Kit and method for detecting human adenoviruses 3 and 7

Abstract

The invention provides a kit and a method for detecting human adenoviruses 3 and 7, and belongs to the technical field of virus detection. According to the present invention, the MCDA amplification technology and the CRISPR / Cas12b detection technology are combined, and the MCDA amplification primer, the gRNA and the probe molecule are designed according to the Hexon gene of the HAdv-3 and the HAdv-7; the kit and the detection method provided by the invention have the advantages of high sensitivity, good specificity, high detection efficiency, accurate and objective interpretation and good repeatability, and can realize accurate detection of HAdv-3 and HAdv-7; and the detection method has low requirements on equipment, is less limited by places, is simple and convenient to operate, and can realize on-site and real-time detection of HAdv-3 and HAdv-7.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and in particular relates to a kit and a method for detecting human adenovirus types 3 and 7. Background Art

[0002] Human adenovirus (HAdv) is a non-enveloped double-stranded DNA virus that can cause a variety of diseases in humans, affecting multiple organ systems, including the respiratory tract, gastrointestinal tract, cornea, urinary system, cardiovascular system, and nervous system. HAdv infection can quickly cause an epidemic outbreak in crowded places.

[0003] To date, HAdv has been divided into seven subgenera (A to G) with at least 113 serotypes. Among them, acute respiratory tract infections (ARTIs) caused by subgroups B, C, and E are the most prevalent, accounting for approximately 5-10% of ARITs in children and 1-7% in adults. Pathogenic surveillance studies have shown that serotypes 3 and 7 of subspecies B have experienced outbreaks in many countries and regions around the world. Compared with HAdv-3, HAdv-7 has been reported to cause more severe clinical consequences, including severe pneumonia, toxic encephalopathy, sepsis, respiratory failure, and high mortality; in addition, it may also cause long-term pulmonary sequelae. Therefore, rapid and accurate identification of HAdv-3 and HAdv-7 can help to quickly implement appropriate surveillance measures, effectively control the spread of the epidemic, and develop effective care strategies in the early stages of infection.

[0004] A variety of technologies have been developed for the diagnosis of HAdv. However, traditional virus isolation and culture methods are laborious and time-consuming, and usually require weeks to obtain results. Although antigen detection methods such as immunofluorescence, enzyme immunoassay, immunochromatography, and latex agglutination are relatively fast, their sensitivity is generally lower than that of cell culture and molecular diagnostic methods. In the past few years, the development of molecular diagnostic methods (including polymerase chain reaction (PCR)-based technology and nucleic acid isothermal amplification (NIA) technology) has significantly improved the sensitivity, specificity, and turnaround time of detection. However, PCR-based technology requires complex thermal cycling equipment, professional operators, and 2-4 hours of operation time, which limits its application in the field of field diagnosis and its widespread use in resource-limited areas.

[0005] Therefore, it is necessary to develop a method for detecting adenovirus types 3 and 7 that has low equipment requirements, simple and convenient operation, high detection efficiency, and low cost. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a kit and method for detecting human adenovirus type 3 and type 7. The kit and the detection method have high sensitivity, good specificity, high detection efficiency, accurate and objective interpretation, good repeatability, and can achieve accurate detection of HAdv-3 and HAdv-7; and the detection method has low requirements for equipment, is less restricted by the location, is simple and convenient to operate, and can achieve on-site and immediate detection of HAdv-3 and HAdv-7.

[0007] To achieve this object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a kit for detecting human adenovirus type 3 and type 7, the kit comprising: MCDA primer, gRNA, CRISPR-Cas12b protein and probe; Wherein, the MCDA primers include a replacement primer pair, a cross primer pair, a first amplification primer pair, a second amplification primer pair and a third amplification primer pair; The replacement primer pair includes a forward replacement primer F1 and a reverse replacement primer F2; the sequence of the forward replacement primer F1 is shown in SEQ ID NO.1, and the sequence of the reverse replacement primer F2 is shown in SEQ ID NO.2; The cross primer pair includes a forward cross primer CP1 and a reverse cross primer CP2; the sequence of the forward cross primer CP1 is shown in SEQ ID NO.3, and the sequence of the reverse cross primer CP2 is shown in SEQ ID NO.4; The first amplification primer pair includes a first reverse amplification primer D1 and a first forward amplification primer D2; the sequence of the first reverse amplification primer D1 is shown in SEQ ID NO.5, and the sequence of the first forward amplification primer D2 is shown in SEQ ID NO.6; The second amplification primer pair includes a second reverse amplification primer C1 and a second forward amplification primer C2; the sequence of the second reverse amplification primer C1 is shown in SEQ ID NO.7, and the sequence of the second forward amplification primer C2 is shown in SEQ ID NO.8; The third amplification primer pair includes a third reverse amplification primer R1 and a third forward amplification primer R2; the sequence of the third reverse amplification primer R1 is shown in SEQ ID NO.9, and the sequence of the third forward amplification primer R2 is shown in SEQ ID NO.10; The sequence of the gRNA is shown in SEQ ID NO.11; The probe is ssDNA with fluorescence / quenching dual labeling.

