A kit and method for detecting human adenovirus type 3 and type 7

Abstract

The present invention provides a kit and method for detecting human adenovirus type 3 and type 7, belonging to the technical field of virus detection. The present invention combines the MCDA amplification technology with the CRISPR / Cas12b detection technology, and designs a set of MCDA amplification primers, gRNA, and probe molecules for the Hexon gene of HAdv-3 and HAdv-7 at the same time. 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; moreover, this detection method has low requirements for equipment, is less restricted by the venue, is simple and convenient to operate, and can achieve on-site and instant detection of HAdv-3 and HAdv-7.

Description

Technical Field

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

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

[0003] To date, HAdv has been divided into 7 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 childhood ARITs and 1 - 7% of adult ARITs. Etiological surveillance studies have shown that serotypes 3 and 7 of subgroup B have had outbreak epidemics 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 helps to quickly implement appropriate monitoring measures, effectively control the spread of the epidemic, and develop effective care strategies in the early stage of infection.

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

[0005] Therefore, there is a need to develop a detection method for adenovirus type 3 and type 7 that has low requirements for equipment, is simple and convenient to operate, has high detection efficiency, and low cost. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a kit and method for detecting human adenovirus type 3 and type 7. The kit and 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; moreover, the detection method has low requirements for equipment, is less restricted by the venue, is simple and convenient to operate, and can achieve on-site and instant detection of HAdv-3 and HAdv-7.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a kit for detecting human adenovirus type 3 and type 7, which includes: MCDA primers, gRNA, CRISPR-Cas12b protein and probes;

[0009] 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;

[0010] The displacement primer pair includes a forward displacement primer F1 and a reverse displacement primer F2; the sequence of the forward displacement primer F1 is as shown in SEQ ID NO.1, and the sequence of the reverse displacement primer F2 is as shown in SEQ ID NO.2;

[0011] 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 as shown in SEQ ID NO.3, and the sequence of the reverse cross primer CP2 is as shown in SEQ ID NO.4;

[0012] 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 as shown in SEQ ID NO.5, and the sequence of the first forward amplification primer D2 is as shown in SEQ ID NO.6;

[0013] 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 as shown in SEQ ID NO.7, and the sequence of the second forward amplification primer C2 is as shown in SEQ ID NO.8;

[0014] 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 as shown in SEQ ID NO.9, and the sequence of the third forward amplification primer R2 is as shown in SEQ ID NO.10;

[0015] The sequence of the gRNA is as shown in SEQ ID NO.11;

[0016] The probe is ssDNA with fluorescence / quenching dual labeling.

[0017] In some embodiments of the present invention, the sequence of the ssDNA is as shown in SEQ ID NO.12.

[0018] In some embodiments of the present invention, the fluorescent label in the probe is FAM (carboxyfluorescein), and the quenching label is BHQ1 (a member of the black hole quencher family).

[0019] In some embodiments of the present invention, the kit further comprises a DNA polymerase with strand displacement activity, an amplification reaction buffer, a buffer for Cas12 protein trans-cleavage reaction, and double-distilled water;

[0020] Wherein, the amplification reaction buffer contains tris(hydroxymethyl)aminomethane hydrochloride, KCl, MgSO4, (NH4)2SO4, polysorbate 20, betaine, and deoxyribonucleotides.

[0021] In some embodiments of the present invention, the DNA polymerase with strand displacement activity is Bst 2.0 DNA polymerase.

[0022] In a second aspect, the present invention provides a method for detecting human adenovirus type 3 and type 7. The method is carried out using the kit as described in the first aspect, and the method comprises the following steps:

[0023] Extract the viral genomic DNA from the sample to be tested;

[0024] Use the multi-cross displacement amplification method and amplify the viral genomic DNA with MCDA primers to obtain an amplification product;

[0025] Complex the CRISPR-Cas12b protein with the gRNA to form a CRISPR-Cas12b-gRNA binary complex;

[0026] Incubate the amplification product, the CRISPR-Cas12b-gRNA binary complex with the probe to obtain an incubation product;

[0027] Perform fluorescence detection on the incubation product, and judge whether human adenovirus type 3 and human adenovirus type 7 exist in the sample to be tested according to the detected fluorescence signal.

