Probe and primer composition for avian infectious bronchitis virus typing detection, kit containing composition and application

Abstract

The invention provides a primer and probe composition for typing detection of an infectious bronchitis virus. The composition is used for detecting genotypes of 4 / 91, TC07-2, LDT3, QX, LSC-99I and TW. The invention also provides a detection kit containing the primer and probe composition and a detection method. According to the invention, the detection of six genotypes is integrated into only two reaction tubes, the reaction steps are obviously reduced, the flux is improved, and a great leap is realized in multiple detection capability and detection efficiency, so that the kit is suitable for large-scale clinical screening and epidemiological investigation. The invention provides an unprecedented convenient and accurate tool for the diagnosis of complex clinical mixed infection by effectively identifying and combining the mixed infection of a plurality of genotypes through one-time reaction.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to probe and primer compositions for typing and detecting chicken infectious bronchitis virus based on multiplex quantitative PCR, as well as a kit containing the probe and primer compositions, and the application of the kit. Background Technology

[0002] Infectious Bronchitis Virus (IBV) is an acute, highly contagious respiratory disease caused by coronaviruses, characterized by rapid spread, high pathogenicity, and diverse genotypes. In recent years, IBV has spread widely globally, and dozens of genotypes have been identified, including 4 / 91, TC07-2, LDT3, QX, LSC-99I, and TW. Different genotypes of IBV exhibit significant differences in antigenicity, pathogenicity, and tissue tropism, resulting in limited cross-protection against the virus and increasing the difficulty of prevention and control. Infection with different IBV subtypes can lead to different symptoms in chicken flocks. Furthermore, mixed infections of different genotypes are common, further exacerbating the complexity of the disease and the difficulty of its control.

[0003] Currently, the main methods for detecting IBV include virus isolation, serological detection (such as ELISA and neutralization assays), and molecular biological methods (such as conventional PCR and quantitative real-time PCR). However, these methods have many drawbacks, such as insufficient sensitivity and specificity, long virus isolation time (requiring 5-7 days), low success rate (dependent on live virus), and inability to distinguish genotypes. Conventional PCR can only detect a single genotype and requires multiple independent reactions to complete virus typing. Although existing single-channel quantitative real-time PCR methods can quantitatively detect viral nucleic acid, they cannot achieve simultaneous detection of multiple genotypes, and improper primer and probe design can easily lead to cross-reactions (such as non-specific binding of QX type and 4 / 91 type primers), resulting in false positives or missed detections. In addition, there are also drawbacks such as poor reagent stability and sensitivity to storage conditions.

[0004] For example, Chinese invention patent application CN105463136A discloses a kit for RT-PCR typing detection of infectious bronchitis virus (IBV) in chickens. This kit, based on conventional RT-PCR and gel electrophoresis, can identify three types / genotypes (respiratory / M41, renal, and 4 / 91 types) of IBV by the number and size of bands in a single reaction. However, on the one hand, its typing ability is limited, only identifying three types, failing to cover many currently prevalent important genotypes (such as QX, LDT3, TW, etc.); on the other hand, its sensitivity and accuracy are low because it relies on endpoint gel electrophoresis and cannot achieve quantitative detection, resulting in lower sensitivity than quantitative real-time PCR, and it is prone to aerosol contamination leading to false positives. Furthermore, this kit cannot detect mixed infections; a single reaction can only provide a judgment of one dominant type, making it difficult to effectively identify mixed infections with other genotypes.

[0005] For example, Chinese invention patent application CN116769965A discloses a genotyping detection kit for IBV. However, this technical solution is based on five independent RT-PCR reactions and detects five genotypes by gel electrophoresis. Therefore, it is cumbersome to operate: it requires five independent PCR reactions for each sample, and the throughput is low, making it unsuitable for rapid screening of large-scale clinical samples.

[0006] Therefore, there is an urgent need to develop an efficient and accurate IBV multiplex quantitative PCR genotyping detection technology. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an efficient and reliable technical tool for the accurate typing and control of IBV by combining multiplex quantitative PCR technology with a highly stable reagent formulation. This is expected to enable accurate clinical pathogen monitoring and rapid disease diagnosis, significantly reduce economic losses caused by IBV in the aquaculture industry, and provide key data support for vaccine development and epidemiological research.

