0056] Example 1: Rapid HIV-1 / HIV-2 Total Nucleic Acid Detection Kit (Isothermal Amplification-CRISPR Fluorescence Method)

[0057] 1. The specific components of the nucleic acid detection kit for HIV-1 / HIV-2 total nucleic acid POL and LTR genes based on Cas12a or Cas13a fluorescence detection technology are as follows:

[0058] Reagent section

[0059] 1.1 Extraction reagents: Total nucleic acids (including RNA and DNA) were extracted from the samples using the Total Nucleic Acid Co-extraction Kit (MagMAX™ Sequential DNA / RNA Kit, Thermo Fisher).

[0060] 1.2 The composition of the Cas12a-CRISPR fluorescence detection system is as follows:

[0061] RT-isothermal amplification: Isothermal amplification was performed using a PCR reaction vessel. The eight-well array contained pre-filled magnesium ion lyophilized reagent (Table 2) and the following lyophilized RT-isothermal amplification reagent beads (Table 1): Well 1: RT-isothermal amplification lyophilized reagent specifically targeting HIV-1-POL total nucleic acid; Well 2: RT-isothermal amplification lyophilized reagent specifically targeting HIV-1-LTR total nucleic acid; Well 3: RT-isothermal amplification lyophilized reagent specifically targeting HIV-2-POL total nucleic acid; Well 4: RT-isothermal amplification lyophilized reagent specifically targeting HIV-2-LTR total nucleic acid; Well 5: RT-isothermal amplification lyophilized reagent targeting the internal control sequence; Well 6: RT-isothermal amplification lyophilized reagent without primers (control).

[0062] Table 1: RT-Isothermal Amplification Lyophilized Reagents

[0063]

[0064] Table 2: Magnesium Ion Lyophilized Reagents

[0065]

[0066] CRISPR Detection: CRISPR detection was performed using PCR reaction containers. The following lyophilized CRISPR detection reagent balls were pre-filled into eight wells (Table 3): Well 1: Lyophilized CRISPR detection reagent targeting HIV-1-POL total nucleic acid amplification products; Well 2: Lyophilized CRISPR detection reagent targeting HIV-1-LTR total nucleic acid amplification products; Well 3: Lyophilized CRISPR detection reagent targeting HIV-2-POL total nucleic acid specific target total nucleic acid amplification products; Well 4: Lyophilized CRISPR detection reagent targeting HIV-2-LTR total nucleic acid specific target total nucleic acid amplification products; Well 5: Lyophilized CRISPR detection reagent targeting the internal control sequence; Well 6: Lyophilized CRISPR detection reagent without crRNA (control).

[0067] Table 3: CRISPR Detection Lyophilized Reagents

[0068]

[0069] 1.3 The composition of the Cas13a-CRISPR fluorescence detection system is as follows:

[0070] The RT-isothermal amplification system is the same as in 1.2.

[0071] CRISPR Detection: CRISPR detection was performed using a PCR reaction vessel. The following lyophilized CRISPR detection reagent balls were pre-filled into the eight-well array (Table 4): Well 1: Lyophilized CRISPR detection reagent for HIV-1-POL total nucleic acid amplification products; Well 2: Lyophilized CRISPR detection reagent for HIV-1-LTR total nucleic acid amplification products; Well 3: Lyophilized CRISPR detection reagent for HIV-2-POL total nucleic acid amplification products; Well 4: Lyophilized CRISPR detection reagent for HIV-2-LTR total nucleic acid amplification products; Well 5: Lyophilized CRISPR detection reagent for the internal control sequence; Well 6: Lyophilized CRISPR detection reagent without crRNA (control).

[0072] Table 4: CRISPR Detection Lyophilized Reagents

[0073]

[0074] 1.4 The specific information on the primers and crRNA for isothermal amplification-CRISPR is as follows:

[0075] Following the design principles of primers and crRNA, primer lengths were maintained at 28-32 bp, and crRNA lengths at 18-25 bp. The designed primers and crRNAs were BLAST-aligned to ensure primer-probe specificity. The isothermal amplification primer and crRNA sequences for the Cas12a-CRISPR HIV-1 POL, LTR, and HIV-2 POL, LTR genes are shown in Table 5.