[0028] The applicant has established a new isothermal nucleic acid amplification technology (CN104946744A), named multiple cross displacement amplification (MCDA). This amplification technology does not require template thermal denaturation and the support of expensive thermal cycling equipment. It only needs a metal bath or water bath that can maintain a constant temperature for operation. The amplification system only requires one isothermal displacement enzyme and can achieve nucleic acid amplification within 40 minutes. It has the advantages of fast amplification reaction, high sensitivity, and high specificity. In the MCDA system, there are a total of 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 three pairs of amplification primers (D1, C1, R1, D2, C2, and R2), thus ensuring the specificity of the amplification reaction. And it uses a single DNA polymerase with strand displacement activity to amplify the target template at a constant temperature, with low requirements for equipment and high amplification efficiency. Conventionally, gel electrophoresis, colorimetry, and turbidimetry are usually used to determine the MCDA amplification products. However, these methods cannot distinguish non-specific amplification and are prone to false positive results, thus affecting the specificity of detection.

[0029] 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, combined with the "collateral activity" exhibited by Cas proteins (such as Cas12a, Cas12b, Cas13), so it can accurately identify the target nucleic acid sequence and cleave the fluorescence / quenching dual-labeled single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA), thereby releasing a large amount of detectable fluorescence 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, thus rapidly and non-specifically cleaving the surrounding non-target single-stranded DNA (ssDNA). The broken ssDNA releases a large amount of fluorescence 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 relatively low.

[0030] The present invention combines the MCDA amplification technology with the CRISPR / Cas12b detection technology (named MCDA-CRISPR / Cas12b). Among them, the MCDA amplification technology can solve the shortcoming of the low sensitivity of the CRISPR / Cas12b detection system; at the same time, in the CRISPR / Cas12b system, the target recognition of the 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 its nearby PAM site, the side-chain cleavage activity of the CRISPR-Cas12b protein will be activated. This characteristic can effectively eliminate the false positive results that may occur during the MCDA amplification process, thereby significantly improving the specificity of molecular diagnosis.

[0031] In addition, in order to achieve the detection of HAdv-3 and HAdv-7, the present invention designs a set of MCDA amplification primers, gRNA, and ssDNA probe molecules for their Hexon genes. 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 brought by the lack of natural PAM sites in the target gene and increasing the target sequence selection range.

[0032] Clinical experimental verification shows 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 the accurate detection of HAdv-3 and HAdv-7. And because this detection method has low requirements for equipment, is less restricted by the venue, and is simple and convenient to operate, it can realize the on-site and instant detection of HAdv-3 and HAdv-7, which is beneficial to helping doctors carry out 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 stage of infection.

[0033] In some embodiments of the present invention, the amplification step includes:

[0034] Mix the viral genomic DNA, MCDA primers, DNA polymerase with strand displacement activity, amplification reaction buffer, and double-distilled water, and perform an amplification reaction at 62-69 °C (for example, it can be 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, it can be 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, or 60 min, etc.).

[0035] In some embodiments of the present invention, the step of forming the CRISPR-Cas12b-gRNA binary complex includes:

[0036] Mix the CRISPR-Cas12b protein, gRNA, buffer for Cas12 protein trans-cleavage reaction, and double-distilled water, and incubate at 37 °C for 8 - 10 min (e.g., it can be 8 min, 8.5 min, 9 min, 9.5 min, or 10 min, etc.).

[0037] In some embodiments of the present invention, the step of incubating the amplification product, the CRISPR-Cas12b-gRNA binary complex, and the probe includes:

[0038] Mix the amplification product, the CRISPR-Cas12b-gRNA binary complex, the probe, buffer for Cas12 protein trans-cleavage reaction, and double-distilled water, and incubate at 48 - 59 °C (e.g., it can be 48 °C, 50 °C, 52 °C, 53 °C, 55 °C, 56 °C, 58 °C, or 59 °C, etc.) for 5 - 15 min (e.g., it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, etc.).

[0039] In some embodiments of the present invention, the method for fluorescence detection is: using a real-time fluorescence instrument to detect the fluorescence intensity, or observing the fluorescence signal under the irradiation of excitation light.

