A method for identifying three new infectious disease viruses in chicken flocks

Abstract

The application discloses a LAMP primer and probe combination, a kit and a detection method for simultaneously identifying three viruses of chicken parvovirus, chicken infectious anemia virus (CIAV) and fowl adenovirus serotype 4 (FADV4), and comprises three sets of primer groups corresponding to the three viruses and three probes of different colors; a detection reaction process only comprises isothermal amplification and termination, and finally, a triple fluorescence LAMP reaction is completed in a same reaction tube; after the reaction, the three viruses can be identified and diagnosed according to different colors of the three probes; the minimum detection capability is 1 copy / muL of virus DNA; the whole detection process is simple, rapid, low in cost, high in specificity and sensitivity, small in interference, capable of realizing on-site pathogen detection, and suitable for large-scale diagnostic screening of chicken groups.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and particularly to a visualization method that can simultaneously identify three viruses: chicken parvovirus, chicken infectious anemia virus, and avian adenovirus serotype 4, especially a triple fluorescence LAMP combined identification method. Background Technology

[0002] Chicken parvovirus (CHPV), chicken infectious anaemia virus (CIAV), and avian adenovirus serotype 4 (FADV4) are three common viruses causing emerging infectious diseases in chicken flocks in recent years. These three viruses are DNA viruses, primarily infecting chicks under 35 days old, often resulting in mixed infections and various complications. They are major pathogens causing the spread of infectious diseases in poultry farming. CHPV mainly causes avian enteritis syndrome and developmental disorders and dwarfism syndrome, characterized by diarrhea, depression, thermoregulation disorders, growth retardation, and increased feed consumption. Studies show that this disease is widespread in chicken flocks in my country, with an infection rate as high as 38.9%–88.1%, higher in commercial broilers, followed by breeder chickens and laying hens. CIAV can cause atrophy of lymphoid tissue throughout the chicken, especially bone marrow hematopoietic tissue and lymphoid tissue, leading to immunosuppression. Clinical symptoms include growth retardation, anemia, bone marrow regeneration disorders, and thymus atrophy. Chickens of all ages can be infected with CIAV, but clinical symptoms mainly appear in chickens aged 10-14 days. CIAV is prevalent globally, with a CIAV antibody positivity rate as high as 70% in my country. The disease can be transmitted both vertically and horizontally, presenting clinically as typical or asymptomatic infections, and secondary infections with other pathogens can lead to flock mortality. FADV4 mainly causes pericardial effusion-inclusion body hepatitis syndrome, with rapid onset and high mortality. In recent years, there have been continuous outbreaks of avian adenovirus serotype 4 infection in my country, which has had a significant impact on poultry farming.

[0003] Currently, the three main methods for laboratory virus identification rely on traditional methods such as virus isolation, agar diffusion assay, ELISA, and commonly used techniques like DNA sequencing and PCR. These methods are either time-consuming, labor-intensive, and have low sensitivity; or they rely on specialized equipment and are costly; or they can only identify a single virus, or at most two viruses simultaneously. In recent years, the incidence of mixed infections of these three viruses has been on the rise in my country, causing significant economic losses to the poultry industry. Therefore, there is an urgent need to establish rapid, accurate, and combined diagnostic methods to ensure the healthy development of the poultry industry.

[0004] Loop-mediated isothermal amplification (LAMP) technology amplifies the reaction mixture under isothermal conditions (58-67℃), offering advantages such as speed, simplicity, and sensitivity. However, due to inherent technical limitations, significant progress has been slow globally in multiplex LAMP methods. Whether singleton or multiplex, positive results often exhibit identical fluorescence and precipitate colors, making it difficult to pinpoint the specific positive reaction causing the result, thus hindering multiplex differentiation. Furthermore, multiplex fluorescence is limited by the fixed wavelength range of existing fluorescence imaging analysis systems, the inhibition of the reaction by fluorescence-quenching composite probes, and increased competition between primers and probes for the reaction system. Consequently, there are no reports of triple-fluorescence LAMP detection products or methods that simultaneously identify three pathogens. Summary of the Invention

[0005] The purpose of this invention is to provide a triple fluorescence LAMP detection method for simultaneously identifying three viruses: chicken parvovirus (CHPV), chicken infectious anemia virus (CIAV), and avian adenovirus serotype 4 (FADV4).

[0006] To achieve the above objectives, this invention claims protection for a primer and probe composition comprising three sets of primers and probes: outer primers CHPV-F3 and CHPV-B3, inner primers CHPV-FIP and CHPV-BIP, loop primers CHPV-Floop and CHPV-Bloop, and probes CHPV-FD and CHPV-BD designed based on the NS gene sequence of chicken parvovirus, the nucleotide sequences of which are shown in SEQ ID NO:1-8 in the sequence listing; and outer primers CIAV-F3 and CIAV-B3, inner primers CIAV-FIP and CIAV-BIP, loop primers CIAV-Floop and CIAV-Bloop, and probes CIAV-FD and CIAV-BD designed based on the VP1 gene sequence of chicken infectious anemia virus, the nucleotide sequences of which are shown in SEQ ID NO:1-8 in the sequence listing. As shown in NO:9-16; the outer primers FADV4-F3 and FADV4-B3, the inner primers FADV4-FIP and FADV4-BIP, the loop primers FADV4-Floop and FADV4-Bloop, and the probes FADV4-FD and FADV4-BD, designed based on the Hexon gene sequence of avian adenovirus serotype 4, have nucleotide sequences shown in SEQ ID NO:17-24 of the sequence listing.

[0007] The present invention claims a primer and probe composition wherein three sets of primers and probes have, respectively, substitutions, deletions or additions of no more than 5 nucleotides compared with the sequences shown in the corresponding SEQ ID NO:1-24, or have 80%-99.99% sequence identity and have the same function as the sequences in SEQ ID NO:1-24.

[0008] The primer and probe compositions claimed in this invention have quenching groups labeled at the 5' ends of the inner primers CHPV-FIP and CHPV-BIP, CIAV-FIP and CIAV-BIP, and FADV4-FIP and FADV4-BIP sequences, and fluorescent groups labeled at the 3' ends of the probes CHPV-FD and CHPV-BD, CIAV-FD and CIAV-BD, and FADV4-FD and FADV4-BD sequences.

[0009] The primer and probe composition claimed in this invention contains a BHQ series fluorescent quenching group, and fluorescent groups such as Alexa Fluor 488, Cy5, and CY3.

[0010] The primer and probe compositions claimed in this invention, wherein the inner primers CHPV-FIP, CHPV-BIP, CIAV-FIP, CIAV-BIP, FADV4-FIP, FADV4-BIP and the corresponding probes CHPV-FD, CHPV-BD, CIAV-FD, CIAV-BD, FADV4-FD, FADV4-BD have complementary nucleotide sequences, and after annealing, they pair and bind to form non-luminescent fluorescent quenched composite probes CHPV-FIP-FD, CHPV-BIP-BD, CIAV-FIP-FD, CIAV-BIP-BD, FADV4-FIP-FD, FADV4-BIP-BD.

