Detection system for simultaneously detecting circle virus poultry source type 1 and circle virus human source type 1 and application thereof

Abstract

The invention discloses a detection system for simultaneously detecting circle virus poultry source type 1 and circle virus human source type 1 and application of the detection system, and relates to the technical field of biological detection. The detection system comprises an RAA amplification primer pair of GyVg1, an RAA amplification primer pair of GyH1, a 12-crRNA-3 (12-crRNA-3), a 13-crRNA-2 (13-crRNA-2), an ssDNA (single-stranded deoxyribonucleic acid) probe, an ssRNA (single-stranded ribonucleic acid) probe, an LbCas12a nuclease, an LbuCas13a nuclease and a T7 transcriptase. According to the present invention, based on GyVg1 and GyH1 conserved genes, the specific crRNA and the RAA primer are respectively designed, and the RAA amplification technology and the CRISPR-Cas fluorescence detection system are combined so as to establish the detection system for simultaneously detecting GyVg1 and GyH1; the detection system can detect two different subtypes of circle viruses at the same time, the detection efficiency is improved, and a new technical means is provided for dynamic research of virus infection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a detection system and application thereof for simultaneously detecting circovirus avian type 1 and circovirus human type 1. Background Art

[0002] Gyrovirus galga1 (GyVg1) and Gyrovirus homsa1 (GyH1) are the second and third newly discovered circular viruses after Chicken anemia virus (CAV). They are members of the genus Gyrovirus in the family Anelloviridae. They are a type of small, non-enveloped, single-stranded circular DNA virus with a genome length of approximately 2.4 kbp and contain three partially overlapping open reading frames (ORFs), of which ORF1 encodes the backbone protein VP2, ORF2 encodes the apoptosis protein VP3, and ORF3 encodes the capsid protein VP1. The nucleotide similarities of VP1, VP2, and VP3 of GyVg1, GyH1 and CIAV are 42%, 42%, 30% and 43%, 42%, 29%, respectively. The nucleotide similarities of VP1, VP2, and VP3 of GyVg1 and GyH1 are 62%, 63%, and 55%, respectively. GyVg1 and GyH1 mainly infect chickens 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 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 are similar, and the infected people show varying degrees of diarrhea, suggesting that the two new circular viruses have the risk of cross-host transmission.

[0003] GyVg1 and GyVg1 mainly infect chickens. In 2011, Rijsewijk et al. found the genome of a distant relative of CAV in serum samples of sick chickens in Brazil, reported a new member of the genus circovirus, and named it avian circovirus type 2 (AGV2). In 2012, Phan et al. conducted a viral metagenomic analysis of 100 human feces to study the cause of unexplained diarrhea in children with acute gastroenteritis. They identified the presence of circoviruses in feces and discovered a new circovirus species for the first time, avian circovirus type 3 (GyV3), which is the third member of the circovirus family. As of 2024, 16 circoviruses have been identified, and ICTV has reclassified the circovirus family, with the original AGV2 renamed GyVg1 and GyV3 renamed GyH1. At present, there are few research reports on these two newly emerging circoviruses. Their direct involvement in the etiology of specific diseases and the reasons for their ubiquity and host diversity are not fully understood. The GyVg1 and GyH1 subculture system has not yet been found, and the virus cannot be multiplied in large quantities. Their pathogenicity remains largely unexplored. These unknowns have potential impacts on public health and safety and threaten the healthy development of the poultry industry. However, in the more than ten years since the circovirus was first discovered, there has been a lack of prevention and treatment measures for GyVg1 and GyH1, including commercial vaccines, drug treatments, and purification of infected populations, and the viruses are rampant. The first step in preventing and controlling viral infections is to conduct pathogen detection, which can monitor viral infections in a timely manner and provide a basis for subsequent evaluation of epidemics and mutations as well as the effectiveness of vaccines and drug treatments.

