CN114457195A - LAMP and CRISPR-based virus detection kit and method - Google Patents

LAMP and CRISPR-based virus detection kit and method Download PDF

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CN114457195A
CN114457195A CN202210159092.2A CN202210159092A CN114457195A CN 114457195 A CN114457195 A CN 114457195A CN 202210159092 A CN202210159092 A CN 202210159092A CN 114457195 A CN114457195 A CN 114457195A
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曹昊睿
张华�
冉芳
徐鹏奇
汤琳
毛康
晏智
钟理
凯文·托马斯
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Abstract

LAMP and CRISPR-based virus detection kits and methods are disclosed. Specifically disclosed is a primer for specifically detecting a target virus in a sample, which is characterized by comprising two or more primer sets that specifically target different genes of the virus or different regions of the same gene, respectively, and that have different detection limits. Also disclosed are a kit using the primer and a method for detecting a virus.

Description

LAMP and CRISPR-based virus detection kit and method
Technical Field
The invention relates to the field of biological detection, in particular to a kit and a detection method for semi-quantitatively detecting viruses in a sample.
Background
Existing viral assays, such as for the detection of the novel coronavirus (SARS-CoV-2), employ mainly RT-PCR and serological methods. The RT-PCR method has the problems of long reaction time, dependence on precise instruments and equipment and relative lag of reflected information. The serological method has low detection sensitivity, is only suitable for clinic and is not suitable for site in-situ monitoring.
Therefore, a virus detection method which has high sensitivity, strong anti-interference capability, easy operation, rapid detection and is suitable for site in-situ detection is needed.
Disclosure of Invention
To solve one of the above technical problems in the prior art, the present disclosure provides a system and method capable of visually and specifically detecting viruses.
According to one aspect of the present disclosure, there is provided a primer for specifically detecting a virus in a sample, the primer comprising two or more primer sets specifically targeting different genes of the virus or different regions of the same gene, respectively, and the two or more primer sets having different detection limits.
In some embodiments, the primers are used in a loop-mediated isothermal amplification (LAMP) reaction. In some embodiments, the primers are used in a real-time loop-mediated isothermal amplification (RT-LAMP) reaction.
In some embodiments, each primer set comprises an upstream outer primer, a downstream outer primer, an upstream inner primer, and/or a downstream inner primer.
In some embodiments, each primer set comprises an upstream outer primer, a downstream outer primer, an upstream inner primer, a downstream inner primer, an upstream loop primer, and/or a downstream loop primer.
In some embodiments, the virus may be severe acute respiratory syndrome coronavirus (SARS-CoV-2). For example, the virus may be selected from one or more of the Wuhan-Hu-1/2019, Alpha, Beta, Gamma, Delta and Omicron strains of SARS-CoV-2.
In some embodiments, the two or more primer sets each specifically target a different gene of the virus, or target different regions of the same gene. In some embodiments, the two or more primer sets have different detection limits. In a specific embodiment, the two or more primer sets include primer sets 1 to n, where n is an integer greater than 2. In specific embodiments, the 1 st to n-th primer sets each have L1To LnDetection limit of (2), wherein L1To LnAre different from each other.
In some embodiments, the two or more primer sets comprise a first primer set, a second primer, and a third primer set, wherein the first primer set, the second primer, and the third primer set specifically target three different genes of SARS-CoV-2 or different regions of the same gene, respectively. In specific embodiments, the first primer set, the second primer, and the third primer set specifically target the S gene, the N gene, and the E gene, respectively, of SARS-CoV-2. In some embodiments, the first primer set, the second primer, and the third primer set each have a detection limit, L1、L2And L3。
In specific embodiments, the first primer set specifically targets the S gene of SARS-CoV-2. In specific embodiments, the first primer set comprises a first upstream outer primer, a first downstream outer primer, a first upstream inner primer, and a first downstream inner primer. According to a specific embodiment of the present disclosure, the first primer set includes a first upstream outer primer, a first downstream outer primer, a first upstream inner primer, a first downstream inner primer, a first upstream loop portion primer, and a first downstream loop portion primer. In a specific embodiment, the first upstream outer primer has a sequence as set forth in SEQ ID No. 13. In a specific embodiment, the first downstream outer primer has a sequence as shown in SEQ ID No. 14. In a specific embodiment, the first upstream inner primer has a sequence as shown in SEQ ID No. 15. In a specific embodiment, the first downstream inner primer has a sequence as shown in SEQ ID No. 16. In a specific embodiment, the first upstream loop primer has a sequence as shown in SEQ ID No. 17. In a specific embodiment, the first downstream loop primer has a sequence as shown in SEQ ID No. 18.