[0011] This invention claims protection for a method for identifying three emerging infectious disease viruses in chicken flocks, comprising primer and probe compositions of three sets of primers participating in the reaction; further comprising: ① Preparation of fluorescence-quenched annealed composite probes: before the multiplex RT-LAMP reaction, 50 μM FIP and 50 μM FD, 50 μM BIP and 50 μM BD are mixed separately, heated to 98°C for 5 minutes, slowly cooled to room temperature to complete annealing, and stored at -20°C for later use. Following the above method, FIP-FD and BIP-BD annealed composite probes corresponding to CHPV, CIAV, and FADV4 are obtained respectively; ② Preparation of the reaction system: a 20 μL multiplex RT-LAMP reaction system is prepared, comprising 1 μL template, 10 μL WarmStart 2× premix, 16 U Bst 2.0 WarmStart DNA polymerase, 0.66 μM CHPV-FIP, 0.66 μM CIAV-FIP, 0.66 μM FADV4-FIP, 0.66 μM... CHPV-FIP-FD annealed composite probe, 0.66μM CIAV-FIP-FD annealed composite probe, 0.66μM FADV4-FIP-FD annealed composite probe, 0.66μM CHPV-BIP, 0.66μM CIAV-BIP, 0.66μM FADV4-BIP, 0.66μM CHPV-BIP-BD annealed composite probe, 0.66μM CIAV-BIP-BD annealed composite probe, 0.66μM FADV4-BIP-BD annealed composite probe, 0.083μM CHPV-F3, 0.083μM CIAV-F3, 0.083μM FADV4-F3, 0.083μM CHPV-B3, 0.083μM CIAV-B3, 0.083μM FADV4-B3, 0.17μM CHPV-Floop, 0.17μM CIAV-Floop, 0.17μM FADV4-Floop, 0.17μM CHPV-Bloop, 0.17μM CIAV-Bloop, 0.17μM FADV4-Bloop, with the remainder being water; ③ Reaction process: including an extension reaction at 63℃ for 60℃ min and a termination reaction at 80℃ for 5 min; ④ Result detection: including result interpretation using a real-time turbidimeter and an image analyzer under multiple fluorescence channels in two ways; This method is not used for the diagnosis and treatment of diseases.

[0012] This invention claims protection for a kit for identifying three emerging infectious disease viruses in chicken flocks, comprising three primer and probe compositions, WarmStart LAMP colorimetric premix, standards, negative controls, and DNA polymerase.

[0013] The present invention claims a kit for identifying three emerging infectious disease viruses in chicken flocks, wherein the DNA polymerase is Bst 2.0 WarmStart DNA polymerase, and the amount used is 16U per 20μL reaction system.

[0014] The present invention claims a primer and probe composition, a method for identifying three emerging infectious disease viruses in chicken flocks, a kit for identifying three emerging infectious disease viruses in chicken flocks, and its application in identifying emerging infectious diseases in chicken flocks.

[0015] To achieve the goal of simultaneously identifying three viruses—chicken parvovirus (CHPV), chicken infectious anemia virus (CIAV), and avian adenovirus serotype 4 (FADV4)—this application has undergone extensive inventive research.

[0016] Firstly, LAMP detection methods have received special attention due to their more intuitive results and relatively faster detection speed. However, current multiplex LAMP detection methods generally use no more than two sets of primers, with one set for each virus and each set containing at least two primer pairs (outer and inner primers). Furthermore, LAMP detection results can only distinguish between positive and negative results, not which set of primers amplified the positive result. This invention is the first to successfully study and screen three sets of primers and probes targeting three different target fragments. Each primer set includes three pairs of primers (outer, inner, and loop primers) and one pair of probes. Through primer pairing optimization, the primer and probe compositions of the three LAMP primer sets of this invention were obtained. Performance verification showed that the primer and probe compositions of this invention, when applied to the detection kit, exhibited the best specificity and the most stable results.

[0017] The transition from a standard duplex LAMP to a triple LAMP is not a simple matter of stacking elements. Through extensive and creative experiments and adjustments to the reaction system, triple LAMP amplification was ultimately achieved within the same reaction tube.

[0018] Secondly, this invention is the first to successfully screen for a third type of fluorescent substance. Existing fluorescence imaging analysis systems generally have fixed wavelength ranges. For example, the detection wavelength of a multicolor fluorescence imaging analysis system (manufacturer: BIO-RAD, USA, product catalog number Universal HoodⅢ) is 495-750nm, while the wavelength range of blue fluorescence is 422-455nm. This means that most fluorescence imaging analyzers cannot detect blue fluorescence. In the research on the third type of fluorescence, four fluorescent groups (ROX, Alexa Flour 586, CY3, and Texas Red) were compared. The final study found that only CY3 showed color in the 570nm channel and did not overlap with the 520nm (Alexa Fluor 488, green) and 670nm (Cy5, red) channels. However, CY3 fluorescence appears orange, not blue. Since orange and green are difficult to distinguish with the naked eye, the imaging analyzer code was modified by programming to assign blue to CY3, making it the third type of blue fluorescence for triple fluorescence reactions.

[0019] Furthermore, the fluorescence-quenching composite probe directly participates in the reaction, thus inhibiting it. Studies have found that when the fluorescence-quenching composite probe is directly added to the triple fluorescent LAMP reaction, completely replacing the inner primers FIP and BIP, not only does the fluorescence background of the fluorescence-quenching composite probe itself become high, but it also inhibits the LAMP reaction to some extent, or even completely inhibits LAMP. Ultimately, the study found that using an inner primer FIP (or BIP):fluorescence-quenching composite probe FIP-FD (or BIP-BD) ratio of 1:1 can both reduce the fluorescence background and ensure stable amplification.

[0020] Finally, in multiplex fluorescent LAMP reactions, dozens of primers and probes compete for the reaction system, inhibiting each other. Generally, dual-fluorescence LAMP reactions include two sets of primers and probes; increasing to triple fluorescence requires an additional set. In this invention, each primer set includes four groups: an outer primer, an inner primer, a loop primer, and a probe, equivalent to eight primers and probes. Therefore, the entire triple reaction system contains 24 primers and probes, inevitably leading to competition for the reaction system during LAMP. The study ultimately found that without DNA polymerase, the entire reaction was inhibited. Adding 16µbst 2.0 WarmStart DNA polymerase per 20μL of reaction system significantly improved reaction efficiency and achieved amplification equilibrium in the triple reaction, preventing interference between the primers and probes.

