Detection elements, reagent kits, and detection methods for detecting infectious bronchitis virus in chickens.

Abstract

This invention relates to the field of biotechnology, specifically disclosing a detection element, kit, and detection method for detecting infectious bronchitis virus (IBV) in chickens. The detection element is a Toehold Switch element, which is an S4-3 element and / or an S3 element; the nucleotide sequence of the S4-3 element is shown in SEQ ID NO: 1; the nucleotide sequence of the S3 element is shown in SEQ ID NO: 2. The detection element of this invention features high sensitivity and strong specificity, enabling the differentiation of three IBV strains—P65, MH20, and H120—at a single-base resolution.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a detection element, reagent kit, and detection method for detecting infectious bronchitis virus in chickens. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious viral disease of commercial chickens worldwide, caused by IBV coronavirus. Due to its high mutation rate, genetic recombination, and host selection pressures, IBV has rapidly evolved, generating an increasing number of genotypes and serotypes. Protective immunity induced by one IBV serotype offers poor cross-protection against infections of other serotypes, leading to frequent outbreaks of IBV in commercial flocks and causing significant economic losses. Therefore, developing high-resolution genotyping diagnostic technologies is urgently needed to identify differentiated IBV genotypes and develop targeted protective vaccines.

[0003] On the other hand, the *E. coli* cell-free protein expression system (CFS) is an in vitro protein synthesis technology based on cell extracts. This technology is highly favored for its simple genetic background, ease of operation, rapid reaction speed, simple process, and high-throughput processing capabilities. This technology has become a key tool in bioreaction system research. Recently, the advantages of CFS in expressing complex proteins, toxic proteins, and signaling proteins have become increasingly apparent, and its application prospects in the biopharmaceutical industry are particularly broad. However, current CFS systems suffer from problems such as poor activity and low protection efficiency of linear templates. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a detection element for detecting IBV, which has the characteristics of high sensitivity and strong specificity.

[0005] Another technical problem that this invention aims to solve is to provide a Trigger RNA element that enables highly sensitive IBV detection.

[0006] Another technical problem that this invention aims to solve is to provide a LAMP primer for amplifying Trigger RNA elements.

[0007] Another technical problem that this invention aims to solve is to provide a kit for detecting IBV, which has the characteristics of high sensitivity and strong specificity.

[0008] The technical problem that this invention also aims to solve is to provide a non-diagnostic method for detecting IBV, which can achieve highly specific, highly sensitive, and visualized IBV detection.

[0009] To solve the above-mentioned technical problems, the present invention provides a detection element for detecting IBV, wherein the detection element is a Toehold Switch element, which is an S4-3 element and / or an S3 element;

[0010] The nucleotide sequence of the S4-3 element is shown in SEQ ID NO: 1;

[0011] The nucleotide sequence of the S3 element is shown in SEQ ID NO: 2.

[0012] Accordingly, the present invention also discloses a trigger RNA element that is complementary to the Toehold sequence of the above-mentioned Toehold Switch element, wherein the trigger RNA element is a P65-T4-3 element, an MH20-T4-3 element and / or an H120-T4-3 element;

[0013] The P65-T4-3 element is complementary to the S4-3 element and is the test sequence of IBV P65, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0014] The MH20-T4-3 element is complementary to the S3 element and is the sequence to be tested for IBV MH20, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0015] The H120-T4-3 element is complementary to the S3 element and is the test sequence of IBV H120, the nucleotide sequence of which is shown in SEQ ID NO: 5.

[0016] Accordingly, the present invention also discloses a LAMP primer for amplifying the above-mentioned Trigger RNA element, wherein the LAMP primer comprises L96-F3, L96-B3, L96-FIP, L96-BIP, L80-F3, L80-B3, L80-FIP, L80-BIP, L33-F3, L33-B3, L33-FIP, and L33-BIP; the nucleotide sequences of which are shown in SEQ ID NO: 6 to 17, respectively.