In specific embodiments, the second primer set specifically targets the N gene of SARS-CoV-2. In a specific embodiment, the second primer set comprises a second upstream outer primer, a second downstream outer primer, a second upstream inner primer, and a second downstream inner primer. According to a specific embodiment of the present disclosure, the second primer set includes a second upstream outer primer, a second downstream outer primer, a second upstream inner primer, a second downstream inner primer, a second upstream loop portion primer, and a second downstream loop portion primer. In a specific embodiment, the second upstream outer primer has a sequence as shown in SEQ ID No.: 1. In a specific embodiment, the second downstream outer primer has a sequence as shown in SEQ ID No. 2. In a specific embodiment, the second upstream inner primer has a sequence as shown in SEQ ID No.: 4. In a specific embodiment, the second downstream inner primer has a sequence as shown in SEQ ID No. 3. In a specific embodiment, the second upstream loop primer has a sequence as shown in SEQ ID No. 5. In a specific embodiment, the second downstream loop primer has a sequence as shown in SEQ ID No. 6.
In specific embodiments, the third primer set specifically targets the E gene of SARS-CoV-2. In specific embodiments, the third primer set comprises a third upstream outer primer, a third downstream outer primer, a third upstream inner primer, and a third downstream inner primer. According to a specific embodiment of the present disclosure, the third primer set includes a third upstream outer primer, a third downstream outer primer, a third upstream inner primer, a third downstream inner primer, a third upstream loop primer and a third downstream loop primer. In a specific embodiment, the third upstream outer primer has a sequence as shown in SEQ ID No. 7. In a specific embodiment, the third downstream outer primer has a sequence as shown in SEQ ID No. 8. In a specific embodiment, the third upstream inner primer has a sequence as shown in SEQ ID No. 10. In a specific embodiment, the third downstream inner primer has a sequence as shown in SEQ ID No. 9. In a specific embodiment, the third upstream loop primer has a sequence as shown in SEQ ID No. 11. In a specific embodiment, the third downstream loop primer has a sequence as shown in SEQ ID No. 12.
According to another aspect of the present disclosure, a kit for detecting a virus in a sample is provided, the kit comprising the above primer.
In some embodiments, the kit may further comprise a CRISPR system. In particular embodiments, the kit can include a programmable nuclease, and two or more guide rnas (grnas).
In particular embodiments, the programmable nuclease may be selected from Cas12, Cas13, or Cas14 nucleases. In particular embodiments, the programmable nuclease may be a Cas12 nuclease, for example, may be a Cas12a nuclease. In particular embodiments, the Cas12a nuclease may have an amino acid sequence as shown in SEQ ID No. 22.
In a specific embodiment, the two or more grnas include two or three of the first gRNA, the second gRNA, and the third gRNA. In a specific embodiment, the first, second, and third grnas specifically target amplification products of the first, second, and third primer sets, respectively. In a specific embodiment, the first gRNA specifically targets the S gene of SARS-CoV-2. In a specific embodiment, the first gRNA has the sequence shown in SEQ ID No. 21. In a specific embodiment, the second gRNA specifically targets the N gene of SARS-CoV-2. In a specific embodiment, the second gRNA has the sequence shown in SEQ ID No. 19. In a specific embodiment, the third gRNA specifically targets the E gene of SARS-CoV-2. In a specific embodiment, the third gRNA has the sequence shown in SEQ ID No. 20.
In some embodiments, the kit may further comprise a reporter probe that is a single-stranded nucleic acid sequence and carries a detectable group. In specific embodiments, the reporter probe comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues.
In some embodiments, the reporter probe carries a detection group capable of producing a detectable signal. The reporter probe also carries a quenching group. The detection group produces no signal when the reporter probe is not cleaved. In some embodiments, the reporter probe comprises a polypeptide capable of generating a signal. The signal may be a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal (e.g., a fluorescent signal, a luminescent signal, etc.), or a piezoelectric signal. In some embodiments, the detection group is located on one side of the cleavage site of the nucleic acid sequence of the reporter probe. In particular embodiments, the quencher is on the other side of the cleavage site. In some embodiments, the quenching group is 5 'of the cleavage site and the detection group is 3' of the cleavage site. In other embodiments, the detection group is 5 'of the cleavage site and the quencher group is 3' of the cleavage site. In further embodiments, a quenching group is located at the 5' end of the reporter probe. In an alternative embodiment, the quenching group is located at the 3' end of the reporter probe. In further embodiments, a detection group is located at the 5' end of the reporter probe. In an alternative embodiment, the detection group is located at the 3' end of the reporter probe.
