WO2023155495A1 - Virus test kit and method based on lamp and crispr - Google Patents

Abstract

Provided are a virus test kit and method based on LAMP and CRISPR. Specifically, provided is a primer for specifically detecting a target virus in a sample. The primer comprises two or more primer sets, the two or more primer sets specifically target different genes of the virus or different regions of the same gene, respectively, and the two or more primer sets have different limits of detection. Also provided are a test kit using the primer and a method for detecting a virus.

Description

Virus detection kits and methods based on LAMP and CRISPR

This application claims the priority of Chinese patent application 202210159092.2 with a filing date of February 21, 2022 and Chinese patent application 202220349784.9 with a filing date of February 21, 2022, the entire contents of which are incorporated herein by reference.

technical field

The invention relates to the field of biological detection, in particular to a kit and a detection method for semi-quantitative detection of viruses in samples.

Background technique

Existing virus detection, for example, mainly uses RT-PCR and serological methods for the detection of novel coronavirus (SARS-CoV-2). The RT-PCR method has the problems of long reaction time, dependence on precision instruments and equipment, and relatively lagging information reflection. However, the detection sensitivity of serological method is low, and it is only suitable for clinical practice, and it is not suitable for in situ monitoring in the field.

Therefore, there is an urgent need for a virus detection method with high sensitivity, strong anti-interference ability, easy operation, rapid detection, and suitable for field in situ detection.

Contents of the invention

In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure provides a system and method capable of visually and specifically detecting viruses.

The first aspect of the present disclosure provides a primer for specifically detecting viruses in samples, the primers include two or more primer sets, and the two or more primer sets are respectively Specifically targeting different genes of the virus or different regions of the same gene, and the two or more primer sets have 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 includes an upstream outer primer, a downstream outer primer, an upstream inner primer, and/or a downstream inner primer.

In some embodiments, each primer set includes 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 can be severe acute respiratory syndrome coronavirus (SARS-CoV-2). For example, the virus can be selected from one or more of Wuhan-Hu-1/2019, Alpha, Beta, Gamma, Delta and Omicron strains of SARS-CoV-2.

In some embodiments, the two or more primer sets specifically target different genes 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 the first to nth primer sets, wherein n is an integer greater than 2. In a specific embodiment, the first to n primer sets have detection limits of _L1 to _Ln respectively, wherein the detection limits of _L1 to _Ln are different from each other.

In some embodiments, the two or more primer sets include 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 are respectively specific to the target Three different genes of SARS-CoV-2 or different regions of the same gene. In a specific embodiment, the first primer set, the second primer and the third primer set specifically target the S gene, N gene and E gene of SARS-CoV-2, respectively. In some embodiments, the first primer set, the second primer set, and the third primer set have detection limits L ₁ , L ₂ , and L ₃ , respectively.

In a specific embodiment, the first primer set specifically targets the S gene of SARS-CoV-2. In a specific embodiment, the first primer set includes 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 external primer, a first downstream external primer, a first upstream internal primer, a first downstream internal primer, a first upstream loop primer, and a first downstream loop primer. Primer. In a specific embodiment, the first upstream external primer has a sequence as shown in SEQ ID NO.:13. In a specific embodiment, the first downstream external primer has a sequence as shown in SEQ ID NO.:14. In a specific embodiment, the first upstream internal primer has a sequence as shown in SEQ ID NO.:15. In a specific embodiment, the first downstream internal 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 a specific embodiment, the second primer set specifically targets the N gene of SARS-CoV-2. In a specific embodiment, the second primer set includes 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 external primer, a second downstream external primer, a second upstream internal primer, a second downstream internal primer, a second upstream loop primer, and a second downstream loop primer. Primer. In a specific embodiment, the second upstream external primer has a sequence as shown in SEQ ID NO.:1. In a specific embodiment, the second downstream external primer has a sequence as shown in SEQ ID NO.:2. In a specific embodiment, the second upstream internal primer has a sequence as shown in SEQ ID NO.:4. In a specific embodiment, the second downstream internal 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 a specific embodiment, the third primer set specifically targets the E gene of SARS-CoV-2. In a specific embodiment, the third primer set includes 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 external primer, a third downstream external primer, a third upstream internal primer, a third downstream internal primer, a third upstream loop primer, and a third downstream loop primer. Primer. In a specific embodiment, the third upstream external primer has a sequence as shown in SEQ ID NO.:7. In a specific embodiment, the third downstream external primer has a sequence as shown in SEQ ID NO.:8. In a specific embodiment, the third upstream internal primer has a sequence as shown in SEQ ID NO.:10. In a specific embodiment, the third downstream internal 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.

In a second aspect of the present disclosure, a paper chip is provided, the paper chip includes a loading layer made of a hydrophilic material coated with a hydrophobic material, a drainage layer and an adsorption layer; the loading layer is provided with multiple a hydrophilic loading area; the drainage layer is provided with a hydrophilic drainage channel, when the drainage layer and the loading layer are stacked, the loading area overlaps with the drainage channel; on the adsorption layer A hydrophilic adsorption area is provided, and when the adsorption layer overlaps with the drainage layer, the adsorption area (310) overlaps with the drainage channel; an adsorption component for absorbing nucleic acid is arranged at the adsorption area.

In some embodiments, the paper chip also includes an absorption layer made of a hydrophilic material coated with a hydrophobic material, and a hydrophilic absorption area is arranged on the absorption layer. When the absorption layer and the adsorption When the layers are stacked, the absorption area overlaps with the adsorption area.