[0076] Table 5: Primers and crRNA sequences for Cas12a-CRISPR HIV-1 / HIV-2 total nucleic acid detection

[0077]

[0078]

[0079]

[0080]

[0081] Sequence test results:

[0082] High concentration of pseudovirus (1×10) 3 Preliminary screening of amplification primers and crRNAs was conducted using IU / mL. Preliminary experimental results showed that all primers except HIV-1-POL:Cas12a-crRNA-1-2 and HIV-2-POL:Cas12a-crRNA-1-5 could be effectively amplified and cleaved.

[0083] Table 6: Primers and crRNA sequences for Cas13a-CRISPR HIV-1 / HIV-2 total nucleic acid detection

[0084]

[0085]

[0086]

[0087]

[0088] Sequence test results:

[0089] High concentration of pseudovirus (1×10) 3 Preliminary screening of amplification primers and crRNAs was conducted using IU / mL. Preliminary experimental results showed that all primers except HIV-1-POL: Cas13a-crRNA-1-4 and HIV-2-LTR: Cas13a-crRNA-1-1 could be effectively amplified and cleaved.

[0090] 1.5 Other

[0091] Table 7: Internal reference primers and crRNA sequences

[0092]

[0093] (1) The above internal controls all showed good specific cleavage ability in the Cas12a and Cas13a systems. One of the crRNAs was selected.

[0094] (2) Negative nucleic acid tube: 1 tube, containing enzyme-free sterile water.

[0095] (3) Positive nucleic acid tube: 1 tube containing a positive solution of pseudoviruses containing HIV-1 / HIV-2 specific genes.

[0096] (4) Packaging box: 1 piece.

[0097] (5) Instruction manual: 1 copy.

[0098] (6) All lyophilized pellets were sealed in PCR reaction containers.

[0099] 1.6 Signal Detection: The fluorescence signal of each well was detected using a fluorescence PCR instrument. The instrument settings were as follows: excitation / detection wavelength: 495 / 520 nm; detection mode: endpoint fluorescence detection.

[0100] 2. Detection Procedure of HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit

[0101] Using the above-mentioned test kit, follow these steps:

[0102] 2.1 Sample Pretreatment

[0103] (1) Whole blood (WB) sample pretreatment: Invert the WB sample at least seven times [EDTA (light purple cap) or EDTA capillary blood collection tube] to mix the blood.

[0104] (2) Pretreatment of dried blood spot samples: Cut the DBS along the drawn lines. Separate and process the DBS using sterile forceps. When using non-perforated DBS, cut a complete DBS from the filter paper for each sample. Add 500 µL of nucleic acid resuspension to each DBS and vortex for 10 seconds.

[0105] 2.2 Nucleic Acid Extraction: Total nucleic acids (including RNA and DNA) were extracted from the samples using a total nucleic acid co-extraction kit (MagMAX™ Sequential DNA / RNA kit and reagents, Hermo Fisher). Specific steps included: Sample lysis: Blood samples were mixed with lysis buffer to release nucleic acids; Nucleic acid binding: Magnetic bead suspension was added to adsorb nucleic acids onto the magnetic beads; Washing: Impurities were removed using washing buffer; Elution: The purified total nucleic acids were collected using elution buffer; The extracted total nucleic acids were used for subsequent reverse transcription and amplification.

[0106] 2.3 RT-isothermal amplification: 20 μL of the extracted total nucleic acid was added to each of the 6 isothermal amplification wells and reacted at 42 °C for 15 minutes to complete the target amplification.

[0107] 2.4 Signal detection: All isothermal amplification products were transferred to the corresponding detection wells and reacted at 37°C for 15 minutes in a fluorescence PCR instrument. The fluorescence signal was detected by specifically recognizing the target using the CRISPR system.

[0108] 2.5 Result Interpretation Criteria

[0109] (1) Determination of detection validity

[0110] Valid: If the internal control result is positive and the control result is negative, the test is considered valid and the result can be interpreted.

[0111] Invalid: If the internal control result is negative or the control result is positive, the result of this test or the test of this sample is invalid. Retesting is recommended.

[0112] (2) Result Interpretation: Under the premise of satisfying the above-mentioned "valid determination" in section (1), the result shall be interpreted in the following logical order:

[0113] HIV-1 and HIV-2 positive: If at least one HIV-1 target is positive and at least one HIV-2 target is positive, it should be reported as "HIV-1 and HIV-2 total nucleic acid positive".