[0009] To achieve the above objectives, the present invention provides a primer and probe composition for genotyping detection of infectious bronchitis virus (IBV). The composition includes primers and probes for detecting IBV-4 / 91, wherein the upstream primer sequence is SEQ ID NO. 1, the downstream primer sequence is SEQ ID NO. 2, and the probe sequence is SEQ ID NO. 3; primers and probes for detecting IBV-TC07-2, wherein the upstream primer sequence is SEQ ID NO. 4, the downstream primer sequence is SEQ ID NO. 5, and the probe sequence is SEQ ID NO. 6; primers and probes for detecting IBV-LDT3, wherein the upstream primer sequence is SEQ ID NO. 7, the downstream primer sequence is SEQ ID NO. 8, and the probe sequence is SEQ ID NO. 9; and primers and probes for a first internal control gene, wherein the upstream primer sequence is SEQ ID NO. 10, the downstream primer sequence is SEQ ID NO. 11, and the probe sequence is SEQ ID NO. 12; Primers and probes for detecting IBV-QX type, the sequence of the upstream primer is SEQ ID NO. 13, the sequence of the downstream primer is SEQ ID NO. 14, and the sequence of the probe is SEQ ID NO. 15; Primers and probes for detecting IBV-LSC-99I type, the sequence of the upstream primer is SEQ ID NO. 16, the sequence of the downstream primer is SEQ ID NO. 17, and the sequence of the probe is SEQ ID NO. 18; Primers and probes for detecting IBV-TW type, the sequence of the upstream primer is SEQ ID NO. 19, the sequence of the downstream primer is SEQ ID NO. 20, and the sequence of the probe is SEQ ID NO. 21; Primers and probes for the second internal standard gene, the sequence of the upstream primer is SEQ ID NO. 22, the sequence of the downstream primer is SEQ ID NO. 23, and the sequence of the probe is SEQ ID NO. 24.

[0010] In this invention, the probes used to detect IBV-4 / 91 and IBV-QX have fluorescein FAM attached to their 5' ends and fluorescein BHQ1 attached to their 3' ends, respectively; the probes used to detect IBV-TC07-2 and IBV-LSC / 99I have fluorescein HEX attached to their 5' ends and fluorescein BHQ1 attached to their 3' ends, respectively; the probes used to detect IBV-LDT3 and IBV-TW have fluorescein ROX attached to their 5' ends and fluorescein BHQ2 attached to their 3' ends, respectively; and the probes for the first internal standard gene and the second internal standard gene have fluorescein CY5 attached to their 5' ends and fluorescein BHQ2 attached to their 3' ends, respectively.

[0011] The present invention also provides the use of the above composition in the preparation of a kit for detecting chicken infectious bronchitis virus typing, wherein the chicken infectious bronchitis virus includes 4 / 91, TC07-2, LDT3, QX, LSC-99I and TW types.

[0012] On the other hand, the present invention also provides a multiplex quantitative PCR detection kit based on TaqMan probes, the kit comprising a first kit and a second kit, the first kit containing primers and probes as in SEQ ID NO. 1-12, and the second kit containing primers and probes as in SEQ ID NO. 13-24.

[0013] Specifically, in the kits of the present invention, the positive template plasmid of the first kit contains a gene with a sequence as shown in SEQ ID NO. 29, and the positive template plasmid of the second kit contains a gene with a sequence as shown in SEQ ID NO. 34.

[0014] This invention also provides a method for detecting different genotypes of infectious bronchitis virus in chickens using the above-mentioned kit, the method comprising the following steps:

[0015] (1) Extract RNA from the sample to be tested;

[0016] (2) Using the RNA obtained in step (1) as a template, perform real-time RT-PCR using a detection kit;

[0017] (3) The results of quantitative RT-PCR were determined as follows:

[0018] a. Establishment conditions:

[0019] After sample amplification, both the FAM / HEX / ROX channels and the CY5 channel show typical exponential growth curves, while the negative result shows no specific amplification curve, indicating that the experimental conditions are met.