[0008] In some embodiments of the present invention, the sequence of the ssDNA is shown as SEQ ID NO.12.

[0009] In some embodiments of the present invention, the fluorescent marker in the probe is FAM (carboxyfluorescein), and the quenching marker is BHQ1 (a member of the black hole quencher family).

[0010] In some embodiments of the present invention, the kit further comprises a DNA polymerase having strand displacement activity, an amplification reaction buffer, a Cas12 protein trans-cleavage reaction buffer, and double distilled water; The amplification reaction buffer contains tris(hydroxymethyl)aminomethane hydrochloride, KCl, MgSO4, (NH4)2SO4, polyoxyethylene sorbitan monolaurate, (carboxymethyl)trimethylamine inner salt and deoxyribonucleotides.

[0011] In some embodiments of the present invention, the DNA polymerase having strand displacement activity is Bst 2.0 DNA polymerase.

[0012] In a second aspect, the present invention provides a method for detecting human adenovirus type 3 and type 7, the method being performed using the kit as described in the first aspect, the method comprising the following steps: Extracting viral genomic DNA from the sample to be tested; The viral genomic DNA is amplified using MCDA primers using a multiple cross-displacement amplification method to obtain an amplified product; Compounding the CRISPR-Cas12b protein with the gRNA to form a CRISPR-Cas12b-gRNA binary complex; Incubate the amplified product, the CRISPR-Cas12b-gRNA binary complex and the probe to obtain an incubation product; The incubation product is subjected to fluorescence detection, and it is determined whether human adenovirus type 3 and human adenovirus type 7 exist in the sample to be tested according to the detected fluorescence signal.

[0013] The applicant has established a new isothermal nucleic acid amplification technology (CN104946744A), named multiple cross displacement amplification (MCDA). This amplification technology does not require the support of template thermal denaturation and expensive thermal cycling equipment. It only requires a metal bath or water bath that can be heated at a constant temperature to operate. The amplification system only requires a constant temperature displacement enzyme, and nucleic acid amplification can be achieved within 40 minutes. It has the advantages of fast amplification reaction, sensitive reaction, and high specificity. In the MCDA system, there are 10 primers to identify the target sequence, including a pair of cross primers (CP1 and CP2), a pair of displacement primers (F1 and F2) and 3 pairs of amplification primers (D1, C1, R1, D2, C2 and R2), so as to ensure the specificity of the amplification reaction, and a single DNA polymerase with chain displacement activity can be used to amplify the target template at a constant temperature, with low equipment requirements and high amplification efficiency. Gel electrophoresis, colorimetry and turbidity methods are usually used to determine the MCDA amplification product. However, these methods cannot distinguish nonspecific amplification and are prone to false-positive results, thus affecting the specificity of the test.

[0014] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR / Cas) systems have shown significant advantages and potential in the field of molecular detection. The CRISPR / Cas system has inherent programmability, coupled with the "incidental activity" exhibited by Cas proteins (such as Cas12a, Cas12b, and Cas13), so it can accurately identify the target nucleic acid sequence and cut the fluorescent / quenching double-labeled single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA), thereby releasing a large amount of detectable fluorescent signals. For example, the CRISPR-Cas12b protein used in the present invention is an RNA-guided DNA targeting enzyme. When the CRISPR-Cas12b-gRNA binary complex recognizes the complementary target sequence and the adjacent protospacer adjacent motif (PAM) site (TTC) under the guidance of gRNA, the side chain cleavage activity of the Cas12b protein is activated, thereby rapidly and non-specifically cleaving the surrounding non-target single-stranded DNA (ssDNA), and the broken ssDNA releases a large amount of fluorescent signals, and the fluorescence value can be recorded by a real-time fluorescence instrument. However, the detection sensitivity of a single CRISPR / Cas system is low.