[0040] In some embodiments of the present invention, the method for determining whether human adenovirus type 3 and human adenovirus type 7 are present in the test sample according to the detected fluorescence signal is:

[0041] If a fluorescence signal is detected, it is determined that human adenovirus type 3 and / or human adenovirus type 7 are present in the test sample; if no fluorescence signal is detected, it is determined that human adenovirus type 3 and human adenovirus type 7 are not present in the test sample.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention combines the MCDA amplification technology with the 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 simultaneously.

[0044] Clinical experiments have verified 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 accurately detect HAdv-3 and HAdv-7. Moreover, since this detection method has low requirements for equipment, is less restricted by the venue, and is simple and convenient to operate, on-site and immediate detection of HAdv-3 and HAdv-7 can be achieved. This is conducive to helping doctors carry out timely clinical intervention and epidemiological monitoring, thereby effectively controlling the spread of the epidemic and formulating effective nursing strategies in the early stage of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a comparison diagram of the Hexon conserved sequences of HAdv-3 and HAdv-7 with the sequences of other HAdV serotypes;

[0046] Figure 2 It is a diagram showing the positions of the MCDA primer binding sites and gRNA recognition sites in the target sequence;

[0047] Figure 3 It is a schematic diagram of the MCDA amplification reaction principle;

[0048] Figure 4 It is a schematic diagram of the reaction principle of CRISPR / Cas12b detection;

[0049] Figure 5A It is a dynamic turbidity curve diagram of the reaction system during the MCDA amplification process in the feasibility verification experiment of the HAdv-MCDA-CRISPR / Cas12b method;

[0050] Figure 5B It is a dynamic fluorescence intensity curve diagram of the reaction system during the CRISPR / Cas12b detection process in the feasibility verification experiment of the HAdv-MCDA-CRISPR / Cas12b method;

[0051] Figure 6 It is a dynamic turbidity curve diagram of the reaction system at different temperatures in the experiment of the optimal MCDA amplification reaction temperature;

[0052] Figure 7A It is a dynamic fluorescence intensity curve diagram of the reaction system when the cleavage time is 5 min in the experiment of the optimal CRISPR-Cas12b cleavage time;

[0053] Figure 7B It is a dynamic fluorescence intensity curve diagram of the reaction system when the cleavage time is 10 min in the experiment of the optimal CRISPR-Cas12b cleavage time;

[0054] Figure 7CIt is the dynamic fluorescence intensity curve of the reaction system when the cleavage time is 15 min in the optimal CRISPR-Cas12b cleavage time experiment;

[0055] Figure 8A It is the dynamic turbidity curve of the MCDA amplification reaction system at different concentrations of Hexon recombinant plasmid in the sensitivity experiment;

[0056] Figure 8B It is the dynamic fluorescence intensity curve of the reaction system at different concentrations of Hexon recombinant plasmid in the sensitivity experiment;

[0057] Figure 8C It is the fluorescence intensity map of the reaction system when CRISPR-Cas12b cleaves for 5 min at different concentrations of Hexon recombinant plasmid in the sensitivity experiment;

[0058] Figure 9 It is the dynamic fluorescence intensity curve of the reaction system when detecting different nucleic acid templates in the specificity experiment;

[0059] Figure 10 It is the fluorescence intensity heat map after the reaction of detecting clinical samples by the HAdv-MCDA-CRISPR / Cas12b method. Detailed implementation mode

[0060] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation modes. Those skilled in the art should understand that the specific implementation modes are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0061] In the embodiments of the present invention, the reagent combination for detecting HAdv-3 and HAdv-7 based on the MCDA-CRISPR / Cas12b technology includes: MCDA primers targeting the Hexon gene, gRNA corresponding to the amplification product of the Hexon gene primers, ssDNA probe molecules, and CRISPR-Cas12b protein. The process of the method for detecting HAdv-3 and HAdv-7 based on the 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 test sample) through the MCDA technology and obtaining the amplification product; (2) the CRISPR-Cas12b protein binds to the amplification product of the target gene under the guidance of gRNA, activates the side-chain cleavage activity of the CRISPR-Cas12b protein, and cleaves the ssDNA probe molecule, thereby emitting a fluorescence signal; (3) reporting the fluorescence signal through a real-time fluorescence detection system.