[0017] Among them, L96-F3, L96-B3, L96-FIP, and L96-BIP are used to amplify P65-T4-3 elements;

[0018] L80-F3, L80-B3, L80-FIP, and L80-BIP are used to amplify MH20-T4-3 elements;

[0019] L33-F3, L33-B3, L33-FIP, and L33-BIP are used to amplify H120-T4-3 elements.

[0020] Accordingly, the present invention also discloses a kit for detecting IBV, which includes the detection element for detecting IBV described above.

[0021] As an improvement to the above technical solution, the aforementioned LAMP primers are also included.

[0022] As an improvement to the above technical solution, a cell-free protein expression system is also included;

[0023] The cell-free protein expression system includes:

[0024] Escherichia coli cell extract 35-50 vol%, lysis buffer 40-50 vol%, gene template 8-12 vol%, RNase inhibitor 0.1-1 vol%, nuclease-free water 1-15 vol%.

[0025] Preferably, the cell-free protein expression system may also include other adjuvants, which can be selected by those skilled in the art according to specific functions, and will not be listed here.

[0026] Wherein, the *E. coli* is a recombinant bacterium obtained by modifying the recipient *E. coli*, the modification including: (1) knocking out the LacZ gene of the recipient *E. coli*; (2) knocking out the RecBCD operon gene and the ptrA gene of the recipient *E. coli*, and inserting the LyseR gene at the knockout site; specifically, the RecBCD operon gene includes the RecB gene, the RecC gene and the RecD gene;

[0027] The lysis buffer comprises the following components at the following concentrations:

[0028] HEPES 40–60 mM, ATP 1–2 mM, GTP 1–2 mM, CTP 0.5–1.5 mM, UTP 0.5–1.5 mM, tRNA 0.1–0.5 mg / mL, CoA 0.1–0.5 mM, NAD 0.2–0.8 mM, cAMP 0.5–1 mM, leucovorin 0.01–0.1 mM, spermidine 0.5–1.5 mM, 3-PGA 10–50 mM, maltose 10–20 mM, amino acids 20–25 mM, PEG-8000 8–15 mg / mL, potassium glutamate 55–80 mM, magnesium glutamate 0.5–1.5 mM, DTT 0.5–1.5 mM.

[0029] As an improvement to the above technical solution, the CFS includes:

[0030] Escherichia coli cell extract 40 vol%, lysis buffer 44.5 vol%, RNase inhibitor 0.5 vol%, gene template 10 vol%, chlorophenol red-β-D-galactopyranoside (CPRG) 1 vol%, nuclease-free water (depc H2O) 4 vol%.

[0031] The lysis solution comprises components at the following concentrations:

[0032] HEPES 50mM, ATP 1.5mM, GTP 1.5mM, CTP 0.9mM, UTP 0.9mM, tRNA 0.2mg / mL, CoA 0.26mM, NAD 0.33mM, cAMP 0.75mM, leucovorin 0.0678mM, spermidine 1mM, 3-PGA 30mM, maltose 12mM, PEG-8000 11.2mg / mL, potassium glutamate 60.6mM, magnesium glutamate 0.815mM, DTT 1mM, Leucine 20mM, Glycine 24mM, Valine 24mM, Isoleucine 24mM, Methionine 24mM, Proline 24mM, Phenylalanine 24mM, Tyrosine 24mM, Serine 24mM, Threonine 24mM, Tryptophan 24mM, Asparagine 24mM, Glutamine 24mM, Lysine 24mM, Cysteine ​​24mM, Arginine 24mM, Aspartic Acid 24mM, Histidine 24mM, Glutamic Acid 24mM, Alanine 24mM.

[0033] As an improvement to the above technical solution, the CFS and the detection element are freeze-dried on a paper-based material; the freeze-drying temperature is -60 to -40°C.

[0034] Accordingly, the present invention also discloses a method for detecting IBV for non-diagnostic purposes, comprising:

[0035] Obtain the gene template;

[0036] The gene template was amplified using the LAMP primers described above;

[0037] The amplified products were reacted using a reaction system consisting of a cell-free protein expression system and the aforementioned detection element; the results were determined based on color changes.

