Norovirus detection kit and detection method based on photo-thermal isothermal amplification technology

Abstract

The invention is applicable to the technical field of virus detection, and provides a norovirus detection kit based on a photo-thermal isothermal amplification technology and a detection method, the norovirus detection kit comprises a nucleic acid amplification chamber, the nucleic acid amplification chamber is provided with reaction micropores for nucleic acid amplification; an RT-PRA system, a CRISPR-cas12a system, a blank control and a positive control are placed in the reaction micropore, the RT-PRA system comprises RT-PRA solutions R1-R5, and the CRISPR-cas12a system comprises CRISPR-Cas12a solutions C1-C3; and the photo-thermal film covers the nucleic acid amplification chamber and is used for converting sunlight into heat energy and radiating the heat energy to liquid in the reaction micropores so as to provide reaction temperature for nucleic acid amplification. According to the invention, the portable, rapid and accurate detection of norovirus infection can be realized, the detection cost can be effectively reduced, the application scene is wide, workers can be helped to deduce the virus transmission path, and key support is provided for investigation and analysis of norovirus epidemic situations.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus detection, and particularly relates to a norovirus detection kit and a detection method based on photothermal isothermal amplification technology. BACKGROUND

[0002] Norovirus belongs to single-stranded positive-sense RNA virus without envelope, has the characteristics of fast mutation speed, strong environmental resistance, low infection dose, short incubation period, long duration of toxin excretion, and short immune protection period, is widely transmitted, and is susceptible to all human beings, and thus has high infectivity and rapid transmission capacity. Therefore, developing a norovirus detection method with sensitivity and convenience is of great significance to the field of public health and clinical practice.

[0003] At present, the detection methods of norovirus mainly include polymerase chain reaction and enzyme-linked immunosorbent assay, but these methods have the disadvantages of complicated operation process, high requirement for instruments and equipment, long detection time, limited range of applicable detection samples, and complex sample pretreatment steps, and thus cannot meet the actual needs of virus detection in the field environment and resource-limited environment. SUMMARY

[0004] The purpose of the embodiment of the application is to provide a norovirus detection kit based on photothermal isothermal amplification technology, which aims to solve the problems in the background technology.

[0005] The embodiment of the application is implemented in the following manner: the norovirus detection kit based on photothermal isothermal amplification technology comprises: a nucleic acid amplification chamber, which is provided with a reaction microwell on the nucleic acid amplification chamber and is used for nucleic acid amplification; an RT-PRA system, a CRISPR-cas12a system, a blank control, and a positive control, which are placed in the reaction microwell, wherein the RT-PRA system comprises RT-PRA solutions R1 to R5, and the CRISPR-cas12a system comprises CRISPR-Cas12a solutions C1 to C3; a photothermal film, which is covered on the nucleic acid amplification chamber and is used for converting sunlight into heat energy and radiating the heat energy to the liquid in the reaction microwell to provide a reaction temperature for nucleic acid amplification.

[0006] Another purpose of the embodiment of the application is to provide a detection method of the norovirus detection kit based on photothermal isothermal amplification technology, which comprises the following steps: S1, adding a sample into the RT-PRA system and then into the reaction microwell of the nucleic acid amplification chamber; S2, the CRISPR-cas12a system is added to the reaction microwell of the nucleic acid amplification chamber to form an RT-PRA / CRISPR-cas12a detection system; S3, the blank control and the positive control are added to the reaction microwell of the nucleic acid amplification chamber according to steps S1 and S2, respectively; S4, the photothermal film is covered, and irradiation is performed under sunlight for 60 min, detection is performed, and the fluorescence intensity is determined using a fluorescence spectrophotometer; S5, result determination: compared with the blank control, the sample with green fluorescence is positive, the sample without green fluorescence is negative, and the positive control has green fluorescence, indicating that the result is valid, otherwise the result is invalid.

