Detection system for simultaneously detecting avian circovirus type 1 and human circovirus type 1 and its application

Abstract

The present invention discloses a detection system for simultaneously detecting avian gyrovirus type 1 and human gyrovirus type 1, and its application, relating to the technical field of biological detection. The detection system includes an RAA amplification primer pair for GyVg1, an RAA amplification primer pair for GyH1, 12-crRNA-3, 13-crRNA-2, an ssDNA probe, an ssRNA probe, LbCas12a nuclease, LbuCas13a nuclease and T7 transcriptase. Based on the conserved genes of GyVg1 and GyH1, the present invention respectively designs specific crRNAs and RAA primers, and by combining the RAA amplification technology with the CRISPR-Cas fluorescence detection system, a detection system for simultaneously detecting GyVg1 and GyH1 is established. The detection system can simultaneously detect two different subtypes of gyrovirus, improves the detection efficiency, and provides a new technical means for the dynamic study of virus infection.

Description

Technical field

[0001] The present invention relates to the technical field of biological detection, in particular to a detection system for simultaneously detecting Gyrovirus galga1 and Gyrovirus homsa1, and its application. Background technique

[0002] Gyrovirus galga1 (GyVg1) and Gyrovirus homsa1 (GyH1) are the second and third circular viruses newly discovered after Chicken anemia virus (CAV). They belong to the genus Gyrovirus of the family Anelloviridae, and are a class of small, non-enveloped, single-stranded circular DNA viruses. The full-length genome is about 2.4 kbp, containing three partially overlapping open reading frames (ORFs). Among them, ORF1 encodes the scaffold protein VP2, ORF2 encodes the apoptotic protein VP3, and ORF3 encodes the capsid protein VP1. The nucleotide similarities of VP1, VP2, and VP3 between GyVg1, GyH1 and CAV are 42%, 42%, 30% and 43%, 42%, 29% respectively. The nucleotide similarities of VP1, VP2, and VP3 between GyVg1 and GyH1 are 62%, 63%, 55% respectively. GyVg1 and GyH1 mainly co-infect chicken flocks with other pathogens. Infected chickens show symptoms such as bleeding, edema, glandular stomach erosion, and head swelling, which can lead to high morbidity and mortality in chickens. Other reported animals infected with GyVg1 include humans, wild birds, ferrets, dogs, domestic cats, snakes, and ticks. Animals infected with GyH1 include humans, wild birds, ferrets, and domestic cats. The host ranges of the two infections are similar, and the infected individuals all show varying degrees of diarrhea symptoms, suggesting that these two novel Gyroviruses have the risk of cross-host transmission.

[0003] GyVg1 and GyH1 mainly infect chicken flocks. In 2011, Rijsewijk et al. discovered the genomic sequences of distant relatives of CAV in serum samples from diseased chickens in Brazil, reported a new member of the genus Cyclovirus, and named it Avian gyrovirus 2 (AGV2). In 2012, Phan et al. performed viral metagenomic analysis on 100 human fecal samples to study the causes of unexplained diarrhea in children with acute gastroenteritis. Cyclovirus was identified in the feces, and a new cyclovirus species, Avian gyrovirus 3 (GyV3), was discovered for the first time, which is the third member of the cyclovirus family. As of 2024, 16 circular viruses have been identified. The ICTV reclassified the cyclovirus family, and the original AGV2 was renamed GyVg1, and GyV3 was renamed GyH1. Currently, there are few research reports on these two newly discovered cycloviruses. The etiology of their direct involvement in specific diseases, as well as the reasons for their widespread existence and host diversity, are not fully understood. Moreover, no passage culture system for GyVg1 and GyH1 has been found, and large-scale virus propagation cannot be carried out. Their pathogenicity remains largely unexplored, and these unknown factors pose potential public health safety risks and threaten the healthy development of the poultry industry. However, in the more than a decade since the cyclovirus was first discovered, there has been a lack of preventive and therapeutic measures for GyVg1 and GyH1, including commercial vaccines, drug treatments, and the purification of infected populations, allowing the virus to spread unchecked. The first step in preventing and controlling virus infections is to conduct pathogen detection, which can timely monitor the virus infection situation and provide a basis for subsequent assessment of the epidemic and mutation situations, as well as the effectiveness of vaccines and drug treatments.

[0004] Currently, the detection of GyVg1 still relies on molecular detection techniques, including conventional PCR, quantitative real-time PCR, droplet digital PCR, real-time fluorescence recombinase-aided isothermal amplification (RAA), etc. In the clinical detection and epidemiological studies of GyH1, immunological diagnostic methods include indirect ELISA, double antibody sandwich ELISA, and colloidal gold immunochromatographic strip detection methods. Molecular detection techniques include conventional PCR detection, quantitative real-time PCR detection, visual loop-mediated isothermal amplification (LAMP) detection, and real-time fluorescence RAA detection, etc. These methods can specifically detect specific pathogens, but each can only detect one of the cycloviruses at a time, increasing the time and workload of pathogen detection. Based on the similarity of the etiology and clinical manifestations of GyVg1 and GyH1, the present invention aims to establish a duplex detection system for simultaneously detecting the two viruses. Summary of the Invention

[0005] The object of the present invention is to provide a detection system and its application for simultaneously detecting circovirus avian type 1 and circovirus human type 1, so as to solve the problems existing in the above-mentioned prior art. This detection system can simultaneously detect two different subtypes of circovirus, improve the detection efficiency, and provide a new technical means for the dynamic research of virus infection.