[0038] Specifically, the gene template can be a gene template extracted from the sample to be tested, or a gene template prepared by in vitro transcription, but is not limited to these.

[0039] As an improvement to the above technical solution, in the step of amplifying the gene template using the above-mentioned LAMP primers, the reaction procedure is to react at 70°C for 30 minutes.

[0040] In the step of reacting the amplified product with a reaction system consisting of a cell-free protein expression system and the aforementioned detection element, and determining the result based on the color change, the reaction procedure is to react at 30°C for 30–60 minutes.

[0041] Implementing this invention has the following beneficial effects:

[0042] 1. The detection element for detecting IBV in one embodiment of the present invention has the characteristics of high sensitivity and strong specificity. After the method of coupled loop-mediated isothermal amplification (LAMP), it can distinguish three IBV strains, P65, MH20 and H120, with single-base resolution.

[0043] 2. In one embodiment of the present invention, a kit for detecting IBV is developed based on *E. coli* cells. A cell-free fibroblast (CFS) with autolysis and exonuclease activity inhibition or deletion was developed, using a toehold switch coupled with LAMP to highly specifically differentiate three types of IBV within the CFS. The paper-based cell-free sensor diagnostic technology provided by this invention combines the high expression activity and gene stability of CFS, the high specificity and sensitivity of molecular sensors, and the advantages of easy storage, distribution, and colorimetric results of paper-based materials. It is expected to be widely used in the detection of plant and animal pathogens in resource-scarce regions. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the dual-plasmid gene editing principle in Example 1;

[0045] Figure 2 This is a compositional diagram of plasmid pCas12a-λred from Example 1;

[0046] Figure 3 This is a composition diagram of plasmid pUC19-LyseR in Example 1;

[0047] Figure 4 This is a diagram showing the results of CFS expressing three CRISPR proteins in vitro in Example 2;

[0048] Figure 5 This is a screening result diagram of the background leakage expression of the P65 related Switch element in Example 3;

[0049] Figure 6 This is a graph showing the performance test results of the P65-related Switch components in Example 3;

[0050] Figure 7This is a sensitivity diagram of the detection of P65, MH20, and H120 strains using a paper-based Toehold Switch coupled to a LAMP system in Example 3.

[0051] Figure 8 This is a detection specificity diagram of the P65, MH20, and H120 strains using a paper-based Toehold Switch coupled with a LAMP detection system in Example 3. Detailed Implementation

[0052] To facilitate understanding of the present invention, it will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to make the disclosure of the present invention more thorough and complete.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments or examples only and is not intended to limit the invention. The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0054] The following embodiments are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the scope of the invention in any way.

[0055] This invention. Those skilled in the art will understand that variations and other uses within the scope of the claims are included within the spirit and scope of this invention. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available. The promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI, and the specific sequence start positions can be determined from the primers in the primer table. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0056] In this invention, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features.

[0057] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0058] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0059] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0060] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0061] Example 1: CFS Based on Autolysis Procedure and Exonuclease Activity Deficiency

