Norovirus nucleic acid extraction device, detection kit and detection method

Abstract

The invention is applicable to the technical field of biology, and provides a norovirus nucleic acid extraction device, a detection kit and a detection method. According to the detection kit and the detection method, the GII type norovirus is detected on the basis of a reverse transcription recombinase mediated isothermal amplification / CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Cas12a) technology. After nucleic acid extraction is carried out by adopting a pressing column type nucleic acid extraction device, a sample to be detected is added into a reverse transcription recombinase mediated isothermal amplification / CRISPR-Cas12a detection system, a target fragment is amplified by utilizing a reverse transcription recombinase mediated isothermal amplification technology, and meanwhile, a detection signal is subjected to cascade amplification in combination with a CRISPR-Cas12a system, so that qualitative detection of the norovirus is realized. When the prepared kit is used for detecting the norovirus, a sample with the norovirus content larger than 3 copies / microliter can present green fluorescence, and simple, rapid and accurate detection of norovirus infection is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a norovirus nucleic acid extraction device, a detection kit and a detection method. Background Art

[0002] Norovirus is a non-enveloped single-stranded positive-sense RNA virus, which has a fast mutation rate, strong environmental resistance, a low infectious dose, a short incubation period, a long virus excretion time, a short immune protection time after infection, and a variety of transmission routes. The whole population is generally susceptible, with high infectivity and rapid transmission ability. The main manifestations of norovirus infection are diarrhea and / or vomiting, which are the main pathogens of sporadic cases and outbreak epidemics of acute gastroenteritis in China and even globally, bringing a heavy disease burden to the public health system. Therefore, developing a sensitive and simple norovirus detection method is of great significance in public health prevention and control and clinical diagnosis and treatment.

[0003] Currently, the methods for norovirus detection mainly include polymerase chain reaction, enzyme-linked immunosorbent assay, etc., but they have the disadvantages of cumbersome operation, high requirements for instruments and equipment, long detection time, limited detection samples, complex sample pretreatment, etc., and it is difficult to meet the needs of rapid qualitative detection and on-site environmental detection. For this reason, the present invention proposes a norovirus nucleic acid extraction device, a detection kit and a detection method. Summary of the Invention

[0004] The purpose of the present invention is to provide a norovirus nucleic acid extraction device, a detection kit and a detection method, aiming to solve the problems raised in the above background art.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A norovirus nucleic acid extraction device includes a pressing cover, a rubber stopper, a nucleic acid extraction column, a silica gel membrane, a connecting tube and a collection tube; a pushing rod is contained inside the pressing cover; the rubber stopper is connected to the pushing rod of the pressing cover; a silica gel membrane with a pore size of 1 μm is arranged at the bottom inside the tube of the nucleic acid extraction column; the connecting tube is used to connect the nucleic acid extraction column and the collection tube.

[0007] A norovirus detection kit includes the above-mentioned norovirus nucleic acid extraction device, a lysis solution, a rinsing buffer solution, absolute ethanol, an elution buffer solution, a reverse transcription recombinase-mediated isothermal amplification system, a CRISPR-Cas12a system, a negative control and a GⅡ positive control;

[0008] The reverse transcriptase-mediated isothermal amplification system described above includes R1 to R5; R1 is a freeze-dried powder, including recombinase, single-stranded binding protein, DNA polymerase, reverse transcriptase, and dNTPs; R2 is an isothermal amplification buffer solution; R3 is a Forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a Reverse primer with a nucleotide sequence as shown in SEQ ID NO.2; R4 is a reporter probe; R5 is an activator.

[0009] The CRISPR-Cas12a system described above includes C1 to C3; C1 is Cas12a protease; C2 is crRNA, with a nucleotide sequence as shown in SEQ ID NO.3; C3 is a Cas12a digestion buffer solution.

[0010] Furthermore, the lysis solution includes guanidine thiocyanate, Tris-HCl, EDTA, Triton X-100, proteinase K, and carrier RNA.