[0004] At present, the detection of GyVg1 still relies on molecular detection technology, including conventional PCR, fluorescence quantitative PCR, droplet digital PCR, real-time fluorescence recombinase-assisted isothermal amplification (RAA), etc.; in the clinical detection and epidemiological research of GyH1, immunological diagnostic methods include indirect ELISA method, double antibody sandwich ELISA method and colloidal gold immunochromatography test strip detection method, and molecular detection technology includes conventional PCR detection, real-time fluorescence quantitative PCR detection, visualized LAMP detection and real-time fluorescence RAA detection. These methods can specifically detect specific pathogens, but can only detect one of the circle viruses at a time, which increases the time and workload of pathogen detection. Based on the similarity of the etiology and clinical manifestations of GyVg1 and GyH1, the present invention intends to establish a dual detection system that can be used to detect two viruses at the same time. Summary of the invention

[0005] The purpose of the present invention is to provide a detection system and application thereof 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. The detection system can simultaneously detect two different subtypes of circovirus, improve the detection efficiency, and provide a new technical means for the dynamic study of viral infection.

[0006] Recombinase acid amplification (RAA) is an isothermal amplification technology that has emerged in recent years. The primer design is simple and can quickly amplify the target fragment in a short time (20 minutes) under constant temperature (37℃~42℃), but it is prone to spontaneous reaction and non-specific amplification. CRISPR / Cas belongs to the second type of effector protein in the CRISPR system and is a key component of gene editing technology. It uses Cas protein and crRNA with nuclease function to work under the same constant temperature (37℃~42℃). CRISPR / Cas technology has aM-level sensitivity and single nucleotide specificity. However, when the sample target concentration is low, the number of activated Cas proteins is limited, and it cannot generate a large number of detectable signals in a short time to meet the requirements of rapid detection. Therefore, the combined application of CRISPR / Cas system and RAA technology can develop a rapid, sensitive and specific RAA-CRISPR / Cas detection platform. The NUC leaf of Cas12a protein lacks HNH domain, but contains RuvC domain, and can cut single-stranded DNA (ssDNA) indiscriminately after binding with specific crRNA; Cas13a is composed of two higher HEPN domains, and when combined with specific crRNA, its cutting activity is stimulated, causing RNA damage diffusion phenomenon, and RNA can be more widely degraded. The present invention adds CRISPR / Cas12a and CRISPR / Cas13a to the same reaction system, introduces DNA probes labeled with FAM fluorescent groups and RNA probes labeled with ROX fluorescent groups, respectively, CRISPR / Cas12a targets cutting DNA probes, Cas13a targets cutting RNA probes, and realizes fluorescent dual-channel detection of FAM and ROX in a reaction tube, which is similar to dual fluorescent quantitative PCR, and the real-time cutting reaction observes the amplification curve under the corresponding fluorescent channel in the fluorescent quantitative PCR instrument, so as to achieve the purpose of simultaneously distinguishing and detecting GyVg1 and GyH1, and innovatively constructs a constant temperature RAA-CRISPR-Cas12a / Cas13a dual-channel rapid detection method.

[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 circovirus avian type 1 and circovirus human type 1, comprising a RAA amplification primer pair for circovirus avian type 1, a RAA amplification primer pair for circovirus human 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 of the circovirus avian type 1 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.8;

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

[0011] The RAA amplification primer pair of the circle virus human 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 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, the 5' ends of the ssDNA probe and the ssRNA probe are labeled with different fluorescent groups.

[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 use of the above-mentioned RAA-CRISPR-Cas12a / Cas13a dual-channel detection system in the preparation of a product for simultaneously detecting avian-origin type 1 of circle virus and human-origin type 1 of circle virus.

[0019] Furthermore, the product is a kit.

[0020] The present invention also provides a product for simultaneously detecting avian-origin type 1 of circle virus and human-origin type 1 of circle virus, 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 comprises the following steps:

[0024] Using the genomic DNA of the sample to be tested as a template, performing RAA amplification using the RAA amplification primer pair of the avian-origin type 1 of the circle virus and the RAA amplification primer pair of the human-origin type 1 of the circle virus to obtain a RAA amplification product;

[0025] The RAA amplification product was used to prepare a CRISPR-Cas12a / Cas13a dual-channel system reaction solution, and after the CRISPR-Cas12a / Cas13a reaction, the fluorescence intensity was detected.