[0062] 1. Preparation of engineered bacteria

[0063] The RecBCD operon genes (including RecB genes (SEQ ID NO: 18), RecC genes (SEQ ID NO: 19), and RecD genes (SEQ ID NO: 20)) and ptrA genes (SEQ ID NO: 21) in *E. coli* BL21(DE3)-Gold-dLac (Bacterial Strain #99247) were knocked out using a CRISPR / Cas combined with λ-red dual plasmid gene editing system. The LyseR gene was then inserted at this site to obtain the engineered strain BL21(DE3)-Gold-dLac-dRecBCD:LyseR. The schematic diagram is shown below. Figure 1 As shown. The specific steps are as follows: The plasmid pCas12a-λred( Figure 2 (SEQ ID NO: 22) and pUC19-LyseR ( Figure 3(SEQ ID NO: 23) was transformed into Escherichia coli BL21(DE3)-Gold-dLac cells. The cells were cultured in LB medium containing the corresponding antibiotics at 37°C. When the cells grew to OD600 = 0.6, 40 mM arabinose and 0.25 mM IPTG were added to induce the expression of Cas12a protein and λred-related protein, respectively. After culturing for another 5 h, the bacterial culture was diluted 1000 times and spread on agar plates containing 10% sucrose and AmpR resistance, and incubated upside down overnight. On the second day, single colonies were picked for colony PCR verification. Single colonies with the correct bands were selected and cultured and purified on sucrose plates until a single and bright positive target band was shown during colony PCR verification. The gene-edited gRNA sequences include gRNA-Fw (SEQ ID NO: 24) and gRNA-Fw (SEQ ID NO: 25); homologous arms include TYB-Fw (SEQ ID NO: 28) and gRNA-Fw (SEQ ID NO: 29). The PCR primer sequences for subsequent colony validation include argA-F (SEQ ID NO: 26) and ppdB-R (SEQ ID NO: 27). The PCR primers are located at both ends of the gene to be knocked out in the genome.

[0064] 2. Preparation of extracts based on autolysis procedure and exonuclease activity deficiency

[0065] The verified cells were cultured in 2×YTPG medium containing the corresponding antibiotic at 37°C with shaking. When the cells grew to OD600 = 0.6, 1 mM IPTG was added to induce T7 RNA polymerase expression. When the cells grew to OD600 = 3.0, the cells were harvested and the cell pellet was washed three times with S30A buffer (50 mM Tris, 60 mM potassium glutamate, 14 mM magnesium glutamate, final pH adjusted to 7.7, and DTT added on the same day to a final concentration of 2 mM). Finally, S30A buffer was added at a ratio of 2:1 between the volume of S30A buffer and the weight of the cell pellet. After freeze-thaw-vortex cycling, the extract was harvested by high-speed centrifugation (25000 g / 1 h / 4°C).

[0066] 3. Premix preparation

[0067] Prepare the premix according to the recipe in Table 1, mix thoroughly, and then dispense and store in a -80°C freezer.

[0068] Table 1 Premix Composition

[0069]

[0070] The amino acids listed in the table are:

[0071] Leucine 20mM, glycine 24mM, valine 24mM, isoleucine 24mM, methionine 24mM, proline 24mM, phenylalanine 24mM, tyrosine 24mM, serine 24mM, threonine 24mM, tryptophan 24mM, asparagine 24mM, glutamine 24mM, lysine 24mM, cysteine ​​24mM, arginine 24mM, aspartic acid 24mM, histidine 24mM, glutamic acid 24mM, alanine 24mM.

[0072] 4. Preparation of cell-free reaction system

[0073] Prepare the cell-free reaction system according to the specific formula in Table 2:

[0074] Table 2 Composition of the cell-free reaction system

[0075]

[0076] 5. CFS expresses three CRISPR proteins and LacZ protein in vitro.

[0077] In vitro expression experiments were conducted using plasmids pMBP-LbCas12a (addgene, 113431), pET28b-T7-henAsCas12a-NLS-6xHis (addgene, 114072), pC013-Twinstrep-SUMO-huLwCas13a (addgene, 90097), pFN6A-(HQ)-LacZ (SEQ ID NO: 30) and linear gene templates: Linear-LbCas12a, Linear-henAsCas12a, Linear-LwaCas13a, and Linear-LacZ. The linear gene structure was: 5'-T7 promoter-target gene-T7 terminator-3', with 10bp non-coding sequences retained at both ends of the T7 promoter and T7 terminator. Cell-free reaction systems were prepared according to Table 2, and after homogeneous mixing, the mixtures were incubated at 30℃ for 1 h. After cell-free expression, 10 μL of sample was mixed with 30 μL of TBS, and then 40 μL of 2×LD and 4 μL of DTT (1M) were added. After thorough mixing, the mixture was heated at 95℃ for 3 min. 16.8 μL of the mixture was then loaded onto an SDS gel. After protein electrophoresis, the expression effects of plasmid gene templates and linear gene templates in CFS were identified by Coomassie Brilliant Blue staining and Western blot. The results are as follows: Figure 4 As shown in the figure. The results showed that both plasmid genes and linear genes had high expression levels in CFS, with the plasmid gene template showing higher expression levels compared to the linear gene template.