[0006] Recombinase-mediated nucleic acid isothermal amplification (RAA) technology is an isothermal amplification technology that has emerged in recent years. The primer design is simple, and the target fragment can be rapidly amplified within a short time (20 min) under constant temperature (37°C - 42°C) conditions. However, it is prone to spontaneous reaction and there is non-specific amplification. CRISPR / Cas belongs to type II effector proteins in the CRISPR system and is a key component in gene editing technology. It works with Cas proteins with endonuclease function and crRNA under the same constant temperature (37°C - 42°C) conditions. The CRISPR / Cas technology has aM-level sensitivity and single-nucleotide specificity. However, when the target concentration of the sample is low, the number of activated Cas proteins is limited, and a large amount of detectable signals cannot be generated within a short time to meet the requirements of rapid detection. Therefore, by combining the CRISPR / Cas system and RAA technology, a rapid, sensitive, and specific RAA-CRISPR / Cas detection platform can be developed. The NUC lobe of the Cas12a protein lacks the HNH domain but contains the RuvC domain. When combined with a specific crRNA, it can cleave single-stranded DNA (ssDNA) indiscriminately; Cas13a is composed of two high-level HEPN domains. When combined with a specific crRNA, its cleavage activity is stimulated, triggering the RNA damage diffusion phenomenon, and it can degrade RNA more extensively. In the present invention, CRISPR / Cas12a and CRISPR / Cas13a are added to the same reaction system, and DNA probes labeled with FAM fluorescent groups and RNA probes labeled with ROX fluorescent groups are respectively introduced. CRISPR / Cas12a targets and cleaves the DNA probe, and Cas13a targets and cleaves the RNA probe, realizing the fluorescence dual-channel detection of FAM and ROX in one reaction tube, similar to dual fluorescence quantitative PCR. The real-time cleavage reaction observes the amplification curve of the corresponding fluorescence channel under a fluorescence quantitative PCR instrument, so as to achieve the purpose of simultaneously identifying and detecting GyVg1 and GyH1, and innovatively constructing an efficient method for rapid detection with a constant temperature RAA-CRISPR-Cas12a / Cas13a dual channel.

[0007] Based on this, the present invention provides the following solutions:

[0008] The present invention provides a RAA-CRISPR-Cas12a / Cas13a dual-channel detection system for simultaneously detecting avian circovirus type 1 and human circovirus type 1, including a RAA amplification primer pair for avian circovirus type 1, a RAA amplification primer pair for human circovirus type 1, 12-crRNA-3, 13-crRNA-2, ssDNA probe, ssRNA probe, LbCas12a nuclease, LbuCas13a nuclease and T7 transcriptase;

[0009] The RAA amplification primer pair for avian circovirus type 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8;

[0010] The nucleotide sequence of the 12-crRNA-3 is as shown in SEQ ID NO.11;

[0011] The RAA amplification primer pair for human circovirus type 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.14 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.17;

[0012] The nucleotide sequence of the 13-crRNA-2 is as shown in SEQ ID NO.21;

[0013] The nucleotide sequence of the ssDNA probe is TTATT;

[0014] The nucleotide sequence of the ssRNA probe is UUUUUU.

[0015] Furthermore, different fluorescent groups are labeled at the 5'-ends of the ssDNA probe and the ssRNA probe.

[0016] Furthermore, the 5'-end of the ssDNA probe is labeled with a FAM group and the 3'-end is labeled with a BHQ1 group;

[0017] The 5'-end of the ssRNA probe is labeled with a ROX group and the 3'-end is labeled with a BHQ1 group.

[0018] The present invention also provides the application of the above RAA-CRISPR-Cas12a / Cas13a dual-channel detection system in the preparation of a product for simultaneously detecting avian circovirus type 1 and human circovirus type 1.

[0019] Furthermore, the product is a kit.

[0020] The present invention also provides a product for simultaneously detecting avian gyrovirus type 1 and human gyrovirus type 1, including the above-mentioned RAA-CRISPR-Cas12a / Cas13a dual-channel detection system.

[0021] Furthermore, the product is a kit.

[0022] Furthermore, the kit is a fluorescence detection kit.