[0114] HIV-1 positive: If at least one HIV-1 target is positive and all HIV-2 targets are negative, it should be reported as "HIV-1 total nucleic acid positive".

[0115] HIV-2 positive: If all HIV-1 targets are negative and at least one HIV-2 target is positive, it should be reported as "HIV-2 total nucleic acid positive".

[0116] Negative: If all HIV-1 and HIV-2 test targets are negative, the report should be "HIV nucleic acid not detected" or "negative".

[0117] Example 2: Sensitivity Validation of HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Reagent (Isothermal Amplification-CRISPR Fluorescence Assay)

[0118] Sensitivity tests were performed using DNA and RNA references for HIV-1 and HIV-2.

[0119] Reference product information:

[0120] HIV-1 RNA: Human immunodeficiency virus type 1 (HIV-1) RNA standard purchased from the National Institutes for Food and Drug Control, batch number: 220021-20160101.

[0121] HIV-1 DNA: HIV-1 proviral cell content detection standard (8E5 cells) purchased from the China National Institutes for Food and Drug Control, batch number: 220031-202001.

[0122] HIV-2 RNA: International standard HIV-2 RNA procured from NIBSC, NIBSC number: 16 / 296

[0123] HIV-2 DNA: Exported to Qingke Biosynthetic Pseudovirus

[0124] Table 8: Minimum Reference Concentration Settings for HIV-1 / HIV-2

[0125]

[0126] Table 9: Reagent Information and Detection Sensitivity

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] Sensitivity test results:

[0134] HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit (Cas12a-CRISPR Fluorescence Assay)

[0135] (1) HIV-1-POL: In combination 1, Cas12a-crRNA-1-1, Cas12a-crRNA-1-3, and Cas12a-crRNA-1-4 can all be effectively cut, and their specific cutting function is better. Either combination can be selected. In combination 2, Cas12a-crRNA-2-1 and Cas12a-crRNA-2-2 can both be effectively cut, and their specific cutting function is better. Either combination can be selected.

[0136] (2) HIV-1-LTR: In combination 1, Cas12a-crRNA-1-2 and Cas12a-crRNA-1-3 can be effectively cut, and their specific cutting function is better. Either combination can be selected. In combination 2, Cas12a-crRNA-2-1 and Cas12a-crRNA-2-3 can be effectively cut, and their specific cutting function is better. Either combination can be selected.

[0137] (3) HIV-2-POL: In combination 1, Cas12a-crRNA-1-1, Cas12a-crRNA-1-2, Cas12a-crRNA-1-4, and Cas12a-crRNA-1-6 can all be effectively cut, and their specific cutting function is better. Any one of them can be selected. In combination 2, Cas12a-crRNA-2-1, Cas12a-crRNA-2-2, and Cas12a-crRNA-2-3 can all be effectively cut, and their specific cutting function is better. Any one of them can be selected.

[0138] (4) HIV-2-LTR: In combination 1, Cas12a-crRNA-1-1 and Cas12a-crRNA-1-3 can be effectively cut, and their specific cutting function is better. Either combination can be selected. In combination 2, Cas12a-crRNA-2-2, Cas12a-crRNA-2-3 and Cas12a-crRNA-2-4 can be effectively cut, and their specific cutting function is better. Either combination can be selected.

[0139] HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit (Cas13a-CRISPR Fluorescence Assay)

[0140] (1) HIV-1-POL: Among the combinations, Cas13a-crRNA-1-1 and Cas13a-crRNA-1-2 have better specific cutting ability, so one of the crRNAs is selected; among the combinations, Cas13a-crRNA-2-1 and Cas13a-crRNA-2-2 have better specific cutting ability, so one of the crRNAs is selected.

[0141] (2) HIV-1-LTR: Among combination 1, Cas13a-crRNA-1-1 and Cas13a-crRNA-1-2 have better specific cutting ability, so one of the crRNAs is selected; among combination 2, Cas13a-crRNA-2-1 and Cas13a-crRNA-2-2 have better specific cutting ability, so one of the crRNAs is selected.