[0078] Example 2 uses a paper-based Toehold Switch coupled with a LAMP system to specifically differentiate three IBV strains.

[0079] 1. Design of Toehold Switch Components Based on IBV Genome Sequence

[0080] 1.1 Design of Toehold Switch Components

[0081] First, we obtained the genomic RNA sequence of the cell line IBV P65 (Genbank accession number: DQ001339.1). According to literature reports, several structural proteins of IBV have high RNA transcription levels, namely nucleocapsid protein N, membrane protein M, envelope protein E, and spike protein (antigen protein) S. We used the gene sequences of these structural proteins as templates to design Toehold Switch elements. Specifically, the structural protein gene sequences of IBV P65 were segmented in 1000bp units, and the segmented gene sequences were input into the software (NUPACK) to screen for Trigger RNA source sequences. After inputting the source sequences and running the script, the secondary structure of the Toehold Switch was previewed. After checking for errors, the next step of design was carried out, and the overall defect value of different candidate sequences was obtained. Candidate sequences with low defect values ​​were selected for analysis of the secondary structure of Switch RNA and Trigger RNA. Then, combinations of Switch secondary structures that were close to the secondary structure model were selected (specifically, as shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 31-60).

[0082] Specifically, the Switch element contains a T7 promoter, a Switch sequence, a 21nt linker, a LacZ reporter gene, and a T7 terminator. The Switch element is amplified into the vector through two rounds of repeat PCR using designed long primers, followed by homologous recombination to obtain the target plasmid.

[0083] 1.2 In vitro verification experiments of Switch components

[0084] The following section uses pFN6A-S4-1-LacZ as an example to illustrate the construction process of the recombinant plasmid pFN6A-Switch-LacZ. The steps are shown in the table below: Based on the pFN6A-(HQ)-LacZ (SEQ ID NO: 30) vector gene sequence and the candidate Switch RNA gene sequence, long primers were designed using snapgene. The primers include pFN6A-S4-1-F (SEQ ID NO: 61), LacZ-R (SEQ ID NO: 62), pFN6A-S4-1-R1 (SEQ ID NO: 63), pFN6A-S4-1-R2 (SEQ ID NO: 64), and pFN6A-LacZ-F (SEQ ID NO: 65).

[0085] Primers pFN6A-S4-1-R1 and pFN6A-S4-1-R2 contain the Switch sequence. The Switch sequence is ligated into the vector by PCR amplification. The PCR amplification system of the Switch fragment is shown in Table 3 below:

[0086] Table 3. PCR amplification system and conditions

[0087]

[0088] After the PCR amplification is completed as shown in the table above, the product is used as the template for the second round of PCR. The second round of PCR amplification system is shown in Table 4 below. The vector part is also amplified by PCR, and the PCR amplification system and conditions are shown in Table 5.

[0089] Table 4. PCR amplification system and conditions

[0090]

[0091] Table 5. PCR amplification system and conditions

[0092]

[0093] The PCR amplification products in Tables 3 and 4 were digested with DpnI enzyme, then recovered and purified. Homologous recombination was performed to transform into competent cells, and positive plasmids were extracted for enzyme digestion verification. If the sequencing alignment results were correct, it indicated that the plasmid pFN6A-S4-1-LacZ was successfully cloned, and its specific sequence is shown in SEQ ID NO: 66.

[0094] The verification experiment mainly includes the following two parts:

[0095] 1.2.1 Background Leakage Expression Test of P65-Related Switch Components Based on CFS

[0096] Recombinant plasmids were prepared according to the above method. Using recombinant plasmid pFN6A-Switch-LacZ as a template, in vitro expression experiments were conducted at four concentration gradients of 2.5 nM, 5 nM, 7.5 nM, and 10 nM. The color changes at each concentration were recorded after reacting at 30℃ for 1 h.