[0021] The beneficial effects of this invention are:

[0022] Regarding primer and probe sequence design: Based on the conserved regions of the NS gene of chicken parvovirus (CHPV), the VP1 gene of chicken infectious anemia virus (CIAV), and the Hexon gene of avian adenovirus serotype 4 (FADV4), three sets of specific primer and probe combinations targeting three different types of viruses were designed. These combinations can identify and diagnose three emerging infectious diseases in chickens with high sensitivity and specificity.

[0023] Optimization of fluorescent groups: In addition to the conventional inner primers, outer primers, and loop primers for LAMP, three sets of primers and probes were designed, including probes (FD and BD). The regions on the probes are complementary to those on the inner primers, and the 5' end of the inner primer is labeled with a quencher group, while the 3' end of the probe is labeled with a fluorescent group. As mentioned earlier, the color development of the three fluorescent groups on the probes occurs in their respective channels, without cross-contamination or interference. After annealing, the inner primer and probes complementarily bind to form a fluorescently quenched composite probe (i.e., a non-luminescent complex). The annealing reaction is the preparatory stage for the triple fluorescent LAMP reaction. During the triple fluorescent LAMP reaction, the extension reaction causes the probes (FD and BD) to detach, and the detection results can be distinguished based on the fluorescent color of the probes. Using FD and BD dual fluorescence for each virus can greatly improve the specificity and fluorescence increment of the reaction, resulting in better differentiation.

[0024] The reaction process is simple: by introducing three different colored fluorescent groups and completing the annealing preparation before the reaction, the detection process only requires two steps: the extension reaction at 63℃ and the termination reaction at 80℃. The reaction process is simple and can be achieved with a conventional water bath, without the need for complicated instruments and equipment.

[0025] Two Result Determination Methods: The triple fluorescence LAMP isothermal reaction of this invention includes two result determination methods: real-time detection of the reaction precipitate using a real-time turbidimeter and multi-channel determination of fluorescence color using a fluorescence imaging analyzer. First, compared to conventional LAMP which requires opening the tube for gel electrophoresis to observe trapezoidal bands, these two result determination methods eliminate the need for opening the tube, avoiding aerosol contamination of the experimental environment and false positives. Second, the two result determination methods can corroborate each other, making the result determination more accurate and reliable. Third, the introduction of a commercially available LAMP color-changing premix changes the color of the positive reaction product from pink to yellow, allowing for visual determination without ultraviolet irradiation. Furthermore, the positive reaction precipitate can be detected in real-time using a turbidimeter, making it more convenient for on-site quarantine in areas with poor infrastructure. Fourth, the multi-channel fluorescence color determination using a fluorescence imaging analyzer allows for differentiation of virus types and quantities when multiple target fragments (in mixed-infection samples) show nucleic acid amplification. The combined detection of three target viruses using LAMP technology within the same reaction tube is particularly suitable for identifying mixed infections.

[0026] Application of the kit: The kits for three emerging infectious diseases in chickens provided by this invention utilize the three sets of primers and probes designed in this invention, three fluorescent LAMP reaction systems, and a triple fluorescent LAMP detection method. This allows for real-time observation of positive amplification results for CHPV, CIAV, and FADV4 viruses, and also enables specific differentiation of which of these three viruses (CHPV, CIAV, and FADV4) caused the positive amplification, making the results more intuitive.

[0027] Performance advantages: The primer and probe composition, kit, and application of the present invention for identifying three emerging infectious disease viruses in chicken flocks only show positive reactions with the DNA of CHPV, CIAV, and FADV4 viruses, and have no cross-reaction with other common poultry viruses, demonstrating high specificity; for CHPV, CIAV, and FADV4 viruses, each reaction can detect at least one copy of viral DNA, demonstrating high sensitivity.

[0028] In summary, this invention has for the first time successfully established a rapid triple fluorescence LAMP detection method for detecting three target fragments, which is particularly suitable for the rapid identification and diagnosis of three emerging infectious disease viruses in chickens (CHPV, CIAV, and FADV4). This triple fluorescence LAMP method has the advantages of high specificity, high sensitivity, strong anti-interference, low contamination, and convenience. Furthermore, the detection results can be directly observed with the naked eye, qualitatively observed using a real-time turbidimeter, and the fluorescence color of the reaction products can be specifically interpreted using an image analyzer. It is suitable for the rapid clinical diagnosis of the three emerging infectious disease viruses in chickens. The detection process can be completed in 65 minutes using a water bath, and the operation is simple. It is especially suitable for early screening of the three emerging infectious diseases in chickens at grassroots veterinary stations, farms, and border ports. Attached Figure Description

[0029] Figure 1 The results of the specificity test for the method of identifying three emerging infectious disease viruses in chicken flocks are shown in the figure. The sample numbers are: 1 CHPV DNA, 2 CIAV DNA, 3 FADV4 DNA, 4 CHPV+CIAV DNA, 5 CHPV+FADV4 DNA, 6 CIAV+FADV4 DNA, 7 CHPV+CIAV+FADV4 DNA, 8 AIV cDNA, 9 NDV cDNA, 10 IBV cDNA, 11 ARV cDNA, 12 AILTV DNA, 13 APV cDNA, 14 MDV DNA, and 15 negative control (water).

[0030] Figure 2The figure shows the results of a sensitivity test for identifying three emerging infectious disease viruses in chickens. Samples 1-8 are mixed samples of equal amounts of the three viruses (CHPV, CIAV, and FDAV4) DNA at different concentrations. The relationship between the sample number and the corresponding concentration is as follows: 1 = 1 × 10⁻⁶. 7 copies / μl, 2 is 1×10 6 copies / μl, 3 is 1×10 5 copies / μl, 4 is 1×10 4 copies / μl, 5 is 1×10 3 copies / μl, 6 is 1×10 2 copies / μl, 7 is 1×10 1 Sample 8 was 1 copy / μl, and sample 9 was a negative control (water).