[0023] Furthermore, the detection method of the kit includes the following steps:

[0024] Using the genomic DNA of the sample to be tested as a template, performing RAA amplification with the RAA amplification primer pair of avian gyrovirus type 1 and the RAA amplification primer pair of human gyrovirus type 1 to obtain an RAA amplification product;

[0025] Preparing a CRISPR-Cas12a / Cas13a dual-channel system reaction solution with the RAA amplification product, performing a CRISPR-Cas12a / Cas13a reaction, and then detecting the fluorescence intensity.

[0026] The present invention discloses the following technical effects:

[0027] Based on the conserved genes of GyVg1 and GyH1, the present invention respectively designs specific crRNAs and RAA primers. By combining the RAA amplification technology with the CRISPR-Cas fluorescence detection system, an RAA-CRISPR / Cas12a-Cas13a dual-channel detection system for simultaneously detecting GyVg1 and GyH1 is established. It can detect the presence of GyVg1 and GyH1 within 1 hour, and can specifically distinguish GyVg1 and GyH1. It is convenient to operate, has good specificity and high sensitivity, and the lowest detection limit can reach 1.5 copies / μL. The established RAA-CRISPR / Cas12a-Cas13a dual-channel detection system of the present invention can simultaneously detect two different subtypes of gyroviruses, improving the detection efficiency and providing a new technical means for the dynamic study of virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1Workflow diagram of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection for GyVg1 and GyH1;

[0030] Figure 2 Results diagram of the screening experiment for RAA-CRISPR / Cas12a primers and crRNAs; among them, A shows the results of different RAA primer combinations for crRNA1; B shows the results of different RAA primer combinations for crRNA2; C shows the results of different RAA primer combinations for crRNA3;

[0031] Figure 3 Results diagram of the screening experiment for RAA-CRISPR / Cas13a primers and crRNAs; among them, A shows the results of different RAA primer combinations for crRNA1; B shows the results of different RAA primer combinations for crRNA2; C shows the results of different RAA primer combinations for crRNA3;

[0032] Figure 4 Results diagram of the optimization experiment for the RAA-CRISPR / Cas12a system of GyVg1; among them, A shows the results of different RAA primer concentrations; B shows the results of different crRNA concentrations; C shows the results of different ssDNA concentrations;

[0033] Figure 5 Results diagram of the optimization experiment for the RAA-CRISPR / Cas13a system of GyH1; among them, A shows the results of different RAA primer concentrations; B shows the results of different Cas13a concentrations; C shows the results of different crRNA concentrations; D shows the results of different ssRNA concentrations; E shows the results of different volumes of T7 transcriptase;

[0034] Figure 6 Results diagram of the optimization of the RAA-CRISPR-Cas12a / Cas13a dual-channel system for GyVg1 and GyH1;

[0035] Figure 7 Results diagram of the specificity test for the RAA-CRISPR-Cas12a / Cas13a dual-channel detection;

[0036] Figure 8 Results diagram of the sensitivity test for the RAA-CRISPR-Cas12a / Cas13a dual-channel detection; among them, A shows the results of the sensitivity test for GyVg1 in the FAM channel; B shows the results of the sensitivity test for GyH1 in the ROX channel. Specific implementation manners

[0037] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Samples

[0045] One GyVg1-positive sample was from the liver tissue of diseased chickens with neurological symptoms, one GyH1-positive sample was from the glandular stomach tissue of healthy chickens in the live poultry market, and one sample positive for mixed infection with GyVg1 and GyH1 was from the homogenate of the visceral tissues of chicks with tumor symptoms. The three positive samples were confirmed by PCR and verified by sequencing. The other 12 non-target reference strains were used for specificity tests, including Chicken infectious anemia virus (CIAV), Fowl adenovirus type 4 (FAdV4), Avian influenza virus subtype H9 (H9-AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), Infectious bursal disease virus (IBDV), Chicken parvovirus (ChPV), Avian leukosis virus (ALV), Avian nephritis virus (ANV), Chicken astrovirus (CAstV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS), which were provided by the Biological Laboratory of Guangxi Zhuang Autonomous Region Veterinary Research Institute. 192 clinical samples were collected from live poultry markets and small-scale chicken farms in Guangxi, including 116 chicken oral and anal swabs, 28 environmental samples, and 48 visceral tissue samples of pecking shell embryos, and stored at -80 °C.

[0046] 1.2 Reagents and Instruments

[0047] All GyVg1 primers and GyH1 PCR primers used in this invention were synthesized by Shanghai Sangon Biotech Co., Ltd. The upstream GyH1 RAA primer containing the T7 promoter, the downstream RAA primer, crRNA, ssDNA fluorescent reporter probe, and ssRNA fluorescent reporter probe were synthesized by Guangzhou Aidy Gene Technology Co., Ltd. The RAA-basic kit was purchased from Nanning Zhuangbo Biotechnology Co., Ltd. LbCas12a Nuclease, LbuCas13a Nuclease, and T7 transcription kit were purchased from Guangzhou Aidy Gene Technology Co., Ltd. RNase Inhibitor was purchased from BBI Life Sciences Co., Ltd. Fluorescence readings were recorded by the QuantStudio5 fluorescence quantitative PCR instrument of Thermo Fisher Scientific Co., Ltd.

