Primer for detecting and distinguishing genotype of infectious bronchitis virus and application

Abstract

The invention discloses a primer for detecting and distinguishing genotypes of avian infectious bronchitis viruses and application, and belongs to the technical field of biological detection. The primer combination comprises a universal primer and a probe group I for detecting an IBV total group, a specific primer and a probe combination II for detecting a GI-19 genotype, and a specific primer and a probe combination III for detecting a GVI-1 genotype, and nucleotide sequences are respectively shown as SEQ ID NO.1-9; different fluorophores are respectively marked at 5'ends of each group of probes, and corresponding quenching groups are marked at 3 'ends of each group of probes. The primer combination provided by the invention has high specificity and high sensitivity, rapid detection of the IBV and accurate distinguishing of main epidemic genotypes (GVI-1 and GI-19) can be completed in a single reaction, complex sequencing and analysis steps in a traditional method are omitted, the detection efficiency is greatly improved, and the primer combination has a good application prospect. And a convenient tool is provided for farm epidemic situation monitoring and clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a triple fluorescent RT-PCR primer combination and its application for detecting chicken infectious bronchitis virus and distinguishing between GI-19 and GVI-1 genotypes. Background Technology

[0002] Infectious bronchitis (IB) is an acute and highly contagious viral disease that can affect chickens of all ages. The main symptoms include tracheal rales, coughing, and sneezing. It often leads to decreased growth performance, increased mortality, and reduced egg production and shell quality in laying hens and breeder chickens. This disease causes severe economic losses to the poultry industry and is one of the most significant infectious diseases in poultry farming. Currently, IB has become a global disease, widely distributed worldwide, and poses a major challenge to the global poultry industry.

[0003] Infectious Bronchitis Virus (IBV) is the pathogen that causes IB, belonging to the family Coronaviridae and the genus Gamma-coronavirus. The IBV genome is a single-stranded positive-sense RNA, approximately 27.6 kb in length. IBV exhibits extremely high variability, resulting in a complex and diverse range of serotypes and genotypes, posing significant challenges to vaccine immunization and clinical control. Therefore, monitoring the epidemiology and molecular genetic variations of IBV has become an important component of effective IBV control.

[0004] The IBV genome is transcribed into six subgenomic mRNAs (mRNA1-mRNA6) within the cell via a discontinuous transcription mechanism. These mRNAs contain at least 10 open reading frames (ORFs), encoding IBV polyproteins, structural proteins, and accessory proteins. Among these, the spike protein (S) encoded by mRNA2 binds to the viral receptor on the host cell membrane and is cleaved by host cell proteases into the N-terminal S1 protein and the C-terminal S2 protein. The S1 protein is the main protein responsible for IBV infectivity, pathogenicity, and tissue tropism. Different IBV strains exhibit variations in these proteins. S1 Significant genetic variation exists. Currently, based on... S1 Phylogenetic analysis of genes is an important basis for the classification of IBV genotypes. According to the internationally accepted classification standards, IBV can be divided into 9 genotypes GI-GIX, among which the GI genotype contains 30 lineages (GI-1 to GI-30). Currently, GI-19 (also known as QX type) and GVI-1 type are the most widespread and harmful in China, and have become the two IBV genotypes that need to be monitored in the breeding process.

[0005] Traditional IBV detection methods include virus isolation and identification, serological testing, and conventional PCR. However, these methods are cumbersome, time-consuming, have low throughput, and cannot quickly distinguish between different IBV genotypes. In recent years, fluorescent RT-PCR has been widely used in veterinary clinical pathogen detection due to its advantages of high specificity, sensitivity, and high throughput. Existing studies have developed fluorescent RT-PCR analysis methods targeting the universal IBV sequence, such as the TaqMan real-time RT-PCR method developed by Callison et al., which targets the 5′-UTR region of IBV to achieve detection of universal IBV types. Some studies have also achieved specific detection of single genotypes, such as patent document CN111471801A, which discloses the use of fluorescent RT-PCR to detect TC07-2 type IBV.