[0020] b. Qualitative analysis to determine the positive / negative status of each channel in FAM / HEX / ROX:

[0021] Samples with a CT value ≤35 in the detection channel and a CT value ≤35 in the internal standard channel, and exhibiting a typical exponential growth curve, are judged as positive.

[0022] For the detection channel with a CT value of 35 < CT value ≤ 40 and the internal standard channel with a CT value ≤ 35, it is recommended to repeat the test. If the CT value is still 35 < CT value ≤ 40 and a typical exponential growth curve is shown, it is considered positive. If the detection channel has no CT value or an atypical amplification curve and the internal standard channel has a CT value ≤ 35, it is considered negative.

[0023] For detection channels with a CT value > 40 and no specific amplification curve, or with no CT value and an internal standard channel CT value ≤ 35, the result is considered negative.

[0024] For any value or no CT value in the detection channel, and for the internal standard channel with a CT value >35 or no CT value, the result is considered invalid.

[0025] c. Based on the above qualitative results, further quantitative analysis should be performed on viral nucleic acid positive or weakly positive results:

[0026] Standard curves for different viral copy numbers were established using positive plasmid standards. Based on the established viral copy number standard curves, the copy numbers of different viral nucleic acids in the samples to be tested were calculated using the CT values ​​of the samples.

[0027] In this invention, the detection system for real-time RT-PCR is as follows:

[0028] First reagent kit composition: 10 μL 2.5×One-step RT-qPCR buffer II (Primer & ProbePlus) + 2 μL One-Step RT-qPCR Enzyme Mix II + 5 μL Template + 8 μL RNase-free water, total composition 25 μL;

[0029] Second kit composition: 10 μL 2.5×One-step RT-qPCR buffer II (Primer & ProbePlus) + 2 μL One-Step RT-qPCR Enzyme Mix II + 5 μL Template + 8 μL RNase free water, total 25 μL.

[0030] Specifically, the standard curves for each viral genotype are as follows:

[0031] IBV-4 / 91 standard curve: y = -3.414x + 41.58;

[0032] IBV-TC07-2 standard curve: y = -3.535x + 41.743;

[0033] IBV-LDT3 standard curve: y = -3.511x + 40.522;

[0034] IBV-QX standard curve: y = -3.489x + 41.984;

[0035] IBV-LSC-99I standard curve: y = -3.301x + 40.502;

[0036] IBV-TW standard curve: y = -3.657x + 44.63.

[0037] This invention experimentally verifies that the above-mentioned kit can be used as a useful tool for rapid detection and diagnosis of different genotypes of infectious bronchitis in chickens in clinical practice, and provides key data support for vaccine development and epidemiological research.

[0038] Compared with existing technologies, this invention uses TaqMan probe-based multiplex real-time quantitative PCR, which differs from existing technologies that rely on gel electrophoresis, and achieves real-time monitoring and precise quantification of the PCR process.

[0039] On the other hand, this invention integrates the detection of six genotypes (4 / 91, TC07-2, LDT3, QX, LSC-99I, TW) into only two reaction tubes, significantly reducing reaction steps and increasing throughput. It achieves a great leap in multiplex detection capability and detection efficiency, thus adapting to large-scale clinical screening and epidemiological surveys.

[0040] Furthermore, this invention provides an unprecedentedly convenient and accurate tool for the diagnosis of complex clinical mixed infections, enabling the effective identification of mixed infections with multiple genotypes through a single reaction. Attached Figure Description

[0042] 1. Figure 1 The image shows the results of amplifying positive plasmid standards using two different kits.

[0043] A shows the results of amplifying the positive plasmid standard using kit 1; B shows the results of amplifying the positive plasmid standard using kit 2.

[0044] 2. Figure 2 This is a schematic diagram showing the detection results of positive plasmid sensitivity and the establishment of standard curves for the two reagent kits.

[0045] Figure A shows the sensitivity detection results for IBV-4 / 91 positive plasmid amplification. Curves 1-8 represent the results for template concentrations of 6.18 × 10⁻⁶. 7 -6.18×10 0 Amplification curve of template positive plasmid copies / μL in kit 1; the standard curve is shown on the right.

[0046] B shows the sensitivity detection results for IBV-TC07-2 positive plasmid amplification. Curves 1-8 represent the results for template concentrations of 6.86 × 10⁻⁶. 7 -6.86×10 0 Amplification curve of template positive plasmid copies / μL in kit 1; the standard curve is shown on the right.