[0015] The present invention combines MCDA amplification technology with CRISPR / Cas12b detection technology (named MCDA-CRISPR / Cas12b). Among them, MCDA amplification technology can solve the disadvantage of low sensitivity of CRISPR / Cas12b detection system; at the same time, in the CRISPR / Cas12b system, the targeted recognition of CRISPR-Cas12b protein depends on the gRNA to recognize the complementary DNA target sequence and the PAM near the DNA target sequence, that is, only after the gRNA binds to the complementary target sequence and the PAM site near it, will the side chain cleavage activity of CRISPR-Cas12b protein be stimulated. This feature can effectively eliminate the false positive results that may occur during MCDA amplification, thereby significantly improving the specificity of molecular diagnosis.

[0016] In addition, in order to detect HAdv-3 and HAdv-7, the present invention designs a set of MCDA amplification primers, gRNA, and ssDNA probe molecules for the Hexon genes of the two. Among them, the present invention integrates the PAM site sequence (TTC) into the connection region of the MCDA forward cross primer CP1, thereby overcoming the limitation caused by the lack of natural PAM sites in the target gene and increasing the selection range of the target sequence.

[0017] Clinical experiments have shown that the kit and detection method provided by the present invention have high sensitivity, good specificity, high detection efficiency, accurate and objective interpretation, good repeatability, and can achieve accurate detection of HAdv-3 and HAdv-7. And because the detection method has low requirements for equipment, is less restricted by the location, and is simple and convenient to operate, it can achieve on-site and immediate detection of HAdv-3 and HAdv-7, which is conducive to helping doctors to conduct clinical intervention and epidemiological monitoring in a timely manner, thereby effectively controlling the spread of the epidemic and formulating effective nursing strategies in the early stages of infection.

[0018] In some embodiments of the present invention, the step of amplifying comprises: The viral genomic DNA, MCDA primer, DNA polymerase with strand displacement activity, amplification reaction buffer and double distilled water are mixed, and an amplification reaction is carried out at 62-69° C. (for example, 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C. or 69° C., etc., preferably 66° C.) for 40-60 min (for example, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min or 60 min, etc.).

[0019] In some embodiments of the present invention, the step of forming a CRISPR-Cas12b-gRNA binary complex comprises: Mix the CRISPR-Cas12b protein, gRNA, Cas12 protein trans-cleavage reaction buffer and double distilled water, and incubate at 37°C for 8-10 min (for example, 8 min, 8.5 min, 9 min, 9.5 min or 10 min, etc.).

[0020] In some embodiments of the present invention, the step of incubating the amplified product, the CRISPR-Cas12b-gRNA binary complex and the probe comprises: The amplified product, the CRISPR-Cas12b-gRNA binary complex, the probe, the Cas12 protein trans-cleavage reaction buffer and double distilled water are mixed, and incubated at 48-59° C. (for example, 48° C., 50° C., 52° C., 53° C., 55° C., 56° C., 58° C. or 59° C., etc.) for 5-15 min (for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.).

[0021] In some embodiments of the present invention, the fluorescence detection method is: using a real-time fluorescence instrument to detect the fluorescence intensity, or observing the fluorescence signal under the irradiation of excitation light.

[0022] In some embodiments of the present invention, the method for determining whether human adenovirus type 3 and human adenovirus type 7 exist in the sample to be tested according to the detected fluorescent signal is: If the fluorescent signal is detected, it is determined that human adenovirus type 3 and / or human adenovirus type 7 exist in the sample to be tested; if no fluorescent signal is detected, it is determined that human adenovirus type 3 and human adenovirus type 7 do not exist in the sample to be tested.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention combines MCDA amplification technology with CRISPR / Cas12b detection technology (named MCDA-CRISPR / Cas12b), and designs a set of MCDA amplification primers, gRNA, and ssDNA probe molecules for the Hexon genes of HAdv-3 and HAdv-7.