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

[0027] The present invention is based on the conserved genes of GyVg1 and GyH1, designs specific crRNA and RAA primers respectively, and establishes a RAA-CRISPR / Cas12a-Cas13a dual-channel detection system for simultaneously detecting GyVg1 and GyH1 by combining RAA amplification technology with the CRISPR-Cas fluorescence detection system. The system can detect the presence of GyVg1 and GyH1 within 1 hour, and can specifically distinguish GyVg1 and GyH1. It is easy to operate, has good specificity and high sensitivity, and the minimum detection limit can reach 1.5 copies / μL. The RAA-CRISPR / Cas12a-Cas13a dual-channel detection system established by the present invention can simultaneously detect two different subtypes of circle viruses, improve the detection efficiency, and provide a new technical means for the dynamic study of viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0030] Figure 2 Result diagram of the screening experiment of RAA-CRISPR / Cas12a primers and crRNA; wherein A is the result of different RAA primer combinations of crRNA1; B is the result of different RAA primer combinations of crRNA2; C is the result of different RAA primer combinations of crRNA3;

[0031] Figure 3 Result diagram of the screening experiment of RAA-CRISPR / Cas13a primers and crRNA; wherein A is the result of different RAA primer combinations of crRNA1; B is the result of different RAA primer combinations of crRNA2; C is the result of different RAA primer combinations of crRNA3;

[0032] Figure 4 The results of the RAA-CRISPR / Cas12a system optimization experiment of GyVg1 are shown in Figure 1; A is the result of different RAA primer concentrations; B is the result of different crRNA concentrations; C is the result of different ssDNA concentrations;

[0033] Figure 5 The results of the RAA-CRISPR / Cas13a system optimization experiment of GyH1 are shown in Figure 1; A is the result of different RAA primer concentrations; B is the result of different Cas13a concentrations; C is the result of different crRNA concentrations; D is the result of different ssRNA concentrations; E is the result of different T7 transcriptase volumes;

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

[0035] Figure 7 This is a graph showing the results of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection specificity test;

[0036] Figure 8 The figure shows the results of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection sensitivity test; A is the result of the GyVg1 sensitivity test under the FAM channel; B is the result of the GyH1 sensitivity test under the ROX channel. DETAILED DESCRIPTION

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

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Sample

[0045] One GyVg1-positive sample came from the liver tissue of a sick chicken with neurological symptoms, one GyH1-positive sample came from the glandular stomach tissue of a healthy chicken in a live poultry market, and one GyVg1 and GyH1 mixed infection-positive sample came from the visceral tissue homogenate of a chicken with tumor symptoms. Three positive samples were confirmed by PCR and sequenced. The other 12 non-target reference strains were used for specific tests, including chicken infectious anemia virus (CIAV), avian adenovirus type 4 (FAdV4), H9 subtype avian influenza virus (H9-AIV), Newcastle disease virus (NDV), avian Infectious bronchitis virus (IBV), infectious bursal virus (IBDV), chicken parvovirus (ChPV), avian leukemia virus (ALV), avian nephritis virus (ANV), chicken astrovirus (CAstV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were provided by the Biological Laboratory of Guangxi Veterinary Research Institute; 192 clinical samples were collected from live poultry markets and small-scale chicken farms in Guangxi, including 116 chicken oroanal swabs, 28 environmental samples, and 48 shell-pecked embryo visceral tissue samples, and frozen at -80°C.

[0046] 1.2 Reagents and instruments

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

[0048] 1.3 Design of primers and gRNA

[0049] 1.3.1 Design and synthesis of primers and crRNAs targeting GyVg1

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

[0051] 1.3.1 Design and synthesis of primers and crRNAs targeting GyH1

[0052] First, three crRNAs were designed to screen out the crRNA with the best activity for the establishment of the CRISPR / Cas13a system. As in the above method, a conservative target sequence was found, and a reverse complementary base sequence was designed according to the target region, with a size of 20 bp. Note that the flanking sequence at the 3' end of the target region preferably does not contain base G; a stem-loop structure was added upstream of the target region, and its sequence information was: 5'-GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC-3', thereby determining the crRNA sequence. Then, three upstream RAA primers and three downstream RAA primers were designed outside the crRNA, with a primer length of 28-32 bp, and the 5' end of the upstream primer needed to add a T7 promoter sequence (5'-GGACCACCCCAAAAAUGAAGGGGACCAAAAC-3') to facilitate subsequent in vitro transcription purification. The amplicon length is about 300 bp, and the primer sequence should also avoid pairing with the crRNA sequence. Finally, a pair of PCR primers for preparing plasmid standards were designed outside the amplicon. All sequences are shown in Table 1.