[0011] The washing buffer solution includes Tris-HCl and ethanol.

[0012] The elution buffer solution is sterile and enzyme-free pure water.

[0013] The negative control is sterile and enzyme-free ultrapure water.

[0014] The GⅡ positive control is a GⅡ norovirus target nucleic acid fragment.

[0015] A detection method for norovirus, using the detection kit as described above, includes the following steps:

[0016] Use a norovirus nucleic acid extraction device and nucleic acid reagents to extract viral RNA from clinical samples, add the extracted viral RNA to the reverse transcriptase-mediated isothermal amplification / CRISPR-Cas12a detection system, react at 37 - 42 °C for 30 minutes, and then observe the fluorescence under a hand-held ultraviolet light and measure the fluorescence intensity with a fluorescence spectrophotometer.

[0017] Furthermore, the specific process of the step of using a norovirus nucleic acid extraction device and nucleic acid reagents to extract viral RNA from clinical samples is as follows:

[0018] Dilute the clinical sample to 10 times its volume with phosphate buffer solution, and then centrifuge at 6000 rpm for 5 minutes. After centrifugation, take 200 μL of the supernatant and add 400 μL of lysis solution. Incubate the resulting mixture at room temperature for 2 minutes, and then add the incubated mixture to the center of the nucleic acid extraction column. Press the pressing cap, rinse the nucleic acid extraction column twice with 200 μL of rinsing buffer solution, and then rinse the nucleic acid extraction column once with 200 μL of absolute ethanol. After completing the washing, transfer the nucleic acid extraction column to the detection system, elute the silica gel membrane on the nucleic acid extraction column with 20 μL of elution buffer solution, and inject it into the collection tube.

[0019] Further, in the step of adding the extracted viral RNA to the reverse transcription recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system, nucleic acid amplification and detection are performed.

[0020] Further, in the step of observing fluorescence under a handheld ultraviolet lamp and measuring the fluorescence intensity with a fluorescence spectrophotometer, the test sample is first observed for fluorescence under a handheld ultraviolet lamp and compared with the negative control. A sample with green fluorescence is positive, and a sample without green fluorescence is negative. The GⅡ positive control has green fluorescence, indicating that the result is valid; otherwise, the experimental result is considered invalid. Then, measure the fluorescence intensity with a fluorescence spectrophotometer. The fluorescence intensity measured for each sample is compared with the negative control, and a two-independent-sample t-test is performed. When the p-value of the hypothesis test < 0.05, it is determined as a positive sample; when the p-value > 0.05, it is determined as a negative sample.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention provides a pressing column type nucleic acid extraction device, as well as a kit and a detection method for detecting GII norovirus by using reverse transcriptase-mediated isothermal amplification / CRISPR-Cas12a technology. After nucleic acid extraction is carried out by using the pressing column type nucleic acid extraction device, a sample to be detected is added to a reverse transcriptase-mediated isothermal amplification / CRISPR-Cas12a detection system, and the target fragment is amplified by using the reverse transcriptase-mediated isothermal amplification technology. At the same time, the detection signal is cascaded and amplified by combining with the CRISPR-Cas12a system to realize the qualitative detection of norovirus. When the kit prepared by the present invention is used for detecting norovirus, samples with a norovirus content greater than 3 copies / μL can show green fluorescence, and the fluorescence intensity has a statistically significant difference compared with the negative control (P < 0.05). Based on this, whether there is a fluorescence signal can be used to quickly judge whether there is norovirus in the fecal sample, realizing the simple, rapid and accurate detection of norovirus infection. To sum up, the present invention can quickly and timely extract norovirus in clinical samples, and carry out sensitive and specific detection, with a short time and high accuracy, which is helpful for inferring the transmission route and epidemic investigation. In addition, the present invention can directly detect GII norovirus in feces, greatly shortening the detection time and reducing the detection cost, providing a new idea for the rapid on-site detection of norovirus in clinical samples, and having broad application prospects. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the nucleic acid extraction device of the present invention.