[0097] 1.2.2 Verification of the detection capability of the Toehold Switch component

[0098] Screening was conducted based on the background leakage level of each recombinant plasmid at different concentrations. Recombinant plasmids with high background leakage levels were selected for further experiments at 2.5 nM or 5 nM, while those with low background leakage levels were selected at 10 nM. ssDNA sequences corresponding to the trigger RNA sequences were synthesized, and their detection capability against the switch was validated using ssDNA. Building upon section 1.2.1, 1 μL of 10 μM ssDNA was added to the positive control group instead of 1 μL of depcH2O to the negative control group. The reaction was carried out at 30°C for 30 min, and the color change of the reaction product was observed. The fold change value was calculated from the measured absorbance value OD570. If the addition of ssDNA resulted in a significant purple change compared to the switch alone, and the fold change value was large, it indicated a high matching value between the ssDNA and the switch, meaning that this group of switches possessed detection capability. Specific results are as follows: Figure 6 As shown. In the in vitro validation experiment, the combination with the highest fold change value and the most obvious color difference between the positive and negative groups was selected as the effective Toehold Switch element, namely S4-3 and P65-T4-3.

[0099] Next, based on the sequence differences between MH20 (Genbank accession number: ON260867.1) and H120 (Genbank accession number: MK937831.1), point mutations were performed on element S4-3. The two point-mutated switch elements were then tandemly connected to S4-3 via 17nt and 11nt linkers to obtain S3 (SEQ ID NO: 2), MH20-T4-3 element (SEQ ID NO: 4), and H120-T4-3 element (SEQ ID NO: 5).

[0100] 2. Design LAMP amplification primers targeting effective toehold switch element sequences.

[0101] Based on the genomic RNA sequences of P65, MH20, and H120, highly conserved fragments were selected in the upstream and downstream regions of the trigger RNA. Primers were designed using PrimerExplorer V5 software in accordance with the LAMP primer design principles. In the FIP primers of the candidate LAMP primer set, a reverse complementary T7 promoter sequence was added at the junction of FIC and F2, while the other primers remained unchanged. The selected LAMP primer sequences are: L96-F3 (SEQ ID NO: 6), L96-B3 (SEQ ID NO: 7), L96-FIP (SEQ ID NO: 8), L96-BIP (SEQ ID NO: 9), L80-F3 (SEQ ID NO: 10), L80-B3 (SEQ ID NO: 11), L80-FIP (SEQ ID NO: 12), L80-BIP (SEQ ID NO: 13), L33-F3 (SEQ ID NO: 14), L33-B3 (SEQ ID NO: 15), L33-FIP (SEQ ID NO: 16), and L33-BIP (SEQ ID NO: 17).

[0102] 4. Preparation of gene templates

[0103] PCR primers were designed 1500 bp upstream and downstream of the location of the IBV-selected trigger RNA element sequence. Using plasmids carrying the relevant genes as templates, RNA templates for three IBV strains were obtained through PCR amplification, in vitro transcription, and RNA purification. The RNA length was 3000 bp. After determining the RNA concentration, it was diluted 10-fold to obtain concentration gradients of 500 pM, 50 pM, 5 pM, 500 fM, 50 fM, 5 fM, 500 aM, 50 aM, 50 aM, and 500 zM. PCR primers include IVT3000bp-P65-T4-3-F (SEQ ID NO: 67), IVT3000bp-P65-T4-3-R (SEQ ID NO: 68), IVT3000bp-MH20-T4-3-F (SEQ ID NO: 69), IVT3000bp-MH20-T4-3-R (SEQ ID NO: 68) NO:70), IVT3000bp-H120-T4-3-F (SEQ ID NO:71) and IVT3000bp-H120-T4-3-R (SEQ ID NO:72).

[0104] 5. Construction of the Toehold Switch Reaction System

[0105] (1) Construction of recombinant plasmid pFN6A-Switch-LacZ (Switch plasmid)

[0106] Refer to the above construction process of pFN6A-S4-1-LacZ plasmid.