[0062] The following is a detailed description of the R & D process of the present invention.

[0063] 1. Preparation of target DNA and clinical samples

[0064] The Hexon gene sequences of HAdV types 3 and 7 were obtained from the National Center for Biotechnology Information (NCBI) of the United States and aligned using MEGA11 software to identify highly conserved regions. And through sequence alignment analysis on a computer, the target sequences of HAdv-3 and HAdv-7 were compared with other HAdV serotypes (as shown in Figure 1 ) to evaluate sequence specificity. The obtained DNA sequences were integrated into the pUC57 vector using existing conventional methods to obtain standard plasmids (synthesized by Beijing Tianyi Huiyuan Company, China). The standard plasmids were serially diluted 10-fold, with dilution factors ranging from 500 pg per microliter to 0.5 fg, for primer screening for MCDA amplification, exploration of the optimal temperature, and evaluation of sensitivity. And 24 non-HAdv templates extracted from other common respiratory pathogens were used to evaluate the specificity of the HAdv-MCDA-CRISPR / Cas12b method. And 88 clinical throat swab samples of acute respiratory infections were collected, and after DNA extraction, to evaluate the practicability of the HAdv-MCDA-CRISPR / Cas12b method in the detection of clinical samples.

[0065] 2. Design of MCDA primers and gRNA

[0066] A set of 10 primers was designed using PREMIER 5.0 software according to the MCDA amplification principle, and the gRNA sequence was designed according to the principle of the CRISPR / Cas12b system.

[0067] Among them, the MCDA primers include displacement primer pairs, crossover primer pairs, first amplification primer pairs, second amplification primer pairs, and third amplification primer pairs. The displacement primer pair includes F1 (forward displacement primer) and F2 (reverse displacement primer); the crossover primer pair includes CP1 (forward crossover primer) and CP2 (reverse crossover primer). To meet the requirements of the CRISPR / Cas12b system, the present invention modified the PAM site (-TTC-) at the middle junction of the forward crossover 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 the gRNA was synthesized and purified by Genscript Biotech Co., Ltd.

[0068] The MCDA primer binding sites and gRNA recognition sites are as shown in Figure 1 , Figure 2 . The sequences of the MCDA primers, gRNAs and probes are shown in Table 1. Figure 1 and Figure 2 . In Figure 1 , the right arrow and the left arrow represent the primer consistent with the sense strand and the primer reverse complementary to the sense strand respectively, and P1 and P2 are respectively a part of CP1 and CP2.

[0069] Table 1

[0070]

[0071] 3. HAdv-MCDA-CRISPR / Cas12b method

[0072] The HAdv-MCDA-CRISPR / Cas12b method adopted in the embodiments of the present invention includes the following steps:

[0073] (1) Obtain the DNA of the target gene (Hexon recombinant plasmid or viral genomic DNA in the sample to be tested).

[0074] (2) MCDA amplification

[0075] Use a 25 μL reaction system to mix the MCDA primers (final concentrations are: 0.4 μM for each of primers F1 and F2, 1.6 μM for each of primers CP1 and CP2, 0.8 μM for each of primers C1, C2, D1, D2, R1 and R2), 12.5 μL of 2× amplification reaction buffer, Bst 1.0 μL of 2.0 DNA polymerase, 1.0 μL of the obtained DNA sample and 8.3 μL of double-distilled water, and amplify at 62 - 69 °C for 40 min in a constant temperature device, and use a real-time turbidimeter (LA-320C) to measure the turbidity generated by the accumulation of magnesium pyrophosphate (the white precipitate formed during the MCDA reaction). After the amplification is completed, the MCDA amplification product is obtained. The principle of the MCDA amplification reaction is as shown in Figure 3 . Since CP1 modifies the PAM site, after the MCDA amplification is completed, the CRISPR-Cas12b protein recognition site is constructed on the target amplicon.