[0031] Figure 3 The figure shows the results of an interference experiment for identifying three emerging infectious disease viruses in chickens. Samples 1-8 are mixed samples of DNA from the three viruses (CHPV, CIAV, and FADV4) at different concentrations. The relationship between the sample number and the virus concentration is as follows: 1 = 10. 2 CHPV+10 2 CIAV+10 9 FADV4,2 is 10 2 CHPV+10 3 CIAV+10 8 FADV4,3 is 10 2 CHPV+10 4 CIAV+10 7 FADV4, 4 is 10 2 CHPV+10 5 CIAV+10 6 FADV4,5 is 10 2 CHPV+10 6 CIAV+10 5 FADV4,6 is 10 2 CHPV+10 7 CIAV+10 4 FADV4, 7 is 10 2 CHPV+10 8 CIAV+10 3 FADV4, 8 is 10 2 CHPV+10 9 CIAV+10 2 FADV4, concentration unit is copies / μl. Detailed Implementation

[0032] The exemplary embodiments of the present invention are described in detail below with reference to practical applications. In the following detailed description, many specific details are set forth for ease of explanation to provide a full understanding of the disclosed embodiments. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. However, it is apparent that one or more embodiments may be implemented without these specific details. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0034] LAMP color-changing premix (containing UDG) and DNA polymerase (Bst 2.0) DNAPolymerase was purchased from NEB (New England Biolabs, Inc.); DNA / RNA co-extraction kit ( Viral DNA / RNA Kit and Plasmid Mini-Extraction Kit The Plasmid MiniPrep Kit was purchased from Axygen Scientific, Inc. (USA); the Loopamp LA-32OC real-time turbidimeter was purchased from Eiken Chemical Co., Ltd. (Tokyo, Japan); the image analyzer was purchased from Bio-Rad Laboratories, Inc. (USA); the NanoDrop 2000 nucleic acid analyzer was purchased from ThermoFisher Scientific (USA); the reverse transcription kit (PrimeScript™ 1st Strand cDNA Synthesis Kit) and the PMD-18T vector were purchased from Baori Biotechnology Co., Ltd.; primers were synthesized by Baori Biotechnology Co., Ltd.

[0035] Six strains of chicken parvovirus (CHPV) (Genbank accession numbers: KX084399, KX084401, KX133426, KU523900, KX133415, KX133418), five strains of chicken infectious anemia virus (CIAV) (Genbank accession numbers: MN649254, MN649256, MK484614, MN103402, MN103404), five strains of avian adenovirus group 4 (FADV4) (Genbank accession numbers: MN577977, MN577978, MN577984, MW439040, MW439043), and three types of avian influenza virus (Avianinfluenza) were detected. Avian infectious bronchitis virus (IBV) strains H3N2, H6N6, and H9N2; two Newcastle disease virus (NDV) strains, F48E9 and LaSota; IBV strain Mass 41; Avian reovirus (ARV) strain S1133; Avian infectious laryngotracheitis virus (AILTV) strain Beijing strain; and Avian metapneumovirus (AMV). Metapneumovirus (APV) strain MN-10 was preserved by the Key Laboratory of Biotechnology, Guangxi Zhuang Autonomous Region Veterinary Research Institute; Marek's disease MDV live vaccine virus (CVI988 strain) was purchased from Nanjing Merial Animal Health Products Co., Ltd.; 70 clinical samples were obtained from cotton swabs collected from the oral cavity and cloaca of live poultry collected from Nanning live poultry market. The clinical samples have been identified by routine PCR testing, and positive results were sent to BGI Genomics for sequencing identification; RNA and DNA were extracted from different viruses and clinical samples according to the instructions in the EasyPure Viral DNA / RNA Kit purchased from Beijing TransGen Biotechnology Co., Ltd. The RNA virus was reverse transcribed into cDNA according to the instructions in the reverse transcription kit purchased from Baori Biotechnology Co., Ltd. The cDNA / DNA template was stored at -30℃ for later use. All the above-mentioned viral strains are known viruses and can be obtained from the applicant by the public under the condition of complying with biosafety procedures. They can only be used to repeat the experiments of this invention and must not be used for other purposes.

[0036] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0037] This invention only relates to the detection and identification of biological information from cotton swabs taken from the mouth and cloaca of chickens. It does not directly apply to living animals and does not include the steps of comparing the detected biological information with benchmark data or drawing specific diagnostic conclusions based on the biological detection information. Therefore, this invention does not belong to the diagnostic methods for diseases and meets the basic requirements of the Patent Law for the subject matter of patent protection.

[0038] This invention provides a method for identifying three emerging infectious disease viruses in chickens. Based on the overall concept of this invention, it specifically discloses primer and probe compositions, kits, detection methods, and applications for identifying these three viruses. The primer and probe compositions include three sets of primers and probes corresponding to the three viruses. Each set of primers contains an inner primer, an outer primer, a loop primer, and a probe. Their nucleotide sequences are shown in SEQ ID No. 1-24 of the sequence listing. The 5' end of the inner primer is labeled with a quenching group, and the 3' end of the probe is labeled with a fluorescent group. The nucleotide sequences of the inner primer and the corresponding probe have complementary pairing regions, allowing them to combine to form a non-luminescent fluorescent quenched composite probe. A fluorescent annealing composite probe is prepared before the reaction. The reaction procedure is: amplification at 63℃ for 60 minutes, followed by inactivation at 85℃ for 5 minutes, and then termination of the reaction. The kit includes WarmStart LAMP color-changing premix, standards, and Bst 2.0 WarmStart DNA polymerase. The primer and probe compositions, detection methods, and kits described herein can be used to identify emerging infectious diseases in chickens.

[0039] In a specific embodiment of the present invention, the sample to be tested is specifically a cotton swab from the oral cavity and cloaca. The nucleic acid extracted from the sample, the virus to be tested, or the pathogen to be tested is obtained using an RNA / DNA co-extraction kit. The nucleic acid can be DNA, RNA, or a mixture of DNA and RNA. When the nucleic acid contains RNA, the RNA is first reverse transcribed into cDNA. The prepared cDNA / DNA template is stored at -30°C for later use in the LAMP amplification reaction. During the LAMP amplification reaction, the primer set includes outer primers, inner primers, loop primers, and probes. The outer primers are named with the suffixes F3 and B3, the inner primers with the suffixes FIP and BIP, the loop primers with the suffixes Floop and Bloop, and the probes with the suffixes FD and BD. Each primer set is named according to the different virus types: CHPV, CIAV, and FADV4.

[0040] The following describes specific embodiments of the method for identifying three emerging infectious disease viruses in chicken flocks according to the present invention. The details are as follows:

[0041] Example 1: Design of primer and probe compositions

[0042] Based on the conserved regions of the NS gene sequence of chicken parvovirus (CHPV), the VP1 gene sequence of chicken infectious anemia virus (CIAV), and the Hexon gene sequence of avian adenovirus serotype 4 (FADV4) in GenBank, three sets of primer and probe compositions targeting these three emerging infectious disease viruses in chicken flocks were designed. Each primer set includes outer primers F3 and B3, inner primers FIP and BIP, loop primers Floop and Bloop, and probes FD and BD. The primer sequences are detailed in SEQ ID NO:1-24 in the sequence listing. The inner primers FIP and BIP and the corresponding probes FD and BD have complementary nucleotide sequences, which can pair and bind after annealing to form non-luminescent fluorescent quenched composite probes. The 5' ends of the inner primers FIP and BIP are labeled with BHQ series fluorescent quenching groups, and the 3' ends of the probes FD and BD are labeled with three different fluorescent groups: Alexa Fluor 488, Cy5, and CY3. The primer and probe compositions were synthesized by Baori Biotechnology Co., Ltd.