[0107] (2) Configuration of the Toehold Switch reaction system

[0108] Prepare the reaction system according to the specific formula in Table 6:

[0109] Table 6. Composition of the Toehold Switch reaction system

[0110]

[0111] The Toehold Switch reaction system was frozen using the following steps:

[0112] (1) Use a handheld puncher of different shapes with a diameter of 0.9cm to punch holes in cellulose quantitative filter paper (Whatman, 1442-042), collect the small filter paper pieces cut from the puncher, and place them at the bottom of a 96-well plate;

[0113] (2) The Toehold Switch reaction system was added dropwise at a volume of 9.6 μL per well to the bottom of the wells of a 96-well plate containing filter paper.

[0114] (3) The paper-based material containing the Toehold Switch reaction system was freeze-dried in a freeze dryer. The freeze-drying program was set to freeze at -50℃ for 5 hours and dry at -50℃ for 20 hours.

[0115] 6. LAMP amplification reaction

[0116] Prepare the reaction system according to the specific formula in Table 7:

[0117] Table 7 Composition of the LAMP reaction system

[0118]

[0119] Sensitivity and specificity detection experiments were conducted using the prepared gene templates. Specifically, in the sensitivity test, the concentrations of the gene templates were 500 fM, 50 fM, 5 fM, 500 aM, 50 aM, 5 aM, and 500 zM, respectively; in the specificity test, the concentration of the gene templates was 500 pM.

[0120] 7. Color reaction

[0121] 9.6 μL of nuclease-free water was mixed thoroughly with 0.4 μL of LAMP amplification product and then added dropwise to paper-based material loaded with the Toehold Switch reaction system to activate the lyophilized system. The system was incubated at 30 °C for 30–60 min, and the color change of the reaction product and its absorbance at 570 nm were recorded. The results are shown below. Figure 7 and Figure 8 The results show that the sensitivity of P65 is 5 aM, while the sensitivities of MH20 and H120 are both 50 aM; the trigger RNA element sequences of the three strains differ by only one nucleotide. Figure 8 This experiment demonstrates that the P65, MH20, and H120 strains can be genotyped at a single-base resolution, indicating that the paper-based Toehold Switch coupled LAMP system has extremely high detection resolution.

[0122] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above embodiments only illustrate several implementation methods of the present invention to facilitate a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0123] It should be understood that any technical solutions obtained by those skilled in the art based on the technical solutions provided in this invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A detection element for detecting IBV, characterized in that, The detection element is a Toehold Switch element, which is an S4-3 element and / or an S3 element; The nucleotide sequence of the S4-3 element is shown in SEQ ID NO: 1; The nucleotide sequence of the S3 element is shown in SEQ ID NO:

2.

2. A triggerRNA element complementary to the Toehold sequence in the Toehold Switch element of claim 1, characterized in that, The trigger RNA element is a P65-T4-3 element, an MH20-T4-3 element, and / or an H120-T4-3 element; The P65-T4-3 element is complementary to the S4-3 element and is the test sequence of IBV P65, the nucleotide sequence of which is shown in SEQ ID NO: 3; The MH20-T4-3 element is complementary to the S3 element and is the sequence to be tested for IBV MH20, the nucleotide sequence of which is shown in SEQ ID NO: 4; The H120-T4-3 element is complementary to the S3 element and is the test sequence of IBV H120, the nucleotide sequence of which is shown in SEQ ID NO:

5.

3. A LAMP primer for amplifying the Trigger RNA element as described in claim 2, characterized in that, The LAMP primers include L96-F3, L96-B3, L96-FIP, L96-BIP, L80-F3, L80-B3, L80-FIP, L80-BIP, L33-F3, L33-B3, L33-FIP, and L33-BIP; their nucleotide sequences are shown in SEQ ID NO: 6–17, respectively. Among them, L96-F3, L96-B3, L96-FIP, and L96-BIP are used to amplify P65-T4-3 elements; L80-F3, L80-B3, L80-FIP, and L80-BIP are used to amplify MH20-T4-3 elements; L33-F3, L33-B3, L33-FIP, and L33-BIP are used to amplify H120-T4-3 elements.