[0047] C represents the sensitivity detection results for IBV-LDT3 positive plasmid amplification. Curves 1-8 represent template concentrations of 1.03 × 10⁻⁶.7 -1.03×10 0 Amplification curve of template positive plasmid copies / μL in kit 1; the standard curve is shown on the right.

[0048] D is the sensitivity detection result of IBV-QX positive plasmid amplification. Curves 1-8 represent the results for template concentrations of 1.45 × 10⁻⁸. 7 -1.45×10 0 Amplification curve of template positive plasmid copies / μL in kit 2; the standard curve is shown on the right.

[0049] E is the sensitivity detection result of IBV-LSC-99I positive plasmid amplification. Curves 1-8 represent the results when the template concentration is 7.19 × 10⁻⁶. 7 -7.19×10 0 Amplification curve of template positive plasmid copies / μL in kit 2; the standard curve is shown on the right.

[0050] F shows the sensitivity detection results for IBV-TW positive plasmid amplification. Curves 1-7 represent template concentrations of 8.02 × 10⁻⁶. 7 -8.02×10 1 Amplification curve of template positive plasmid copies / μL in kit 2; the standard curve is shown on the right.

[0051] 3. Figure 3 Figure 1 shows the results of specificity experiments for the two reagent kits.

[0052] A is the amplification curve of reagent kit 1 against 4 / 91, TC07-2, LDT3, QX, LSC-99I, and TW virus strains;

[0053] B shows the amplification curves of the 4 / 91, TC07-2, LDT3, QX, LSC-99I, and TW virus strains from kit 2. Detailed Implementation

[0055] Example 1: Design and synthesis of primer sets and probes

[0056] Seventy IBV sequences were downloaded from NCBI, including the S1 gene sequences of representative strains of major IBV genotypes currently prevalent in China and vaccine strains, as well as seven IBV sequences isolated and preserved by Qilu Animal Health Products Co., Ltd. Sequence analysis software was used to compare and analyze their S1 gene sequences. Primers and probes were designed for regions with significant sequence differences among different genotypes (4 / 91, TC07-2, LDT3, IBV-QX, LSC-99I, and TW) and relatively conserved regions among different strains within the same genotype.

[0057] Based on the primers and probes described above, a matrix of primer and probe sequence crossovers was established. Primer synthesis software was used to analyze the mismatch free energy and number of mismatched bases between primers, probes, and between primers and probes. The final confirmed primer and probe combinations for detecting IBV-4 / 91 are shown in SEQ ID Nos. 1-3; for IBV-TC07-2, as shown in SEQ ID Nos. 4-6; for IBV-LDT3, as shown in SEQ ID Nos. 7-9; for the first internal standard gene, as shown in SEQ ID Nos. 10-12; for IBV-QX, as shown in SEQ ID Nos. 13-15; for IBV-LSC-99I, as shown in SEQ ID Nos. 16-18; and for IBV-TW, as shown in SEQ ID Nos. 10-12. As shown in SEQ ID Nos. 22-24, the primer and probe combinations for the second internal control gene are as shown in SEQ ID Nos. 19-21. All of the above primers and probes were artificially synthesized.

[0058] Example 2

[0059] The TaqMan probe-based multiplex quantitative PCR detection kit includes the primer combination described in Example 1, 2.5×One-step RT-qPCR buffer II (Primer & Probe Plus) for establishing the quantitative PCR reaction system, and One-Step RT-qPCR Enzyme Mix II.

[0060] The primer and probe combinations for detecting IBV-4 / 91, IBV-TC07-2, and IBV-LDT3, as well as the primer and probe combination for the first internal control gene, were designed in the first kit. The remaining primer and probe combinations were designed in the second kit. Multiplexed quantitative RT-PCR detection methods were established for each kit.

[0061] (I) Preparation of positive plasmid standards

[0062] The conserved region sequences obtained from the S1 gene analysis of IBV-4 / 91, TC07-2, and LDT3, along with the first internal standard gene, were inserted into the pUC57 cloning vector to synthesize plasmid standards for each genotype.