[0053] Table 1 Primers, 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 oral and anal cotton swab samples were soaked and squeezed with PBS, and the tissue samples were soaked and ground with PBS, and then frozen and thawed three times at -80°C. The viral DNA was extracted using a pre-packaged viral RNA / DNA extraction kit. 200 μL of viral supernatant was added to the sample well of the 96-well extraction kit, and the kit was placed in a fully automatic nucleic acid extraction and purification instrument for automatic extraction. After 18 minutes, the genomic DNA of the sample was directly aspirated from the nucleic acid well and stored at -20°C for later use.

[0059] Take a portion of GyVg1 and GyH1 positive DNA template, use GyVg1-F, GyVg1-R and GyH1-F, GyH1-R to amplify and prepare positive plasmid standards, measure the plasmid concentration with an ultraviolet spectrophotometer, calculate the plasmid copy concentration, and store at -20℃ for future use.

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

[0061] Standard RAA is used for primer pair screening. In the 50μL RAA system, refer to the instructions of the RAA basic kit, add different primer sets to the detection unit tube, and incubate in a constant temperature metal bath at 39°C for 30 minutes to complete the target sequence amplification. The standard CRISPR / Cas system is used to screen gRNA. In the 20μL CRISPR / Cas system, refer to the instructions of LbCas12aNuclease and LbuCas13a Nuclease and T7 transcription kit to configure the CRISPR / Cas12a reaction system of GyVg1 and the CRISPR / Cas13a reaction system of GyH1, incubate at room temperature for 15 minutes, wait for gRNA to bind to Cas protein to form a CRISPR / Cas complex, add 2μL RAA amplification product to the unit tube cap, centrifuge instantaneously, mix, and centrifuge again. Use QuantStudio5 fluorescence PCR instrument at 37°C to collect fluorescence signals every 1 minute. The most suitable primers and gRNA are selected 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] Taking GyVg1 positive nucleic acid as a template, the primer concentration, 12-crRNA concentration, and ssDNAReporter were optimized and adjusted based on the standard RAA and standard CRISPR / Cas12a systems to achieve ideal RAA-CRISPR / Cas12a reaction efficiency. The primer concentration (200nM, 300nM, 400nM, 500nM, 600nM), 12-crRNA concentration (8.3nM, 16.7nM, 25nM, 33nM, 41.3nM, 50nM, 58nM) (under the premise that the concentration of the LbCas12a Nuclease standard system is 33nM), and ssDNAReporter concentration (200nM, 300nM, 400nM, 500nM, 600nM) were changed in sequence, and the ideal working concentration was selected according to the FAM channel fluorescence value.

[0065] 1.6.2GyH1 RAA-CRISPR / Cas13a system optimization

[0066] Taking GyH1 positive nucleic acid as template, the primer concentration, Cas13a enzyme concentration, 13-crRNA concentration, ssRNA Reporter and T7 transcriptase were optimized and adjusted based on the standard RAA and standard CRISPR / Cas13a system to achieve ideal RAA-CRISPR / Cas13a reaction efficiency. Change the primer concentration (200nM, 300nM, 400nM, 500nM, 600nM), Cas13a enzyme concentration (25nM, 50nM, 75nM, 100nM, 125nM), 13-crRNA concentration (50nM, 75nM, 100nM, 125nM, 150nM, 175nM, 200nM), ssRNAReporter concentration (200nM, 300nM, 400nM, 500nM, 600nM) and T7 transcriptase volume (0.3μL, 0.4μL, 0.5μL, 0.6μL, 0.7μL, 0.8μL, 0.9μL, 1.0μL) in turn to select the ideal working concentration according to the ROX channel fluorescence value.

[0067] 1.6.3 Optimization of the RAA-CRISPR-Cas12a / Cas13a dual-channel system of GyVg1 and GyH1

[0068] Take one GyVg1 positive nucleic acid sample, one GyH1 positive nucleic acid sample and one GyVg1, GyH1 mixed positive nucleic acid sample as templates, according to the RAA-CRISPR / Cas12a and RAA-CRISPR / Cas13a optimization results, in the same RAA system, GyVg1-F, GyVg1-R and GyH1-F, GyH1-R were combined for amplification; 2μL of amplification product was used for CRISPR-Cas12a / Cas13a cleavage system optimization. According to the CRISPR / Cas12a and CRISPR / Cas13a optimization systems, each component was adjusted in equal proportions to make the end point fluorescence intensity of the FAM channel and the ROX channel equivalent, which was used as the final dual-channel system optimization result for subsequent experiments.