[0007] The norovirus detection kit based on the photothermal isothermal amplification technology provided by the embodiment of the application adopts efficient photothermal conversion of the photothermal film, adds the sample to be detected into the RT-PRA / CRISPR-cas12a reaction system, uses the PRA to amplify the target fragment, and uses the CRISPR-cas12a system to cascade amplify the detection signal, so that the qualitative detection of the norovirus is realized; the norovirus detection kit prepared by the embodiment of the application can make the sample of a single copy of the norovirus present green fluorescence, and has good differentiation from the blank control, so that the patient infected with the norovirus in the patient with diarrhea can be quickly identified, and the sensitive, specific, simple, rapid and accurate detection of the norovirus is realized. The embodiment of the application can not only greatly shorten the detection period, but also effectively reduce the detection cost, and has a wide application scenario, and can help the staff to infer the virus transmission route and provide key support for the investigation and analysis of the norovirus epidemic. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The norovirus detection kit based on the photothermal isothermal amplification technology and the schematic diagram of the detection process provided by the embodiment of the application are shown in the figure; Figure 2 The photothermal performance evaluation results of the photothermal film provided by the embodiment of the application (a is the result of irradiating the glass and the photothermal film using 1 solar intensity light, respectively; b is the result of irradiating the glass and the photothermal film using 0.5 solar intensity light, respectively); Figure 3 The detection limit results provided by the embodiment of the application (** p < 0.01, NC: blank control); Figure 4 The ROC curve and AUC results provided by the embodiment of the application are shown in the figure.

[0009] In the attached diagram: 1-RT-PRA system; 2-CRISPR-cas12a system; 3-Photothermal film; 4-Nucleic acid amplification chamber; 5-Reaction microwell. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0011] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0012] Example 1: Norovirus detection kit based on photothermal isothermal amplification technology, such as... Figure 1 As shown, it includes: Nucleic acid amplification chamber 4, wherein the nucleic acid amplification chamber 4 is provided with reaction microwells 5 for nucleic acid amplification; RT-PRA system 1, CRISPR-Cas12a system 2, blank control, and positive control are placed in reaction microwell 5. RT-PRA system 1 contains RT-PRA solutions R1~R5, and CRISPR-Cas12a system 2 contains CRISPR-Cas12a solutions C1~C3. A photothermal film 3, covering the nucleic acid amplification chamber 4, is used to convert sunlight into heat energy and radiate it to the liquid in the reaction microwell 5 to provide the reaction temperature for nucleic acid amplification. In the RT-PRA solutions R1-R5, R1 is a lyophilized powder (including recombinase, single-stranded binding protein, DNA polymerase, reverse transcriptase, and dNTPs), R2 is an isothermal amplification buffer, R3 is the forward and reverse primers, the sequences of which are shown in SEQ ID NO: 1-2, R4 is the reporter probe (5'FAM-TTATT-3'BHQ1), and R5 is the activator (Mg). 2+ ).

[0013] In the CRISPR-Cas12a solution C1~C3, C1 is Cas12a protease, C2 is crRNA with the sequence shown in SEQ ID NO: 3, and C3 is Cas12a digestion buffer. The blank control was sterile, enzyme-free ultrapure water; The positive control is a target nucleic acid fragment of type GII norovirus; The photothermal film 3 is prepared by uniformly dispersing carbon black nanoparticles in polydimethylsiloxane (0.1~10% w / w), uniformly coating it on glass, and curing it at 85℃ for 2h. The preparation method of the nucleic acid amplification chamber 4 is as follows: the polydimethylsiloxane component and the curing agent are uniformly mixed at a ratio of 10:1, added to a mold, and cured at 85 DEG C for 2h; The reaction micro-hole 5 has a diameter of 5 mm and a height of 6 mm, which ensures good heat radiation efficiency.

[0014] In example 2, the photothermal performance of the photothermal film 3 is evaluated by using a xenon lamp to simulate sunlight, and the specific steps are as follows: The glass and the photothermal film 3 are irradiated in turn using 1 sun intensity and 0.5 sun intensity respectively, and the real-time temperature signal is collected by using an infrared thermal imager after 10 min, and the results are shown in Figure 1 As shown in Figure 1 It can be seen that the photothermal film 3 used in the embodiment of the present application has good heat generation capacity under different solar irradiation.