[0006] Currently, there is a lack of multiplex fluorescent RT-PCR methods capable of simultaneously detecting IBV and rapidly distinguishing its main prevalent genotypes, GI-19 and GVI-1, especially regarding GVI-1 genotyping, which has relatively few reported studies. Therefore, it is of great significance to develop a rapid, sensitive, specific, high-throughput, and genotyping-capable multiplex fluorescent RT-PCR technique. Summary of the Invention

[0007] The purpose of this invention is to provide a detection method that can rapidly, sensitively, and specifically detect IBV and differentiate between the major circulating genotypes GI-19 and GVI-1. This method is suitable for rapid on-site screening, epidemiological investigations, and virus typing studies.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention downloads all 805 publicly available IBV whole-genome sequences from the NCBI viruses database. After alignment using a multiple sequence alignment algorithm, a program is designed to enumerate highly conserved regions within the genome. While meeting the primer design criteria for quantitative real-time PCR, target regions are screened with the goal of maximizing theoretical coverage. Through multi-target specificity analysis and synergistic optimization of primer-probe interactions, the optimal primer and probe sequence combinations across the entire genome are designed and screened. Similarly, 234 IBV GI-19 subtype sequences and 47 S1 gene sequences of the GVI-1 subtype published on NCBI are downloaded, and the optimal primer and probe sequence combinations within the S1 gene range are designed for each. Using these primer and probe combinations, IBV detection and identification of the GI-19 or GVI-1 genotype are simultaneously achieved in a single quantitative real-time PCR reaction system.

[0009] Specifically, the present invention provides a triple fluorescent RT-PCR primer set for detecting IBV and distinguishing between GI-19 and GVI-1 genotypes. The primer set includes a universal primer and probe set I for detecting the IBV total population, a specific primer and probe set II for detecting the GI-19 genotype, and a specific primer and probe set III for detecting the GVI-1 genotype. The universal primer and probe set I for detecting the total IBV population includes an upstream primer IBV-F, a downstream primer IBV-R, and a probe IBV-P. The nucleotide sequence of the upstream primer IBV-F is: 5′-CATCCGTTGCTTGGGCTACCTAG-3′ (SEQ ID NO. 1); the nucleotide sequence of the downstream primer IBV-R is: 5′-GCCATGTTGTCACTGTCTATTGTATGTCTG-3′ (SEQ ID NO. 2); and the nucleotide sequence of the probe IBV-P is: 5′-CTAAACACCACCAGAACCTGTCACCTCAGGGGTTG-3′ (SEQ ID NO. 3). The specific primer and probe combination II for detecting the GI-19 genotype includes an upstream primer GI-19-F, a downstream primer GI-19-R, and a probe GI-19-P. The nucleotide sequence of the upstream primer GI-19-F is: 5′-TTATGTGTACTACTACCAAAGTGCCTTTAGGC-3′ (SEQ ID NO.4); the nucleotide sequence of the downstream primer GI-19-R is: 5′-ACCTGCTTTAAAATACACACCTGCTGAC-3′ (SEQ ID NO.5); and the nucleotide sequence of the probe GI-19-P is: 5′-TTACAGTGTGCACTACAAAATTGTG-3′ (SEQ ID NO.6). The specific primer and probe combination III for detecting the GVI-1 genotype includes an upstream primer GVI-1-F, a downstream primer GVI-1-R, and a probe GVI-1-P. The nucleotide sequence of the upstream primer GVI-1-F is 5′-ATGGTGGTGCGTATGCAGTAGAAC-3′ (SEQ ID NO.7); the nucleotide sequence of the downstream primer GVI-1-R is 5′-TGTAACAAAAACAACAATTGTACTAAAATTGCAATG-3′ (SEQ ID NO.8); and the nucleotide sequence of the probe GVI-1-P is 5′-TGCAAGTGCAGTTGCGATTACTACACCTGTTAATGGCATG-3′ (SEQ ID NO.9). The 5′ ends of probes IBV-P, GI-19-P, and GVI-1-P are each labeled with different fluorescent groups, and their 3′ ends are labeled with quenching groups corresponding to the fluorescent groups.

[0010] The target region detected by the primer and probe combination provided in this invention is obtained based on the whole genome screening of global IBV, achieving the highest coverage among all candidates. Validated through clinical samples, the primer and probe combination provided in this invention exhibits good performance and can specifically distinguish the target lineage. The constructed triplet real-time PCR reaction system is stable, with no primer interactions and no byproducts such as dimers or hairpin structures generated.