[0043] Example 2: Establishment of a triple fluorescence LAMP detection platform for identifying three emerging infectious disease viruses in chicken flocks.

[0044] 2.1 Template Extraction

[0045] RNA and / or DNA were extracted from 26 different viral strains and 70 clinical samples according to the instructions in the EasyPure Viral DNA / RNA Kit. The RNA viruses were first reverse transcribed into cDNA, and the cDNA / DNA templates were stored at -30°C for later use.

[0046] 2.2 Standard Products

[0047] The NS gene from CHPV, the VP1 gene from CIAV, and the Hexon gene from FAVD-4 were cloned into the PMD-18T vector, respectively, to obtain PMD-18T-NS, PMD-18T-VP1, and PMD-18T-Hexon recombinant plasmids. Plasmids from positive recombinant bacteria were extracted using a plasmid micro-extraction kit. The plasmid concentration was determined using a NanoDrop-2000 nucleic acid detector, and the concentration was converted to copy number based on the Avogadro number: copy number (copies / μL) = plasmid concentration (g / μL) × 10⁻⁶. -9×6.02×1023 / 660×total plasmid length. Dilute the calculated copy number of plasmid DNA for each virus to 3.3×10⁻⁶. 9 Three different concentrations of standards corresponding to the three viruses were obtained by mixing ~1 copies / μL; then, plasmid DNA of the same concentration was used to prepare equal volumes of two-mix and three-mix standards according to the design, finally preparing standards with different concentrations of DNA single sample, two-mix sample, and three-mix standards, with a DNA concentration of 1×10⁻⁶. 9 1 copy / μL - 1 copy / μL, store at -30℃ for later use.

[0048] 2.3 Reaction System

[0049] Before the reaction, a fluorescence quenching annealing composite probe was prepared: 50 μM FIP and 50 μM FD were mixed, and 50 μM BIP and 50 μM MBD were mixed. The mixture was heated to 98 °C for 5 minutes, and then slowly cooled to room temperature to complete the annealing. The probe was then stored at -20 °C for later use. Following the above method, CHPV-FIP-FD and BIP-BD annealing composite probes, FADV4-FIP-FD and BIP-BD annealing composite probes, and CIAV-FIP-FD and BIP-BD annealing composite probes were obtained, respectively.

[0050] Preparation of the reaction system: The reaction system for multiplex RT-LAMP was set at 20 μL, including 1 μL template, 10 μL WarmStart 2× premix, 16 U Bst 2.0 WarmStart DNA polymerase, 0.66 μM CHPV-FIP, 0.66 μM MCIAV-FIP, 0.66 μM FADV4-FIP, 0.66 μM CHPV-FIP-FD annealing probe (containing 0.33 μM CHPV-FIP and 0.33 μM fluorescent CHPV-FD), 0.66 μM CIAV-FIP-FD annealing probe (containing 0.33 μM CIAV-FIP and 0.33 μM MCIAV-FD), 0.66 μM FADV4-FIP-FD annealing probe (containing 0.33 μM FADV4-FIP and 0.33 μM FADV4-FD), 0.66 μM CHPV-BIP, and 0.66 μM... CIAV-BIP, 0.66μM FADV4-BIP, 0.66μM CHPV-BIP-BD annealed composite probe (containing 0.33μM CHPV-BIP and 0.33μM CHPV-BD), 0.66μM CIAV-BIP-BD annealed composite probe (containing 0.33μM CIAV-BIP and 0.33μM CIAV-BD), 0.66μM FADV4-BIP-BD annealed composite probe (containing 0.33μM FADV4-BIP and 0.33μM FADV4-BD), 0.083μM CHPV-F3, 0.083μM CIAV-F3, 0.083μM FADV4-F3, 0.083μM CHPV-B3, 0.083μM CIAV-B3, 0.083μM FADV4-B3, 0.17μM CHPV-Floop, 0.17μM MCIAV-Floop, 0.17μM FADV4-Floop, 0.17μM CHPV-Bloop, 0.17μM CIAV-Bloop, 0.17μM MADV4-Bloop, with the remainder being water.

[0051] 2.4 Reaction Conditions

[0052] Place the prepared 20 μL reaction tube in a water bath (or LA-320C real-time turbidimeter), react at 63℃ for 60 min, and inactivate at 80℃ for 5 min to complete the LAMP extension amplification reaction.

[0053] 2.5 Result Judgment

[0054] After the experiment, the amplification results of this invention include two determination methods.

[0055] Method 1 for determining the result: This is a visually perceptible positive / negative result determination. Based on the magnesium phosphate byproduct of LAMP amplification, the result is determined visually according to the LAMP color-changing premix kit instructions: the LAMP reaction product changes color from pink to yellow, indicating a positive result; if no color change occurs, the result is negative. This method eliminates the need for electrophoresis and the addition of fluorescent agents, reducing laboratory contamination from reaction products. The result is determined by the color reaction of the product, significantly shortening the detection time. Furthermore, the LA-320C real-time turbidimeter provides a graph where the horizontal axis represents reaction time and the vertical axis represents turbidity intensity (i.e., the amount of white precipitate of magnesium pyrophosphate, a byproduct of RT-LAMP). This allows observation of the real-time amplification peak of positive LAMP amplification, identifying the earliest entry into the exponential growth phase, and optimizing specific reaction conditions such as LAMP reaction temperature and time. However, this method cannot distinguish which primers triggered the positive amplification of which type of viral nucleic acid, i.e., it cannot identify the positive pathogen.

[0056] Result determination method two: Use an image analyzer to analyze the triple fluorescence colorimetric results. In this invention, the 3' ends of the probes corresponding to CHPV, CIAV, and FADV4 are labeled with Alexa Fluor 488, Cy5, and CY3, respectively. Therefore, CHPV positive (Alexa Fluor 488 labeled) appears green in channel 520, CIAV positive (Cy5 labeled) appears red in channel 670, and FADV4 positive (CY3 labeled) appears blue in channel 570 (CY3's original color is orange, but the image analyzer assigns it blue, which does not affect the result interpretation). This method interprets the positive results of the three specific fluorescent signals by detecting the color of the fluorescent group carried by the reaction tube in the corresponding channel of the image analyzer. Based on the different fluorescence acceptance in different channels, it can intuitively distinguish which type of viral nucleic acid caused the positive result by which set of primers. That is, it can clearly distinguish whether the positive result of identifying the three emerging infectious disease viruses in chicken flocks is caused by one or more specific virus types of CHPV, CIAV, and FADV4, thereby enabling the joint detection of three target viruses using LAMP technology in the same reaction tube.