In some embodiments, the loading layer, drainage layer, adsorption layer and absorption layer are connected in a set order or are independent of each other;

In some embodiments, the adsorption layer includes a connected first adsorption layer and a second adsorption layer, and the first adsorption layer and the second adsorption layer are provided with the adsorption layer at corresponding positions. zone, the adsorption component is arranged between the first adsorption layer and the second adsorption layer.

In some embodiments, the absorbent member is made of fiberglass.

In some embodiments, the loading layer, drainage layer, adsorption layer, and absorption layer are made of wax-coated filter paper.

In a third aspect of the present disclosure, a paper microfluidic device is provided, the paper microfluidic device includes the above-mentioned paper chip and a detection board; The detection area corresponding to the loading area.

In some embodiments, the detection plate is made of acrylic material.

The paper chip and the paper microfluidic device provided by the present disclosure can separate and detect the nucleic acid in the liquid to be detected containing the pathogen by stacking each layer of the paper chip. The device has a simple structure and strong operability, and does not require professionals. And equipment; can cooperate with constant temperature amplification technology, combined with the high efficiency of constant temperature amplification and low requirements for detection conditions, the detection can be completed within one hour, and can be carried out under site conditions, thus effectively avoiding data loss caused by time Distortion, and can meet the needs of infectious disease monitoring.

The fourth aspect of the present disclosure provides a kit for detecting viruses in samples, the kit including the primers described in the first aspect of the present disclosure.

In some embodiments, the kit can also include a CRISPR system. In a specific embodiment, the kit may include a programmable nuclease and two or more guide RNAs (gRNAs).

In a specific embodiment, the programmable nuclease may be selected from Cas12, Cas13 or Cas14 nucleases. In a specific embodiment, the programmable nuclease may be Cas12 nuclease, such as Cas12a nuclease. In a specific embodiment, the Cas12a nuclease can 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 gRNA, the second gRNA and the third gRNA specifically target the first primer set, the second primer set and the third primer set respectively amplified product. In a specific embodiment, the first gRNA specifically targets the S gene of SARS-CoV-2. In a specific embodiment, the first gRNA has a sequence as 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 a sequence as 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 a sequence as shown in SEQ ID NO.:20.

In some embodiments, the kit may further include a reporter probe, which 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 bears a detection group capable of producing a detectable signal. The reporter probe also bears a quenching group. The detection group produces no signal until the reporter probe is 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, a current signal, an optical signal (eg, 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 specific embodiments, the quenching group is on the opposite side of the cleavage site. In some embodiments, the quenching group is 5' to the cleavage site and the detection group is 3' to the cleavage site. In other embodiments, the detection group is 5' to the cleavage site and the quencher group is 3' to the cleavage site. In a further embodiment, the 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 a further embodiment, the 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 a specific embodiment, the detection group can be selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, AlexaFluor or ATTO TM633. The quenching group may be selected from Iowa Black RQ, Iowa Black FQ or Black Hole quenchers.

When the detection group generates a detectable signal, it indicates that the programmable nuclease has cleaved, that is to say, the target nucleic acid exists in the sample.

In some embodiments, the reporter probe has biotin on one side. In a further embodiment, the other side of the reporter probe has fluorescein, 6-FAM fluorescein, FITC, IRDYE 700, TYE 665, AlexaFluor or ATTO TM633. Detection can 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 the control line. When the reporter probe is cleaved, the released biotin passes over the control line and is captured by the test line 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 some embodiments, the kit of the present disclosure may further include a paper microfluidic device including a paper chip and a detection plate. The paper chip is prepared by paraffin dipping and a heating plate, and a hydrophobic region and a hydrophilic region are formed on the qualitative filter paper through paraffin dipping to control the flow direction of the fluid.

In some embodiments, the paper microfluidic device is selected from the paper microfluidic device described in the third aspect of the present disclosure.

In the CRISPR-Cas effector family, Cas12 is an RNA-guided DNase belonging to the class II class V-A system that induces nonspecific single-stranded DNA (ssDNA) side-chain cleavage after target recognition. This leads to the degradation of ssDNA reporter probes, which emit a fluorescent signal upon cleavage or can be detected in a portable fashion on paper strips (by lateral flow). Therefore, CRISPR-Cas12-based kits have the potential for rapid, in situ detection of SARS-CoV-2 virus.

In a fifth aspect of the present disclosure, a method for detecting a target virus in a sample is provided, the method comprising: incubating two or more than two primer sets of the present disclosure with the sample, and performing loop-mediated constant temperature amplification ( LAMP) reaction to obtain a reaction product; in the reaction product, add programmable nuclease and guide RNA (gRNA) complex and reporter probe to incubate. If a signal is detected, it indicates that the programmable nuclease cuts the reporter probe, and the reaction product includes the amplification product of the virus, that is, the sample includes the virus to be detected. If no signal is detected, it indicates that the programmable nuclease does not cut the reporter probe, and the reaction product does not include the amplification product of the virus, that is, the sample does not contain the virus to be detected . When a signal is detected in a detection product, 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° C. for 20-40 minutes.

In some embodiments, two or more primer sets are used for simultaneous detection in the method, wherein the two or more primer sets have different detection limits. The two or more primer sets include 1 to n primer sets, wherein n is an integer greater than 1. In a specific embodiment, the 1 to n primer sets have detection limits of L ₁ to L _n respectively, wherein the detection limits of L ₁ to L _n are different from each other. By using these primer sets with different detection limits, the method of the present disclosure can perform semi-quantitative detection of the virus to be tested in the sample.