[0011] In this invention, one end of the probe is labeled with a fluorescent group, and the other end with a quencher group. When the probe is intact, the energy of the fluorescent group is absorbed by the quencher group, and no fluorescent signal is generated. During the amplification reaction, the probe specifically binds to the target sequence. Under the action of DNA polymerase 5′→3′ exonuclease activity, the probe is cleaved, the fluorescent group separates from the quencher group, and the fluorescent group releases and emits a fluorescent signal of a specific wavelength. A real-time quantitative PCR instrument monitors each fluorescent signal through different optical channels, achieving real-time monitoring of the corresponding amplification products. In this invention, three sets of probes (IBV-P, GI-19-P, and GVI-1-P) are labeled with three fluorescent groups with high spectral resolution, thereby achieving multiplex detection in the same fluorescent RT-PCR system.

[0012] Preferably, the probe IBV-P has a FAM group labeled at its 5′ end and a BHQ1 group labeled at its 3′ end; the probe GI-19-P has a VIC group labeled at its 5′ end and an MGB group labeled at its 3′ end; and the probe GVI-1-P has a ROX group labeled at its 5′ end and a BHQ2 group labeled at its 3′ end. These three sets of fluorescent labels offer high spectral separation, facilitating simultaneous detection in different channels, avoiding signal interference, and providing good instrument compatibility, sensitivity, and stability. Matching BHQ1, MGB, and BHQ2 with the fluorescent groups effectively quenches background signals, improves specificity and signal-to-noise ratio, and MGB also increases the probe's melting temperature, ensuring good specificity even when identifying short target sequences.

[0013] Another object of the present invention is to provide a kit for detecting IBV and distinguishing between GI-19 and GVI-1 genotypes, the kit comprising the aforementioned triple fluorescent RT-PCR primer combination.

[0014] Preferably, the kit further comprises a premix for establishing a fluorescence quantitative PCR reaction system, and the premix contains a PCR buffer, a DNA polymerase, dNTPs, and magnesium ions. Specifically, any commercial premix applicable to the fluorescence quantitative PCR reaction using the multiplex probe method can be directly selected.

[0015] Preferably, the final concentration of each primer in the fluorescence quantitative PCR reaction system is 0.2 μM, and the final concentration of each probe is 0.1 μM.

[0016] Preferably, the kit further includes a positive control and a negative control. Specifically, the positive control includes the recombinant plasmid pIBV containing the IBV total population specific fragment, the recombinant plasmid pGI-19 containing the GI-19 specific fragment, and the recombinant plasmid pGVI-1 containing the GVI-1 specific fragment; the negative control is chicken-derived negative sample nucleic acid or nuclease-free water. The nucleotide sequence of the IBV total population specific fragment is as shown in SEQ ID NO.10, the nucleotide sequence of the GI-19 specific fragment is as shown in SEQ ID NO.11, and the nucleotide sequence of the GVI-1 specific fragment is as shown in SEQ ID NO.12.

[0017] The present invention also provides a method for detecting IBV and differentiating between GI-19 and GVI-1 genotypes using the kit, comprising the following steps: (1) Extract the RNA of the test sample and reverse-transcribe it into cDNA; (2) Using the reverse-transcribed cDNA as a template, establish a fluorescence quantitative PCR reaction system using the kit and perform fluorescence quantitative PCR amplification; (3) During the fluorescence quantitative PCR reaction, detect the fluorescence signal in the corresponding fluorescence channel in real time, and judge whether it is infected with IBV and differentiate the genotype according to the amplification result; The judgment basis is: when the threshold cycle number Ct ≤ 37, it is judged as positive; when 37 < Ct ≤ 40, it is judged as suspicious; when Ct > 40 or there is no amplification signal, it is judged as negative; When the fluorescence signal carried by the probe IBV-P is positive, it is judged that the test sample is positive for IBV; If the fluorescence signal carried by probe IBV-P is positive, and the fluorescence signal carried by probe GI-19-P is positive while the fluorescence signal carried by probe GVI-1-P is negative, then the sample is determined to be of the GI-19 genotype; if the fluorescence signal carried by probe GVI-1-P is positive while the fluorescence signal carried by probe GI-19-P is negative, then the sample is determined to be of the GVI-1 genotype; if both the fluorescence signals carried by probe GI-19-P and probe GVI-1-P are positive, then the sample is determined to be a mixed infection of GI-19 and GVI-1; if both the fluorescence signals carried by probe GI-19-P and probe GVI-1-P are negative, then the sample is determined to be an IBV-positive untyped sample.