A schematic diagram of the detection principle according to some embodiments of the present disclosure is shown in fig. 1.
In particular embodiments, the detection moiety may be selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM 633. The quenching group may be selected from an Iowa Black RQ, Iowa Black FQ, or Black Hole quencher.
When the detection group produces a detectable signal, it indicates that the programmable nuclease has cleaved, i.e., that the target nucleic acid is present in the sample.
In some embodiments, the reporter probe carries biotin on one side. In further embodiments, the other side of the reporter probe carries fluorescein, 6-FAM fluorescein, FITC, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM 633. The detection may be performed using a lateral flow assay device. The lateral flow assay device includes a control line and a test line. When the reporter probe is not cleaved, the reporter probe is bound by a control line. When the reporter probe is cleaved, the released biotin crosses the control line and is captured by the test line, allowing it to develop a color.
In a specific embodiment, the reporter probe comprises the sequence 5 '- (6-FAM) -TTATT- (BHQ1) -3', wherein 6-FAM is 6-fluorescein. In a specific embodiment, the reporter probe comprises the sequence 5 '- (6-FAM) -TTATTATT- (Bio) -3', wherein Bio is biotin.
In the CRISPR-Cas effector family, Cas12 is an RNA-guided DNase belonging to class II class V-a systems that induces non-specific single-stranded dna (ssdna) side chain cleavage upon target recognition. This can lead to degradation of ssDNA reporter probes that emit fluorescent signals upon cleavage or can be detected in a portable manner on a paper strip (by lateral flow). Therefore, the kit based on CRISPR-Cas12 has the potential of rapidly detecting SARS-CoV-2 virus in situ.
According to yet another aspect of the present disclosure, there is provided a method of detecting a target virus in a sample, the method comprising: incubating two or more than two primer groups disclosed by the invention with a sample, and carrying out loop-mediated isothermal amplification (LAMP) reaction to obtain a reaction product; in the reaction product, a programmable nuclease is added for incubation with a guide rna (grna) complex and a reporter probe. If a signal is detected, it is an indication that the programmable nuclease cleaves the reporter probe and the amplification product of the virus is included in the reaction product, i.e., the sample includes the virus to be detected. If no signal is detected, it is an indication that the programmable nuclease does not cleave the reporter probe and that the amplification product of the virus is not included in the reaction product, i.e., the sample does not contain the virus to be detected. When a signal is detected in one of the detection products, it is judged that the sample contains a virus concentration corresponding to the detection limit of the primer set used.
In some embodiments, the LAMP reaction is performed at about 62-65 ℃ for 20-40 min.
In some embodiments, the method uses two or more primer sets for simultaneous detection, wherein the two or more primer sets have different detection limits. The two or more primer sets include 1 to n primer sets, where n is an integer greater than 1. In a specific embodiment, the 1 to n primer sets each have L1To LnDetection limit of (2), wherein L1To LnAre different from each other. By using these primer sets with different detection limits, the methods of the present disclosure enable semi-quantitative detection of the virus to be detected in a sample.
Taking three primer sets as an example, the detection limit of the first primer set is L1And the detection limit of the second primer set is L2And the detection limit of the third primer set is L3And L is1<L2<L3. When the amplification products of the three primer sets can not be detected by the method, the amount of the virus to be detected in the sample is judged to be less than L1. When the amplification product of the first primer set can be detected but the amplification products of the second primer set and the third primer set cannot be detected by the above method, it is determined that the amount of the virus to be detected contained in the sample is L1～L2Between (L)1Not more than virus<L2). When the amplification products of the first primer set and the second primer set can be detected but the amplification product of the third primer set cannot be detected by the above method, it is determined that the amount of the virus to be detected contained in the sample is L2～L3Between (L)2Not more than virus<L3). When the amplification products of the three primer sets are detected by the above method, the amplification products of the three primer sets are detectedJudging that the amount of the virus to be detected contained in the sample reaches L3(Virus. gtoreq.L3)。
Therefore, the primer group combinations with different detection limits can be set according to the viruses to be detected and requirements so as to carry out semi-quantitative detection on different samples and obtain corresponding detection results.