[0063] Among them, the conserved region sequence of the S1 gene of IBV-4 / 91, P-IBV-4 / 91, is shown in SEQ ID NO.25; P-IBV-TC07-2 is shown in SEQ ID NO.26; P-IBV-LDT3 is shown in SEQ ID NO.27; and the first internal standard gene, P-internal standard gene, Pb-56, is shown in SEQ ID NO.28.

[0064] The conserved region sequences obtained from the analysis of IBV-4 / 91, TC07-2, LDT3, and the internal standard gene Pb-56 were sequentially ligated, and DNA was synthesized by Hunan Aikerui Biotechnology Co., Ltd., as shown in SEQ ID NO.29. After synthesis, the template plasmid was ligated into the multiple cloning site of the pUC57 plasmid, and the resulting plasmid is the positive template plasmid of the first kit.

[0065] Similarly, the conserved region sequences obtained from the S1 gene analysis of IBV-QX, LSC-99I, and TW, along with the second internal standard gene, were inserted into the pUC57 cloning vector to synthesize plasmid standards for each genotype.

[0066] Among them, the conserved region sequence of the S1 gene of IBV-QX is shown in SEQ ID NO.30, P-IBV-LSC-99I is shown in SEQ ID NO.31, P-IBV-TW is shown in SEQ ID NO.32, and the second internal standard gene P-internal standard gene Pb-56 is shown in SEQ ID NO.33.

[0067] The conserved regions of IBV-QX, LSC-99I, TW, and the second internal standard gene Pb-56 were sequentially ligated, and DNA was synthesized by Hunan Aikerui Biotechnology Co., Ltd., with the sequence shown in SEQ ID NO.34. After synthesis, the template plasmid was ligated into the multiple cloning site of the pUC57 plasmid, and the resulting plasmid served as the positive template plasmid for the second kit.

[0068] The copy number of plasmid standards is calculated using the following formula: copy number / μL = 6.02 × 10⁻⁶ 23 × plasmid concentration (ng / μL) × 10 -9 / Plasmid length (bp) / 660.

[0069] In the first kit: the copy numbers of plasmids P-IBV-4 / 91, P-IBV-TC07-2, and P-IBV-LDT3 were 6.18 × 10⁻⁶. 10 6.86×10 10 1.03×10 11 The copy number of the tandem plasmid was 2.70 × 10⁻⁶. 10 .

[0070] In the second kit: the copy numbers of P-IBV-QX, P-IBV-LSC-99I, and P-IBV-TW plasmids were 1.45 × 10⁻⁶. 11 7.19×10 10 8.02×10 10 The copy number of the tandem plasmid was 2.70 × 10⁻⁶. 10 .

[0071] (II) Optimization of reagent kit reaction system and conditions

[0072] Using the positive plasmid standard prepared in step (I) as a template, the primer concentration, probe concentration, reaction time, and annealing temperature of the two kit reaction systems were optimized and screened. Finally, the total reaction volume of the two kits was confirmed to be 25 μL, including 10 μL of 2.5×One-step RT-qPCR buffer II (Primer & Probe Plus), 2 μL of One-StepRT-qPCR Enzyme Mix II, 8 μL of RNase free water, and 5 μL of the template to be tested.

[0073] In the premix of the first kit, the final concentrations of primers IBV-4 / 91-F / R, IBV-TC07-2-F / R, IBV-LDT3-F / R, and internal control gene-F / R were all 0.2 μM, and the final concentrations of probes IBV-4 / 91-P, IBV-TC07-2-P, IBV-LDT3-P, and internal control gene-P were all 0.4 μM.

[0074] In the premix of the second kit, the final concentrations of primers IBV-QX-F / R, IBV-LSC-99I-F / R, IBV-TW-F / R, and internal control gene-F / R were all 0.2 μM, and the final concentrations of probes IBV-QX-P, IBV-LSC-99I-P, IBV-TW-P, and internal control gene-P were all 0.4 μM.

[0075] The reaction procedure was as follows: UNG enzyme treatment at 25℃ for 10 min, reverse reaction at 50℃ for 15 min, pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 5 s, annealing at 60℃ for 30 s, for 45 cycles. Fluorescence signals were collected during the annealing stage to obtain the highest amplification efficiency.