[0018] The reaction procedure for the quantitative PCR amplification is as follows: Step 1, 95℃ for 2 min; Step 2, 95℃ for 10 s, 58℃ for 10 s, 72℃ for 50 s, for 45 cycles.

[0019] The beneficial effects of this invention are as follows: (1) The present invention uses a triple fluorescent probe system, which can simultaneously detect IBV and its GI-19 and GVI-1 genotypes in one reaction without the need for repeated RNA extraction or multiple reactions, greatly improving efficiency and reducing reagent and consumable costs, and avoiding the complex process of traditional genotyping that relies on sequencing.

[0020] (2) The triple fluorescent RT-PCR primer combination provided by the present invention has good specificity and can simultaneously identify IBV virus and its two major circulating genotypes, GI-19 and GVI-1, without cross-reacting with common avian respiratory pathogens such as NDV, AIV, ILTV or other IBV genotypes.

[0021] (3) The triple fluorescent RT-PCR primer combination provided by the present invention has high detection sensitivity and can detect viral loads as low as 10 copies / μL. It can be used for early infection detection in clinical samples with low viral loads, which helps in early warning and rapid treatment.

[0022] (4) The kit of the present invention is used for real-time PCR amplification. It has high batch-to-batch stability and uniform amplification efficiency, providing a convenient tool for disease monitoring and clinical diagnosis in aquaculture farms. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the amplification curve of a GI-19 sample detected using the triple fluorescence RT-PCR method.

[0024] Figure 2 This is a schematic diagram of the amplification curve of a GVI-1 sample detected using the triple fluorescence RT-PCR method.

[0025] Figure 3 This is a schematic diagram of the amplification curve of a GI-1 type sample using the triple fluorescence RT-PCR detection method (taking the H120 vaccine strain as an example).

[0026] Figure 4 This is a schematic diagram of the amplification curve for the absence of IBV virus using the triple fluorescent RT-PCR detection method.

[0027] Figure 5 This is a FAM channel diagram for specific detection using the triple fluorescence RT-PCR method (detecting the total IBV population). In the diagram, 1-12: SH20241122-1 (GI-19), SH20241122-2 (GI-19), SH20241120 (GVI-1), IBVYDH (GVI-1), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1); 13-20: AIV, NDV, ILTV, IBDV, MDV, ALV, and 2 negative results.

[0028] Figure 6 This is a VIC channel diagram for specific detection using triple fluorescence RT-PCR (detection of GI-19). In the diagram, 1: SH20241122-1 (GI-19), 2: SH20241122-2 (GI-19), 3-20: SH20241120 (GVI-1), IBV YDH (GVI-1), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1), AIV, NDV, ILTV, IBDV, MDV, ALV, and 2 negative results.

[0029] Figure 7This is a ROX channel diagram for specific detection using triple fluorescence RT-PCR (detection of GVI-1 type). In the diagram, 3: SH20241120 (GVI-1), 4: IBV YDH (GVI-1), 1-2, 5-20: SH20241122-1 (GI-19), SH20241122-2 (GI-19), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1), AIV, NDV, ILTV, IBDV, MDV, ALV, and 2 negative results.

[0030] Figure 8 This is a graph showing the sensitivity detection results of pIBV plasmid amplification using the triple fluorescent RT-PCR method. Curves 1-5 in the graph represent plasmid concentrations of 10⁻⁶. 5 10 4 10 3 10 2 and 10 1 copies / μL.

[0031] Figure 9 This is a graph showing the sensitivity detection results of pGI-19 plasmid amplification using triple fluorescent RT-PCR. Curves 1-5 in the graph represent plasmid concentrations of 10⁻⁶ and 10⁻⁶, respectively. 5 10 4 10 3 10 2 and 10 1 copies / μL.