In particular embodiments of the present disclosure, the amount of SARS-CoV-2 in a community sewage sample is detected. By using a first primer set with a detection limit of about 10 copies/mL for the targeted S gene, a second primer set with a detection limit of about 25 copies/mL for the targeted N gene, and a third primer set with a detection limit of about 310 copies/mL for the targeted E gene, the concentration of SARS-CoV-2 in the community wastewater sample can be semi-quantitatively detected. When the concentration of SARS-CoV-2 in the sewage is lower than 10 copies/mL, the signals aiming at the three genes are all negative, and the community is indicated to be in a low risk state; when the concentration in the sewage is 10-25 copies/mL, the signal for the S gene is positive, and the signals for the N gene and the E gene are negative, indicating that sporadic patients may exist in the community and are in a medium risk state; when the sewage concentration is 25-310 copies/ml, signals aiming at the S gene and the N gene are positive, and the community is at high risk; when the sewage concentration is more than 310 copies/mL, signals aiming at the three genes are all positive, and the community is at extremely high risk.
In general, the method relates to various primer group combinations, and further combines with a CRISPR system (HF-RT-LAMP), so that the method has great flexibility in visualization, and on the other hand, the detection method is simple and convenient to operate, is suitable for in-situ qualitative and semi-quantitative detection, and can realize fine risk early warning on novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 outside a laboratory.
To further simplify the detection step, the kits of the present disclosure may further comprise a paper microfluidic device comprising a paper chip and a detection plate. The paper chip is prepared by utilizing paraffin dip-dyeing and a heating plate, and a hydrophobic area and a hydrophilic area are formed on qualitative filter paper through the paraffin dip-dyeing so as to control the flow direction of fluid.
The following examples and figures are provided to aid in the understanding of the present invention. It is to be understood that these examples and drawings are illustrative of the invention and are not to be construed as limiting in any way. The actual scope of the invention is set forth in the following claims. It is to be understood that any modifications and variations may be made without departing from the spirit of the invention.
Drawings
A schematic diagram of the detection principle according to some embodiments of the present disclosure is shown in fig. 1.
FIG. 2 shows the results of RT-LAMP testing for the N, E, S gene of SARS-CoV-2. FIGS. 2a to 2c show the electrophoresis of RT-LAMP amplicon of N, E, S gene, wherein lane 1 of a, b, c is marker and lane 2 is negative control; lanes 3 to 12 of FIG. 2a are 1.5X 10 in ten-fold increasing concentration order0To 1.5X 109Copies/. mu.L of sample amplicon; lanes 3 to 12 of FIG. 2b are 2.6X 10 in ten-fold increasing concentration order0To 2.6X 109Copies/. mu.L of sample amplicon; lanes 3 to 12 of FIG. 2c are 5X 10 in ten-fold increasing concentration order-1To 5X 108Copies/. mu.L of sample amplicon. FIGS. 2d to 2f are graphs showing the change of the normalized end-point fluorescence signals of N, E, S genes amplified by RT-LAMP at different concentrations. FIGS. 2g to 2i show RT-LAMP quantitation graphs of N, E, S gene.
FIG. 3 shows normalized profiles of visual detection of HF-RT-LAMP and ImageJ extraction data. FIGS. 3a to 3c are the visualization results of the detection of new coronavirus of different concentrations using fluorescent probe in combination with CRISPR/Cas12a using RT-LAMP and the intensity information graph extracted using ImageJ. FIGS. 3d to 3f are the visualization result of using biotin probe to detect new coronavirus with different concentrations by using RT-LAMP in combination with CRISPR/Cas12a, and the intensity information graph extracted by using ImageJ.
Fig. 4 shows a schematic diagram of the design and detection of a paper chip. FIG. 4a shows a schematic design diagram of a paper chip, FIG. 4b shows a schematic diagram of RNA purification using a paper chip, FIG. 4c shows a schematic diagram of SARS-CoV-2 visual detection using a paper chip, FIG. 4d shows a schematic diagram of a detection result of a lateral flow method, and FIG. 4e shows a schematic diagram of a detection result of a fluorescence method.
Figure 5 shows the results of the double-blind experimental tests.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not constitute any limitation on the invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in numerous publications.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purpose of explaining the present specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.
As used herein, the expressions "a" and "an" include plural references unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means within ± 20% of the value following. In some embodiments, the term "about" means a range of ± 10% of the value following it. In some embodiments, the term "about" means a range of ± 5% of the value following.
The term "detection limit" as used herein refers to the lowest concentration of a target in a sample that can be detected using a primer set.