[0024] Figure 2 It is a flow chart of norovirus nucleic acid extraction and detection in the present invention.

[0025] Figure 3 It is the detection limit result of the detection method of the present invention (**p < 0.01, NC: negative control).

[0026] Figure 4 It is the ROC curve and AUC of the detection result of the kit of the present invention.

[0027] In the figure: pressing cover 1, rubber stopper 2, nucleic acid extraction column 3, silica gel membrane 4, connecting tube 5, collecting tube 6. Detailed Embodiments

[0028] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be understood as a limitation to the implementable scope of the present invention.

[0029] The present invention provides a nucleic acid extraction device and a supporting reagent, including a pressing column type nucleic acid extraction device, a lysis solution, a washing buffer solution, and an elution buffer solution. The device and the reagent can efficiently extract norovirus nucleic acid.

[0030] 1) Pressing column type nucleic acid extraction device;

[0031] As Figure 1 shown, the pressing column type nucleic acid extraction device includes a pressing cap 1, a rubber stopper 2, a nucleic acid extraction column 3, a silica gel membrane 4, a connecting tube 5, and a collection tube 6; a pushing rod is contained inside the pressing cap 1, and the pushing rod is connected to the rubber stopper 2; a silica gel membrane 4 with a pore diameter of 1 μm is arranged at the bottom inside the nucleic acid extraction column 3; the connecting tube 5 connects the nucleic acid extraction column 3 and the collection tube 6.

[0032] 2) Reagent;

[0033] Solution 1: Lysis solution (guanidine thiocyanate, Tris-HCl, EDTA, Triton X-100, proteinase K, carrier RNA);

[0034] Solution 2: Washing buffer solution (Tris-HCl, ethanol);

[0035] Solution 3: Absolute ethanol;

[0036] Solution 4: Elution buffer solution (sterile and enzyme-free pure water).

[0037] The present invention provides a reverse transcription recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system, including a reverse transcription recombinase-mediated isothermal amplification system (R1 to R5), a CRISPR-Cas12a system (C1 to C3), a negative control, and a GII positive control.

[0038] 1) Reverse transcription recombinase-mediated isothermal amplification system, wherein:

[0039] R1 is a freeze-dried powder, including: recombinase, single-stranded binding protein, DNA polymerase, reverse transcriptase, dNTPs;

[0040] R2 is an isothermal amplification buffer solution;

[0041] R3 is Forward primer (5’-TTACGTGCCCAGACAAGAGCCAATGTTCAGA-3’, as shown in SEQ ID NO.1) and Reverse primer (5’-GCCATAACCTCATTGTTGACCTCTGGGACGAG-3’, as shown in SEQ ID NO.2);

[0042] R4 is a reporting probe (5’FAM-TTATT-3’BHQ1);

[0043] R5 is an activator (Mg 2+ ).

[0044] 2) The CRISPR-Cas12a system, wherein:

[0045] C1 is Cas12a protease;

[0046] C2 is crRNA (5’-AAUUUCUACUAAGUGUAGAUUGAAUGAAGAUGGCGUCGAA-3’, as shown in SEQ ID NO.3);

[0047] C3 is Cas12a digestion buffer.

[0048] 3) Negative control: Sterile and enzyme-free ultrapure water.

[0049] 4) GⅡ positive control: GⅡ norovirus target nucleic acid fragment.