[0076] (3) Synthesis of the CRISPR-Cas12b-gRNA binary complex

[0077] ​Using a 100 µL reaction system, mix 1.5 µL of AapCas12b solution (10 µM, Cas12b protein from Alicyclobacillus acidophilus), 2 µL of gRNA solution (10 µM), 50 µL of 2× buffer for Cas12 protein trans-cleavage reaction, and 46.5 µL of double-distilled water, and incubate at 37 °C for 8 minutes to obtain the CRISPR-Cas12b-gRNA binary complex. The prepared complex is stored at low temperature (0 - 4 °C) for no more than 12 hours.

[0078] (4)CRISPR / Cas12b Detection

[0079] Using a 25 µL reaction system, mix 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× buffer for Cas12 protein trans-cleavage reaction, and 6 µL of double-distilled water, and incubate at 50 °C for 5 - 15 minutes. Set the ABI7500 real-time fluorescence system to record the fluorescence value once every 30 seconds.

[0080] The reaction principle of CRISPR / Cas12b detection is as Figure 4 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, the side-chain cleavage activity of the Cas12b protein is activated, cutting the ssDNA probe molecule and emitting a fluorescence signal; when the target product does not exist in the system, the ssDNA probe molecule cannot be cut and no fluorescence signal is released.

[0081] In the invention examples, the sources of some raw materials used are as follows: The isothermal DNA amplification kit, purchased from Tianjin Huidexin Technology Development Co., Ltd., contains Bst 2.0 DNA polymerase and amplification reaction buffer, purchased from Tianjin Huidexin Technology Development Co., Ltd.; Cas nuclease (AapCas12b), purchased from Tianjin Huidexin Technology Development Co., Ltd., contains AapCas12b solution and buffer for Cas12 protein trans-cleavage reaction.

[0082] 4. Feasibility Verification of the HAdv-MCDA-CRISPR / Cas12b Method

[0083] To verify the effectiveness and specificity of the MCDA primers and gRNA in the HAdv-MCDA-CRISPR / Cas12b method, the Hexon recombinant plasmid (500 fg / µL), Epstein-Barr virus genomic DNA, and distilled water (DW) were used as the 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".

[0084] The reaction conditions for MCDA amplification were 40 minutes of amplification at 67 °C. During the reaction, a real-time turbidimeter was used to monitor the turbidity of the reaction system, and the results are as Figure 5A shown. Figure 5A It was shown that the turbidity increased in the PC group, while there was no turbidity in the NC and BC groups.

[0085] 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 as Figure 5B shown. Figure 5B It was shown that the fluorescence intensities of the BC and NC groups were 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 and NC groups.

[0086] The above experimental results indicate 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, and preliminarily confirm the effectiveness of the HAdv-MCDA-CRISPR / Cas12b method established by the present invention.

[0087] 5. Optimization of the MCDA amplification reaction temperature

[0088] Temperature is crucial for the MCDA amplification efficiency and the final amplification result, and an appropriate temperature will greatly improve the amplification efficiency. In this experiment, 8 constant amplification reaction temperatures were set, ranging from 62 °C to 69 °C, at intervals of 1 °C. The 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 carried out according to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method". A turbidimeter (LA-320C) was used to monitor the turbidity of the reaction system, and a turbidity dynamic curve was obtained. The results are as Figure 6 shown.

[0089] Figure 6 It was shown that at a temperature of 66 °C, the amplification efficiency was the highest compared to other temperatures. Therefore, 66 °C was determined to be the optimal temperature for the MCDA amplification reaction. In the subsequent MCDA amplification step, the conditions were set as 66 °C for 40 minutes.

[0090] 6. Optimization of CRISPR-Cas12b Cleavage Time

[0091] To determine the shortest time required for the CRISPR-Cas12b protein to cleave the target product, this experiment set three different reaction times for CRISPR / Cas12b detection (5, 10, and 15 minutes), used the 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 above-determined optimal MCDA reaction conditions were used for the experiment, and the fluorescence values were detected by the ABI7500 real-time fluorescence system. The results are as Figures 7A - 7C shown. In Figures 7A - 7C , 1-7 represent the concentrations of the Hexon recombinant plasmid as 500 pg / µL, 50 pg / µL, 5 pg / µL, 500 fg / µL, 50 fg / µL, 5 fg / µL, 0.5 fg / µL respectively, and 8 represents the blank control group using double-distilled water.