[0069] 1.7 Specificity and sensitivity determination

[0070] The specificity of the reaction was evaluated by detecting other emerging gastrointestinal infectious disease pathogens related to GyVg1 and GyH1 and other common poultry disease pathogens, including CIAV, FAdV4, H9-AIV, NDV, IBV, IBDV, ChPV, ALV, ANV, CAstV, MG, and MS. The results were based on the fluorescence values ​​collected in the FAM and ROX channels of the fluorescence quantitative PCR instrument.

[0071] The GyVg1 positive plasmid and the GyH1 positive plasmid were diluted to 3×10 9 The sensitivity of the RAA-CRISPR-Cas12a / Cas13a dual-channel system was evaluated by serial dilution of the mixed plasmid. 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 The positive plasmid with 10 copies / μL was used as a template, ddH2O was used as a negative control, and the optimized ideal system was used for RAA-CRISPR-Cas12a / Cas13a reaction. After the reaction, the fluorescence signal was collected 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 testing

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

[0074] 2. Results

[0075] 2.1 Study plan

[0076] First, RAA was amplified at 37°C for 30 minutes to obtain sufficient targets, and then crRNA, Cas12a, and Cas13a were added to the CRISPR premix system to form crRNA / Cas12a and crRNA / Cas13a complexes, respectively. Finally, RAA-specific amplification targets and T7 transcriptase were added to the premix system and reacted at 37°C for 30 minutes. At this time, RAA-CRISPR / Cas12a cut ssDNAReporter, and the transcribed RAA-CRISPR / Cas13a cut ssRNAReporter, thereby completing signal amplification detection. After the reaction, the fluorescence signals under the FAM channel and ROX channel were collected respectively, realizing the rapid and simultaneous detection of GyVg1 and GyH1 in one reaction.

[0077] 2.2 RAA primers and crRNA screening

[0078] Three upstream primers, three downstream primers, and three crRNAs were arranged and combined, with a total of 27 experimental groups, and 27 corresponding negative control groups. The 54 combinations were simultaneously placed in a fluorescent quantitative PCR instrument for detection, and the ideal crRNA and primers were screened out based on the intensity of the collected fluorescent signals.

[0079] GyVg1 primer and crRNA screening results are as follows Figure 1 As shown, only some experimental groups produced fluorescent signals using crRNA1 and crRNA2 ( Figure 2 In A and B), all experimental groups using crRNA3 produced fluorescent signals, and the corresponding negative control group did not produce fluorescent signals ( Figure 2 In C), according to the curve appearance time and fluorescence intensity, the ideal primers and crRNAs 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] GyH1 primer and crRNA screening results are as follows Figure 2 As shown, neither crRNA1 nor crRNA3 produced fluorescent signals ( Figure 3In A and B), all experimental groups using crRNA3 produced fluorescent signals, and the corresponding negative control group did not produce fluorescent signals ( Figure 3 In C), according to the curve appearance time and fluorescence intensity, the ideal primers and crRNAs 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 Ideal reaction conditions for RAA-CRISPR-Cas12a / Cas13a dual channels

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

[0083] from Figure 4 It can be seen that when the primer concentration is 500nM ( Figure 4 A), 12-crRNA concentration was 25nM ( Figure 4 B), ssDNAReporter concentration is 500nM ( Figure 4 In (C), the fluorescence appears earliest, the fluorescence value is the highest, and the overall reaction efficiency is the highest, that is, the ideal RAA-CRISPR / Cas12a system is determined as follows: the first step RAA amplification reaction includes ABuffer 25μL, GyVg1 primer (10μM) 2.5μL each, DNA template 5μL, B Buffer 2.5μL, and sterile water is added to 50μL; the second step CRISPR / Cas12a reaction includes 10×Cleavage Buffer 2μL, LbCas12aNuclease (1μM) 1μL, 12-crRNA (500nM) 1μL, ssDNAReporter (4μM) 2.5μL, RAA amplification product 2μL, and sterile water is added to 20μL.