Taking three primer sets as an example, the detection limit of the first primer set is L ₁ , the detection limit of the second primer set is L ₂ , the detection limit of the third primer set is L ₃ , and L ₁ <L ₂ < _L3 . When the amplification products of the three primer sets cannot be detected by the above method, it is judged that the amount of the virus to be tested contained in the sample is less than L ₁ . When the amplification products of the first primer set can be detected by the above method, but the amplification products of the second primer set and the third primer set cannot be detected, it is judged that the amount of the virus to be tested contained in the sample is between L ₁ ~ Between L ₂ (L ₁ ≤ virus < L ₂ ). When the amplified products of the first primer set and the second primer set can be detected by the above method, but the amplified products of the third primer set cannot be detected, it is judged that the amount of the virus to be tested contained in the sample is between L ₂ ~ Between L ₃ (L ₂ ≤ virus < L ₃ ). When the amplification products of the three primer sets can all be detected by the above method, it is determined that the amount of the virus to be tested contained in the sample reaches L ₃ (virus ≥ L ₃ ).

Therefore, primer set combinations with different detection limits can be set according to the virus to be detected and requirements, so as to perform semi-quantitative detection on different samples and obtain corresponding detection results.

In specific embodiments of the present disclosure, the amount of SARS-CoV-2 is detected in community sewage samples. By using the first primer set with a detection limit of about 10 copies/mL targeting the S gene, the second primer set with a detection limit of about 25 copies/mL targeting the N gene, and the detection limit of the E gene The third primer set with a limit of about 310 copies/mL can semi-quantitatively detect the concentration of SARS-CoV-2 in community sewage samples. When the concentration of SARS-CoV-2 in sewage is lower than 10 copies/mL, the signals for the three genes are all negative, indicating that the community is in a low-risk state; when the concentration in sewage is between 10 and 25 copies/mL, the signals for S The signal of the gene is positive, and the signal of the N gene and E gene is negative, indicating that there may be sporadic patients in the community and in a state of medium risk; when the sewage concentration is between 25 and 310 copies/ml, the signal of the S gene and N gene All positive, the community is at high risk; when the sewage concentration is greater than 310 copies/mL, the signals for the three genes are all positive, and the community is at extremely high risk.

Generally speaking, the present disclosure involves a variety of primer set combinations, and further combined with the CRISPR system (HF-RT-LAMP), which has great flexibility in visualization. On the other hand, the detection method of the present disclosure is easy to operate and suitable for In-situ qualitative and semi-quantitative detection can realize refined risk early warning of the new coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 outside the laboratory.

The following examples and figures are provided to aid understanding of the present invention. However, it should be understood that these embodiments and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention.

Description of drawings

Fig. 1 shows a schematic diagram of the detection principle according to some embodiments of the present disclosure.

Figure 2 shows the results of RT-LAMP testing the N, E, and S genes of SARS-CoV-2. Figures 2a-2c show the RT-LAMP amplicon electrophoresis images of N, E, and S genes, in which the first lane of a, b, and c is a marker, and the second lane is a negative control; the third to 12th lanes of Figure 2a The lanes are sample amplicons from 1.5×10 ⁰ to 1.5×10 ⁹ copies/μL placed according to the ten-fold increasing order of concentration; the 3rd to 12th lanes in Figure 2b are 2.6×10 ⁰ placed according to the ten-fold increasing order of concentration 2.6×10 ⁹ copies/μL of sample amplicons; the 3rd to 12th lanes of Figure 2c are the sample amplicons of 5×10 ^-1 to 5×10 ⁸ copies/μL placed in order of ten-fold increasing concentration. Figures 2d-2f show the changes in the normalized endpoint fluorescence signals of N, E, and S genes amplified by RT-LAMP at different concentrations. Figures 2g-2i show the RT-LAMP quantification curves of N, E, and S genes.

Figure 3 shows the normalized distribution diagram of the visual detection of HF-RT-LAMP and the data extracted by ImageJ. Figures 3a to 3c are the visualization results of using RT-LAMP combined with CRISPR/Cas12a to detect different concentrations of new coronaviruses with fluorescent probes, and the intensity information extracted by ImageJ. Figures 3d to 3f are the visualization results of using RT-LAMP combined with CRISPR/Cas12a to detect different concentrations of new coronaviruses using biotin probes, and the intensity information maps extracted using ImageJ.

Figure 4 shows a schematic diagram of the paper chip design. Fig. 4a, Fig. 4b and Fig. 4c are respectively a schematic diagram of unfolded plan, a schematic diagram of a three-dimensional structure and a schematic diagram of a stacked state of one paper chip, and Fig. 4d is a schematic diagram of a three-dimensional structure of another paper chip.

Fig. 5 shows a schematic structural diagram of one of the detection boards.

Fig. 6 shows a schematic diagram of the design and detection of the paper chip in the constructed paper microfluidic device. Figure 6a shows a schematic diagram of the design of a paper chip, Figure 6b shows a schematic diagram of RNA purification using a paper chip, Figure 6c shows a schematic diagram of the visual detection of SARS-CoV-2 using a paper chip, and Figure 6d shows a schematic diagram of the detection results of flow measurement , 6e shows a schematic diagram of the detection results of the fluorescence method.

Figure 7 shows the test results of the double-blind experiment.

The reference signs are in order:

1. Loading layer, 11. Loading area, 2. Drainage layer, 21. Drainage channel, 3. Adsorption layer, 31. First adsorption layer, 32. First adsorption layer, 310. Adsorption area, 4. Absorption layer , 41, absorption area, 100, paper chip, 200, detection board, 210, detection area.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. The specific embodiments described here are only used to explain the present invention, and are not intended to constitute any limitation to the present invention. Also, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such structures and techniques are also described in numerous publications.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the field to which this invention belongs. For the purpose of interpreting this 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 referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, and the like.