[0142] (3) HIV-2-POL: Among the combinations, Cas13a-crRNA-1-1 and Cas13a-crRNA-1-3 have better specific cutting ability, so one of the crRNAs is selected; among the combinations, Cas13a-crRNA-2-1 and Cas13a-crRNA-2-2 have better specific cutting ability, so one of the crRNAs is selected.

[0143] (4) HIV-2-LTR: Among combination 1, Cas13a-crRNA-1-2 and Cas13a-crRNA-1-3 have better heterogeneous splicing ability, so one of the crRNAs is selected; among combination 2, Cas13a-crRNA-2-1 and Cas13a-crRNA-2-2 have better specific splicing ability, so one of the crRNAs is selected.

[0144] The HIV-1 / HIV-2 total nucleic acid rapid test kits (Cas12a-CRISPR fluorescence method) and HIV-1 / HIV-2 total nucleic acid rapid test kits (Cas13a-CRISPR fluorescence method) with the above sequences can detect DNA and RNA at the lower limit of reference, up to S3, which is the target concentration lower limit.

[0145] Example 3: Specificity assessment of the rapid HIV-1 / HIV-2 total nucleic acid detection reagent (isothermal amplification-CRISPR fluorescence method)

[0146] Analytical Specificity Validation: The analytical specificity of the test reagent is mainly examined from two aspects: cross-reactivity and interference testing. Cross-reactivity refers to whether false positive results are produced between pathogens that easily produce cross-reactivity and pathogens that may cause similar clinical symptoms, effectively reducing the probability of false positives in clinical testing. This test reagent tested 15 cross-reactive pathogens, details of which are shown in Table 10. Interference testing refers to the interference caused by endogenous and exogenous residual substances (such as various therapeutic drugs) on sample detection, leading to false negatives. Through interference testing, the influence of various endogenous and exogenous substances on sample detection results is eliminated, thereby improving the accuracy of the reagent's test results. The interference testing of this reagent was conducted under the potential maximum concentration ("worst-case condition") of each interfering substance. The names of the tested interfering substances are shown in Tables 11 and 12.

[0147] Table 10: Pathogenic microorganisms used for cross-reactivity

[0148]

[0149] Table 11: Endogenous Interfering Substances

[0150]

[0151] Table 12: Exogenous Interfering Substances

[0152]

[0153] Verification results:

[0154] Table 13: Results of Cross-Specificity Reactivity Test

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] "-" represents negative.

[0164] Table 14: Test Results on Whether Endogenous and Exogenous Substances Interfere with the Detection of Low-Concentration Samples

[0165]

[0166]

[0167]

[0168]

[0169]

[0170] "+" represents a positive result.

[0171] After testing against 15 pathogenic microorganisms with potential cross-reactivity and 12 interfering substances, this test reagent showed negative results for other pathogenic microorganisms and was unaffected by interfering substances, thus ensuring accuracy and efficiency in clinical sample testing.

[0172] Example 4: Rapid HIV-1 / HIV-2 Total Nucleic Acid Detection Kit (Isothermal Amplification-CRISPR Fluorescence Assay) for Co-detection of RNA and DNA

[0173] Based on the results of the sensitivity and specificity of different primer crRNA, 700 types of HIV-1 / HIV-2 total nucleic acid rapid detection kits (Cas12a--CRISPR) can be developed. Given the large number of combinations, duplicate and overlapping combinations were eliminated, resulting in 7 kit formats covering all detection systems, as shown in Tables 15 and 16.

[0174] Based on the results of different primer crRNA sensitivity and specificity, 256 rapid HIV-1 / HIV-2 total nucleic acid detection kits (Cas13a-CRISPR) were developed. Given the large number of combinations, duplicate and overlapping combinations were removed, resulting in four kit formats covering all detection systems, as shown in Tables 15 and 16.

[0175] Table 15: Cas12a-CRISP and Cas13a-CRISPR sequence combination numbers

[0176]

[0177]

[0178] Table 16: Different Combinations of Cas12a-CRISP and Cas13a-CRISPR Kits

[0179]

[0180] Send out synthetic HIV-1-POL DNA and RNA pseudoviruses, and HIV-1-LTR DNA and RNA pseudoviruses: Mix and dilute HIV-1-POL DNA pseudoviruses and HIV-1-LTR RNA pseudoviruses to the minimum limit of detection (MRD) concentration, and mix and dilute HIV-1-LTR DNA pseudoviruses and HIV-1-POL RNA pseudoviruses to the MRD concentration. Perform sensitivity testing using the kit; the HIV-2 protocol is the same as for HIV-1.