The information on the main reagents used in the examples is as follows:
HiScribe T7 Quick High Yield RNA synthesis kit (E2050S), NEB WarmStart LAMP 2X Master Mix (E1700L), Buffer2.1 (B7202S) were all purchased from NEB; enzyme-free sterile water (10977023), TAE buffer (B49), Evagreen, RiboLock RNase inhibitor (EO0381) purchased from Saimerfei; gel recovery kit (D4002) was purchased from Zymo Research; VAHTS RNA Clean Beads (N412-01) were purchased from Vazyme Biotech; plasmid containing N or E gene (SARS-CoV-2-5) is synthesized by Jinzhi; the S gene-containing plasmid (2019-nCovS) was synthesized by Shanghai; qualitative square filter paper (1001 + 917), glass fiber (1825 + 047) from whatman; paraffin wax was purchased from Xerox; primers, DNA probes and gRNAs are all synthesized in Shanghai, and specific sequences are shown in Table 1.
TABLE 1 primer and gRNA sequences
Figure BDA0003513658650000071
Figure BDA0003513658650000081
Example 1 RT-LAMP reaction targeting N, E, S three Gene fragments of SARS-Cov-2
N, E, S gene RNA was prepared by in vitro transcription. In brief, the plasmids containing N, E and S genes are respectively used as templates for PCR amplification, the segments containing N, E and S genes are respectively amplified, and a gel recovery kit is adopted to extract and purify the PCR amplicon. Then, 0.5. mu.g of each PCR amplicon was used as a template, and in vitro transcription was performed using HiScribe T7 Quick High Yield RNA synthesis kit, DNA in the transcription system was removed using DNase I, and RNA was purified using VAHTS RNA Clean Beads. The purified RNA is diluted, subpackaged and stored in a refrigerator at the temperature of minus 80 ℃.
RT-LAMP reaction used 20. mu.L system, containing 0.1. mu. M F3, 0.1. mu. M B3, 0.8. mu.M FIP, 0.8. mu.M BIP, 0.6. mu.M LF, 0.6. mu.M LB, 10. mu.L of LNEB WarmStart LAMP 2 XTER Mix, 2. mu.L of RNA sample, 1. mu.L of 20 × Eventreen (to avoid interference with FAM fluorescent signal, no fluorescent dye was added in the DETECTOR reaction), 4. mu.L of enzyme-free sterile water, 65 ℃ incubation for 30 min.
The RNA sample prepared by in vitro transcription was diluted ten orders of magnitude stepwise, and the concentration of the RNA sample for the N gene was 1.5X 100To 1.5X 109Copy/. mu.L ten-fold increments; RNA sample concentration of E Gene 2.6×100To 2.6X 109Copy/. mu.L ten-fold increments; the RNA sample concentration of the S gene was 5X 10-1To 5X 108Copy/. mu.L was ten times increased. The amplified fluorescence data was read in real time using ABI7500, and the amplified products were further confirmed by agarose gel electrophoresis, the results are shown in fig. 2.
Since the RT-LAMP amplicons are a group of amplicon mixtures with different sizes, the electrophoresis bands of the RT-LAMP amplicons are in a ladder shape, as shown in FIGS. 2a to 2 c. N, E, S the three gene amplicons showed a stepwise pattern in lanes 4 (FIG. 2a), 5 (FIG. 2b) and 4 (FIG. 2c), which respectively correspond to the N gene sample containing 15 copies/. mu.L, the E gene sample containing 260 copies/. mu.L and the S gene sample containing 5 copies/. mu.L. Therefore, it can be seen from this result that the detection limits of the LAMP system used in this example for these three genes were 15 copies/. mu.L, 260 copies/. mu.L, and 5 copies/. mu.L, respectively, and all achieved aM-level sensitivity.
The comparison of the fluorescence signal intensities at the end points of the LAMP amplification reactions was further performed, and the results are shown in FIGS. 2d to 2 f. As can be seen from the results of FIGS. 2d to 2f, the fluorescence signal intensity does not increase with the increase of the concentration of the target gene in the sample. From the real-time fluorescence curve of the binding reaction, the saturation of each concentration group was reached by the end of the reaction. Therefore, RT-LAMP can only be used for end-point characterization but not for end-point quantification.
To further investigate whether quantification can be performed using real-time fluorescence, the time to reach the threshold was fitted to the logarithm of the corresponding target concentration using 25% of the maximum fluorescence intensity as the threshold, R2The quantitative determination method has the advantages that the quantitative determination method reaches more than 0.99, and E and S even reach 0.999, so that RT-LAMP has excellent real-time quantitative capability, the linear range can reach 6-8 orders of magnitude, and the results are shown in FIGS. 2 g-2 i.