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

[0085] from Figure 5 It can be seen that when the primer concentration is 500nM ( Figure 5 A), Cas13a concentration is 100nM ( Figure 5 B), 13-crRNA concentration was 100nM ( Figure 5 C), ssRNAReporter concentration is 600nM ( Figure 5 D), T7 transcriptase at 0.5 μL ( Figure 5Middle E) The fluorescence appears earliest, the fluorescence value is the highest, and the overall reaction efficiency is the highest, that is, the ideal RAA-CRISPR / Cas13a system is determined as follows: the first step RAA amplification reaction includes A Buffer 25μL, GyH1 primer (10μM) 2.5μL each, DNA template 5μL, B Buffer 2.5μL, and sterile water is added to 50μL; the second step CRISPR / Cas13a reaction includes 5×CleavageBuffer 4μL, LbuCas13a Nuclease (5μM) 0.4μL, crRNA (500nM) 4μL, ssRNA Reporter (4μM) 3μL, 40U RNase Inhibitor 0.5μL, NTP mix 0.8μL, 5×T7 Transcription Reaction Buffer 1μL and T7 Transcription Enzyme mix 0.5μL, and finally, 2μL of RAA amplification product is added and sterile water is added to 20μL.

[0086] 2.3.3 Optimization of the RAA-CRISPR-Cas12a / Cas13a dual-channel system of 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 500nM, Cas12a concentration was 50nM, 12-crRNA concentration was 20nM, ssDNAReporter concentration was 400nM, Cas13a concentration was 100nM, 13-crRNA concentration was 100nM, ssRNAReporter concentration was 600nM, and T7 transcriptase was 0.5μL, the endpoint fluorescence intensity collected by the FAM channel and the ROX channel was equivalent ( Figure 6 ), the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system has a good effect, that is, the determined dual-channel system is shown in Table 2, and subsequent experiments are carried out with this system.

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

[0089]

[0090]

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

[0092] The established RAA-CRISPR-Cas12a / Cas13a dual-channel method was used to detect 15 reference strain nucleic acids, and the results showed that ( Figure 7 ): 2 GyVg1, 2 GyH1 and 1 GyVg1 and GyH1 mixed infection positive samples all had corresponding amplification reactions, and typical fluorescence amplification curves appeared, while the other 12 avian pathogens (CIAV, FadV4, AIV-H9, NDV, IBV, IBDV, ARV, ALV, CAstV, ANV, MG, MS) had no amplification like ddH2O, and did not produce fluorescent signals, indicating that the two sets of primers and crRNA designed by 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 by 10-fold serial dilutions of the GyVg1 and GyH1 plasmid DNA mixture, which is equivalent to an actual template working concentration of 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, see Figure 7 When the concentration of GyVg1 plasmid DNA was 1.5 copies / μL, the “FAM channel” could still detect the fluorescence signal ( Figure 8 Middle A); When the concentration of GyH1 plasmid DNA is 1.5 copies / μL, the "ROX channel" can still detect fluorescence signals ( Figure 8 (B) The established RAA-CRISPR-Cas12a / Cas13a dual-channel method has a minimum detection limit of 1.5 copies / μL and high sensitivity.

[0094] 2.5 Clinical sample testing

[0095] To further evaluate the clinical performance of the RRAA-CRISPR-Cas12a / Cas13a dual-channel detection method, the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method and the conventional PCR detection method were used to detect 192 samples collected in the laboratory, and 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%), and 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%), and the total coincidence rate of the two detection methods was 97.40%, which was highly consistent. It shows that the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method established in the present invention has high accuracy and reliable detection results.

[0096] Table 3 Comparison of the results of the two methods applied to clinical sample testing

[0097]

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

[0099] The present invention places CRISPR / Cas12a and CRISPR / Cas13a systems in one system, labels DNA probes with FAM reporter groups, and labels RNA probes with ROX reporter groups, so that CRISPR / Cas12a reaction results are displayed under FAM channels, and CRISPR / Cas13a reaction results are displayed under ROX channels, realizing dual-channel detection of two targets in a single system, i.e., double CRISPR / Cas detection. In order to obtain sufficient targets required for CRISPR / Cas reactions, the present invention uses amplification efficiency higher, shorter reaction time isothermal amplification technology to meet the front-end amplification requirements, and common isothermal amplification technologies include loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP), recombinase polymerase amplification technology (also called RPA, RAA, MIRA), rolling circle amplification technology (Rolling circle amplification, RCA), enzymatic isothermal amplification technology (Enzymatic Recombinase Amplification, ERA) and cross primer amplification technology (Crossing-primeramplification, CPA). Among them, RAA primers are simple in design, have a mild reaction temperature (37-42°C), and a fast reaction speed (within 30 minutes). It is an excellent nucleic acid amplification technology. The study combines RAA amplification technology with 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 specific identification of the enzyme-cut target sequence and relies on reporter probes to visualize the detection results, greatly improving the detection sensitivity and specificity.