[0048] 1.3 Primer and gRNA Design

[0049] 1.3.1 Design and Synthesis of Primers and crRNAs for GyVg1

[0050] First, design 3 crRNAs to screen out the crRNA with the best activity for the establishment of the CRISPR / Cas12a system. Obtain all the existing sequences of GyVg1 from the NCBI database, perform multiple sequence alignments using the Bioedit program of the DNAStar software to confirm the conserved target sequences, find the 5'-TTTN-3' PAM on the target sequence or the complementary strand of the target sequence (PAM is the protospacer adjacent motif, and N represents A / T / C / G), and take the 20 bases downstream of the PAM sequence as the spacer sequence of the crRNA. Note that the first 8 bases of the spacer sequence are the seed region and need to be highly conserved. Previously, Doudna et al. have obtained the optimal direct repeat sequence: 5’-UAAUUUCUACUAAGUGUAGAU-3’. The crRNA consists of two parts: the direct repeat sequence and the spacer sequence, and thus the crRNA sequence is determined. Then, design 3 upstream RAA primers and 3 downstream RAA primers outside the crRNA. The primer length is 28 - 32 bp, and the amplicon length is about 300 bp. The primer sequences should avoid pairing with the crRNA sequence. Finally, design a pair of PCR primers outside the amplicon for preparing plasmid standards. All sequences are shown in Table 1.

[0051] 1.3.1 Primer and crRNA Design and Synthesis for GyH1

[0052] First, design 3 crRNAs to screen out the crRNA with the best activity for the establishment of the CRISPR / Cas13a system. Find the conserved target sequences in the same way as above, design the reverse complementary base sequence according to the target region, with a size of 20 bp. Note that the flanking sequence at the 3' end of the target region is preferably free of base G; add a stem-loop structure upstream of the target region, and its sequence information is: 5’-GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC-3’. Thus, the crRNA sequence is determined. Then, design 3 upstream RAA primers and 3 downstream RAA primers outside the crRNA. The primer length is 28 - 32 bp, and the 5' end of the upstream primer needs to add the T7 promoter sequence (5’-GGACCACCCCAAAAAUGAAGGGGACCAAAAC-3’) to facilitate subsequent in vitro transcription and purification. The amplicon length is about 300 bp. Similarly, the primer sequences should avoid pairing with the crRNA sequence. Finally, design a pair of PCR primers outside the amplicon for preparing plasmid standards. All sequences are shown in Table 1.

[0053] Table 1 Primer, crRNA, and ssDNA Reporter Probe Sequences

[0054]

[0055]

[0056] Note: The "" part is the promoter sequence; the bold part of the crRNA sequence represents the direct repeat sequence of Cas12a or Cas13a.

[0057] 1.4 Genomic DNA extraction

[0058] The taken oral and anal swab samples were soaked and squeezed with PBS, and the tissue samples were soaked and ground with PBS, and repeatedly frozen and thawed 3 times at -80°C. A pre-packaged virus RNA / DNA extraction kit was used to extract viral DNA. Take 200 μL of virus supernatant, add it to the sample well of the 96-well extraction kit, and place the kit in an automatic nucleic acid extraction and purification instrument for automatic extraction. After 18 minutes, directly aspirate the genomic DNA of the sample from the nucleic acid well and store it at -20°C for later use.

[0059] Take one copy of GyVg1 and GyH1 positive DNA templates, and use GyVg1-F, GyVg1-R and GyH1-F, GyH1-R to amplify and prepare positive plasmid standards respectively. Measure the plasmid concentration with a UV spectrophotometer, calculate the plasmid copy concentration, and store it at -20°C for later use.

[0060] 1.5 Establishment of standard RAA and CRISPR / Cas systems

[0061] Standard RAA is used for primer pair screening. In a 50 μL RAA system, refer to the RAA basic kit instructions, add different primer sets to the detection unit tube, and incubate in a 39°C constant temperature metal bath for 30 min to complete the amplification of the target sequence. The standard CRISPR / Cas system is used for the screening of gRNA. In a 20 μL CRISPR / Cas system, configure the CRISPR / Cas12a reaction system of GyVg1 and the CRISPR / Cas13a reaction system of GyH1 respectively according to the LbCas12a Nuclease instructions and the LbuCas13a Nuclease and T7 transcription kit instructions, incubate at room temperature for 15 min, wait for the gRNA to bind to the Cas protein to form a CRISPR / Cas complex, add 2 μL of RAA amplification product to the tube cap of the unit tube, centrifuge transiently, mix well, and centrifuge again. Use a QuantStudio5 fluorescence PCR instrument to collect fluorescence signals every 1 min at 37°C. Select the optimal primers and gRNA according to the fluorescence intensity for subsequent experiments.