[0181] Reference product information:

[0182]

[0183] Table 17: Minimum Reference Standards for HIV-1 / HIV-2 DNA and RNA Co-detection

[0184]

[0185] Table 18: Reagent Test Results

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] The CRISPR detection kit can detect both DNA and RNA as minimum detection reference samples. This kit can achieve the purpose of nucleic acid co-detection.

[0192] Example 5: Clinical Applicability of the HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit

[0193] Following the kit operation steps in Example 1, the HIV-1 / HIV-2 total nucleic acid rapid detection kit (Cas12a-CRISPR fluorescence method) and HIV-1 / HIV-2 total nucleic acid rapid detection kit (Cas13a-CRISPR fluorescence method) developed in Example 4 were compared with Cypert Xpert-HIV-1-Qual-XC and DaAn commercial kits: Human Immunodeficiency Virus 1 (HIV-1) Nucleic Acid Assay Kit (PCR-fluorescent probe method) and Human Immunodeficiency Virus 1 (HIV-1) DNA Detection Kit (PCR-fluorescent probe method). 100 whole blood samples and 100 dried blood spot samples collected clinically for HIV-1 were tested, and all samples had clinical diagnostic results. The detection performance of the different kits was compared.

[0194] Table 19: Summary of HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit Results for Clinical Samples

[0195]

[0196] in conclusion:

[0197] As shown in Table 19, the CRISPR test kit demonstrates superior performance in both whole blood and dried blood spot sample types. The test results are comparable to those of commercially available PCR kits. When used in conjunction with a fluorescent PCR instrument, this kit provides strong support and a reliable guarantee for primary healthcare practices.

[0198] Example 6: Application of the HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit in HIV-2 Detection

[0199] Since HIV-2 positive patients are relatively rare clinically, we used spurious virus administration on 50 negative whole blood samples to verify the accuracy of this test kit. The results were compared with Tianlong's commercially available kit: Hepatitis B Virus, Hepatitis C Virus, and Human Immunodeficiency Virus (Type 1+2) Nucleic Acid Detection Kit (PCR-Fluorescence Method). The results are summarized in Table 20 below:

[0200] Table 20: Summary of HIV-1 / HIV-2 Total Nucleic Acid Rapid Detection Kit Results in HIV-2 Simulated Samples

[0201]

[0202] in conclusion:

[0203] As shown in Table 20, the CRISPR test kits demonstrated superior performance in HIV-2 simulated whole blood samples, with test results comparable to those of commercially available PCR kits, providing strong support and reliability for medical practice.

[0204] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0205] The embodiments of the present invention have been described above, but the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and these forms are all within the protection scope of the present invention.

Claims

1. A total nucleic acid detection kit for HIV1 / 2 based on isothermal amplification + CRISPR, characterized in that, This includes lyophilized reagents for RT-isothermal amplification of HIV-1-POL total nucleic acid specific targets, lyophilized reagents for RT-isothermal amplification of HIV-1-LTR total nucleic acid specific targets, lyophilized reagents for RT-isothermal amplification of HIV-2-POL total nucleic acid specific targets, lyophilized reagents for RT-isothermal amplification of HIV-2-LTR total nucleic acid specific targets, lyophilized reagents for RT-isothermal amplification of internal reference sequences, and lyophilized reagents for RT-isothermal amplification without primers; as well as lyophilized reagents for CRISPR detection targeting HIV-1-POL total nucleic acid amplification products, lyophilized reagents for CRISPR detection targeting HIV-1-LTR total nucleic acid amplification products, lyophilized reagents for CRISPR detection targeting HIV-2-POL total nucleic acid amplification products, lyophilized reagents for CRISPR detection targeting HIV-2-LTR total nucleic acid amplification products, lyophilized reagents for CRISPR detection targeting internal reference sequences, and lyophilized reagents for CRISPR detection without crRNA; The RT-isothermal amplification lyophilized reagent for HIV-1-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO.1-2 and SEQ ID NO.9-10, and the CRISPR detection lyophilized reagent for HIV-1-POL total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO.3, SEQ ID NO.5-6 and SEQ ID NO.11-12; The RT-isothermal amplification lyophilized reagent for HIV-1-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 13-14 and SEQ ID NO. 20-21, and the CRISPR detection lyophilized reagent for HIV-1-LTR total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 16-17, SEQ ID NO. 22 and SEQ ID NO.