[0050] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0051] Example 1: Extraction of viral RNA from clinical fecal samples;

[0052] Use a pressing-column nucleic acid extraction device and reagents to extract viral RNA from 3 clinical samples. The specific steps are as follows (see Figure 2 ):

[0053] Dilute the fecal clinical sample to 10 times its volume with phosphate buffer, and then centrifuge at 6000 rpm for 5 minutes. After centrifugation, take 200 μL of the supernatant and add 400 μL of Solution 1. Incubate the resulting mixture at room temperature for 2 minutes, then add the incubated mixture to the center of the nucleic acid extraction column 3, press the pressing cover 1, so that the mixture passes through the silica gel membrane 4, and the nucleic acid is adsorbed on the membrane. Rinse the nucleic acid extraction column 3 twice with 200 μL of Solution 2 and once with 200 μL of Solution 3. After washing, transfer the nucleic acid extraction column 3 to the detection system. Elute the silica gel membrane 4 on the nucleic acid extraction column 3 with 20 μL of Solution 4 and inject it into the collection tube 6.

[0054] Use an ultraviolet-visible spectrophotometer to measure the total concentration and purity of the RNA extracted by the method of the present invention and the RNA extracted by the magnetic bead method above. Among them, the purity measurement index is OD260 / 280, and each is measured 5 times. Calculate the average value and record it in Table 1.

[0055] Table 1 Comparison of the effects of extracting RNA by the method of the present invention and the magnetic bead method

[0056]

[0057]

[0058] As can be seen from the data in Table 1, compared with the magnetic bead method, the method of the present invention shows significant advantages: the total concentration of RNA extracted by the method of the present invention is high, and the OD260 / 280 of the extracted RNA is between 1.96 and 2.03. It can be seen that the RNA extracted by the method of the present invention has a high concentration and good purity.

[0059] Example 2: Sensitive detection of GII norovirus plasmid based on reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a technology;

[0060] Taking the gene sequences of the conserved regions for genotyping of GII norovirus (NoV), the gene sequences of the region encoding RNA-dependent RNA polymerase on open reading frame 1 and the region encoding the major structural protein VP1 on open reading frame 2 as target sequences, a GII norovirus plasmid was constructed and detected by this detection method. The specific steps are as follows:

[0061] The total volume of the reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system is 50 μL, and this system consists of two parts:

[0062] The first part is a 15-μL reverse transcriptase recombinase-mediated isothermal amplification system: R1 is dissolved with R2, and R3, R4 and R5 are added in sequence. 2.5 μL of GII norovirus plasmids with different concentrations (concentrations are 3.07, 30.7×10 1 、30.7×10 2 、30.7×10 3 、30.7×10 4 、30.7×10 5 copies / μL) are added, and a negative control is set at the same time.

[0063] The second part is a 35-μL CRISPR-Cas12a system: components C1 to C3 of the CRISPR-Cas12a system are added in sequence.

[0064] After completing the configuration of the above two systems, 15 μL of the reverse transcriptase recombinase-mediated isothermal amplification system is added to 35 μL of the CRISPR-Cas12a system, and then the mixed system is placed at 37 - 42 °C for reaction for 30 minutes. Three parallel samples are set for each concentration of plasmid, and the average value and standard deviation are calculated. After the reaction is completed, the fluorescence intensity of the above samples is measured using a fluorescence spectrophotometer, and the results are as Figure 3 shown.

[0065] From Figure 3 It can be seen that the detection method of the present invention is stable. The qualitative detection limit of norovirus is 3 copies / μL, and the detection time is 30 minutes, meeting the requirements of rapid detection.

[0066] Example 3: Specific detection of GII norovirus plasmid and other diarrhea virus plasmids based on reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a technology;

[0067] The operation steps of the reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system are the same as those in Example 2. Among them, 2.5 μL of GII norovirus plasmid (10 4 copies / μL), enterovirus common plasmid (10 5 copies / μL), adenovirus plasmid (10 5 copies / μL), rotavirus plasmid (10 5 copies / μL), negative control and GII positive control were added to the reverse transcriptase recombinase-mediated isothermal amplification system. Three parallel samples were set for each virus plasmid, and the average value and standard deviation were calculated. The fluorescence intensity was measured using a fluorescence spectrophotometer, and the results are shown in Table 2.