[0057] Example 3: Performance verification of a method for identifying three emerging infectious disease viruses in chicken flocks

[0058] 3.1 Specificity

[0059] A 20 μL reaction system was prepared using a reaction temperature of 63 °C, the concentration of the primer and probe combination described above, and the determined ratio. Single or mixed templates of CHPV, CIAV, and FADV4, as well as nucleic acid samples of other common avian control viruses AIV, NDV, IBV, ARV, AILTV, APV, and MDV, were amplified at an isothermal temperature for 65 minutes to verify their specificity. The 26 pre-extracted viral strains are: CHPV strains: KX084399, KX084401, KX133426, KU523900, KX133415, KX133418; CIAV strains: MN649254, MN649256, MK484614, MN103402, MN103404; FADV4 strains: MN577977, MN577978, MN577984, MW439040, MW439043; AIV strains: H3N2, H6N6, and H9N2; NDV strains: F48E9 and LaSota; IBV strain: Mass 41; ARV strain: S1133; AILTV Beijing strain; and APV strains. Using cDNA or DNA from MN-10 strain and MDVCVI988 strain as templates, and RNase-free water as a negative control, the specificity of the reaction system was verified after the reaction was completed.

[0060] like Figure 1 The results of the specificity verification test are shown below: All 15 labeled reaction tubes were monitored using a real-time turbidimeter (multi-channel). The results, as shown in the multi-channel display, show that only reaction tubes containing one or more of CHPV, CIAV, and FADV4 (labeled 1-7) exhibited significant amplification and were positive. Reaction tubes labeled 1-7, representing common avian control viruses and negative controls, were negative. This demonstrates that the present invention can identify three emerging infectious disease viruses in chickens (CHPV, CIAV, FADV4) without cross-reactivity to common avian viruses other than these three.

[0061] Using an image analyzer, the color interpretation results of the fluorescent groups carried by the reaction tubes were detected in the corresponding channels. The three target viruses in the mixed sample were accurately distinguished, and their corresponding colors were displayed in the appropriate channels. Specifically, six CHPV strains (channel 520) isolated from Guangxi, five CIAV strains (channel 670) isolated from Guangxi, and five FADV4 strains (channel 570) all showed positive amplification. Simultaneously, no non-specific fluorescent positive information was found in any of the three channels for other control viruses (AIV, NDV, IBV, ARV, AILTV, APV, and MDV) and the negative control tube; all detection results were negative. Therefore, this result indicates that the triple fluorescence method established in this invention can not only identify three emerging infectious disease viruses in chickens but also distinguish which of CHPV, CIAV, or FADV4, or a mixture of them, exhibiting good specificity.

[0062] 3.2 Sensitivity

[0063] A 20 μL reaction system was prepared using a reaction temperature of 63 °C, the concentration of the primer and probe composition described above, and the determined ratio. A mixed sample of pre-prepared standards of different concentrations was amplified and reacted for 65 minutes to verify its sensitivity.

[0064] A real-time turbidimeter (multi-channel) and an image analyzer (including 520 channels for green, 670 channels for red, and 570 channels for blue) were used to monitor the sensitivity of all nine labeled reaction tubes in their respective channels. The results are as follows: Figure 2 As shown: Samples 1-8 are mixtures of CHPV, CIAV, and FADV4 viral standards with different DNA concentrations in equal proportions, where: 1 is 1×10 7 copies / μl, 2 is 1×10 6 copies / μl, 3 is 1×10 5 copies / μl, 4 is 1×10 4 copies / μl, 5 is 1×10 3 copies / μl, 6 is 1×10 2 copies / μl, 7 is 1×10 1Sample 8 was 1 copy / μl, and sample 9 was a negative control (ddH2O). Results showed that, except for the negative control (sample 9), all other samples (samples 1-8) were positive. The corresponding lowest concentration tube, sample 8, represents the lowest detection limit for CHPV, CIAV, and FADV4. The DNA concentration of each viral standard in the equal-proportion mixture of these three viral standards in this tube was 1 copy / μl. Therefore, the sensitivity of this method for the three viral DNA samples is 1 copy / μl. This means that the triple fluorescence method for identifying three emerging infectious diseases in chicken flocks can detect at least one copy of viral DNA per reaction, demonstrating high sensitivity.

[0065] 3.3 Interference

[0066] A 20 μL reaction system was prepared using a reaction temperature of 63 °C, the concentration of the primer and probe composition described above, and the determined ratio. Mixed samples of different concentrations of three pre-prepared viral plasmid DNA standards were amplified and reacted for 65 minutes to verify whether the high concentration of target nucleic acid when different viruses are mixed would inhibit the specific amplification process of the low concentration of target nucleic acid, that is, to verify whether there is interference between different concentrations of target nucleic acid.

[0067] A real-time turbidimeter (multi-channel) and an image analyzer (including 520 channels for green, 670 channels for red, and 570 channels for blue) were used to monitor the interference amplification of all eight labeled reaction tubes in their respective channels. The results are as follows: Figure 3 As shown: Samples 1-8 represent individual standard samples corresponding to the three viruses mixed at different concentrations to form mixtures of the three viral target nucleic acids at different concentrations, simulating the preparation of mixed infection samples at different concentrations. Specifically, 1 represents 10... 2 CHPV+10 2 CIAV+10 9 FADV4,2 is 10 2 CHPV+10 3 CIAV+10 8 FADV4,3 is 10 2 CHPV+10 4 CIAV+10 7 FADV4, 4 is 10 2 CHPV+10 5 CIAV+10 6 FADV4,5 is 10 2 CHPV+10 6 CIAV+10 5 FADV4,6 is 10 2 CHPV+10 7 CIAV+10 4FADV4, 7 is 10 2 CHPV+10 8 CIAV+10 3 FADV4, 8 is 10 2 CHPV+10 9 CIAV+10 2 FADV4, in which the concentration units of the three virus standards were all copies / μl, and no negative control was set. The results showed that all tests labeled 1-8 were positive, and there were no significant differences in positive results between different channels or between different concentration ratios.

[0068] Theoretically, when multiple sets of primers compete simultaneously in a reaction tube, a high concentration of the target nucleic acid template may rapidly acquire components of the reaction system at the start of the reaction, thereby inhibiting the specific amplification of a low concentration of the target nucleic acid template. However, the standard constructed in this invention simulates test samples infected with mixed viral loads at different concentrations, even when the highest concentration of a certain virus reaches 10... 9 Copies / μl will not inhibit the minimum concentration of 10 in the template. 2 Specific amplification of copies / μl, although the concentration ratio between different viruses is as high as 10. 7 :1. However, experiments have shown that no interference or inhibition was observed between the primer and probe compositions, reaction systems, and reaction conditions described in this invention, and between the target nucleic acid mixtures of different concentrations of viruses. In other words, the method for identifying three emerging infectious disease viruses in chicken flocks described in this invention has strong anti-interference capabilities.