4. A kit for detecting IBV, characterized in that, Includes the detection element for detecting IBV as described in claim 1.

5. The kit for IBV as described in claim 4, characterized in that, It also includes the LAMP primers as described in claim 3.

6. The kit for detecting IBV as described in claim 4 or 5, characterized in that, It also includes cell-free protein expression systems; The cell-free protein expression system includes: Escherichia coli cell extract 35-50 vol%, lysis buffer 40-50 vol%, RNase inhibitor 0.1-1 vol%, gene template 8-12 vol%, chromogenic agent 0.5-1.5 vol%, nuclease-free water 1-5 vol%. Wherein, the Escherichia coli is a recombinant bacterium obtained by modifying the recipient Escherichia coli, the modification including: (1) knocking out the LacZ gene of the recipient Escherichia coli; (2) knocking out the RecBCD operon gene and the ptrA gene of the recipient Escherichia coli, and inserting the LyseR gene at the knockout site; The lysis buffer comprises the following components at the following concentrations: HEPES 40–60 mM, ATP 1–2 mM, GTP 1–2 mM, CTP 0.5–1.5 mM, UTP 0.5–1.5 mM, tRNA 0.1–0.5 mg / mL, CoA 0.1–0.5 mM, NAD 0.2–0.8 mM, cAMP 0.5–1 mM, folinic acid 0.01–0.1 mM, spermidine 0.5–1.5 mM, 3-PGA 10–50 mM, maltose 10–20 mM, amino acids 20–25 mM, PEG-8000 8–15 mg / mL, potassium glutamate 55–80 mM, magnesium glutamate 0.5–1.5 mM, DTT 0.5–1.5 mM.

7. The kit for detecting IBV as described in claim 6, characterized in that, The CFS includes: Escherichia coli cell extract 40 vol%, lysis buffer 44.5 vol%, RNase inhibitor 0.5 vol%, chlorophenol red-β-D-galactopyranoside 1 vol%, gene template 10 vol%, nuclease-free water 4 vol%. The lysis solution comprises components at the following concentrations: HEPES 50mM, ATP 1.5mM, GTP 1.5mM, CTP 0.9mM, UTP 0.9mM, tRNA 0.2mg / mL, CoA 0.26mM, NAD 0.33mM, cAMP 0.75mM, leucovorin 0.0678mM, spermidine 1mM, 3-PGA 30mM, maltose 12mM, PEG-8000 11.2mg / mL, potassium glutamate 60.6mM, magnesium glutamate 0.815mM, DTT 1mM, Leucine 20mM, Glycine 24mM, Valine 24mM, Isoleucine 24mM, Methionine 24mM, Proline 24mM, Phenylalanine 24mM, Tyrosine 24mM, Serine 24mM, Threonine 24mM, Tryptophan 24mM, Asparagine 24mM, Glutamine 24mM, Lysine 24mM, Cysteine ​​24mM, Arginine 24mM, Aspartic Acid 24mM, Histidine 24mM, Glutamic Acid 24mM, Alanine 24mM.

8. The kit for detecting IBV as described in claim 6, characterized in that, The CFS and the detection element are freeze-dried on a paper-based material; the freeze-drying temperature is -60 to -40°C.

9. A method for detecting IBV for non-diagnostic purposes, characterized in that, include: Obtain the gene template; The gene template was amplified using the LAMP primers as described in claim 3; The amplified products are reacted using a cell-free protein expression system and a reaction system consisting of the detection element as described in claim 1; and the results are determined based on the color change.

10. The method for detecting IBV for non-diagnostic purposes as described in claim 9, characterized in that, In the step of amplifying the gene template using the LAMP primers as described in claim 3, the reaction procedure is to react at 70°C for 30 minutes. In the step of reacting the amplified product with a reaction system consisting of a cell-free protein expression system and the detection element as described in claim 1, and determining the result based on the color change, the reaction procedure is to react at 30°C for 30–60 min.

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