However, in the field qualitative detection aspect, the detection is interfered by the false positive problem caused by non-specific amplification. As shown in FIGS. 2 a-2 c, the negative control in lane 2 and the sample below the detection limit in lane 3 also show an amplified band similar to dimer, while the non-specific amplification in lane 4 of FIG. 2b is more severe, and the real-time fluorescence curve also shows the presence of false positive.
Example 2 construction of paper microfluidic devices
To simplify the detection of viruses in samples outside the laboratory, a paper microfluidic device was introduced, comprising a paper chip and a detection plate. The paper chip and the detection board are arranged and detected as shown in fig. 4a to 4 c.
Briefly, a paper chip was prepared by the following steps:
(1) printing a corresponding pattern on qualitative filter paper by using a paraffin printer Colorqube 8570, and leaving hydrophilic adsorption areas (white areas in FIGS. 4a and 4 b);
(2) baking the filter paper for 1-2 min at 120 ℃ by using a heating plate to completely dip the filter paper in paraffin;
(3) adding glass fiber at a specific position (shown by arrows in fig. 4a and 4 b), and folding in a manner shown in fig. 4 a;
(4) add 50. mu.L of ddH to paper chips2And O, testing the flow rate at different apertures and optimizing.
The detection plate is printed by a laser cutting machine (LC-1390), the plate is made of black acrylic material, the thickness of the plate is 5mm, the cutting power is 94% of the maximum power, and the cutting speed is 0.48 mm/s. The pore diameter is the same as the hydrophilic pore diameter of the paper chip.
And loading the sample on an adsorption area of the paper chip, so that the nucleic acid in the sample is adsorbed by the glass fiber, injecting washing liquid into the adsorption area, and eluting impurities. Then, injecting eluent into the glass fiber through the adsorption area, so that the nucleic acid adsorbed on the glass fiber is eluted, and the nucleic acid flows into the corresponding hole of the detection plate from the hole of the bottom layer of the paper chip for detection.
When a fluorescent probe is used, a color reaction can be directly performed in the detection plate of the paper microfluidic chip. When a biotin probe is used, a lateral flow test strip may be placed in the reaction solution in the detection plate to perform a color reaction.
Example 3 visual detection of RT-LAMP reaction in combination with CRISPR-Cas12a System
In this example, CRISPER-Cas12a system was introduced and combined with LAMP in example 1 to construct HF-RT-LAMP. Interference from non-specific amplification is excluded by introducing Cas12a to recognize specific amplification products due to their different sequences from the specific amplification products. In a simple way, a gRNA of a target amplification section is selected firstly to construct a Cas12a enzyme digestion system, and then the enzyme digestion system is combined with RT-LAMP, so that the construction of HF-RT-LAMP reaction is completed. The specific method comprises the following steps:
about 10mL of each sample was taken and concentrated by membrane adsorption-elution. Briefly, large particles in the sample were first removed by filtration through a 3 μm filter, the pH was then adjusted to neutral, and Mg was added2+The final concentration was 25mM, and then filtered through a 0.45 μm filter, and then 40 μ L GluSCN lysate was added to the filter, followed by incubation at room temperature for 10 min. Then, the lysate is absorbed and loaded on a paper chip for purification, 10 mu L of enzyme-free sterile water is used for elution, and then the paper chip is added into the reaction hole of the microfluidic plate by using a puncher for reaction. The negative control is sterile water without enzyme, and the positive control adopts a sample labeling method.
The final concentration of 100nM Cas12a, 125nM gRNA, 500nMCAS12a fluorescent probe in 1 XNEBuffer2.1 buffer, 37 degrees C, incubation for 10min, formed the enzyme cutting premix. Then, 2. mu.L of RT-LAMP amplicon obtained in example 1 was added to 18. mu.L of the premix and incubated at 37 ℃ for 30 min. Fluorescence signals excited by 480nm blue light were recorded using ABI7500 (negative control was colorless, positive sample appeared bright green), and color information was extracted using ImagineJ, the results are shown in fig. 3a to 3 c.
As can be seen from the fluorescence results shown in fig. 3 a-3 c, the introduction of Cas12a enzyme can be visualized for the detection of the sample. The positive sample contains the gRNA-targeted amplification product, thereby activating the Cas12a enzyme. The activated Cas12a enzyme cleaves the Cas12a fluorescent probe to release the fluorescent molecule, so that a bright green fluorescent signal is seen under blue light excitation.