[0032] Figure 10 This is a graph showing the sensitivity detection results of pGVI-1 plasmid amplification using triple fluorescent RT-PCR. Curves 1-5 in the graph represent plasmid concentrations of 10⁻⁶ and 10⁻⁶, respectively. 5 10 4 10 3 10 2 and 10 1 copies / μL.

[0033] Figure 11 This image shows the results of clinical sample testing using the triple fluorescence RT-PCR detection method. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] Example 1: Design and Synthesis of Primers and Probes 1. IBV primer and probe design Download all IBV whole genome sequences from the NCBI Viruses database up to July 2025, covering multiple epidemic years and geographical origins, and encompassing multiple domestic and international genotypes (GI-GIX), for multiple sequence alignment. Using a programmed algorithm, code was designed to enumerate the highly conserved regions with the highest theoretical coverage across the entire genome. The resulting conserved sequences are shown in Table 1. Using SH20241122-2 (GenBank accession number: PX388002) as an example, the start position, region length, and specific sequence of the conserved sequence are illustrated. Following the design principles of quantitative real-time primers and probes, primers and probes with the highest theoretical coverage were designed within the conserved regions, targeting the 5′UTR region of the IBV genome.

[0037] Table 1. Highly conserved sequences with the highest theoretical coverage across the entire genome of IBV strains

[0038] 2. Design of GI-19 primers and probes All published GI-19 IBV S1 gene sequences were downloaded from the NCBI database, covering multiple epidemic years, geographical origins, and domestic and international genotypes. Multiple sequence alignment was performed to analyze sequence variation sites. A program algorithm was designed to enumerate the highly conserved regions with the highest theoretical coverage within the S1 sequence range. Specific regions that can be clearly distinguished from non-GI-19 types were then screened. The resulting conserved sequences are shown in Table 2. Taking GI-19 SH20241122-1 (GenBank accession number: PX388001) as an example, the start position, interval length, and specific sequence of the conserved sequence are illustrated. Following the design principles of quantitative real-time primers and probes, primers and probes with the highest theoretical coverage for GI-19 types were designed within the conserved regions to target the S1 region of the IBV genome.

[0039] Table 2. Highly conserved sequences with the highest theoretical coverage within the S1 gene region of GI-19.

[0040] 3. GVI-1 Primer and Probe Design: All published GVI-1 IBV S1 gene sequences in the NCBI database were collected and subjected to multiple sequence alignment. Sequence variation sites were analyzed, and a program algorithm was used to enumerate the highly conserved regions with the highest theoretical coverage within the S1 sequence range. Specific regions that could be clearly distinguished from non-GVI-1 sequences were screened. The resulting conserved sequences are shown in Table 3. Using GVI-1 sequence GX-NN09032 (GenBank accession number: JX897900.1) as an example, the start position, interval length, and specific sequence of the conserved sequence are illustrated. Following the design principles of quantitative real-time primers and probes, GVI-1 primers and probes with the highest theoretical coverage were designed within the conserved regions to target the S1 region of the IBV genome.

[0041] Table 3. Highly conserved sequences with the highest theoretical coverage within the S1 genome of GVI-1 type.

[0042] 4. To ensure the stability and specificity of the multiplex reaction system, complementarity and dimerization analyses were performed on the three sets of primers and probes to ensure the absence of dimers, hairpin structures, or complementary binding. The final confirmed primer and probe sequences are shown in SEQ ID NO.1-SEQ ID NO.9. The probes were synthesized by Anhui General Biotechnology Co., Ltd., and the primers were synthesized by Zhejiang Shangya Biotechnology Co., Ltd. Details are as follows: IBV-F: 5′-CATCCGTTGCTTGGGCTACCTAG-3′ (SEQ ID NO. 1); IBV-R: 5′-GCCATGTTGTCACTGTCTATTGTATGTCTG-3′ (SEQ ID NO. 2); IBV-P: 5′-CTAAACACCACCAGAACCTGTCACCTCAGGGGTTG-3′ (SEQ ID NO. 3); GI-19-F: 5′-TTATGTGTACTACTACCAAAGTGCCTTTAGGC-3′ (SEQ ID NO. 4); GI-19-R: 5′-ACCTGCTTTAAAATACACACCTGCTGAC-3′ (SEQ ID NO. 5); GI-19-P: 5′-TTACAGTGTGCACTACAAAATTGTG-3′ (SEQ ID NO. 6); GVI-1-F: 5'-ATGGTGGTGCGTATGCAGTAGAAC-3' (SEQ ID NO. 7); GVI-1-R: 5′-TGTAACAAAAACAACAATTGTACTAAAATTGCAATG-3′ (SEQ ID NO. 8); GVI-1-P: 5′-TGCAAGTGCAGTTGCGATTACTACACCTGTTAATGGCATG-3′ (SEQ ID NO. 9).