[0062] 1.6 Optimization of RAA-CRISPR-Cas12a / Cas13a dual-channel reaction conditions

[0063] 1.6.1 Optimization of the RAA-CRISPR / Cas12a system for GyVg1

[0064] Using the positive nucleic acid of GyVg1 as a template, on the basis of the standard RAA and standard CRISPR / Cas12a systems, the primer concentration, 12-crRNA concentration, and ssDNAReporter were optimized and adjusted to achieve an ideal RAA-CRISPR / Cas12a reaction efficiency. The primer concentration (200 nM, 300 nM, 400 nM, 500 nM, 600 nM), 12-crRNA concentration (8.3 nM, 16.7 nM, 25 nM, 33 nM, 41.3 nM, 50 nM, 58 nM) (under the premise that the standard concentration of LbCas12a Nuclease is 33 nM), and ssDNAReporter concentration (200 nM, 300 nM, 400 nM, 500 nM, 600 nM) were sequentially changed, and the ideal working concentration was selected according to the fluorescence value of the FAM channel.

[0065] 1.6.2 Optimization of the RAA-CRISPR / Cas13a System for GyH1

[0066] Using the positive nucleic acid of GyH1 as a template, on the basis of the standard RAA and standard CRISPR / Cas13a systems, the primer concentration, Cas13a enzyme concentration, 13-crRNA concentration, ssRNA Reporter, and T7 transcriptase were optimized and adjusted to achieve an ideal RAA-CRISPR / Cas13a reaction efficiency. The primer concentration (200 nM, 300 nM, 400 nM, 500 nM, 600 nM), Cas13a enzyme concentration (25 nM, 50 nM, 75 nM, 100 nM, 125 nM), 13-crRNA concentration (50 nM, 75 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM), ssRNAReporter concentration (200 nM, 300 nM, 400 nM, 500 nM, 600 nM), and T7 transcriptase volume (0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, 0.8 μL, 0.9 μL, 1.0 μL) were sequentially changed, and the ideal working concentration was selected according to the fluorescence value of the ROX channel.

[0067] 1.6.3 Optimization of the RAA-CRISPR-Cas12a / Cas13a Dual-Channel System for GyVg1 and GyH1

[0068] Take a GyVg1-positive nucleic acid sample, a GyH1-positive nucleic acid sample, and a mixed GyVg1 and GyH1-positive nucleic acid sample as templates. According to the optimization results of RAA-CRISPR / Cas12a and RAA-CRISPR / Cas13a, in the same RAA system, combine and amplify GyVg1-F, GyVg1-R, GyH1-F, and GyH1-R; take 2 μL of the amplification product for the optimization of the CRISPR-Cas12a / Cas13a cleavage system. Adjust each component proportionally according to the optimized CRISPR / Cas12a and CRISPR / Cas13a systems to make the end fluorescence intensities in the FAM channel and the ROX channel equivalent, which is regarded as the final optimization result of the dual-channel system for subsequent experiments.

[0069] 1.7 Specificity and sensitivity determination

[0070] Evaluate the specificity of the reaction by detecting other pathogens of newly emerging digestive tract infectious diseases related to GyVg1 and GyH1 and other common avian disease pathogens, including CIAV, FAdV4, H9-AIV, NDV, IBV, IBDV, ChPV, ALV, ANV, CAstV, MG, and MS. The results are based on the fluorescence values collected in the FAM and ROX channels of the fluorescence quantitative PCR instrument.

[0071] Dilute both the GyVg1 positive plasmid and the GyH1 positive plasmid to 3×10 9 copies / μL and mix well. Use the continuously diluted mixed plasmid to evaluate the sensitivity of the RAA-CRISPR-Cas12a / Cas13a dual-channel system. Select positive plasmids with 3×10 0 copies / μL, 3×10 1 copies / μL, 3×10 2 copies / μL, 3×10 3 copies / μL, 3×10 4 copies / μL, 3×10 5 copies / μL as templates, set ddH2O as the negative control, and use the optimized ideal system for the RAA-CRISPR-Cas12a / Cas13a reaction. After the reaction, collect the fluorescence signal to determine the minimum detection limit of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system for GyVg1 and GyH1.

[0072] 1.8 Clinical sample detection

[0073] The established RAA-CRISPR-Cas12a / Cas13a dual-channel detection method of the present invention was used to detect 192 clinical samples collected in the laboratory, and the detection results were compared with the PCR method to evaluate the consistency of the two detection methods.

[0074] 2. Results

[0075] 2.1 Research plan

[0076] First, RAA was amplified at a constant temperature of 37°C for 30 min to obtain a sufficient amount of target. Then, crRNA, Cas12a, and Cas13a were added to the CRISPR premix system to form crRNA / Cas12a and crRNA / Cas13a complexes respectively. Finally, the RAA-specific amplified target and T7 transcriptase were added to the premix system and reacted at 37°C for 30 min. At this time, RAA-CRISPR / Cas12a cleaved ssDNAReporter, and the transcribed RAA-CRISPR / Cas13a cleaved ssRNAReporter, thus completing the signal amplification detection. After the reaction, the fluorescence signals in the FAM channel and ROX channel were collected respectively to achieve the rapid simultaneous detection of GyVg1 and GyH1 in one reaction.