[0100] The conserved regions of GyVg1 and GyH1 were selected in the overlapping regions of VP2 and VP3 genes, and specific RAA primers and crRNA were designed to first ensure the specificity of target cutting, and then screen out primers and probes with the best reaction efficiency through the reaction results of different primer pairs with crRNA. The RAA-CRISPR / Cas12a system and the RAA-CRISPR / Cas13a system were optimized separately, and then the RAA-CRISPR / Cas12a system was combined with the RAA-CRISPR / Cas13a system and the ratio of the two systems was adjusted for optimization, and finally an ideal RAA-CRISPR-Cas12a / Cas13a dual-channel detection method was established. The research results show that the fluorescence results under the dual channels of mixed target amplification are basically consistent with the fluorescence results under the dual channels of single GyVg1 or GyH1 target amplification, indicating that the primers of GyVg1 and GyH1 are placed in the same RAA amplification system for amplification, and there is no inhibition of amplification; GyVg1-CRISPR / Cas12a and GyH1-CRISPR / Cas13a are placed in the same CRISPR / Cas system, and there is no inhibition of cutting; the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method of GyVg1 and GyH1 established by the present invention is feasible. The inventors previously developed a real-time fluorescence RAA detection method for GyVg1 and a GyH1, and tried 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 does play an important role in specificity and anti-interference. In addition, the working temperature of the experiment of the present invention is 37°C throughout the whole process, which ensures that all enzymes can function normally. Subsequently, the present invention tested the specificity and sensitivity of the detection method. The RAA-CRISPR-Cas12a / Cas13a dual-channel detection method does not cross-react with other non-target nucleic acids, and the sensitivity of detecting GyVg1 and GyH1 targets can reach 1.5 copies / μL, indicating that the RAA-CRISPR-Cas12a / Cas13a dual-channel detection method is a reliable choice for the development of dual diagnosis technology, enriching the research gap of CRISPR multiple diagnosis technology, and has broad application prospects.

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 simultaneous detection of avian-origin type 1 of circle virus and human-origin type 1 of circle virus, characterized in that: Including RAA amplification primer pair of avian-origin type 1 of circle virus, RAA amplification primer pair of human-origin type 1 of circle virus, 12-crRNA-3, 13-crRNA-2, ssDNA probe, ssRNA probe, LbCas12a nuclease, LbuCas13a nuclease and T7 transcriptase; The RAA amplification primer pair of the circovirus avian type 1 comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.8; The nucleotide sequence of the 12-crRNA-3 is shown in SEQ ID NO.11; The RAA amplification primer pair of the circle virus human 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 shown in SEQ ID NO.21; The nucleotide sequence of the ssDNA probe is TTATT; The nucleotide sequence of the ssRNA probe is UUUUUU.

2. The RAA-CRISPR-Cas12a / Cas13a dual-channel detection system according to claim 1, characterized in that The 5' ends of the ssDNA probe and the ssRNA probe are labeled with different fluorescent groups.

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

4. An application of the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system as described in any one of claims 1 to 3 in the preparation of a product for simultaneously detecting avian-origin type 1 of circle virus and human-origin type 1 of circle virus.

5. The use according to claim 4, characterized in that: The product is a test kit.

6. A product for simultaneously detecting circovirus avian type 1 and circovirus human type 1, characterized in that: Comprising the RAA-CRISPR-Cas12a / Cas13a dual-channel detection system according to any one of claims 1 to 3.

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

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

9. The kit according to claim 8, characterized in that The detection method of the kit comprises the following steps: Using the genomic DNA of the sample to be tested as a template, performing RAA amplification using the RAA amplification primer pair of the avian-origin type 1 of the circle virus and the RAA amplification primer pair of the human-origin type 1 of the circle virus to obtain a RAA amplification product; The RAA amplification product was used to prepare a CRISPR-Cas12a / Cas13a dual-channel system reaction solution, and after the CRISPR-Cas12a / Cas13a reaction, the fluorescence intensity was detected.

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