[0015] In example 3, the RT-PRA / CRISPR-cas12a technology based on the photothermal device is used for sensitive detection of GII type norovirus plasmid: Based on the gene sequence of the conserved region of norovirus genotype typing, the region encoding RNA-dependent RNA polymerase in open reading frame 1 and the region encoding major structural protein VP1 in open reading frame 2 are selected as target sequences, the target sequences are synthesized into pUC57 cloning plasmid, and GII type norovirus plasmid is constructed, and the detection is carried out, as shown in Figure 1 The specific operation steps are as follows: The total volume of the RT-PRA / CRISPR-cas12a detection system is 50 muL: 15 muL of RT-PRA system: freeze-dried powder R1 is dissolved in R2 buffer solution, and R3, R4 and R5 are added in turn, and 2.5 muL of different concentrations of GII type norovirus plasmid is added; 35 muL of CRISPR-cas12a system: C1~C3 components of CRISPR-Cas12a system are added in turn; The RT-PRA system and the CRISPR-cas12a system are added to the reaction micro-hole 5 of the nucleic acid amplification chamber 4, and the photothermal film 3 is covered, and irradiated under sunlight for 60 minutes; Three parallel samples are set for each concentration of plasmid, and the average value and standard deviation are calculated; The fluorescence intensity of the above sample is determined by using a fluorescence spectrophotometer, and the results are shown in Figure 2 As shown in Figure 2 It can be seen that the detection method provided by the embodiment of the present application is stable, the qualitative detection limit of norovirus is single copy, and has good sensitivity.

[0016] Example 4, RT-PRA / CRIPSR-Cas12a technology based on photothermal equipment for specific detection of GII type norovirus plasmid and other diarrhea virus plasmids: The operation steps of the RT-PRA / CRISPR-cas12a detection system are used in Example 3. In the RT-PRA system, 2.5 μL of GII type norovirus plasmid (10 4 copies / μL), enterovirus (10 5 copies / μL), adenovirus (10 5 copies / μL), rotavirus plasmid (10 5 copies / μL), blank control and positive control (the virus plasmid construction step is the same as Example 3, and the preparation is completed by Shengong Bioengineering (Shanghai) Co., Ltd.) were added, respectively. Three parallel samples were set for each virus plasmid, and the average value and standard deviation were calculated, and the results are shown in Table 1: Table 1 Specific detection results of norovirus

[0017] As can be seen from Table 2, when the concentration of non-target is 10 times that of the target, only GII norovirus shows a positive result, and the others are negative, proving that the detection method established in the example has good specificity.

[0018] Example 5, application of norovirus nucleic acid extraction and detection based on RT-PRA / CRIPSR-Cas12a technology of photothermal equipment in clinical samples: The operation steps of the RT-PRA / CRISPR-cas12a detection system are used in Example 3, and virus RNA in the clinical sample is added in the RT-PRA system, and the detection of negative control and positive control is carried out. Result determination standard: the detection sample is observed under the hand-held ultraviolet light and the fluorescence intensity is measured by the fluorescence spectrophotometer, the fluorescence is observed under the hand-held ultraviolet light, compared with the blank control, the sample with green fluorescence is positive, the sample without green fluorescence is negative, and the positive control has green fluorescence, indicating that the result is effective, otherwise the experimental result is invalid. Among the 16 clinical samples, samples 1-12 have obvious green fluorescence under ultraviolet irradiation, and the blank control has almost no fluorescence. The detection results are consistent with the existing gold standard real-time RT-qPCR detection results of norovirus detection. The detection result is compared with the RT-qPCR result by sensitivity, specificity, receiver operating characteristic curve (ROC curve) and area under curve (AUC), wherein the sensitivity = true positive / (true positive + false negative), the specificity = true negative / (true negative + false positive), the consistency rate = (true positive + true negative) / (true positive + false positive + false negative + true negative), and the results are shown in Table 2 and Figure 4 Table 2 Comparison of detection results

[0019] As shown in Table 2 and Figure 4 The method provided by the embodiment of the present application has high detection accuracy, and the detection effect is equivalent to that of the real-time RT-qPCR detection result, the sensitivity is 100%, the specificity is 100%, the consistency rate is 100%, and the AUC = 1.