[0077] 2.2 Screening of RAA primers and crRNA

[0078] Three upstream primers, three downstream primers, and three crRNAs were arranged in combination, with a total of 27 experimental groups, and 27 negative control groups were set correspondingly. The 54 combinations were simultaneously detected in a fluorescence quantitative PCR instrument, and the ideal crRNA and primers were screened according to the intensity of the collected fluorescence signals.

[0079] The screening results of GyVg1 primers and crRNA are as Figure 1 shown. When using crRNA1 and crRNA2, only some experimental groups produced fluorescence signals ( Figure 2 A and B in it), and when using crRNA3, all experimental groups produced fluorescence signals, and the corresponding negative control groups did not produce fluorescence signals ( Figure 2 C in it). According to the appearance time of the curve and the fluorescence intensity, the ideal primers and crRNA were determined to be GyVg1-F3, GyVg1-R3, and 12-crRNA-3 respectively. GyVg1-F3, GyVg1-R3, and 12-crRNA-3 were used for subsequent experiments.

[0080] The screening results of GyH1 primers and crRNA are as Figure 2 shown. When using crRNA1 and crRNA3, no fluorescence signals were produced ( Figure 3In A and B), fluorescence signals were generated in all experimental groups using crRNA3, and no fluorescence signal was generated in the corresponding negative control group. Figure 3 In C), according to the appearance time of the curve and fluorescence intensity, the ideal primers and crRNA were determined to be GyH1-F1, GyH1-R1, and 13-crRNA-2 respectively. GyH1-F1, GyH1-R1, and 13-crRNA-2 were used for subsequent experiments.

[0081] 2.3 Optimal reaction conditions for the RAA-CRISPR-Cas12a / Cas13a dual-channel

[0082] 2.3.1 Optimization of the RAA-CRISPR / Cas12a system for GyVg1

[0083] From Figure 4 it can be seen that when the primer concentration is 500 nM ( Figure 4 in A), the 12-crRNA concentration is 25 nM ( Figure 4 in B), and the ssDNAReporter concentration is 500 nM ( Figure 4 in C), the fluorescence appearance time is the earliest, the fluorescence value is the highest, and the comprehensive reaction efficiency is the highest. That is, the determined ideal RAA-CRISPR / Cas12a system is as follows: The first-step RAA amplification reaction includes 25 μL of A Buffer, 2.5 μL of each GyVg1 primer (10 μM), 5 μL of DNA template, 2.5 μL of B Buffer, and sterilized water is added to make up to 50 μL; the second-step CRISPR / Cas12a reaction includes 2 μL of 10×Cleavage Buffer, 1 μL of LbCas12a Nuclease (1 μM), 1 μL of 12-crRNA (500 nM), 2.5 μL of ssDNAReporter (4 μM), 2 μL of RAA amplification product, and sterilized water is added to make up to 20 μL.

[0084] 2.3.2 Optimization of the RAA-CRISPR / Cas13a system for GyH1

[0085] From Figure 5 it can be seen that when the primer concentration is 500 nM ( Figure 5 in A), the Cas13a concentration is 100 nM ( Figure 5 in B), the 13-crRNA concentration is 100 nM ( Figure 5 in C), the ssRNAReporter concentration is 600 nM ( Figure 5 in D), and the T7 transcriptase is 0.5 μL ( Figure 5In group E), the fluorescence appeared earliest, had the highest fluorescence value, and the highest comprehensive reaction efficiency. That is, the determined ideal RAA-CRISPR / Cas13a system is as follows: The first-step RAA amplification reaction includes 25 μL of A Buffer, 2.5 μL each of GyH1 primers (10 μM), 5 μL of DNA template, 2.5 μL of B Buffer, and sterilized water is added to make up to 50 μL; The second-step CRISPR / Cas13a reaction includes 4 μL of 5×Cleavage Buffer, 0.4 μL of LbuCas13a Nuclease (5 μM), 4 μL of crRNA (500 nM), 3 μL of ssRNA Reporter (4 μM), 0.5 μL of 40 U RNase Inhibitor, 0.8 μL of NTP mix, 1 μL of 5×T7 Transcription Reaction Buffer, and 0.5 μL of T7 Transcription Enzyme mix. Finally, 2 μL of the RAA amplification product is added, and sterilized water is added to make up to 20 μL.