[0024] Clinical experiments have shown that the kit and detection method provided by the present invention have high sensitivity, good specificity, high detection efficiency, accurate and objective interpretation, good repeatability, and can achieve accurate detection of HAdv-3 and HAdv-7. And because the detection method has low requirements for equipment, is less restricted by the location, and is simple and convenient to operate, it can achieve on-site and immediate detection of HAdv-3 and HAdv-7, which is conducive to helping doctors to conduct clinical intervention and epidemiological monitoring in a timely manner, thereby effectively controlling the spread of the epidemic and formulating effective nursing strategies in the early stages of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a comparison diagram of the conserved Hexon sequences of HAdv-3 and HAdv-7 with the sequences of other HAdV serotypes; Figure 2 A map showing the location of the MCDA primer binding site and gRNA recognition site in the target sequence; Figure 3 This is the schematic diagram of the McdA amplification reaction; Figure 4 Schematic diagram of the reaction for CRISPR / Cas12b detection; Figure 5A This is a dynamic curve diagram of the turbidity of the reaction system during the MCDA amplification process in the feasibility verification experiment of the HAdv-MCDA-CRISPR / Cas12b method; Figure 5B This is a dynamic curve diagram of the fluorescence intensity of the reaction system during the CRISPR / Cas12b detection process in the feasibility verification experiment of the HAdv-MCDA-CRISPR / Cas12b method; Figure 6 This is the turbidity dynamic curve of the reaction system at different temperatures in the optimal MCDA amplification reaction temperature experiment; Fig. 7A This is a dynamic curve of the fluorescence intensity of the reaction system when the shearing time is 5 minutes in the optimal CRISPR-Cas12b shearing time experiment; Figure 7B This is a dynamic curve of the fluorescence intensity of the reaction system when the shearing time is 10 minutes in the optimal CRISPR-Cas12b shearing time experiment; Figure 7C This is a dynamic curve of the fluorescence intensity of the reaction system when the shearing time is 15 minutes in the optimal CRISPR-Cas12b shearing time experiment; Fig. 8A This is the turbidity dynamic curve of the MCDA amplification reaction system at different Hexon recombinant plasmid concentrations in the sensitivity experiment; Figure 8BThis is a dynamic curve diagram of the fluorescence intensity of the reaction system at different Hexon recombinant plasmid concentrations in the sensitivity experiment; Figure 8C This is the fluorescence intensity diagram of the reaction system when CRISPR-Cas12b shears for 5 minutes at different Hexon recombinant plasmid concentrations in the sensitivity experiment; Fig. 9 It is a dynamic curve diagram of the fluorescence intensity of the reaction system when detecting different nucleic acid templates in the specificity experiment; Fig.10 This is a heat map of the fluorescence intensity after the reaction of clinical samples detected by the HAdv-MCDA-CRISPR / Cas12b method is completed. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0027] In an embodiment of the present invention, a reagent combination for detecting HAdv-3 and HAdv-7 based on MCDA-CRISPR / Cas12b technology includes: MCDA primers for Hexon genes, gRNA corresponding to the amplification products of Hexon gene primers, ssDNA probe molecules, and CRISPR-Cas12b proteins. The process of the method for detecting HAdv-3 and HAdv-7 based on MCDA-CRISPR / Cas12b technology (hereinafter referred to as HAdv-MCDA-CRISPR / Cas12b) includes: (1) amplifying the DNA of the target gene (Hexon gene DNA, or viral genomic DNA in the sample to be tested) by MCDA technology, and obtaining an amplification product; (2) CRISPR-Cas12b protein binds to the amplification product of the target gene under the guidance of gRNA, stimulates the side chain cleavage activity of CRISPR-Cas12b protein, cuts the ssDNA probe molecule, and emits a fluorescent signal; (3) reporting the fluorescent signal by a real-time fluorescence detection system.

[0028] The following is a detailed description of the development process of the present invention.

[0029] 1. Preparation of target DNA and clinical samples The Hexon gene sequences of HAdV 3 and 7 were obtained from the National Center for Biotechnology Information (NCBI) and aligned using MEGA11 software to identify the highly conserved regions. The target sequences of HAdv-3 and HAdv-7 were compared with other HAdV serotypes by sequence alignment analysis on a computer (e.g. Figure 1As shown in the figure, the sequence specificity was evaluated. The obtained DNA sequence was integrated into the pUC57 vector using existing conventional methods to obtain a standard plasmid (synthesized by Tianyi Huiyuan Company, Beijing, China). The standard plasmid was diluted 10 times in a row, from 500 pg to 0.5 fg per microliter, for primer screening, optimal temperature exploration and sensitivity evaluation of MCDA amplification. The specificity of the HAdv-MCDA-CRISPR / Cas12b method was evaluated using 24 non-HAdv templates extracted from other common respiratory pathogens. In addition, 88 clinical throat swab samples of acute respiratory infection were collected, and DNA was extracted to evaluate the practicality of the HAdv-MCDA-CRISPR / Cas12b method in clinical sample detection.