[0020] In summary, the embodiment of the present application relies on the photothermal material to convert sunlight into heat energy, which is used for reverse transcription recombinase-mediated isothermal amplification technology to complete the amplification of norovirus nucleic acid fragments; meanwhile, the specificity recognition ability and cascade signal amplification function of the CRISPR-Cas12a "nucleic acid scissors" are combined, and finally a norovirus detection kit and method suitable for both laboratory and resource-limited field environment are developed. The kit can qualitatively judge whether norovirus exists in the fecal sample by judging whether a fluorescent signal appears, so as to realize simple, rapid and accurate detection of samples in resource-limited field environment detection and clinical work.

[0021] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A norovirus detection kit based on the technique of photothermally isothermal amplification, characterized in that, The kit comprises: a nucleic acid amplification chamber (4) provided with a reaction microwell (5) for nucleic acid amplification; an RT-PRA system (1), a CRISPR-cas12a system (2), a blank control, and a positive control, which are placed in the reaction microwell (5), wherein the RT-PRA system (1) comprises RT-PRA solutions R1-R5, and the CRISPR-cas12a system (2) comprises CRISPR-Cas12a solutions C1-C3; a photothermal film (3) covering the nucleic acid amplification chamber (4) and used for converting sunlight into heat energy and radiating to the liquid in the reaction microwell (5) to provide a reaction temperature for nucleic acid amplification.

2. The test kit for detecting norovirus based on the photothermoisothermal amplification technique according to claim 1, characterized by, In the RT-PRA solutions R1-R5, R1 is a freeze-dried powder, R2 is an isothermal amplification buffer solution, R3 is a forward primer and a reverse primer, the sequences of which are shown in SEQ ID NOs: 1-2, R4 is a reporter probe, and R5 is an activator. 3.The norovirus detection kit based on the photothermally-mediated isothermal amplification technique according to claim 1, characterized in that, In the CRISPR-Cas12a solutions C1-C3, C1 is a Cas12a protease, C2 is a crRNA, the sequence of which is shown in SEQ ID NO: 3, and C3 is a Cas12a enzyme digestion buffer. 4.The norovirus detection kit based on the photothermally-mediated isothermal amplification technique according to claim 1, characterized in that, The blank control is sterile enzyme-free ultrapure water. 5.The norovirus detection kit based on the photothermally-mediated isothermal amplification technique according to claim 1, characterized in that, The positive control is a GII-type norovirus target nucleic acid fragment, the sequence of which is shown in SEQ ID NO:

4. 6.The norovirus detection kit based on the photothermally-mediated isothermal amplification technique according to claim 1, characterized in that, The preparation method of the photothermal film (3) is as follows: uniformly dispersing carbon black nanoparticles in polydimethylsiloxane, uniformly coating the glass, and curing at 85°C for 2h. 7.The norovirus detection kit based on the photothermally-mediated isothermal amplification technique according to claim 1, characterized in that, The preparation method of the nucleic acid amplification chamber (4) is as follows: uniformly mixing polydimethylsiloxane components and a curing agent at a ratio of 10:1, adding them to a mold, and curing at 85°C for 2h.

8. A method for detecting a norovirus using a test kit based on the photothermally isothermal amplification technique according to any one of claims 1 to 7, characterized by, The method comprises the following steps: S1, adding a sample into the RT-PRA system (1) and then into the reaction microwell (5) of the nucleic acid amplification chamber (4); S2, adding the CRISPR-cas12a system (2) into the reaction microwell (5) of the nucleic acid amplification chamber (4) to form an RT-PRA / CRISPR-cas12a detection system; S3, adding the blank control and the positive control into the reaction microwell (5) of the nucleic acid amplification chamber (4) according to steps S1 and S2; S4, covering the photothermal film (3) and irradiating under sunlight for 60min for detection, and determining the fluorescence intensity using a fluorescence spectrophotometer; S5, result determination: compared with the blank control, the sample with green fluorescence is positive, the sample without green fluorescence is negative, and the positive control has green fluorescence, indicating that the result is valid, otherwise the result is invalid.

Images