[0069] 3.4. Clinical Sample Consistency Verification

[0070] A 20 μL reaction system was prepared using a reaction temperature of 63°C, the concentration of the primer and probe composition described above, and the determined ratio. cDNA / DNA from 70 pre-prepared clinical samples from Example 2 was amplified, and the reaction time was 65 minutes to verify the consistency between real clinical samples and classic PCR sequencing results, and the reliability of this method. A real-time turbidimeter (multi-channel) and an image analyzer (containing 520 green channels, 670 red channels, and 570 blue channels) were used to monitor the interference amplification in all eight labeled reaction tubes under the corresponding channels. Samples with positive results from triple fluorescence LAMP were sent for sequencing identification. The results are shown in Table 2 below: 14 samples were CHPV positive (positive rate 20.0%), 22 samples were CIAV positive (31.4%), 6 samples were FADV4 positive (8.5%), 4 samples were positive for CHPV+CIAV mixed infection (5.7%), and 1 sample was positive for CHPV+FADV4 mixed infection (1.4%). The positive results identified by triple fluorescence LAMP were confirmed by PCR sequencing. Results: The detection results of the method for identifying three emerging infectious disease viruses in chicken flocks described in this invention were confirmed by the classic PCR sequencing method. The positive results were completely consistent, with a positive concordance rate of 100%, and the consistency results were good.

[0071] Table 2 Comparison of PCR sequencing results and triple fluorescence LAMP identification results

[0072]

[0073] Note: The clinical samples were 70 cotton swabs from the oral cavity and cloaca of chickens collected from the Nanning live poultry market. The cDNA / DNA was obtained after nucleic acid extraction. The blank space in the lower left corner of the table represents duplicate content, and the results are not marked.

[0074] This invention discloses a method for identifying three emerging infectious disease viruses in chickens. The method fully discloses the LAMP primer sets, standards, kits, detection methods, and applications used. It includes three sets of primer and probe compositions corresponding to the three viruses. The inner primers are labeled with quenching groups, and the probes are labeled with three different colored fluorescent groups. The nucleotide sequences of the inner primers and corresponding probes have complementary pairing regions. After high-temperature annealing, they can pair and bind to form non-luminescent fluorescent quenched composite probes. After LAMP isothermal amplification and termination, the results can be obtained through a single LAMP reaction in a single reaction tube. Two result observation methods are used: a real-time turbidimeter (multi-channel) and an image analyzer (including 520 channels of green, 670 channels of red, and 570 channels of blue). This allows for real-time quantitative observation of positive results and differentiation of positive pathogens. After performance verification and clinical sample validation, the method demonstrates high specificity, high sensitivity, and low interference. It can detect as little as one mixed template copy / reaction, effectively suppressing false positive results and exhibiting good clinical detection performance. The detection method provided by this invention is simple, fast, requires no expensive instruments, and is low in cost. It can realize on-site detection of pathogens and is suitable for large-scale epidemiological surveys of CHPV, CIAV, and FADV4 in chicken flocks.

[0075] In actual research, at least two sets of primers were designed based on the conserved regions of each virus (inappropriate primer sequences will not be described in detail here). In addition to ensuring a certain level of sensitivity and specific positive amplification results for the DNA of the three viruses CHPV, CIAV, and FADV4, it is also necessary to ensure that there is no cross-interference between the primer sets of the three selected viruses. During the research and development process, primer and probe sequences for primer and probe compositions targeting the purpose of this invention were finally screened through different combinations. Furthermore, a series of technical problems encountered in triple fluorescence LAMP detection reactions, such as the limitation of fluorescence wavelength range by the fluorescence imaging analyzer, the inhibition of the reaction by the fluorescence quenching composite probe, and the increased competition between primers and probes for the reaction system, were solved. Therefore, the beneficial effects described in this invention were obtained through extensive creative work in numerous experimental designs and verification processes. Specificity verification with clinical samples has also proven that the primer and probe compositions, kits, and detection methods described in this invention have good specificity, sensitivity, and anti-interference properties. They can be used for initial screening of emerging infectious diseases in chicken flocks, and negative results can also serve as a reference for the exclusion of these three viral infection sources. In the future, they can also be extended to reference research on joint identification platforms for other three viruses.

[0076] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, various modifications or substitutions may be made to those details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof. sequence list <110> Guangxi Zhuang Autonomous Region Veterinary Research Institute <120> A method for identifying three emerging infectious disease viruses in chicken flocks <130> 20211208 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 agaggaggaa cccccctat 19 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 cgcttgcggt gaagtctg 18 <210> 3 <211> 40 <212> DNA <213> Artificial sequence <400> 3 acggggataaa tccctgggac cttggttctg aatccgggct 40 <210> 4 <211> 42 <212> DNA <213> Artificial sequence <400> 4 acagagacca tcgagctcct gggctcgtct ggaaatccac tc 42 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <400> 5 tcttaccttc gttggctttt tcaa 24 <210> 6 <211> twenty two <212> DNA <213> Artificial sequence <400> 6 agacggagat cctcagcgaa tc 22 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <400> 7 aggtcccagg gatttatccc gt 22 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <400> 8 ccaggagctc gatggtctct gt 22 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <400> 9 aggccaccaa caagttcac 19 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 ggttgatcgg tcctcaagtc 20 <210> 11 <211> 40 <212> DNA <213> Artificial sequence <400> 11 gcagccacac agcgatagag tgccgttgga aacccctcac 40 <210> 12 <211> 40 <212> DNA <213> Artificial sequence <400> 12 cgcgctccca cgctaagatc cggcacattc ttgaaaccag 40 <210> 13 <211> 25 <212> DNA <213> Artificial sequence <400> 13 attgtaattg cagcgatacc aatcc 25 <210> 14 <211> twenty two <212> DNA <213> Artificial sequence <400> 14 actgcggaca attcagaaag ca 22 <210> 15 <211> twenty two <212> DNA <213> Artificial sequence <400> 15 cactctatcg ctgtgtggct gc 22 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 gatcttagcg tgggagcgcg 20 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <400> 17 gaggtgaacc tcatggcc 18 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <400> 18 ttgatgcgag tgaaggacc 19 <210> 19 <211> 42 <212> DNA <213> Artificial sequence <400> 19 tggtggcgtt tctcagcatc agacttcatg cccatggatc ac 42 <210> 20 <211> 39 <212> DNA <213> Artificial sequence <400> 20 acgctctata ctcggtgccc ggtgcgagcg ggaatgttg 39 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <400> twenty one ctcgagctgg ttactggtgt tg 22 <210> twenty two <211> 18 <212> DNA <213> Artificial sequence <400> twenty two ctccaccgcc ctcaccat 18 <210> twenty three <211> twenty two <212> DNA <213> Artificial sequence <400> twenty three ctgatgctga gaaacgccac ca 22 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <400> twenty four cgggcaccga gtatagagcg t 21