[0086] 2.3.3 Optimization of the RAA-CRISPR-Cas12a / Cas13a dual-channel system for GyVg1 and GyH1

[0087] The proportions of the components of the CRISPR / Cas12a and CRISPR / Cas13a optimization systems were adjusted as a whole. When GyVg1-F / R and GyH1-F / R were 500 nM, the Cas12a concentration was 50 nM, the 12-crRNA concentration was 20 nM, the ssDNA Reporter concentration was 400 nM, the Cas13a concentration was 100 nM, the 13-crRNA concentration was 100 nM, the ssRNA Reporter concentration was 600 nM, and the T7 transcriptase was 0.5 μL, the end-point fluorescence intensities collected by the FAM channel and the ROX channel were equivalent ( Figure 6 ), and the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system had good effects. That is, the determined dual-channel system is shown in Table 2, and subsequent experiments were carried out with this system.

[0088] Table 2 RAA-CRISPR-Cas12a / Cas13a dual-channel optimization system

[0089]

[0090]

[0091] 2.4 Sensitivity and specificity of RAA-CRISPR-Cas12a / Cas13a

[0092] Using the established RAA-CRISPR-Cas12a / Cas13a dual-channel method to detect the nucleic acids of 15 reference strains, the results showed ( Figure 7 ): For 2 GyVg1 samples, 2 GyH1 samples, and 1 sample with mixed infection of GyVg1 and GyH1, corresponding amplification reactions occurred, showing typical fluorescence amplification curves. However, for the other 12 avian pathogens (CIAV, FadV4, AIV-H9, NDV, IBV, IBDV, ARV, ALV, CAstV, ANV, MG, MS), there was no amplification, and no fluorescence signal was generated, just like in ddH2O. This indicates that the two sets of primers and crRNAs designed in the present invention have good specificity, and the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method has strong specificity.

[0093] The sensitivity of the RAA-CRISPR-Cas12a / Cas13a dual-channel method was evaluated using a 10-fold serial dilution of a mixture of GyVg1 and GyH1 plasmid DNAs. The actual template working concentrations of GyVg1 and GyH1 for the experiment were 1.5×10 0 copies / μL, 1.5×10 1 copies / μL, 1.5×10 2 copies / μL, 1.5×10 3 copies / μL, 1.5×10 4 copies / μL, 1.5×10 5 copies / μL. The results are shown in Figure 7 . When the concentration of GyVg1 plasmid DNA was 1.5 copies / μL, a fluorescence signal could still be detected in the "FAM channel" ( Figure 8 A in); when the concentration of GyH1 plasmid DNA was 1.5 copies / μL, a fluorescence signal could still be detected in the "ROX channel" ( Figure 8 B in). That is, the lowest detection limit of the established RAA-CRISPR-Cas12a / Cas13a dual-channel method is 1.5 copies / μL, and it has high sensitivity.

[0094] 2.5 Clinical sample detection

[0095] To further evaluate the clinical performance of the RRAA-CRISPR-Cas12a / Cas13a dual-channel detection method, 192 samples collected in the laboratory were detected using both the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method and the conventional PCR detection method. The positive samples detected by PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The results showed (Table 3) that the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method identified 8 GyVg1 positive samples (4.17%), and the PCR detection method identified 6 GyVg1 positive samples (3.13%). The total coincidence rate of the two detection methods was 98.96%. At the same time, the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method identified 6 GyH1 positive samples (3.13%), and the PCR detection method identified 1 GyH1 positive sample (0.52%). The total coincidence rate of the two detection methods was 97.40%, showing a high degree of consistency. This indicates that the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method established in the present invention has a high accuracy and reliable detection results.

[0096] Table 3 Comparison of the results of two methods applied to the detection of clinical samples

[0097]

[0098] In summary, the present invention successfully established an RAA-CRISPR-Cas12a / Cas13a dual-channel detection method for GyVg1 and GyH1. The design of the primers and crRNAs of this method is simple, the detection operation is convenient, it can sensitively and accurately identify and detect GyVg1 and GyH1 in clinical samples, reduce the detection cost and workload, and is suitable for the rapid detection of GyVg1 and GyH1 and disease diagnosis in production.

[0099] The present invention combines the CRISPR / Cas12a and CRISPR / Cas13a systems in one system, labels DNA probes with the FAM reporter group, and labels RNA probes with the ROX reporter group, so that the reaction results of CRISPR / Cas12a are displayed in the FAM channel, and the reaction results of CRISPR / Cas13a are displayed in the ROX channel, realizing dual-channel detection of two targets in a single system, that is, dual CRISPR / Cas detection. In order to obtain a sufficient amount of targets required for CRISPR / Cas reactions, the present invention selects isothermal amplification technologies with higher amplification efficiency and shorter reaction time to meet the front-end amplification requirements. Common isothermal amplification technologies include loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (also known as RPA, RAA, MIRA), rolling circle amplification (RCA), enzymatic recombinase amplification (ERA), and cross-priming amplification (CPA). Among them, RAA has simple primer design, mild reaction temperature (37-42 °C), and fast reaction speed (within 30 minutes), and is an excellent nucleic acid amplification technology. The study combines the RAA amplification technology with the CRISPR / Cas technology. RAA is responsible for isothermal amplification of the target sequence to increase the copy number of the target sequence, and Cas12a / Cas13a is responsible for specifically identifying and cleaving the target sequence and visualizing the detection results depending on the reporter probe, greatly improving the detection sensitivity and specificity.