[0076] (III) Evaluation of the sensitivity of positive plasmids and establishment of standard curves

[0077] The plasmid standard prepared in step (I) was serially diluted 10-fold as an experimental template. Amplification was performed using the real-time PCR reaction system and conditions established in step (II), with a negative control group included. Signals from four fluorescence channels were collected using a real-time PCR instrument to obtain amplification curves, and a standard curve was plotted. The results are as follows: Figure 2 As shown:

[0078] (1) The limit of detection for the IBV-4 / 91 positive plasmid standard is 6.18 copies. Figure 2 A) The linear equation of the standard curve is: y = -3.414x + 41.58, and its correlation coefficient (R²) is... 2 The value is 0.999;

[0079] (2) The limit of detection for the IBV-TC07-2 positive plasmid standard is 6.86 copies. Figure 2 B), the linear equation of the standard curve is: y = -3.535x + 41.743, and its correlation coefficient (R²) is... 2 The value is 0.999;

[0080] (3) The limit of detection for the IBV-LDT3 positive plasmid standard is 1.03 copies. Figure 2 C), the linear equation of the standard curve is: y = -3.511x + 40.522, and its correlation coefficient (R²) is... 2 The value is 0.999;

[0081] (4) The limit of detection for IBV-QX positive plasmid standards is 1.45 copies. Figure 2 D), the linear equation of the standard curve is: y = -3.489x + 41.984, and its correlation coefficient (R²) is... 2 The value is 0.999;

[0082] (5) The limit of detection for the IBV-LSC-99I positive plasmid standard is 7.19 copies. Figure 2 E), the linear equation of the standard curve is: y = -3.301x + 40.502, and its correlation coefficient (R) 2 The value is 0.998;

[0083] (6) The limit of detection for IBV-TW positive plasmid standards is 80.2 copies. Figure 2 F), the linear equation of the standard curve is: y = -3.657x + 44.63, and its correlation coefficient (R) 2 The value is 0.999;

[0084] Therefore, it can be seen that the reagent kit of the present invention has extremely high sensitivity, and its lowest detection limit is significantly better than that described in the prior art.

[0085] (iv) Specificity evaluation of the kit

[0086] Nucleic acid was extracted from the preserved IBV strains of different genotypes (4 / 91, TC07-2, LDT3, QX, LSC-99I, TW, MASS) using the first and second kits in step (II), and then detected. The amplification curves are shown below. Figure 3 As shown.

[0087] The results of quantitative RT-PCR were interpreted as follows:

[0088] After sample amplification, both the FAM / HEX / ROX channels and the CY5 channel (internal standard channel) exhibit typical exponential growth curves ("S"-shaped amplification curves), while negative amplification curves show no specificity. This indicates that the experimental conditions are met.

[0089] Samples with a CT value ≤35 in the detection channel and a CT value ≤35 in the internal standard channel, and exhibiting a typical exponential growth curve (“S”-shaped amplification curve), are judged as positive.

[0090] For the detection channel with a CT value of 35 < CT value ≤ 40 and the internal standard channel with a CT value ≤ 35, it is recommended to repeat the test. If the CT value is still 35 < CT value ≤ 40 and a typical exponential growth curve is shown, it is considered positive. If the detection channel has no CT value or an atypical amplification curve and the internal standard channel has a CT value ≤ 35, it is considered negative.

[0091] For detection channels with a CT value > 40 and no specific amplification curve or no CT value, and internal standard channels with a CT value ≤ 35, the result is considered negative.

[0092] For any value or no CT value in the detection channel, and for the internal standard channel with a CT value > 35 or no CT value, the result is considered invalid and needs to be re-detected.

[0093] The results showed that the kit established in this invention can effectively detect IBV virus strains of different genotypes. The first kit can effectively amplify IBV-4 / 91, TC07-2, and LDT3 virus strains, but cannot amplify QX, LSC-99I, TW, and MASS virus strains. Therefore, it can be used to detect IBV-4 / 91, TC07-2, and LDT3 types. Figure 3 As shown in A. The second kit can effectively amplify IBV-QX, LSC-99I, and TW virus strains, but cannot amplify 4 / 91, TC07-2, LDT3, and MASS virus strains. Therefore, it can be used to detect IBV-QX, LSC-99I, and TW types, such as... Figure 3 As shown in B.