[0030] 2. Design of MCDA primers and gRNA A set of 10 primers was designed using PREMIER 5.0 software based on the MCDA amplification principle, and the gRNA sequence was designed based on the principle of the CRISPR / Cas12b system.

[0031] Among them, the MCDA primers include a displacement primer pair, a cross primer pair, a first amplification primer pair, a second amplification primer pair, and a third amplification primer pair. The displacement primer pair includes F1 (forward displacement primer) and F2 (reverse displacement primer); the cross primer pair includes CP1 (forward cross primer) and CP2 (reverse cross primer). In order to meet the requirements of the CRISPR / Cas12b system, the present invention modifies the PAM site (-TTC-) at the middle connection of the forward cross primer CP1; the first amplification primer pair includes D1 (first reverse amplification primer) and D2 (first forward amplification primer); the second amplification primer pair includes C1 (second reverse amplification primer) and C2 (second forward amplification primer); the third amplification primer pair includes R1 (third reverse amplification primer) and R2 (third forward amplification primer). All MCDA primers were synthesized and purified by DIA-UP Biotech Co., Ltd., and gRNA was synthesized and purified by Genscript Biotech Co., Ltd.

[0032] McdA primer binding site and gRNA recognition site as Figure 1 , Figure 2 The sequences of MCDA primers, gRNA, and probe are shown in Table 1. Figure 1 and Figure 2 In the figure, the right arrow and the left arrow represent the primer consistent with the positive chain and the primer reverse complementary to the positive chain, respectively. P1 and P2 are parts of CP1 and CP2, respectively. Figure 1 The boxed part in the figure represents the gRNA recognition site (gRNA forms a complementary pair with the amplicon of the target gene).

[0033] Table 1

[0034] 3. HAdv-MCDA-CRISPR / Cas12b method The HAdv-MCDA-CRISPR / Cas12b method used in the embodiment of the present invention comprises the following steps: (1) Obtain the DNA of the target gene (Hexon recombinant plasmid, or viral genomic DNA in the sample to be tested).

[0035] (2) McdA amplification In a 25 µL reaction system, MCDA primers (final concentrations: primers F1 and F2 0.4 µM each, primers CP1 and CP2 1.6 µM each, primers C1, C2, D1, D2, R1 and R2 0.8 µM each), 2× amplification reaction buffer 12.5 µL, Bst 2.0 DNA polymerase 1.0µL, DNA sample 1.0µL and double distilled water 8.3µL were mixed, and the amplification reaction was carried out at 62-69℃ in a constant temperature device for 40 min. The turbidity generated by the accumulation of magnesium pyrophosphate (white precipitate formed during the MCDA reaction) was measured using a real-time turbidity meter (LA-320C). After the amplification was completed, the MCDA amplification product was obtained. The principle of MCDA amplification reaction is as follows Figure 3 As shown, since CP1 modified the PAM site, a CRISPR-Cas12b protein recognition site was constructed on the target amplicon after completing the MCDA amplification.

[0036] (3) Synthesis of CRISPR-Cas12b-gRNA binary complex Using a 100µL reaction system, 1.5µL of AapCas12b solution (10 µM, Cas12b protein from Alicyclobacillus acidophilus), 2 µL of gRNA solution (10 µM), 50µL of 2×Cas12 protein trans-cleavage reaction buffer, and 46.5µL of double distilled water were mixed and incubated at 37°C for 8 minutes to obtain a CRISPR-Cas12b-gRNA binary complex. The prepared complex was stored at low temperature (0-4°C) for no more than 12 hours.

[0037] (4) CRISPR / Cas12b detection Using a 25-µL reaction system, 2 µL of MCDA amplification product, 4 µL of CRISPR-Cas12a-gRNA binary complex, 0.5 µL of probe solution (100 µM), 12.5 µL of 2×Cas12 protein trans-cleavage reaction buffer, and 6 µL of double distilled water were mixed, incubated at 50°C for 5-15 minutes, and the ABI7500 real-time fluorescence system was set to record the fluorescence value every 30 seconds.

[0038] The reaction principle of CRISPR / Cas12b detection is as follows Figure 4 As shown, when the target product exists in the system, the Cas12b-gRNA binary complex recognizes the complementary target sequence and the adjacent PAM site under the guidance of gRNA, and the side chain cleavage activity of the Cas12b protein is activated, cutting the ssDNA probe molecule and emitting a fluorescent signal; when the target product does not exist in the system, the ssDNA probe molecule cannot be cut and no fluorescent signal is released.