As used herein, the term "about" means a range of ±20% of the numerical value that follows. In some embodiments, the term "about" indicates a range of ±10% of the numerical value that follows. In some embodiments, the term "about" indicates a range of ±5% of the numerical value that follows.

As used herein, the term "limit of detection" refers to the lowest concentration of a target in a sample that can be detected using a primer set.

The information of main reagent used in each embodiment is as follows:

HiScribe T7 Quick High Yield RNA Synthesis Kit (E2050S), NEB WarmStart LAMP 2X Master Mix (E1700L), Buffer 2.1 (B7202S) were purchased from NEB Company; enzyme-free sterile water (10977023), TAE buffer (B49), Evagreen, RiboLock RNase Inhibitor (EO0381) was purchased from Thermo Fisher; Gel Recovery Kit (D4002) was purchased from Zymo Research; VAHTS RNA Clean Beads (N412-01) was purchased from Vazyme Biotech; plasmids containing N or E genes (SARS -CoV-2-5) was synthesized by Jinweizhi; the plasmid containing the S gene (2019-nCovS) was synthesized by Shanghai Sangong; qualitative square filter paper (1001-917), glass fiber (1825-047) was purchased from whatman; paraffin was purchased From Xerox; primers, DNA probes, and gRNA were all synthesized by Shanghai Sangon, and the specific sequences are shown in Table 1.

Table 1. Primers and gRNA sequences

Example 1. Targeting the RT-LAMP reaction of the N, E, and S three gene fragments of SARS-Cov-2

RNAs of N, E, and S genes were prepared by in vitro transcription. To put it simply, plasmids containing N, E, and S genes were used as templates for PCR amplification, and fragments containing N, E, and S genes were amplified, and PCR amplicons were extracted and purified using a gel recovery kit. Then, 0.5 μg of PCR amplicons were taken as templates, and HiScribe T7 Quick High Yield RNA Synthesis Kit was used for in vitro transcription, DNA in the transcription system was removed by DNase I, and RNA was purified by VAHTS RNA Clean Beads. The purified RNA was diluted and aliquoted and stored in a -80°C refrigerator.

RT-LAMP reaction uses 20 μL system, including 0.1 μM F3, 0.1 μM B3, 0.8 μM FIP, 0.8 μM BIP, 0.6 μM LF, 0.6 μM LB, 10 μL NEB WarmStart LAMP 2X Master Mix, 2 μL RNA sample, 1 μL 20×Evagreen (for To avoid interference to the FAM fluorescence signal, no fluorescent dye was added to the DETECTOR reaction), 4 μL of enzyme-free sterile water, and incubated at 65°C for 30 minutes.

The RNA samples prepared by in vitro transcription were serially diluted by ten orders of magnitude. The RNA sample concentration of the N gene was 1.5×10 ⁰ to 1.5×10 ⁹ copies/μL in ten-fold increments; the RNA sample concentration of the E gene was 2.6× Ten-fold increments from 10 ⁰ to 2.6×10 ⁹ copies/μL; the RNA sample concentration of the S gene ranged from 5×10 ^-1 to 5×10 ⁸ copies/μL in ten-fold increments. ABI7500 was used to read the amplification fluorescence data in real time, and the amplified product was further confirmed by agarose gel electrophoresis, and the results are shown in Figure 2.

Since the RT-LAMP amplicon is a mixture of amplicons of different sizes, its electrophoretic bands present a ladder shape, as shown in Figures 2a-2c. Three gene amplicons of N, E, and S appeared ladder-like bands in lane 4 (Fig. 2a), lane 5 (Fig. 2b), and lane 4 (Fig. 2c), respectively, corresponding to μL of the N gene sample, the E gene sample containing 260 copies/μL, and the S gene sample containing 5 copies/μL. Therefore, from this result, it can be concluded that the detection limits of the LAMP system used in this example for these three genes are 15 copies/μL, 260 copies/μL and 5 copies/μL, respectively, all reaching aM level sensitivity.

The fluorescence signal intensity at the end point of the LAMP amplification reaction was further compared, and the results are shown in Figures 2d-2f. It can be seen from the results in Figures 2d-2f that the fluorescence signal intensity does not increase with the increase in the concentration of the target gene in the sample. From the real-time fluorescence curve of the binding reaction, when the reaction reaches the end point, each concentration group has reached saturation. Therefore, RT-LAMP can only be used for end-point qualitative but not end-point quantification.

In order to further explore whether real-time fluorescence can be used for quantification, 25% of the maximum fluorescence intensity was used as the threshold, and the time to reach the threshold was fitted with the logarithm of the corresponding target concentration. ^R2 reached above 0.99, and E and S even reached 0.999. It shows that RT-LAMP has excellent real-time quantitative ability, and the linear range can reach 6-8 orders of magnitude, the results are shown in Fig. 2g-2i.

However, in the field of qualitative detection, the problem of false positives caused by non-specific amplification will interfere with the detection. As shown in Figures 2a to 2c, the negative control in the second lane and the sample below the detection limit in the third lane also have amplified bands similar to dimers, especially in the fourth lane in Figure 2b For severe non-specific amplification, the real-time fluorescence curve also proves the existence of false positives.

Embodiment 2 constructs paper chip and detection plate

This embodiment provides a paper chip. FIGS. 4a to 4c respectively show a schematic diagram of the unfolded plane, a schematic diagram of a three-dimensional structure, and a schematic diagram of a stacked state of the paper chip.

As shown in FIG. 4 a , the paper chip 100 provided in this embodiment includes: a loading layer 1 made of a hydrophilic material coated with a hydrophobic material, a drainage layer 2 and an adsorption layer 3 .