Example 4 visual detection of HF-RT-LAMP Using probes with FAM/Biotin
In this example, probes (called "Cas 12a biotin probe") with FAM and biotin at the 5 'end and 3' end of the probe, respectively, were used instead of the Cas12a fluorescent probe in example 3, and HF-RT-LAMP detection was performed on N, E, S genes, respectively. mu.L of the purified sample obtained in example 3 was added to 18. mu.L of the premix, and 80. mu.L of 1 XNEBuffer2.1 was added and incubated at 37 ℃ for 30 min. Then, a lateral flow test strip (Milenia HybriDetect 1, twist Dx) was inserted into the reaction tube, and after about two minutes, when the test strip had only one band near the loading side, it was indicated as negative; a positive is indicated when the sample has two bands or only one band near the top of the strip (biotin). The banding information was extracted using ImagineJ, and the results are shown in FIGS. 3d to 3 f.
As can be seen from FIGS. 3d to 3f, the detection method using Cas12a biotin probe in this example has similar effect to the Cas12a fluorescent probe. Because a very weak strip exists in a detection line (close to the top of the test paper) in the flow detection test paper, the test paper can well judge whether the detection line is a background strip of the test paper or a strip generated by an amplicon by means of ImageJ. Moreover, the problem of false positive is effectively solved. And calculating the lateral flow method gray value threshold of the negative sample to be 150 according to the negative ImageJ extraction result, thereby avoiding the background color generated by the lateral flow method. In addition, from the results of FIG. 3, it can be seen that the detection limit for S, N, E gene can be 10 copies/mL, 25 copies/mL and 310 copies/mL, respectively, as reduced to the original volume of the sample (10mL), and both reach the zM order close to the single molecule level.
Example 5 evaluation of detection Effect by Using paper chip in combination with HF-RT-LAMP
In this example, a double-blind experiment of random labeling of wastewater was performed by combining a paper chip with HF-RT-LAMP in a wastewater labeling manner, and then SARS-CoV-2 was qualitatively and semi-quantitatively detected.
SARS-CoV-2 samples collected from general mountain Hospital, Guizhou, were added to the SARS-CoV-2 free wastewater. In order to simulate community sewage in different development stages of epidemic as truly as possible, 50 sewage samples with different concentrations and the concentration of 0-500 copies/mL are prepared. A sample combination consisting of 44 spiked wastewater samples and 6 negative samples randomly grouped was tested using a paper chip in combination with HF-RT-LAMP.
As shown in Table 2 and FIG. 5, 43 of the 44 spiked samples successfully detected SARS-CoV-2 virus and no false positive appeared, and the half-quantitative results of 41 samples were consistent with those of the true samples with 100% specificity. These results demonstrate that using three sets of detection primers, performing HF-RT-LAMP on a paper chip can not only resist the interference of complex media of sewage, successfully distinguish between labeled sewage and unlabeled sewage, but also detect labeled sewage as low as 10 copies/mL, and has a semi-quantitative capability within a concentration range of 10-310 copies/mL.
TABLE 2 double-blind experimental test results
Figure BDA0003513658650000111
Figure BDA0003513658650000121
Note: p indicates a positive result, and N indicates a negative result.
In conclusion, the paper chip not only successfully solves the problem of nucleic acid extraction and purification under site conditions, but also successfully overcomes the semi-quantitative problem by means of multi-channel detection, realizes revolutionary crossing of rapid detection and rapid visual assessment of the risk from pathogens, and also provides a new method for realizing semi-quantitative detection of rapid detection equipment.
The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.
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Claims (13)

1. A primer for specifically detecting a target virus in a sample, wherein the primer comprises two or more primer sets, wherein the two or more primer sets specifically target different genes of the virus or different regions of the same gene, respectively, and wherein the two or more primer sets have different detection limits.
2. The primer according to claim 1, wherein the primer is used for a loop-mediated isothermal amplification reaction, preferably for a real-time loop-mediated isothermal amplification reaction.
3. The primer according to claim 1 or 2, wherein each of the primer sets comprises an upstream outer primer, a downstream outer primer, an upstream inner primer and/or a downstream inner primer,
preferably, the primer sets each comprise an upstream outer primer, a downstream outer primer, an upstream inner primer, a downstream inner primer, an upstream loop primer and/or a downstream loop primer.