[0039] In the examples of the invention, some of the raw materials used were from the following sources: DNA isothermal amplification kit, purchased from Tianjin Huidexin Technology Development Co., Ltd., including Bst 2.0 DNA polymerase and amplification reaction buffer were purchased from Tianjin Huidexin Technology Development Co., Ltd.; Cas nuclease (AapCas12b) was purchased from Tianjin Huidexin Technology Development Co., Ltd., including AapCas12b solution and Cas12 protein trans-cleavage reaction buffer.

[0040] 4. Feasibility verification of HAdv-MCDA-CRISPR / Cas12b method In order to verify the effectiveness and specificity of MCDA primers and gRNA in the HAdv-MCDA-CRISPR / Cas12b method, Hexon recombinant plasmid (500 fg / µL), Epstein-Barr virus genomic DNA and distilled water (DW) were used as positive control (PC), negative control (NC) and blank control (BC), respectively, and the experiment was carried out according to the steps in “3. HAdv-MCDA-CRISPR / Cas12b method”.

[0041] The reaction conditions for MCDA amplification were 67°C for 40 minutes. During the reaction, a real-time turbidity meter was used to monitor the turbidity of the reaction system. Figure 5A shown. Figure 5A The PC group showed increased turbidity, while the NC and BC groups showed no turbidity.

[0042] The reaction conditions for CRISPR / Cas12b detection were incubation at 50°C for 5 minutes. The detection results of the real-time fluorescence system are shown in Figure 5B shown. Figure 5BIt was shown that the fluorescence intensity of the BC group and the NC group was almost 0, and the fluorescence intensity of the reaction tube containing the target MCDA product (PC group) was significantly higher than that of the BC group and the NC group.

[0043] The above experimental results show that the MCDA primers and gRNA used in the embodiments of the present invention can be used for the detection of the target Hexon gene, which preliminarily confirms the effectiveness of the HAdv-MCDA-CRISPR / Cas12b method established in the present invention.

[0044] 5. Optimization of MCDA amplification reaction temperature Temperature has a critical effect on MCDA amplification efficiency and the final amplification results. A suitable temperature will greatly improve the amplification efficiency. This experiment set 8 constant amplification reaction temperatures ranging from 62°C to 69°C, with an interval of 1°C. Hexon recombinant plasmid (1 pg / µL) was used as the positive control (PC), and double distilled water was used as the blank control (BC). MCDA amplification was performed according to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method". A turbidity meter (LA-320C) was used to monitor the turbidity of the reaction system and obtain a turbidity dynamic curve. The results are shown in Figure 2. Figure 6 shown.

[0045] Figure 6 It was shown that at 66°C, the amplification efficiency was the highest compared to other temperatures, so 66°C was determined to be the optimal temperature for the MCDA amplification reaction. In the subsequent MCDA amplification step, the conditions were set at 66°C for 40 minutes.

[0046] 6. Optimization of CRISPR-Cas12b shearing time In order to determine the shortest time required for CRISPR-Cas12b protein to cut the target product, this experiment set three different CRISPR / Cas12b detection reaction times (5, 10 and 15 minutes), using Hexon recombinant plasmid (7 different concentrations) as the positive control (PC), and double distilled water as the blank control (BC). According to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method", the optimal MCDA reaction conditions determined above were used for the experiment, and the fluorescence value was detected by ABI7500 real-time fluorescence system. The results are shown in the figure. Figure 7A to Figure 7C As shown. Figure 7A to Figure 7C In the table, 1-7 represent the concentrations of Hexon recombinant plasmids of 500pg / µL, 50pg / µL, 5pg / µL, 500fg / µL, 50fg / µL, 5fg / µL, and 0.5fg / µL, respectively, and 8 represents the blank control group using double distilled water.

[0047] Figure 7A to Figure 7CIt was shown that a 5-minute reaction time was sufficient to complete the trans-cleavage step of the ssDNA probe. In the subsequent CRISPR / Cas12b detection step, the reaction conditions were set at 50°C for 5 minutes.