The loading layer 1 is provided with a plurality of hydrophilic loading regions 11; the drainage layer 2 is provided with hydrophilic drainage channels 21, and when the drainage layer 2 and the loading layer 1 are stacked, the loading regions 11 and the drainage channels 21 overlap. The shape and quantity of the loading areas 11 and their arrangement in the loading layer 11 can be determined as required. Moreover, the shape, size and arrangement of the drainage channels 21 on the drainage layer 2 are determined according to the shape, quantity and arrangement of the loading regions 11 on the loading layer 1, so that when the drainage layer 2 and the loading layer 1 overlap Therefore, the loading area 11 disposed on the loading layer 1 can completely overlap the drainage channel 21 , that is, be located on the drainage channel 21 . In this way, the nucleic acid in the liquid to be detected containing the pathogen can be distributed to each loading area 11 through the drainage channel 21 after being eluted.

The adsorption layer 3 is provided with a hydrophilic adsorption region 310. When the adsorption layer 3 and the drainage layer 2 overlap, the adsorption region 310 overlaps with the hydrophilic channel 21, so that the nucleic acid in the liquid to be detected can enter the drainage after elution. Channel 21. An adsorption member (not shown in the figure) for adsorbing nucleic acid and separating it from other substances is provided at the adsorption region 310 . The adsorption member may be a glass fiber sheet having the same shape as the adsorption region 310, and the glass fiber is used to specifically adsorb nucleic acid. In order to facilitate the fixing of the adsorption parts, thereby obtaining better adsorption effect, the adsorption layer 3 includes a connected first adsorption layer 31 and a second adsorption layer 32, and the first adsorption layer 31 and the second adsorption layer 32 are corresponding to each other. Adsorption area 310 is arranged on the position, the first adsorption layer 31 and the second adsorption layer 32 overlap to sandwich the adsorption components, and the adsorption components are arranged on the first adsorption layer 31 and the second adsorption layer 32 The upper adsorption regions 310 overlap.

The paper chip 100 is also provided with an absorption layer 4 made of a hydrophilic material coated with a hydrophobic material. The absorption layer 4 is provided with a hydrophilic absorption area 41. When the absorption layer 4 is stacked with the absorption layer 3 , the absorption area 41 overlaps with the adsorption area 310 .

The loading layer 1, the drainage layer 2, the adsorption layer 3 and the absorption layer 4 can be made of filter paper coated with wax, and the area coated with wax on each layer has a hydrophobic property; the area not coated with wax has a hydrophilic property. The characteristics of, for example, the loading area 11, the drainage channel 21, the adsorption area 31 and the absorption area 41. That is, by printing hydrophobic wax on the filter paper to restrict the fluid flow channels and flow directions on each layer. The loading layer 1, the drainage layer 2, the adsorption layer 3 and the absorption layer 4 can be connected in a set order, and the corresponding layers can be stacked together by folding; they can also be set independently of each other.

As shown in Figures 4b to 4c, the loading layer 1, the drainage layer 2, the first adsorption layer 31, the second adsorption layer 32 and the absorption layer 4 are squares of 3cm*3cm, and are connected in sequence. Layers on top of each other. In the center of the loading layer 1 there are five evenly arranged circular loading zones 11 with a diameter of 4 mm. A starfish-shaped drainage channel 21 is correspondingly provided on the drainage layer 2 , which includes five drainage branch channels with a width of 4 mm, and each drainage branch channel corresponds to a circular loading area 11 . When the drainage layer 2 and the loading layer 1 are stacked up and down, the end of each drainage branch channel overlaps with a corresponding circular loading area 11 up and down. At the center of the first adsorption layer 31 and the second adsorption layer 32, a circular adsorption area 310 is provided. When the first adsorption layer 31 and the second adsorption layer 32 are stacked up and down by folding, Between the two adsorption regions 310 on the first adsorption layer 31 and the second adsorption layer 32, a glass fiber sheet with the same shape and size as the adsorption region 310 is placed as an adsorption component. When the first adsorption layer 31, the second adsorption layer 32 and the drainage layer 2 are stacked up and down together, the adsorption part and the two adsorption regions 310 overlap with the center of the starfish-shaped drainage channel 21, so that the suction part flows out from the adsorption part. The liquid is evenly transported to the five circular loading areas 11 through the five drainage branch channels of the drainage channel 21 . A circular absorbing region 41 is arranged on the absorbing layer 4 .

When using the paper chip, the adsorption layer 3 with the adsorption component and the absorption layer 4 are first stacked up and down, and then the liquid to be detected containing the pathogen is poured on the adsorption area 310 on the first adsorption layer 31 of the adsorption layer 3, After the liquid to be detected is filtered through the adsorption region 310 of the first adsorption layer 31, it flows to the adsorption component, so that the nucleic acid in the liquid to be detected is retained in the adsorption component, separated from other substances, and passed through the second adsorption The absorbing area 41 on the absorbing layer 4 on the layer 32 absorbs non-nucleic acid substances in the liquid to be tested. Then add washing liquid to the adsorption part through the adsorption area 310 on the first adsorption layer 31 to further wash away the impurities in the adsorption part, and pass through the absorption area 41 placed on the absorption layer 4 on the second adsorption layer 32 surface Absorbs washing liquid and waste. Then the adsorption layer 3 sandwiching the adsorption component is stacked up and down with the drainage layer 2 and the loading layer 1, and the eluent is injected into the adsorption component through the adsorption region 310 on the first adsorption layer 31 of the adsorption layer 3, so that the adsorption is absorbed on the adsorption layer 3. The nucleic acid in the component is separated from the adsorption component, and flows into the drainage channel 21 through the adsorption area 310 on the second adsorption layer 32 , and is distributed to the five circular loading areas 11 through the five drainage branch channels of the drainage channel 21 .