[0092] Figures 7A - 7C It shows that a 5-minute reaction time is sufficient to complete the trans-cleavage step of the ssDNA probe. In the subsequent CRISPR / Cas12b detection step, the reaction conditions were set as 50 °C for 5 minutes.

[0093] 7. Sensitivity Test of HAdv-MCDA-CRISPR / Cas12b Method

[0094] To further determine the sensitivity of the HAdv-MCDA-CRISPR / Cas12b method and clarify the lowest concentration of the standard plasmid it can detect, this experiment serially diluted the Hexon recombinant plasmid 10-fold, with concentrations of 500 pg / µL, 50 pg / µL, 5 pg / µL, 500 fg / µL, 50 fg / µL, 5 fg / µL, 0.5 fg / µL respectively. 1 µL of each diluted standard plasmid was added to the reaction system, and at the same time, distilled water was set as the blank control. According to the steps in "3. HAdv-MCDA-CRISPR / Cas12b Method", the above-determined optimal reaction conditions were used for the experiment. The turbidity of the MCDA amplification reaction system was detected using a LA-320C turbidimeter, and the results are as Figure 8A shown; the fluorescence values were detected by the ABI7500 real-time fluorescence system, and the results are as Figure 8B , Figure 8C shown. Among them, 1-7 represent the concentrations of the Hexon recombinant plasmid as 500 pg / µL, 50 pg / µL, 5 pg / µL, 500 fg / µL, 50 fg / µL, 5 fg / µL, 0.5 fg / µL respectively, and 8 represents the blank control group using double-distilled water.

[0095] Figures 8A - 8C It shows that when the concentration of the Hexon recombinant plasmid is 5 fg / µL or higher, strong fluorescence signals can be detected; when the concentration of the Hexon recombinant plasmid is 0.5 fg / µL and in the blank control group, no fluorescence signals are detected. Therefore, the HAdv-MCDA-CRISPR / Cas12b method provided in the embodiments of the present invention can detect as low as 5 fg / µL of the Hexon recombinant plasmid per reaction.

[0096] 8. Specificity test of the HAdv-MCDA-CRISPR / Cas12b method

[0097] To study the specificity of the HAdv-MCDA-CRISPR / Cas12b method for detection, nucleic acid detection was performed on 1 Hexon recombinant plasmid and 24 non-HAdv nucleic acid extraction templates (the samples to be detected are shown in Table 2). According to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method", the optimal reaction conditions determined above were used for the experiment. The fluorescence values were detected by the ABI7500 real-time fluorescence system, and the results are as Figure 9 shown.

[0098] Figure 9 It shows that positive results only appear in the reaction tubes containing the Hexon recombinant plasmid, while negative results appear in the reaction tubes of non-HAdv. This indicates that the HAdv-MCDA-CRISPR / Cas12b method provided by the present invention has strong specificity for HAdv and meets the application requirements.

[0099] Table 2

[0100]

[0101] In Table 2, CIP: Capital Institute of Pediatrics; CDC: Chinese Center for Disease Control and Prevention; N: Negative; P: Positive.

[0102] 9. Feasibility verification of the HAdv-MCDA-CRISPR / Cas12b method for clinical samples

[0103] To further verify the feasibility of the HAdv-MCDA-CRISPR / Cas12b method for detecting HAdv-3 and HAdv-7 infections in a clinical setting, 88 throat swabs suspected of human adenovirus infection were collected from Children's Hospital at Capital Institute of Pediatrics. The nucleic acids of the collected samples were first extracted using the TransGen Biotech EasyPure Viral Genomic DNA Kit, and then detected according to the steps in "3. HAdv-MCDA-CRISPR / Cas12b method" using the optimal reaction conditions determined above. At the same time, to verify the accuracy of the HAdv-MCDA-CRISPR / Cas12b method, these clinical samples were also detected using the real-time PCR method, and the results were compared with those of the HAdv-MCDA-CRISPR / Cas12b method. The primers and probes for real-time PCR were from a published study (X. Lu, E. Trujillo-Lopez, L. Lott, D.D. Erdman, Quantitative Real-TimePcr Assay Panel for Detection and Type-Specific Identification of EpidemicRespiratory Human Adenoviruses, J. Clin. Microbiol. 51 (2013) 1089-1093.), and their sequences are shown in Table 3.