[0048] 7. Sensitivity test of HAdv-MCDA-CRISPR / Cas12b method In order to further determine the sensitivity of the HAdv-MCDA-CRISPR / Cas12b method and clarify the lowest concentration of the standard plasmid that can be detected, this experiment diluted the Hexon recombinant plasmid 10 times in succession, with concentrations of 500pg / µL, 50pg / µL, 5pg / µL, 500fg / µL, 50fg / µL, 5fg / µL, and 0.5fg / µL, respectively. 1µL of each diluted standard plasmid was added to the reaction system, and distilled water was set as a blank control at the same time. According to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method", the experiment was carried out using the optimal reaction conditions determined above. The turbidity of the MCDA amplification reaction system was detected using a LA-320C turbidity meter, and the results are as follows: Fig. 8A As shown; the fluorescence value was detected by ABI7500 real-time fluorescence system, and the results were as follows Figure 8B , Figure 8C As shown. Among them, 1-7 represent the concentrations of Hexon recombinant plasmids of 500pg / µL, 50pg / µL, 5pg / µL, 500fg / µL, 50fg / µL, 5fg / µL, and 0.5fg / µL, respectively, and 8 represents the blank control group using double distilled water.

[0049] Figure 8A to Figure 8C It is shown that when the concentration of Hexon recombinant plasmid is 5fg / µL and above, a strong fluorescence signal can be detected; when the concentration of Hexon recombinant plasmid is 0.5fg / µL, and in the blank control group, no fluorescence signal is detected. Therefore, the HAdv-MCDA-CRISPR / Cas12b method provided in the embodiment of the present invention can detect Hexon recombinant plasmid as low as 5 fg / µL per reaction.

[0050] 8. Specificity test of HAdv-MCDA-CRISPR / Cas12b method In order to study the specificity of the HAdv-MCDA-CRISPR / Cas12b method, this experiment conducted nucleic acid detection on 1 Hexon recombinant plasmid and 24 non-HAdv extracted nucleic acid templates (see Table 2 for samples to be tested). According to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method", the experiment was carried out using the optimal reaction conditions of the above determination. The fluorescence value was detected by the ABI7500 real-time fluorescence system. The results are shown in Fig. 9shown.

[0051] Fig. 9 It shows that positive results only appear in the reaction tube containing the Hexon recombinant plasmid, and negative results appear in the reaction tube without HAdv. This shows that the HAdv-MCDA-CRISPR / Cas12b method provided by the present invention has strong specificity for HAdv and meets the application requirements.

[0052] Table 2

[0053] In Table 2, CIP: Capital Institute of Pediatrics; CDC: Chinese Center for Disease Control and Prevention; N: negative; P: positive.

[0054] 9. Feasibility verification of HAdv-MCDA-CRISPR / Cas12b method for clinical samples To further verify the feasibility of the HAdv-MCDA-CRISPR / Cas12b method for detecting HAdv-3 and HAdv-7 infections in clinical settings, 88 pharyngeal swabs suspected of being infected with human adenovirus were collected from the Children's Hospital affiliated to the Capital Institute of Pediatrics. The collected samples were first extracted with the All-Gold EasyPure Viral Genomic DNA Kit, and then tested using the optimal reaction conditions of the above assay according to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method". At the same time, in order to verify the accuracy of the HAdv-MCDA-CRISPR / Cas12b method, these clinical samples were tested using the real-time PCR method, and the results were compared with the HAdv-MCDA-CRISPR / Cas12b method. The primers and probes for real-time PCR were from published studies (X. Lu, E. Trujillo-Lopez, L. Lott, DD Erdman, Quantitative Real-Time PCR Assay Panel for Detection and Type-Specific Identification of Epidemic Respiratory Human Adenoviruses, J. Clin. Microbiol. 51 (2013) 1089-1093.), and their sequences are shown in Table 3.

[0055] Table 3

[0056] The fluorescence intensity of all clinical samples tested by the HAdv-MCDA-CRISPR / Cas12b method after the reaction was completed was recorded and a heat map was drawn. The results are as follows Fig.10 As shown, 47 of these samples were determined to be positive and 41 were determined to be negative. The results of the real-time PCR test are shown in Table 4.

[0057] Table 4

[0058] All positive samples detected by the HAdv-MCDA-CRISPR / Cas12b method were confirmed to belong to HAdv-3 by real-time PCR, which may be due to the fact that HAdv-3 was the main epidemic strain during the sample collection period. The detection results of the HAdv-MCDA-CRISPR / Cas12b method were consistent with the detection results of real-time PCR. These results show that the HAdv-MCDA-CRISPR / Cas12b method provided in the embodiment of the present invention can accurately reflect the infection status of clinical samples.