As shown in Fig. 4d, three circular loading areas 11 arranged in a straight line and equidistant can also be set on the loading layer 1 of the paper chip of the present disclosure, and the drainage channel 21 arranged on the drainage layer 2 is in a straight line. When the drainage layer 2 and the loading layer 1 are stacked up and down, the straight drainage channel 21 and the three circular loading regions 11 overlap up and down.

In addition, this embodiment also provides a test board 200 , as shown in FIG. 5 , the test board 200 is provided with a test area 210 corresponding to the loading area 11 on the loading layer 1 of the paper chip 100 . The detection area 210 is used to detect the nucleic acid in the liquid to be detected adsorbed in the loading area 11.

The detection board 200 may be made of acrylic material, and processed by laser cutting to have an outer dimension of 3cm*3cm*0.5cm. When the paper chip is the structure shown in Figures 4a to 4c, five square plates (as shown in Figure 5) with a hole-like detection area 210 with a diameter of 4mm are provided on the top surface of the detection plate 200; when the paper chip is In the structure shown in FIG. 4 d , three hole-shaped detection areas 210 (as shown in FIG. 6 a ) are opened on the top surface of the detection plate 200 .

During detection, the loading area 11 on the loading layer 1 of the paper chip 100 is separated from the loading layer 1 . Placed in the detection area 210 of the detection plate 200, for example, punch the loading area 11 into the detection area 210 with a hole puncher, and then add the reaction reagent in the detection area 210 to obtain the detection result.

Example 3. Construction of paper microfluidic devices

To simplify virus detection of samples outside the laboratory, a paper microfluidic device was introduced, which consists of a paper chip and a detection plate. The setup and detection process of the paper chip and detection board are shown in Figures 6a-6c. Briefly, a paper chip is prepared by the following steps:

(1) Use the paraffin printer Colorqube 8570 to print the corresponding pattern on the qualitative filter paper, leaving the hydrophilic adsorption area (the white area in Figure 6a and 6b);

(2) Use a heating plate to bake the filter paper at 120°C for 1-2 minutes to completely impregnate the filter paper with paraffin;

(3) Add glass fibers (shown by arrows in Figures 6a and 6b) at specific positions, and then fold them, as shown in Figure 6a for the folding method;

(4) Add 50 μL ddH ₂ O to the paper chip to test and optimize the flow rate under different pore sizes.

The test board is printed by a laser cutting machine (Shandong leapion machine co.ltd; LC-1390). The board is made of black acrylic material, the thickness of the board is 5mm, the cutting power is 94% of the maximum power, and the cutting speed is 0.48mm/s. The pore size is the same as the hydrophilic pore size of the paper chip.

The sample is loaded on the adsorption area of the paper chip, so that the nucleic acid in the sample is absorbed by the glass fiber, and the washing liquid is injected into the adsorption area to elute the impurities. Then, the eluent is injected into the glass fiber through the adsorption area, so that the nucleic acid adsorbed on the glass fiber is eluted, and flows from the hole at the bottom of the paper chip into the corresponding hole of the detection plate for detection.

As shown in Figure 6c, the paper microfluidic device of the present disclosure can be used for visual detection of the new coronavirus. As shown in Figure 6e, when fluorescent probes are used, the color reaction can be directly carried out in the detection plate of the paper microfluidic device. As shown in Figure 6d, when a biotin probe is used, the flow detection test paper can be put into the reaction solution in the detection plate for color reaction.

Example 4. RT-LAMP reaction combined with CRISPR-Cas12a system for visual detection

In this example, the CRISPER-Cas12a system was introduced and combined with the LAMP in Example 1 to construct HF-RT-LAMP. Since the sequence of the non-specific amplification product is different from that of the specific amplification product, Cas12a is introduced to identify the specific amplification product, thereby eliminating the interference caused by the non-specific amplification. To put it simply, first select the gRNA targeting the amplified segment, construct the Cas12a enzyme digestion system, and then combine it with RT-LAMP to complete the construction of the HF-RT-LAMP reaction. The specific method is as follows:

About 10 mL of each sample was taken and concentrated by the membrane adsorption-elution method. To put it simply, first use a 3μm filter membrane to remove large particles in the sample, then adjust the pH to neutral, add Mg ²⁺ to make the final concentration 25mM, then use a 0.45μm filter membrane to filter, and then filter on the filter membrane Add 40 μL of GuSCN lysate and incubate at room temperature for 10 min. Then, the lysate was aspirated and loaded onto the paper chip for purification, and eluted with 10 μL of enzyme-free sterile water, and then the paper chip was added to the reaction well of the microfluidic plate using a puncher for reaction. The negative control is enzyme-free sterile water, and the positive control is the method of sample addition.

The final concentration of 100nM Cas12a, 125nM gRNA, and 500nMCas12a fluorescent probe was incubated in 1×NEBuffer2.1 buffer at 37°C for 10 minutes to form an enzyme digestion master mix. Then, 2 μL of the RT-LAMP amplicon obtained in Example 1 was added to 18 μL of the master mix, and incubated at 37° C. for 30 min. ABI7500 was used to record the fluorescence signal excited by 480nm blue light (the negative control was colorless, and the positive sample was bright green), and the color information was extracted by ImagineJ. The results are shown in Figures 3a-3c.

From the fluorescence results shown in Figures 3a-3c, it can be seen that the introduction of Cas12a enzyme can be the detection visualization of the sample. The Cas12a enzyme is activated in the positive sample due to the amplification product targeted by the gRNA. The activated Cas12a enzyme cleaves the Cas12a fluorescent probe to release fluorescent molecules, so that a bright green fluorescent signal can be seen under blue light excitation.