[0043] The universal probe IBV-P is labeled with FAM fluorescein at the 5′ end and BHQ1 quencher at the 3′ end; the GI-19 probe GI-19-P is labeled with VIC fluorescein at the 5′ end and MGB quencher at the 3′ end; the GVI-1 probe GVI-1-P is labeled with ROX fluorescein at the 5′ end and BHQ2 quencher at the 3′ end.

[0044] Example 2: Triple fluorescent RT-PCR detection This example demonstrates the results of triple fluorescent RT-PCR detection using the primer and probe composition provided in Example 1 in four different detection subjects, including GI-19, GVI-1, GI-1, and the absence of IBV virus.

[0045] The tested samples were SH20241122-1 (GI-19), SH20241120 (GVI-1), and H120 (GI-1), all of which were identified and preserved by the Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs, Zhejiang University.

[0046] The triplet fluorescent RT-PCR reaction system consisted of: 10 μL of 2× probe qPCR Master Mix (General Biotech Co., Ltd. Cat#KQMU3001), with a final concentration of 0.2 μM for each primer, a final concentration of 0.1 μM for each probe, 0.5 μL of DNA template, and double-distilled water to a final volume of 20 μL.

[0047] The triplet fluorescent RT-PCR amplification reaction program was as follows: 95℃ for 2 min; 95℃ for 10 s, 58℃ for 10 s, and 72℃ for 50 s, for 45 cycles. Fluorescence signals were detected in real-time using the FAM, VIC, and ROX channels during the quantitative PCR reaction.

[0048] Figure 1 This is a schematic diagram of the amplification results of the GI-19 sample. The FAM channel (detecting IBV) and VIC channel (detecting GI-19) both show typical S-shaped amplification curves, while the ROX channel (detecting GVI-1) shows no amplification signal.

[0049] Figure 2 This is a schematic diagram of the amplification results of the GVI-1 sample. Amplification curves are shown in both the FAM channel (for detecting IBV) and the ROX channel (for detecting GVI-1), while no amplification signal is shown in the VIC channel (for detecting GI-19).

[0050] Figure 3 This is a schematic diagram of the amplification results for the GI-1 type sample. Only the FAM channel (detecting IBV) shows an amplification curve, while the VIC channel (detecting GI-19 type) and the ROX channel (detecting GVI-1 type) show no amplification signal.

[0051] Figure 4 This is a schematic diagram of amplification in a negative control without IBV virus. No amplification signal was observed in any of the three channels (FAM, VIC, ROX).

[0052] Therefore, the triple fluorescent RT-PCR detection system established in this invention can simultaneously detect the total IBV population and accurately distinguish between GI-19 and GVI-1 types in a single reaction, which is completely consistent with the S1 gene sequencing results. The judgment logic is clear and the results are accurate and reliable.

[0053] Example 3: Specificity Detection This embodiment tested the specificity of three sets of RT-PCR primer and probe combinations, and the specific methods are as follows: Various avian viruses identified and preserved by the Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs, Zhejiang University, including avian influenza virus (AIV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV), avian leukosis virus (ALV), and isolates and vaccine strains of different IBV genotypes: H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1), SH20241122-1 (GI-19), SH20241122-2 (GI-19), SH20241120 (GVI-1), and IBV were used. YDH (GVI-1), all of the above samples were detected according to the triple fluorescent RT-PCR reaction system established in Example 2.

[0054] Figure 5The image shows the FAM channel diagram (for detecting IBV). Numbers 1-12 represent: SH20241122-1 (GI-19), SH20241122-2 (GI-19), SH20241120 (GVI-1), IBV YDH (GVI-1), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), and QD0716 (GV-1). Numbers 13-20 represent: AIV, NDV, ILTV, IBDV, MDV, ALV, and nucleic acid extracts from uninfected IBV chick tissue samples, and two negative controls: nuclease-free water. The results showed that typical S-type amplification curves were generated in all IBV positive samples, while no amplification signals were observed in other avian viruses, uninfected chicken tissue, and negative controls.