[0059] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for detecting human adenovirus type 3 and type 7, characterized in that: The kit includes: MCDA primer, gRNA, CRISPR-Cas12b protein and probe; Wherein, the MCDA primers include a replacement primer pair, a cross primer pair, a first amplification primer pair, a second amplification primer pair and a third amplification primer pair; The replacement primer pair includes a forward replacement primer F1 and a reverse replacement primer F2; the sequence of the forward replacement primer F1 is shown in SEQ ID NO.1, and the sequence of the reverse replacement primer F2 is shown in SEQ ID NO.2; The cross primer pair includes a forward cross primer CP1 and a reverse cross primer CP2; the sequence of the forward cross primer CP1 is shown in SEQ ID NO.3, and the sequence of the reverse cross primer CP2 is shown in SEQ ID NO.4; The first amplification primer pair includes a first reverse amplification primer D1 and a first forward amplification primer D2; the sequence of the first reverse amplification primer D1 is shown in SEQ ID NO.5, and the sequence of the first forward amplification primer D2 is shown in SEQ ID NO.6; The second amplification primer pair includes a second reverse amplification primer C1 and a second forward amplification primer C2; the sequence of the second reverse amplification primer C1 is shown in SEQ ID NO.7, and the sequence of the second forward amplification primer C2 is shown in SEQ ID NO.8; The third amplification primer pair includes a third reverse amplification primer R1 and a third forward amplification primer R2; the sequence of the third reverse amplification primer R1 is shown in SEQ ID NO.9, and the sequence of the third forward amplification primer R2 is shown in SEQ ID NO.10; The sequence of the gRNA is shown in SEQ ID NO.11; The probe is ssDNA with fluorescence / quenching dual labeling.

2. The kit according to claim 1, characterized in that The sequence of the ssDNA is shown in SEQ ID NO.12; And / or, the fluorescent marker in the probe is FAM, and the quenching marker is BHQ1.

3. The kit according to claim 1 or 2, characterized in that The kit also includes a DNA polymerase with strand displacement activity, an amplification reaction buffer, a Cas12 protein trans-cleavage reaction buffer, and double distilled water; The amplification reaction buffer contains tris(hydroxymethyl)aminomethane hydrochloride, KCl, MgSO4, (NH4)2SO4, polyoxyethylene sorbitan monolaurate, (carboxymethyl)trimethylamine inner salt and deoxyribonucleotides.

4. The kit according to claim 3, characterized in that The DNA polymerase having strand displacement activity is Bst 2.0 DNA polymerase.

5. A method for detecting human adenovirus type 3 and type 7, characterized in that: The method is carried out using the kit according to any one of claims 1 to 4, and the method comprises the following steps: Extracting viral genomic DNA from the sample to be tested; The viral genomic DNA is amplified using MCDA primers using a multiple cross-displacement amplification method to obtain an amplified product; The CRISPR-Cas12b protein is complexed with the gRNA to form a CRISPR-Cas12b-gRNA binary complex; Incubate the amplified product, the CRISPR-Cas12b-gRNA binary complex and the probe to obtain an incubation product; The incubation product is subjected to fluorescence detection, and it is determined whether human adenovirus type 3 and human adenovirus type 7 exist in the sample to be tested according to the detected fluorescence signal.

6. The method according to claim 5, characterized in that The amplification step comprises: The viral genomic DNA, MCDA primer, DNA polymerase with strand displacement activity, amplification reaction buffer and double distilled water are mixed, and an amplification reaction is carried out at 62-69° C. for 40-60 min.

7. The method according to claim 5, characterized in that The steps of forming the CRISPR-Cas12b-gRNA binary complex include: Mix CRISPR-Cas12b protein, gRNA, Cas12 protein trans-cleavage reaction buffer and double-distilled water and incubate at 37°C for 8-10 min.

8. The method according to claim 5, characterized in that The step of incubating the amplified product, the CRISPR-Cas12b-gRNA binary complex and the probe comprises: The amplified product, the CRISPR-Cas12b-gRNA binary complex, the probe, the Cas12 protein trans-cleavage reaction buffer and double distilled water were mixed and incubated at 48-59° C. for 5-15 min.

9. The method according to claim 5, characterized in that The fluorescence detection method is: using a real-time fluorescence instrument to detect the fluorescence intensity, or observing the fluorescence signal under the irradiation of excitation light.

10. The method according to claim 5, characterized in that The method for judging whether human adenovirus type 3 and human adenovirus type 7 exist in the sample to be tested according to the detected fluorescent signal is: If the fluorescent signal is detected, it is determined that human adenovirus type 3 and / or human adenovirus type 7 exist in the sample to be tested; if no fluorescent signal is detected, it is determined that human adenovirus type 3 and human adenovirus type 7 do not exist in the sample to be tested.

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