[0094] The above results show that the probe and primer composition and kit provided by the present invention can effectively detect the infection status of common clinical IBV genotypes, with good specificity and anti-interference properties, and no cross-reaction between different genotype strains occurs.

[0095] (v) Repeatability evaluation

[0096] The recombinant plasmid standard was serially diluted 10-fold to obtain a concentration of 1×10⁻⁶. 3 copies / μL, 1×10 4 copies / μL and 1×10 5 Using positive plasmids at a concentration of copies / μL as templates, quantitative RT-PCR experiments were performed using both the first and second kits. Each concentration was repeated three times, and the standard deviation (SD) and coefficient of variation (CV) of the Ct values ​​were calculated. Three additional independent experimental dates were selected, and the same dilution gradient was tested three times across different batches to analyze the variability of Ct values ​​between batches. The results are shown in Table 1. The intra- and inter-batch coefficients of variation for both the first and second kits were less than 2%, indicating that the kits of this invention have good reproducibility.

[0097] Table 1. Repeatability tests of the kit

[0098]

[0099] Example 3: Clinical application of the real-time PCR detection kit and method

[0100] Fifty-eight clinical samples, including pharyngeal swabs, tracheal, lung, and kidney tissue, were collected and tested using the kit of this invention. Of these, 23 samples were QX single positive, 5 samples were TC07-2 single positive, 4 samples were 4 / 91 single positive, and 8 samples showed mixed infection with two or more pathogen genotypes.

[0101] Table 2 Results of the kit testing clinical samples

[0102]

[0103] The results showed that for mixed infections with multiple genotypes, the kit of the present invention can directly and clearly identify multiple coexisting genotypes in the sample (such as samples No. 22056, 22247, 24719, etc.) in a single run.

[0104] The above results indicate that the kit of the present invention can be used as a useful tool for rapid detection and diagnosis of different genotypes of infectious bronchitis in chickens in clinical practice, and provides key data support for vaccine development and epidemiological research.

Claims

1. A primer and probe composition for genotyping detection of infectious bronchitis virus (IBV), the composition comprising primers and probes for detecting IBV-4 / 91, wherein the sequence of the upstream primer is SEQ ID NO. 1, the sequence of the downstream primer is SEQ ID NO. 2, and the sequence of the probe is SEQ ID NO. 3; primers and probes for detecting IBV-TC07-2, wherein the sequence of the upstream primer is SEQ ID NO. 4, the sequence of the downstream primer is SEQ ID NO. 5, and the sequence of the probe is SEQ ID NO. 6; primers and probes for detecting IBV-LDT3, wherein the sequence of the upstream primer is SEQ ID NO. 7, the sequence of the downstream primer is SEQ ID NO. 8, and the sequence of the probe is SEQ ID NO. 9; and primers and probes for a first internal control gene, wherein the sequence of the upstream primer is SEQ ID NO. 10, the sequence of the downstream primer is SEQ ID NO. 11, and the sequence of the probe is SEQ ID NO. 12; Primers and probes for detecting IBV-QX type, the sequence of the upstream primer is SEQ ID NO. 13, the sequence of the downstream primer is SEQ ID NO. 14, and the sequence of the probe is SEQ ID NO. 15; Primers and probes for detecting IBV-LSC-99I type, the sequence of the upstream primer is SEQ ID NO. 16, the sequence of the downstream primer is SEQ ID NO. 17, and the sequence of the probe is SEQ ID NO. 18; Primers and probes for detecting IBV-TW type, the sequence of the upstream primer is SEQ ID NO. 19, the sequence of the downstream primer is SEQ ID NO. 20, and the sequence of the probe is SEQ ID NO. 21; Primers and probes for the second internal standard gene, the sequence of the upstream primer is SEQ ID NO. 22, the sequence of the downstream primer is SEQ ID NO. 23, and the sequence of the probe is SEQ ID NO.

24.