[0100] The conserved regions of GyVg1 and GyH1 were both selected in the overlapping region of VP2 and VP3 genes, and specific RAA primers and crRNAs were designed. First, the specificity of the cleavage target was ensured, and then the primers and probes with the best reaction efficiency were screened out through the reaction results of different primer pairs and crRNAs. The RAA-CRISPR / Cas12a system and the RAA-CRISPR / Cas13a system were optimized separately, and then the RAA-CRISPR / Cas12a system and the RAA-CRISPR / Cas13a system were combined and the ratio of the two systems was adjusted for optimization. Finally, an ideal RAA-CRISPR-Cas12a / Cas13a dual-channel detection method was established. The research results showed that the fluorescence results under the amplification of mixed targets in the dual channel were basically the same as those under the amplification of single GyVg1 or GyH1 targets in the dual channel, indicating that when the primers of GyVg1 and the primers of GyH1 were placed in the same RAA amplification system for amplification, there was no inhibition of amplification; when GyVg1-CRISPR / Cas12a and GyH1-CRISPR / Cas13a were placed in the same CRISPR / Cas system, there was no inhibition of cleavage; the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method for GyVg1 and GyH1 established in the present invention was feasible. Previously, the inventors developed a real-time fluorescence RAA detection method for GyVg1 and a real-time fluorescence RAA detection method for GyH1. At that time, an attempt was made to combine the two fluorescence RAA methods into a dual-fluorescence RAA detection, but the results showed that there was strong inhibition between the two fluorescences and subsequent experiments could not be carried out, indicating that the introduction of CRISPR / Cas technology on the basis of RAA amplification indeed played an important role in specificity and anti-interference. In addition, the working temperature of the whole experiment of the present invention was 37 °C, which ensured that various enzymes could function normally. Subsequently, the specificity and sensitivity of the detection method were tested. The RAA-CRISPR-Cas12a / Cas13a dual-channel detection method did not cross-react with other non-target nucleic acids, and the sensitivity of detecting GyVg1 and GyH1 targets could reach 1.5 copies / μL, indicating that the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method was a reliable choice for developing a dual-diagnosis technology, enriching the research gap of CRISPR multiplex diagnosis technology and having broad application prospects.

[0101] The embodiments described above are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A RAA-CRISPR-Cas12a / Cas13a dual-channel detection system for simultaneously detecting avian circovirus type 1 and human circovirus type 1, characterized in that, Including the RAA amplification primer pair for avian-origin circovirus type 1, the RAA amplification primer pair for human-origin circovirus type 1, 12-crRNA-3, 13-crRNA-2, ssDNA probe, ssRNA probe, LbCas12a nuclease, LbuCas13a nuclease, and T7 transcriptase; The RAA amplification primer pair for avian-origin circovirus type 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8; The nucleotide sequence of the 12-crRNA-3 is as shown in SEQ ID NO.11; The RAA amplification primer pair for human-origin circovirus type 1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.14 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.17; The nucleotide sequence of the 13-crRNA-2 is as shown in SEQ ID NO.21; The nucleotide sequence of the ssDNA probe is TTATT; The nucleotide sequence of the ssRNA probe is UUUUUU; The 5'-ends of the ssDNA probe and the ssRNA probe are labeled with different fluorescent groups.

2. The RAA-CRISPR-Cas12a / Cas13a dual-channel detection system according to claim 1, wherein The 5'-end of the ssDNA probe is labeled with the FAM group and the 3'-end is labeled with the BHQ1 group; The 5'-end of the ssRNA probe is labeled with the ROX group and the 3'-end is labeled with the BHQ1 group.

3. Use of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system according to any one of claims 1-2 in the preparation of a product for simultaneously detecting avian-origin circovirus type 1 and human-origin circovirus type 1.

4. The application according to claim 3, characterized in that The product is a kit.

5. A product for simultaneously detecting avian circovirus type 1 and human circovirus type 1, characterized in that, Including the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system according to any one of claims 1-2.

6. The product according to claim 5, characterized in that, The product is a kit.

7. The product according to claim 6, characterized in that, The kit is a fluorescence detection kit.

8. The kit according to claim 7, characterized in that, The detection method of the kit includes the following steps: Using the genomic DNA of the test sample as a template, performing RAA amplification with the RAA amplification primer pair for avian-origin circovirus type 1 and the RAA amplification primer pair for human-origin circovirus type 1 to obtain RAA amplification products; Preparing a CRISPR-Cas12a / Cas13a dual-channel system reaction solution with the RAA amplification products, performing a CRISPR-Cas12a / Cas13a reaction, and then detecting the fluorescence intensity.

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