Claims

1. A primer and probe composition, characterized in that: It includes 3 sets of primers and probes, namely: The outer primers CHPV-F3 and CHPV-B3, the inner primers CHPV-FIP and CHPV-BIP, the fluorescent quenching composite probes CHPV-FIP-FD and CHPV-BIP-BD, and the loop primers CHPV-Floop and CHPV-Bloop were designed based on the NS gene sequence of chicken parvovirus. The outer primers CIAV-F3 and CIAV-B3, the inner primers CIAV-FIP and CIAV-BIP, the fluorescent quenching composite probes CIAV-FIP-FD and CIAV-BIP-BD, and the loop primers CIAV-Floop and CIAV-Bloop were designed based on the VP1 gene sequence of chicken infectious anemia virus. The outer primers FADV4-F3 and FADV4-B3, the inner primers FADV4-FIP and FADV4-BIP, the fluorescent quenching composite probes FADV4-FIP-FD and FADV4-BIP-BD, and the loop primers FADV4-Floop and FADV4-Bloop were designed based on the Hexon gene sequence of avian adenovirus serotype 4. The fluorescence quenching composite probe CHPV-FIP-FD is formed by pairing and binding the inner primer CHPV-FIP and the probe CHPV-FD, which has a complementary region to the inner primer CHPV-FIP, after fluorescence quenching annealing. The fluorescence quenching composite probe CHPV-BIP-BD is formed by pairing and binding the inner primer CHPV-BIP and the probe CHPV-BD, which has a complementary region to the inner primer CHPV-BIP, after fluorescence quenching annealing. The fluorescence quenching composite probe CIAV-FIP-FD is formed by pairing and combining the inner primer CIAV-FIP and the probe CIAV-FD, which has a complementary region to the inner primer CIAV-FIP, after fluorescence quenching annealing. The fluorescence quenching composite probe CIAV-BIP-BD is formed by pairing and combining the inner primer CIAV-BIP and the probe CIAV-BD, which has a complementary region to the inner primer CIAV-BIP, after fluorescence quenching annealing. The fluorescence quenching composite probe FADV4-FIP-FD is formed by pairing and binding the inner primer FADV4-FIP and the probe FADV4-FD, which has a complementary region to the inner primer FADV4-FIP, after fluorescence quenching annealing. The fluorescence quenching composite probe FADV4-BIP-BD is formed by pairing and combining the inner primer FADV4-BIP and the probe FADV4-BD, which has a complementary region to the inner primer FADV4-BIP, after fluorescence quenching annealing. The nucleotide sequences of the outer primers CHPV-F3 and CHPV-B3, the inner primers CHPV-FIP and CHPV-BIP, the loop primers CHPV-Floop and CHPV-Bloop, the probes CHPV-FD and CHPV-BD, the outer primers CIAV-F3 and CIAV-B3, the inner primers CIAV-FIP and CIAV-BIP, the loop primers CIAV-Floop and CIAV-Bloop, the probes CIAV-FD and CIAV-BD, the outer primers FADV4-F3 and FADV4-B3, the inner primers FADV4-FIP and FADV4-BIP, the loop primers FADV4-Floop and FADV4-Bloop, and the probes FADV4-FD and FADV4-BD are as shown in SEQ ID NO:1-24 in the sequence listing. The 5' ends of the inner primers CHPV-FIP and CHPV-BIP, CIAV-FIP and CIAV-BIP, and FADV4-FIP and FADV4-BIP sequences are labeled with quenching groups. The 3' ends of the probe sequences CHPV-FD and CHPV-BD, CIAV-FD and CIAV-BD, and FADV4-FD and FADV4-BD are labeled with different fluorescent groups.

2. The primer and probe composition according to claim 1, characterized in that: The quenching group is a BHQ series fluorescent quenching group, and the fluorescent group is Alexa Fluor 488, Cy5 and CY3.

3. A method for identifying three emerging infectious disease viruses in chicken flocks, characterized in that: The newly emerging infectious disease viruses in the chicken flock are chicken parvovirus, chicken infectious anemia virus, and avian adenovirus serotype 4; The method comprises the primer and probe composition according to any one of claims 1-2 participating in the reaction; the steps include: Preparation of fluorescence quenching composite probes: Before the multiplex LAMP reaction, 50 μM FIP and 50 μM FD, 50 μM BIP and 50 μM BD were mixed separately, heated to 98 °C for 5 minutes, and slowly cooled to room temperature to complete the annealing. They were then stored at -20 °C for later use. Following the above method, the FIP-FD and BIP-BD fluorescence quenching composite probes corresponding to CHPV, CIAV and FADV4 were obtained respectively. Preparation of the reaction system: A 20 μL multiplex LAMP reaction system was prepared, including 1 μL template, 10 μL WarmStart 2× premix, 16 U Bst 2.0 WarmStart DNA polymerase, 0.66 μM CHPV-FIP, 0.66 μM CIAV-FIP, 0.66 μM FADV4-FIP, 0.66 μM CHPV-FIP-FD fluorescence quenching composite probe, 0.66 μM CIAV-FIP-FD fluorescence quenching composite probe, 0.66 μM FADV4-FIP-FD fluorescence quenching composite probe, 0.66 μM CHPV-BIP, 0.66 μM CIAV-BIP, 0.66 μM FADV4-BIP, 0.66 μM CHPV-BIP-BD fluorescence quenching composite probe, 0.66 μM CIAV-BIP-BD fluorescence quenching composite probe, and 0.66 μM... FADV4-BIP-BD fluorescence quenching composite probe, 0.083μM CHPV-F3, 0.083μM MCIAV-F3, 0.083μM FADV4-F3, 0.083μM CHPV-B3, 0.083μM CIAV-B3, 0.083μM FADV4-B3, 0.17μM CHPV-Floop, 0.17μM CIAV-Floop, 0.17μM MADV4-Floop, 0.17μM CHPV-Bloop, 0.17μM CIAV-Bloop, 0.17μM MADV4-Bloop, balance: water; The reaction process includes an extension reaction at 63℃ for 60 minutes and a termination reaction at 80℃ for 5 minutes to inactivate the virus. Results detection: This includes interpreting results in two ways using a real-time turbidimeter and an image analyzer across multiple fluorescence channels; The method described is not intended for the diagnosis or treatment of diseases.

4. A kit for identifying three emerging infectious disease viruses in chicken flocks, characterized in that: It comprises the primer and probe composition as described in any one of claims 1-2, WarmStart LAMP color-changing premix, standards, negative controls, and DNA polymerase.

5. The kit for identifying three emerging infectious disease viruses in chicken flocks according to claim 4, characterized in that: The DNA polymerase used is Bst 2.0 WarmStart DNA polymerase, with a dosage of 16 U per 20 μL reaction system.

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