2. The composition according to claim 1, characterized in that... The probes used to detect IBV-4 / 91 and IBV-QX have fluorescein FAM attached to their 5' ends and fluorescein BHQ1 attached to their 3' ends, respectively. The probes used to detect IBV-TC07-2 and IBV-LSC / 99I have fluorescein HEX attached to their 5' ends and fluorescein BHQ1 attached to their 3' ends, respectively. The probes used to detect IBV-LDT3 and IBV-TW have fluorescein ROX attached to their 5' ends and fluorescein BHQ2 attached to their 3' ends, respectively. The probes used to detect the first internal control gene and the second internal control gene have fluorescein CY5 attached to their 5' ends and fluorescein BHQ2 attached to their 3' ends, respectively.

3. The use of the composition according to claim 1 or 2 in the preparation of a kit for detecting chicken infectious bronchitis virus typing, wherein the chicken infectious bronchitis virus includes 4 / 91, TC07-2, LDT3, QX, LSC-99I and TW types.

4. A TaqMan probe-based multiplex real-time PCR detection kit, the kit comprising a first kit and a second kit, the first kit containing primers and probes of SEQ ID NO. 1-12 according to claim 1 or 2, and the second kit containing primers and probes of SEQ ID NO. 13-24 according to claim 1 or 2.

5. The reagent kit according to claim 4, characterized in that... The positive template plasmid of the first kit contains the gene with the sequence shown in SEQ ID NO.29, and the positive template plasmid of the second kit contains the gene with the sequence shown in SEQ ID NO.

34.

6. A method for detecting different genotypes of infectious bronchitis virus in chickens using the kit described in claim 4, the method comprising the following steps: (1) Extract RNA from the sample to be tested; (2) Using the RNA obtained in step (1) as a template, perform real-time RT-PCR using a detection kit; (3) The results of quantitative RT-PCR were determined as follows: a. Establishment conditions: After sample amplification, both the FAM / HEX / ROX channels and the CY5 channel show typical exponential growth curves, while the negative result shows no specific amplification curve, indicating that the experimental conditions are met. b. Qualitative analysis to determine the positive / negative status of each channel in FAM / HEX / ROX: Samples with a CT value ≤35 in the detection channel and a CT value ≤35 in the internal standard channel, and exhibiting a typical exponential growth curve, are judged as positive. For the detection channel with a CT value of 35 < CT value ≤ 40 and the internal standard channel with a CT value ≤ 35, it is recommended to repeat the test. If the CT value is still 35 < CT value ≤ 40 and a typical exponential growth curve is shown, it is considered positive. If the detection channel has no CT value or an atypical amplification curve and the internal standard channel has a CT value ≤ 35, it is considered negative. For detection channels with a CT value > 40 and no specific amplification curve, or with no CT value and an internal standard channel CT value ≤ 35, the result is considered negative. For any value or no CT value in the detection channel, and for the internal standard channel with a CT value >35 or no CT value, the result is considered invalid. c. Based on the above qualitative results, further quantitative analysis should be performed on viral nucleic acid positive or weakly positive results: Standard curves for different viral copy numbers were established using positive plasmid standards. Based on the established viral copy number standard curves, the copy numbers of different viral nucleic acids in the samples to be tested were calculated using the CT values ​​of the samples.

7. The method according to claim 6, characterized in that... The detection system for real-time RT-PCR is as follows: First reagent kit composition: 10 μL 2.5×One-step RT-qPCR buffer II (Primer & Probe Plus) + 2 μL One-Step RT-qPCR Enzyme Mix II + 5 μL Template + 8 μL RNase free water, total composition 25 μL; Second kit composition: 10 μL 2.5×One-step RT-qPCR buffer II (Primer & Probe Plus) + 2 μL One-Step RT-qPCR Enzyme Mix II + 5 μL Template + 8 μL RNase free water, total 25 μL.

8. The method according to claim 7, characterized in that The standard curves for each viral genotype are as follows: IBV-4 / 91 standard curve: y = -3.414x + 41.58; IBV-TC07-2 standard curve: y = -3.535x + 41.743; IBV-LDT3 standard curve: y = -3.511x + 40.522; IBV-QX standard curve: y = -3.489x + 41.984; IBV-LSC-99I standard curve: y = -3.301x + 40.502; IBV-TW standard curve: y = -3.657x + 44.63.

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