[0068] Table 2 Specific detection results of norovirus

[0069]

[0070] a P value < 0.05.

[0071] It can be seen from Table 2 that when detecting enterovirus common, adenovirus, rotavirus and norovirus plasmids, when the concentration of non-target substances (enterovirus common, adenovirus, rotavirus) is 10 times that of the target substance (GII norovirus), only GII norovirus shows a positive result, and the rest are negative. The results show that the detection method established by the present invention has good specificity for GII norovirus.

[0072] Example 4: Application of norovirus nucleic acid extraction and detection based on reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a technology in clinical samples;

[0073] Preparation of the kit: Nucleic acid extraction device and reagents (including a press-column nucleic acid extraction device, lysis solution, rinsing buffer solution, elution buffer solution). Reverse transcriptase recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system (including reverse transcriptase recombinase-mediated isothermal amplification system (R1 - R5), CRISPR-Cas12a system (C1 - C3), negative control, GII positive control).

[0074] Nucleic acid extraction: The extraction steps of viral RNA from clinical fecal samples were the same as those in Example 1. The extracted viral RNA was injected into a reverse transcription recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system and reacted at 37 - 42 °C for 30 minutes. Then, fluorescence was observed under a hand-held ultraviolet light and the fluorescence intensity was measured using a fluorescence spectrophotometer.

[0075] Detection system: The operating steps of the reverse transcription recombinase-mediated isothermal amplification / CRISPR-Cas12a detection system were the same as those in Example 2. Among them, viral RNA from clinical samples No. 1 - 12 extracted by a pressing column type nucleic acid extraction device was added to the reverse transcription recombinase-mediated isothermal amplification system, and at the same time, negative control and GⅡ positive control were detected.

[0076] Result determination criteria: The test samples were first observed for fluorescence under a hand-held ultraviolet light. Compared with the negative control, samples with green fluorescence were positive, and samples without green fluorescence were negative. And the GⅡ positive control had green fluorescence, indicating that the results were valid; otherwise, the experimental results were considered invalid. Then, the fluorescence intensity was measured using a fluorescence spectrophotometer. The fluorescence intensity measured for each sample was compared with that of the negative control, and a two-sample t-test was performed. When the p-value of the hypothesis test < 0.05, it was determined as a positive sample; when the p-value > 0.05, it was determined as a negative sample. The fluorescence intensity measurement results are shown in Table 3.

[0077] Table 3 Detection results of clinical samples

[0078]

[0079]

[0080] a p-value < 0.05; ct value ≤ 30 was positive, ct value > 30 was negative.

[0081] As shown in Table 3, the fluorescence intensity of samples No. 1 - 9 among the 12 samples was significantly different from that of the negative control. The detection results of this kit were consistent with those of the existing gold standard RT-qPCR kit for norovirus detection.

[0082] The detection results of this kit were compared with those of the RT-qPCR kit through sensitivity, specificity, receiver operating characteristic curve (ROC curve), and area under the curve (AUC), etc. The results are shown in Table 4 and Figure 4 as follows.

[0083] Table 4 Comparison of detection results between the present invention and the RT-qPCR kit

[0084]

[0085] The calculation formulas are as follows: Sensitivity = True positive / (True positive + False negative), Specificity = True negative / (True negative + False positive), Agreement rate = (True positive + True negative) / (True positive + False positive + False negative + True negative).

[0086] As can be seen from Table 4 and Figure 4 the results, the method of the present invention has accurate detection, and the detection effect is comparable to that of the RT-qPCR kit. The sensitivity is 100%, the specificity is 100%, the agreement rate is 100%, and AUC = 1.

[0087] The above is only the preferred implementation mode of the present invention. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. A Norovirus nucleic acid extraction device, characterized in that: It comprises a pressing cover, a rubber plug, a nucleic acid extraction column, a silica gel membrane, a connecting tube and a collecting tube; the pressing cover contains a pushing rod; the rubber plug is connected to the pushing rod of the pressing cover; a silica gel membrane with a pore size of 1 μm is arranged at the bottom of the tube of the nucleic acid extraction column; the connecting tube is used to connect the nucleic acid extraction column and the collecting tube.