[0055] Figure 6 The diagram shows the VIC channel (for detecting GI-19). The numbers in the diagram are: 1: SH20241122-1 (GI-19); 2: SH20241122-2 (GI-19); 3-20: SH20241120 (GVI-1), IBV YDH (GVI-1), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1), AIV, NDV, ILTV, IBDV, MDV, ALV, and nucleic acid extracts from uninfected IBV chick tissue samples and two negative controls (nuclease-free water). The results showed that amplification signals were generated only in GI-19 strain samples, and no amplification signals were generated in other genotypes, non-IBV viruses, uninfected chicken tissues, and negative controls.

[0056] Figure 7The image shows the ROX channel diagram (detecting GVI-1 strain). The channel numbers are: 3: SH20241120 (GVI-1); 4: IBV YDH (GVI-1); 1-2, 5-20: SH20241122-1 (GI-19), SH20241122-2 (GI-19), H120 (GI-1), M41 (GI-1), XC200601 (GI-7), XC201206 (GI-13), SC (GI-22), YC (GI-22), HF220610 (GI-29), QD0716 (GV-1), AIV, NDV, ILTV, IBDV, MDV, ALV, and nucleic acid extracts from uninfected IBV chick tissue samples and two negative controls (nuclease-free water). The results show that amplification signals were only generated in the GVI-1 strain samples; no amplification signals were observed in the other samples.

[0057] The above results indicate that the triple fluorescent RT-PCR system of the present invention can accurately distinguish between IBV total group, GI-19 and GVI-1, and has no cross-reactivity with other common avian viruses, demonstrating good specificity.

[0058] Example 4: Sensitivity Detection This embodiment tested the sensitivity of three sets of RT-PCR primer and probe combinations, and the specific methods are as follows: The amplification products of three sets of primers (IBV-F, IBV-R, GI-19-F, GI-19-R, and GVI-1-F, GVI-1-R) (nucleotide sequences shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively) were ligated into the Topo vector. The recombinant plasmids confirmed by sequencing were named pIBV (containing a total IBV group-specific fragment, amplified from SH20241122-2 strain (GenBank accession number: PX388002), pGI-19 (containing a GI-19-specific fragment, amplified from SH20241122-1 strain (GenBank accession number: PX388001), and pGVI-1 (containing a GVI-1-specific fragment, amplified from SH20241120 strain (GenBank accession number: PX388000), respectively). Plasmid concentration was determined using NanoDrop, copy number was calculated by combining fragment size and full-length vector, and then diluted to 10⁻⁶ with nuclease-free water. 5 10 4 10 3 10 2 and 10 1Ten-fold serial dilutions of copies / μL. Detection was carried out according to the triple fluorescence RT-PCR reaction system and procedure established in Example 2.

[0059] The results were as Figures 8-10 shown. The triple fluorescence RT-PCR system established in the present invention had a good linear range (10 5 ~10 1 copies / μL), and the lowest detection limits of the three channels all reached 10 1 copies / μL, meeting the requirements for rapid and sensitive detection of the total IBV population and GI-19 genotype and GVI-1 genotype in clinical samples.

[0060] Example 5: Detection of clinical samples To verify the applicability of the triple fluorescence RT-PCR system established in the present invention in clinical samples, a total of 119 tissue samples, throat swabs and anal swabs suspected of being infected with IBV clinically were collected and stored in this laboratory. All samples were detected using the triple fluorescence RT-PCR system and procedure of Example 2, and at the same time, the amplification results of the three channels of FAM (total IBV population), VIC (GI-19 type), and ROX (GVI-1 type) were analyzed. The detection results are shown in Figure 11 .

[0061] The following judgment criteria were formulated: Ct ≤ 37 was judged as positive, 37 < Ct ≤ 40 was judged as suspicious, Ct > 40 or no amplification signal was judged as negative; FAM positive was IBV positive; on the premise of FAM positive, if VIC was positive and ROX was negative, it was judged as GI-19; if ROX was positive and VIC was negative, it was judged as GVI-1; if both VIC and ROX were positive, it was judged as a mixed infection of GI-19 and GVI-1; if both VIC and ROX were negative, it was judged as IBV positive but untyped.