24. The RT-isothermal amplification lyophilized reagent for HIV-2-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 25-26 and SEQ ID NO. 34-35, and the CRISPR detection lyophilized reagent for HIV-2-POL total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 27-28, SEQ ID NO. 30, SEQ ID NO. 32 and SEQ ID NO. 36-38. The RT-isothermal amplification lyophilized reagent for HIV-2-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 40-41 and SEQ ID NO. 46-47, and the CRISPR detection lyophilized reagent for HIV-2-LTR total nucleic acid amplification products includes Cas12a-crRNA with sequences SEQ ID NO. 42, SEQ ID NO. 44 and SEQ ID NO. 49-51; The RT-isothermal amplification lyophilized reagent for HIV-1-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 52-53 and SEQ ID NO. 58-59, and the CRISPR detection lyophilized reagent for HIV-1-POL total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 54-55 and SEQ ID NO. 60-61; The RT-isothermal amplification lyophilized reagent for HIV-1-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 63-64 and SEQ ID NO. 69-70, and the CRISPR detection lyophilized reagent for HIV-1-LTR total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 65-66 and SEQ ID NO. 71-72; The RT-isothermal amplification lyophilized reagent for HIV-2-POL total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 74-75 and SEQ ID NO. 80-81, and the CRISPR detection lyophilized reagent for HIV-2-POL total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 76, SEQ ID NO. 78 and SEQ ID NO. 82-83; The RT-isothermal amplification lyophilized reagent for HIV-2-LTR total nucleic acid specific targets includes isothermal amplification primers SEQ ID NO. 85-86 and SEQ ID NO. 91-92, and the CRISPR detection lyophilized reagent for HIV-2-LTR total nucleic acid amplification products includes Cas13a-crRNA with sequences SEQ ID NO. 88-89 and SEQ ID NO. 93-94.

2. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: The combination includes isothermal amplification primers and CRISPR detection crRNA sequence, with sequences SEQ ID NO.1–3, SEQ ID NO.13–14, SEQ ID NO.16, SEQ ID NO.25–27 and SEQ ID NO.40–42; Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, with sequences SEQ ID NO.1–2, SEQ ID NO.5, SEQ ID NO.13–14, SEQ ID NO.17, SEQ ID NO.25–26, SEQ ID NO.28, SEQ ID NO.40–41, and SEQ ID NO.

44. Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, with the sequences being SEQ ID NO.1–2, SEQ ID NO.6, SEQ ID NO.20–22, SEQ ID NO.25–26, SEQ ID NO.30, SEQ ID NO.46–47, and SEQ ID NO.

49. Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO. 9–11, SEQ ID NO. 20–21, SEQ ID NO. 24, SEQ ID NO. 25–26, SEQ ID NO. 32, SEQ ID NO. 46–47 and SEQ ID NO. 50; Alternatively, the isothermal amplification primers and CRISPR detection crRNA sequence combination five, with sequences SEQ ID NO.9–10, SEQ ID NO.12, SEQ ID NO.13–14, SEQ ID NO.16, SEQ ID NO.34–36, SEQ ID NO.46–47 and SEQ ID NO.51; Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.1–3, SEQ ID NO.13–14, SEQ ID NO.17, SEQ ID NO.34–35, SEQ ID NO.37, SEQ ID NO.46–47 and SEQ ID NO.49; Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.1–2, SEQ ID NO.5, SEQ ID NO.20–22, SEQ ID NO.34–35, SEQ ID NO.38, SEQ ID NO.46–47, and SEQ ID NO.

50.

3. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: The combination of isothermal amplification primers and CRISPR detection crRNA sequence is SEQ ID NO.52-54, SEQ ID NO.63-65, SEQ ID NO.74-76, SEQ ID NO.85-86 and SEQ ID NO.88; Alternatively, isothermal amplification primers and CRISPR detection crRNA sequence combination nine, the sequences being SEQ ID NO.52~53, SEQ ID NO.55, SEQ ID NO.63~64, SEQ ID NO.66, SEQ ID NO.74~75, SEQ ID NO.78, SEQ ID NO.85~86 and SEQ ID NO.89; Alternatively, a combination of isothermal amplification primers and CRISPR detection crRNA sequences could be used, with sequences defined as SEQ ID NO. 58–60, SEQ ID NO. 69–71, SEQ ID NO. 80–82, and SEQ ID NO. 91–93. Alternatively, there is a combination of isothermal amplification primers and CRISPR detection crRNA sequences, namely SEQ ID NO.58–59, SEQ ID NO.61, SEQ ID NO.69–70, SEQ ID NO.72, SEQ ID NO.80–81, SEQ ID NO.83, SEQ ID NO.91–92 and SEQ ID NO.

94.

4. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: It also includes isothermal amplification internal reference primers and CRISPR detection internal reference crRNA, with sequences SEQ ID NO. 96~103.

5. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: The following lyophilized reagents are available for RT-isothermal amplification of HIV-1-POL total nucleic acid-specific targets, HIV-1-LTR total nucleic acid-specific targets, HIV-2-POL total nucleic acid-specific targets, HIV-2-LTR total nucleic acid-specific targets, RT-isothermal amplification of internal control sequences, and primer-free RT-isothermal amplification. All reagents contain: trehalose 8-12%, dextran 1-4%, Tris-HCl buffer 40-60 mM, recombinase 150-250 nM, single-stranded DNA binding protein 400-600 nM, DNA polymerase 90-110 nM, dNTPs 0.1-2 mM, ATP 1-6 mM, creatine phosphate 30-40 mM, and creatine kinase 5-15 mM. nM, RT enzyme 0.05U / μL-0.2U / μL, specific primer 0.1-0.5μM, RNase inhibitor 0.2U / μL-0.6U / μL.

6. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: The following CRISPR lyophilized reagents are available for detection: CRISPR lyophilized reagents for HIV-1-POL total nucleic acid amplification products, CRISPR lyophilized reagents for HIV-1-LTR total nucleic acid amplification products, CRISPR lyophilized reagents for HIV-2-POL total nucleic acid amplification products, CRISPR lyophilized reagents for HIV-2-LTR total nucleic acid amplification products, CRISPR lyophilized reagents for internal control sequences, and CRISPR lyophilized reagents without crRNA. All reagents contain: trehalose 10-15%, dextran 4-8%, PEG 8000 0.5-5%, HEPES 20-50 mM, KCl 50-100 mM, DTT 1-7 mM, specific crRNA 10-150 nM, fluorescent probe 50-120 nM, and MgSO4 7-17 mM.

7. The HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in claim 1, characterized in that: It also includes magnesium ion lyophilization reagents: MgCl2 5-20 mM, trehalose 10-30% and dextran 5-15%.

8. The detection procedure of the HIV1 / 2 total nucleic acid detection kit based on isothermal amplification + CRISPR as described in any one of claims 1-7, characterized in that, Includes the following steps: S1, RT-isothermal amplification: The extracted total nucleic acid was added to the PCR reaction container for isothermal amplification. The eight-well array was pre-filled with magnesium ion lyophilization reagent and pre-filled with RT-isothermal amplification lyophilization reagents for HIV-1-POL total nucleic acid specific targets, HIV-1-LTR total nucleic acid specific targets, HIV-2-POL total nucleic acid specific targets, HIV-2-LTR total nucleic acid specific targets, internal control sequence, and primer-free RT-isothermal amplification lyophilization reagent. S2, CRISPR detection: The isothermal amplification products are transferred to another PCR reaction container for CRISPR reaction. The wells are pre-filled with CRISPR detection lyophilized reagents for HIV-1-POL total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-1-LTR total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-2-POL total nucleic acid amplification products, CRISPR detection lyophilized reagents for HIV-2-LTR total nucleic acid amplification products, CRISPR detection lyophilized reagents for internal control sequences, and CRISPR detection lyophilized reagents without crRNA. S3. Signal detection: Use a fluorescence PCR instrument to detect the fluorescence signal of each detection well.

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