Example 5. Visual detection of HF-RT-LAMP using probes with FAM/biotin

In this example, a probe with FAM and biotin at the 5' end and 3' end of the probe (referred to as "Cas12a biotin probe") was used to replace the Cas12a fluorescent probe in Example 4, The N, E, and S genes were detected by HF-RT-LAMP respectively. Take 2 μL of the purified sample obtained in Example 4, add it to 18 μL premix, add 80 μL 1×NEBuffer 2.1, and incubate at 37° C. for 30 min. Then, insert the flow detection test paper (MileniaHybriDetect 1, TwistDx) into the reaction tube. After about two minutes, when the test paper has only one band near the sample loading side, it is negative; when the sample has two bands or When there is only one band (biotin) near the top of the strip, it is considered positive. Band information was extracted using ImagineJ, and the results are shown in Figure 3d-3f.

It can be seen from Figures 3d to 3f that the detection method using the Cas12a biotin probe in this embodiment has a similar effect to that of using the Cas12a fluorescent probe. Since there will be very weak bands in the detection line (near the top of the test paper) in the current flow detection test strip, you can use ImageJ to judge whether it is the background band of the test strip itself or the band generated by the amplicon. Moreover, the problem of false positives is effectively solved. According to the negative ImageJ extraction results, the gray value threshold of the lateral flow method for negative samples was calculated as 150, thus avoiding the background color produced by the lateral flow method itself. In addition, from the results in Figure 3, converted into the original volume of the sample (10mL), it can be seen that the detection limits of the S, N, and E genes can reach 10 copies/mL, 25 copies/mL and 310 copies, respectively. /mL, all reached the zM level close to the single molecule level.

Example 6. Evaluation of detection effect using paper chip combined with HF-RT-LAMP

In this example, a double-blind experiment of random spiked sewage was carried out by using a paper chip combined with HF-RT-LAMP to detect SARS-CoV-2 qualitatively and semi-quantitatively.

SARS-CoV-2 samples collected from Jiangjunshan Hospital in Guizhou were added to SARS-CoV-2-free sewage. In order to simulate community sewage at different development stages of the epidemic as realistically as possible, 50 sewage samples with concentrations ranging from 0 to 500 copies/mL were prepared. Using paper chips combined with HF-RT-LAMP, a sample combination consisting of 44 spiked sewage samples and 6 negative samples randomly grouped was tested.

The test results are shown in Table 2 and Figure 7, 43 of the 44 spiked samples successfully detected the SARS-CoV-2 virus and no false positives occurred, and the semi-quantitative results of 41 samples were consistent with the real samples, with 100% specificity. These results prove that, using three sets of detection primers, HF-RT-LAMP on a paper chip can not only resist the interference of sewage complex media, successfully distinguish between spiked sewage and unspiked sewage, but also can detect as low as 10 copies / mL of spiked sewage, and has semi-quantitative capability in the concentration range of 10-310 copies/mL.

Table 2. Double-blind experimental test results

sample	Concentration (copy/mL)	S gene	N gene		E gene
1	10	P	N		N
2	30	P	P		N
3	15	P	P		N
4	50	P	P		N
5	0	N	N		N
6	5	N	N		N
7	35	P	P		N
8	350	P	P		P
9	120	P	P		N
10	180	P	P		N
11	40	P	P		N
12	20	P	P	N
13	80	P	P	P
14	0	N	N		N
15	25	P	P	N
16	220	P	P	N
17	310	P	P	P
18	0	N	N	N
19	60	P	P		N
20	70	P	P	N
twenty one	90	P	P	N
twenty two	110	P	P	P
twenty three	95	P	P	N
twenty four	55	P	P	N

25	45	P	P	N
26	65	P	P	N
27	95	P	P		N
28	130	P	P	N
29	30	P	N		N
30	0	N	N		N
31	100	P	P		N
32	75	P	P	N
33	25	P	P	N
34	20	P	N		N
35	15	P	N	N
36	0	N	N	N
37	310	P	P	P
38	370	P	P	P
39	280	P	P		P
40	250	P	P		N
41	290	P	P	P
42	125	P	P	N
43	0	N	N	N
44	105	P	P		N
45	50	P	P	N
46	5	P	N	N
47	500	P	P	P
48	360	P	P	P
49	320	P	P		N
50	55	P	P	N

Note: P means positive result, N means negative result.

In summary, the paper chip not only successfully solved the problem of nucleic acid extraction and purification under field conditions, but also successfully overcome the semi-quantitative problem with its multi-channel detection, realizing a revolutionary leap from rapid detection of pathogens to rapid visual assessment of risks , and also provides a new method for the rapid detection equipment to achieve semi-quantitative.

The technical solution of the present invention is not limited to the limitations of the above-mentioned specific embodiments, and any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims (15)

1. A primer for specific detection of a target virus in a sample, including two or more primer sets, the two or more primer sets specifically targeting different genes of the virus or Different regions of the same gene, and the two or more primer sets have different detection limits.
2. The primer according to claim 1, wherein the primer is used for loop-mediated constant temperature amplification reaction, preferably for real-time loop-mediated constant temperature amplification reaction.
3. The primer according to claim 1 or 2, wherein the primer sets each comprise an upstream external primer, a downstream external primer, an upstream internal primer and/or a downstream internal primer,