2. A Norovirus detection kit, characterized in that: It comprises the Norovirus nucleic acid extraction device as claimed in claim 1, a lysate, a rinsing buffer solution, anhydrous ethanol, an elution buffer solution, a reverse transcriptase-mediated isothermal amplification system, a CRISPR-Cas12a system, a negative control and a GⅡ positive control; The reverse transcriptase recombinase-mediated isothermal amplification system includes R1 to R5; R1 is a lyophilized powder, including a recombinase, a single-stranded binding protein, a DNA polymerase, a reverse transcriptase and dNTPs; R2 is an isothermal amplification buffer solution; R3 is a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2; R4 is a reporter probe; and R5 is an activator; The CRISPR-Cas12a system includes C1 to C3; C1 is Cas12a protease; C2 is crRNA, whose nucleotide sequence is shown in SEQ ID NO.3; C3 is Cas12a enzyme cleavage buffer.

3. The norovirus detection kit according to claim 2, characterized in that The lysis solution includes guanidine thiocyanate, Tris-HCl, EDTA, Triton X-100, proteinase K and carrier RNA; The rinse buffer solution includes Tris-HCl and ethanol; The elution buffer solution is sterile enzyme-free pure water; The negative control is sterile enzyme-free ultrapure water; The GⅡ positive control is a GⅡ type Norovirus target nucleic acid fragment.

4. A method for detecting norovirus, characterized in that: The detection kit according to claim 2 comprises the following steps: The viral RNA of clinical samples was extracted using a Norovirus nucleic acid extraction device and nucleic acid reagents. The extracted viral RNA was added to the reverse transcriptase-mediated isothermal amplification / CRISPR-Cas12a detection system and reacted at 37-42°C for 30 minutes. The fluorescence was then observed under a portable UV light and the fluorescence intensity was measured using a fluorescence spectrophotometer.

5. The detection method according to claim 4, characterized in that: The specific process of using the Norovirus nucleic acid extraction device and nucleic acid reagent to extract viral RNA from clinical samples is as follows: The clinical sample was diluted to 10 times volume with phosphate buffer, and then centrifuged at 6000rpm for 5 minutes. After centrifugation, 200μL of supernatant was taken and 400μL of lysis solution was added. The obtained mixture was incubated at room temperature for 2 minutes, and then the incubated mixture was added to the center of the nucleic acid extraction column. The press cover was pressed, and the nucleic acid extraction column was rinsed twice with 200μL of rinsing buffer solution, and then rinsed once with 200μL of anhydrous ethanol. After washing, the nucleic acid extraction column was transferred to the detection system, and the silica gel membrane on the nucleic acid extraction column was eluted with 20μL of elution buffer solution and injected into the collection tube.

6. The detection method according to claim 4, characterized in that: The extracted viral RNA is added to the reverse transcriptase-mediated isothermal amplification / CRISPR-Cas12a detection system step for nucleic acid amplification and detection.

7. The detection method according to claim 4, characterized in that: In the step of observing fluorescence under portable ultraviolet light and measuring fluorescence intensity with a fluorescence spectrophotometer, the test sample is first observed for fluorescence under portable ultraviolet light, and compared with the negative control, the sample with green fluorescence is positive, and the sample without green fluorescence is negative, and the GⅡ positive control has green fluorescence, indicating that the result is valid, otherwise the experimental result is considered invalid; The fluorescence intensity was then measured using a fluorescence spectrophotometer. The fluorescence intensity of each sample was compared with that of the negative control, and a two-independent sample t-test was performed. When the hypothesis test P value was <0.05, it was determined to be a positive sample; when the P value was >0.05, it was determined to be a negative sample.

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