[0062] According to the above judgment criteria, the detection results of 119 clinical samples suspected of being infected with IBV stored in the laboratory were as follows: 101 positive cases (101 / 119, 84.87%) were detected in the FAM channel (detecting the total IBV population), 62 positive cases (62 / 119, 52.10%) were detected in the VIC channel (detecting the GI-19 type), and 24 positive cases (24 / 119, 20.17%) were detected in the ROX channel (detecting the GVI-1 type), and there were 2 cases of mixed infection of GI-19 type and GVI-1 type (2 / 119, 1.68%).

[0063] Consistency verification: First-generation sequencing (Sanger) was performed on the detected positive samples and some negative samples. The sequencing results were analyzed and found to be consistent with the positive, negative and GI-19 and GVI-1 typing of this system, further proving the reliability of this system in clinical samples. The triple fluorescent RT-PCR system established in this invention has good detection performance and typing interpretation ability in clinical samples from different sources, and is suitable for rapid screening, epidemiological monitoring and molecular typing of clinical materials.

[0064] The above description is only a partial embodiment of the present invention. Without departing from the principle of the present invention, those skilled in the art can replace or make equivalent improvements to the sample type, extraction and amplification reagents, and cycling procedures. They can also obtain other embodiments based on this embodiment without creative intent. All of these fall within the protection scope of the present invention.

Claims

1. A triple fluorescent RT-PCR primer set for detecting chicken infectious bronchitis virus and distinguishing between GI-19 and GVI-1 genotypes, characterized in that, The primer combination includes: Universal primer and probe set I for detecting the total IBV population includes upstream primer IBV-F, downstream primer IBV-R, and probe IBV-P, with nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. Specific primer and probe combination II for detecting GI-19 genotype includes upstream primer GI-19-F, downstream primer GI-19-R, and probe GI-19-P, the nucleotide sequences of which are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively; The specific primer and probe combination III for detecting the GVI-1 genotype includes the upstream primer GVI-1-F, the downstream primer GVI-1-R, and the probe GVI-1-P, whose nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. The 5′ ends of probes IBV-P, GI-19-P, and GVI-1-P are each labeled with different fluorescent groups, and their 3′ ends are labeled with quenching groups corresponding to the fluorescent groups.

2. The triple fluorescent RT-PCR primer combination as described in claim 1, characterized in that, The probe IBV-P has a 5′ end labeled with the FAM group and a 3′ end labeled with the BHQ1 group; the probe GI-19-P has a 5′ end labeled with the VIC group and a 3′ end labeled with the MGB group; the probe GVI-1-P has a 5′ end labeled with the ROX group and a 3′ end labeled with the BHQ2 group.

3. A kit for detecting infectious bronchitis virus in chickens and differentiating between GI-19 and GVI-1 genotypes, characterized in that, The kit contains the triple fluorescent RT-PCR primer combination as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that, The kit also includes a premix for establishing a real-time PCR reaction system.

5. The kit according to claim 4, characterized in that, The final concentration of each primer in the quantitative real-time PCR reaction system is 0.2 μM, and the final concentration of each probe is 0.1 μM.

6. The reagent kit as described in claim 3, characterized in that, The kit also includes a positive control and a negative control.

7. The reagent kit according to any one of claims 3-6, characterized in that, A method for detecting chicken infectious bronchitis virus and differentiating between GI-19 and GVI-1 genotypes using the aforementioned kit includes the following steps: (1) Extract RNA from the sample to be tested and reverse transcribe it into cDNA; (2) Using the reverse transcribed cDNA as a template, establish a real-time PCR reaction system using the kit and perform real-time PCR amplification; (3) During the quantitative real-time PCR reaction, the fluorescence signal is detected in the corresponding fluorescence channel in real time. Based on the amplification results, it is determined whether IBV is infected and the genotype is distinguished.

8. The kit according to claim 7, characterized in that, The reaction procedure for real-time PCR amplification is as follows: Step 1, 95℃ for 2 min; Step 2, 95℃ for 10 s, 58℃ for 10 s, 72℃ for 50 s, for 45 cycles.

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