   Preferably, said 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 claim 1 or 2, wherein the target virus is severe acute respiratory syndrome coronavirus (SARS-CoV-2), preferably selected from Wuhan-Hu-1 of SARS-CoV-2 / One or more of the 2019, Alpha, Beta, Gamma, Delta, and Omicron strains.
5. The primer according to claim 4, wherein the primers include two or three of the first primer set, the second primer set and the third primer set, wherein the first primer set, the second primer set The second primer set and the third primer set specifically target the S gene, N gene and E gene of SARS-CoV-2 respectively,

   Preferably, the first primer set comprises:

   The first upstream external primer has a sequence as shown in SEQ ID NO.:13;

   The first downstream external primer has a sequence as shown in SEQ ID NO.:14;

   The first upstream internal primer has a sequence as shown in SEQ ID NO.: 15; and

   The first downstream internal primer has a sequence as shown in SEQ ID NO.:16,

   Preferably, the second primer set comprises:

   The second upstream external primer has a sequence as shown in SEQ ID NO.:1;

   The second downstream external primer has a sequence as shown in SEQ ID NO.:2;

   The second upstream internal primer has a sequence as shown in SEQ ID NO.:4; and

   The second downstream internal primer has a sequence as shown in SEQ ID NO.:3,

   Preferably, the third primer set includes:

   The third upstream external primer has a sequence as shown in SEQ ID NO.:7;

   The third downstream external primer has a sequence as shown in SEQ ID NO.:8;

   The third upstream internal primer has a sequence as shown in SEQ ID NO.:10; and

   The third downstream internal primer has a sequence as shown in SEQ ID NO.:9.
6. primer according to claim 5, is characterized in that,

   The first primer set also includes: 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 shown in SEQ ID NO.:18 ,

   The second primer set also includes: 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 shown in SEQ ID NO.:6 ,

   The third primer set also includes: 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 shown in SEQ ID NO.:12 .
7. A paper chip comprising a loading layer made of a hydrophilic material coated with a hydrophobic material, a drainage layer and an adsorption layer;

   The loading layer is provided with a plurality of hydrophilic loading areas;

   A hydrophilic drainage channel is arranged on the drainage layer, and when the drainage layer overlaps with the loading layer, the loading area overlaps with the drainage channel;

   A hydrophilic adsorption area is provided on the adsorption layer, and when the adsorption layer overlaps with the drainage layer, the adsorption area (310) overlaps with the drainage channel;

   The adsorption area is provided with an adsorption component for adsorbing nucleic acid;

   Preferably, the paper chip also includes an absorption layer made of a hydrophilic material coated with a hydrophobic material, and a hydrophilic absorption area is arranged on the absorption layer. When the absorption layer is stacked with the absorption layer , the absorption area overlaps with the adsorption area;

   Preferably, the loading layer, drainage layer, adsorption layer and absorption layer are connected in a set order or are independent of each other;

   Preferably, the adsorption layer includes a connected first adsorption layer and a second adsorption layer, and the first adsorption layer and the second adsorption layer are provided with the adsorption regions at corresponding positions, so The adsorption component is arranged between the first adsorption layer and the second adsorption layer;

   Preferably, the adsorption member is made of glass fiber;

   Preferably, the loading layer, drainage layer, adsorption layer and absorption layer are made of wax-coated filter paper.
8. A paper microfluidic device, comprising a paper chip and a detection plate according to claim 7; the detection plate is provided with a detection area corresponding to the loading area on the loading layer of the paper chip;

   Preferably, the detection board is made of acrylic material.
9. A kit for detecting viruses in a sample, said kit comprising the primer according to any one of claims 1 to 6.
10. The kit according to claim 9, wherein the kit also includes a programmable nuclease, and two or more gRNAs,

    Preferably, the programmable nuclease is selected from Cas12, Cas13 or Cas14 nuclease,

    More preferably, the programmable nuclease Cas12 nuclease.
11. The kit according to claim 9 or 10, wherein the two or more gRNAs include two or three of the first gRNA, the second gRNA and the third gRNA, wherein the first gRNA One gRNA, the second gRNA and the third gRNA specifically target the S, N and E genes of SARS-CoV-2 respectively,

    Preferably, the first gRNA has a sequence as shown in SEQ ID NO.:21;

    Preferably, the second gRNA has a sequence as shown in SEQ ID NO.:19;

    Preferably, the third gRNA has a sequence as shown in SEQ ID NO.:20.
12. The kit according to claim 9 or 10, wherein the kit also includes a reporter probe, and the reporter probe is a single-stranded nucleic acid sequence;

    Preferably, the reporter probe includes a detection group and a quencher group respectively located at both ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from the group consisting of fluorescein, 6-fluorescein, IRDYE 700, TYE 665, Alexa Fluor or ATTO TM633, the quenching group is selected from Iowa Black RQ, Iowa Black FQ or BlackHole quencher,

    Preferably, the reporter probe comprises biotin.
13. The kit according to claim 12, wherein the reporter probe comprises the sequence 5'-(6-FAM)-TTATT-(BHQ1)-3', or 5'-(6-FAM)-TTATTATT -(Bio)-3'.
14. The kit according to claim 9 or 10, wherein the kit further comprises a paper microfluidic device, and the paper microfluidic device includes a paper chip and a detection plate;

    Preferably, the paper microfluidic device is selected from the paper microfluidic device described in claim 8.
15. A method for semi-quantitative detection of target virus in a sample, said method comprising:

    Incubate two or more than two primer sets according to any one of claims 1 to 6 with the sample respectively, and perform a loop-mediated constant temperature amplification reaction to obtain a corresponding reaction product; in the reaction product In the process, add programmable nuclease and gRNA complexes and reporter probes to incubate respectively to obtain detection products, wherein, when a signal is detected in a detection product, it is judged that the sample contains the primer set corresponding to the used The detection limit of the virus concentration.

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