4. The primer according to any one of claims 1 to 3, wherein the target virus is severe acute respiratory syndrome coronavirus (SARS-CoV-2), preferably one or more selected from the group consisting of Wuhan-Hu-1/2019, Alpha, Beta, Gamma, Delta and Omicron strains of SARS-CoV-2.
5. The primers of claim 4, wherein the primers comprise two or three of a first primer set, a second primer and a third primer set, wherein the first primer set, the second primer and the third primer set specifically target the S gene, the N gene and the E gene of SARS-CoV-2, respectively,
preferably, the first primer set comprises:
a first upstream outer primer having a sequence as set forth in SEQ ID No. 13;
a first downstream outer primer having a sequence as set forth in SEQ ID No. 14;
a first upstream inner primer having a sequence as set forth in SEQ ID No. 15; and
a first downstream inner primer having a sequence as set forth in SEQ ID No.:16,
preferably, the second primer set comprises:
a second upstream outer primer having a sequence as set forth in SEQ ID No.: 1;
a second downstream outer primer having a sequence as set forth in SEQ ID No. 2;
a second upstream inner primer having a sequence as set forth in SEQ ID No. 4; and
a second downstream inner primer having a sequence as shown in SEQ ID No. 3,
preferably, the third primer set comprises:
a third upstream outer primer having a sequence as set forth in SEQ ID No. 7;
a third downstream outer primer having a sequence as set forth in SEQ ID No. 8;
a third upstream inner primer having a sequence as shown in SEQ ID No. 10; and
and the third downstream inner primer has a sequence shown as SEQ ID No. 9.
6. The primer according to claim 5,
the first primer set further comprises: a first upstream loop primer having a sequence as shown in SEQ ID No. 17; and a first downstream loop primer having a sequence as set forth in SEQ ID No. 18,
the second primer set further comprises: a second upstream loop primer having a sequence as shown in SEQ ID No. 5; and a second downstream loop primer having a sequence as set forth in SEQ ID No. 6,
the third primer set further comprises: a third upstream loop primer having a sequence as shown in SEQ ID No. 11; and a third downstream loop primer having a sequence as set forth in SEQ ID No. 12.
7. A kit for detecting a virus in a sample, the kit comprising the primer of any one of claims 1 to 6.
8. The kit of claim 7, further comprising a programmable nuclease, and two or more gRNAs,
preferably, the programmable nuclease is selected from a Cas12, Cas13 or Cas14 nuclease,
more preferably, the programmable nuclease Cas12 nuclease.
9. The kit of claim 7 or 8, wherein the two or more gRNAs include two or three of a first gRNA, a second gRNA, and a third gRNA, wherein the first gRNA, the second gRNA, and the third gRNA specifically target S, N and the E gene of SARS-CoV-2, respectively,
preferably, the first gRNA has the sequence shown in SEQ ID No. 21;
preferably, the second gRNA has the sequence shown in SEQ ID No. 19;
preferably, the third gRNA has the sequence shown in SEQ ID No. 20.
10. The kit of any one of claims 7 to 9, wherein the kit further comprises a reporter probe that is a single-stranded nucleic acid sequence;
preferably, the reporter probe comprises a detection group and a quenching group respectively positioned at two ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, Alexa Fluor or ATTO TM633, and the quenching group is selected from Iowa Black RQ, Iowa Black FQ or Black Hole quencher,
preferably, the reporter probe comprises biotin.
11. The kit of claim 10, wherein the reporter probe comprises the sequence 5 '- (6-FAM) -TTATT- (BHQ1) -3', or 5 '- (6-FAM) -TTATT- (Bio) -3'.
12. A kit as claimed in any one of claims 7 to 11, further comprising a paper microfluidic device comprising a paper chip and a detection plate.
13. A method for semi-quantitatively detecting a target virus in a sample, the method comprising:
respectively incubating two or more primer groups of any one of claims 1 to 6 with the sample to perform a loop-mediated isothermal amplification reaction to obtain corresponding reaction products; and respectively adding programmable nuclease, a gRNA complex and a report probe into the reaction products for incubation to obtain detection products, wherein when a signal is detected in one detection product, the sample is judged to contain the virus concentration corresponding to the detection limit of the used primer group.
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WO2023155495A1 (en) * 2022-02-21 2023-08-24 中国科学院地球化学研究所 Virus test kit and method based on lamp and crispr
CN116024312A (en) * 2022-08-24 2023-04-28 广州国家实验室 Novel coronavirus detection method and application

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