[0104] Table 3

[0105]

[0106] Record the fluorescence intensity after the reaction of all clinical samples detected by the HAdv-MCDA-CRISPR / Cas12b method and draw a heat map. The results are as Figure 10 shown. Among these samples, 47 were determined to be positive and 41 were determined to be negative. The detection results of real-time PCR are shown in Table 4.

[0107] Table 4

[0108]

[0109] All positive samples detected by the HAdv-MCDA-CRISPR / Cas12b method were confirmed to belong to HAdv-3 by real-time PCR, which might be due to HAdv-3 being the main prevalent strain during sample collection. The detection results of the HAdv-MCDA-CRISPR / Cas12b method were consistent with those of real-time PCR. These results indicate that the HAdv-MCDA-CRISPR / Cas12b method provided in the embodiments of the present invention can accurately reflect the infection status of clinical samples.

[0110] The above are only specific embodiments of the present disclosure, enabling those skilled in the art to understand or implement the present disclosure. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can 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 these embodiments described herein, but rather 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 primers, gRNA, CRISPR-Cas12b protein and a probe; Among them, 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 a ssDNA with fluorescence / quenching dual labels.

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

3. The kit according to claim 1 or 2, characterized in that, The kit further includes a DNA polymerase with strand displacement activity, an amplification reaction buffer, a buffer for the trans-cleavage reaction of Cas12 protein, and double-distilled water; Among them, the amplification reaction buffer contains tris(hydroxymethyl)aminomethane hydrochloride, KCl, MgSO4, (NH4)2SO4, polysorbate 20, betaine and deoxyribonucleotides.

4. The kit according to claim 3, wherein The DNA polymerase with strand displacement activity is Bst DNA polymerase 2.

0.

5. A method for detecting human adenovirus type 3 and type 7 for non-disease diagnosis purposes, characterized in that, The method is carried out using the kit according to any one of claims 1-4, and the method includes the following steps: Extract the viral genomic DNA from the sample to be tested; Using the multi-cross displacement amplification method, use the MCDA primers to amplify the viral genomic DNA to obtain an amplification product; Complex the CRISPR-Cas12b protein with the gRNA to form a CRISPR-Cas12b-gRNA binary complex; Incubate the amplification product, the CRISPR-Cas12b-gRNA binary complex with the probe to obtain an incubation product; Perform fluorescence detection on the incubation product, and determine whether human adenovirus type 3 and human adenovirus type 7 are present in the test sample according to the detected fluorescence signal.

6. The method according to claim 5, characterized in that The amplification step includes: Mix the viral genomic DNA, MCDA primers, DNA polymerase with strand displacement activity, amplification reaction buffer, and double-distilled water, and perform an amplification reaction at 62 - 69 °C for 40 - 60 min.

7. The method according to claim 5, characterized in that The step of forming the CRISPR-Cas12b-gRNA binary complex includes: Mix the CRISPR-Cas12b protein, gRNA, buffer for Cas12 protein trans-cleavage reaction, 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 amplification product, the CRISPR-Cas12b-gRNA binary complex with the probe includes: Mix the amplification product, the CRISPR-Cas12b-gRNA binary complex, the probe, buffer for Cas12 protein trans-cleavage reaction, and double-distilled water, and incubate at 48 - 59 °C for 5 - 15 min.

9. The method according to claim 5, wherein The method for fluorescence detection is: use a real-time fluorescence instrument to detect the fluorescence intensity, or observe the fluorescence signal under excitation light irradiation.

10. The method according to claim 5, characterized in that, The method for determining whether human adenovirus type 3 and human adenovirus type 7 are present in the test sample according to the detected fluorescence signal is: If a fluorescence signal is detected, it is determined that human adenovirus type 3 and / or human adenovirus type 7 are present in the test sample; if no fluorescence signal is detected, it is determined that human adenovirus type 3 and human adenovirus type 7 are not present in the test sample.

Images