Detection primer combination of infectious bronchitis virus and application thereof

Abstract

The application discloses a detection primer combination of infectious bronchitis virus, comprising a primer pair and a probe for detecting infectious bronchitis virus subgenomic-N, a primer pair and a probe for detecting infectious bronchitis virus genome and a primer pair and a probe for detecting an internal reference gene. The application also discloses a method for detecting infectious bronchitis virus based on the aforementioned primer combination for non-diagnostic purposes. The primer combination has low detection limit, good sensitivity, good specificity, good repeatability and application prospect to a certain extent, and can determine the infectious period of infectious bronchitis virus.

Description

Technical Field

[0001] This invention belongs to the field of virus detection and relates to a primer combination for detecting infectious bronchitis virus and its application. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious viral respiratory disease in poultry caused by the infectious bronchitis virus (IBV). It is characterized by sneezing, coughing, and tracheal rales in infected chickens. IBV was once listed in the UK as one of the leading infectious poultry diseases causing economic losses to the poultry industry. IBV can lead to decreased feed conversion ratios in chickens; mixed infections are a major cause of broiler culling; infected commercial chickens suffer from enlarged kidneys, forming "mottled kidneys"; infected laying hens experience decreased egg production and quality; and even death, causing huge economic losses to the global poultry industry. For nearly half a century, the main preventative measure for infectious bronchitis has been vaccination, with H120, H52, Ma5, 28 / 86, and IBn vaccine strains being the primary methods of control. In recent years, however, due to the continuous emergence of new variant strains, immunization failures have occurred frequently.

[0003] IBV belongs to the grama coronavirus family, genus Coronavirus, family Coronaviridae. It is a single-stranded, non-segmented, positive-sense RNA virus (genomic RNA, gRNA). During replication and transcription, coronaviruses produce subgenomic RNA (sgRNA). The structures of gRNA and sgRNA are as follows... Figure 1 As shown, coronavirus sgRNA is produced through leader-to-body discontinuous transcription of the replication-transcription complex (RTC). Specifically, the positive-strand sgRNA contains a leader RNA identical to the 5' end of the genome, connected to the body of the sgRNA identical to the 3' end of the downstream genome. Coronavirus sgRNA-N has a high content, good specificity, and the subgenome can reflect the genetic stage of viral replication. Currently, IBV diagnosis mainly uses molecular biology methods such as reverse transcription polymerase chain reaction (RT-PCR) to detect viral genomic RNA (gRNA), but these methods cannot determine viral infectivity or viral replication status.

[0004] Currently, there are no reports on detection methods for infectious bronchitis virus sgRNA, nor are there reports on joint detection of IBV genome and subgenome. Therefore, existing technologies make it difficult to determine the infection period of IBV. Summary of the Invention

[0005] This invention establishes a triple fluorescence quantitative method for detecting genomic RNA (gRNA), subgenomic RNA (sgRNA), and the chicken internal reference β-actin gene of avian infectious bronchitis virus (IBV). This invention enables simultaneous and accurate detection and relative quantification of both the IBV genome and IBV subgenome in a sample. The detection results can be directly used as important indicators for detecting viral infectivity and replication efficiency, and can be used for qualitative and quantitative evaluation of inactivated vaccine quality, the risk of virulence reversion in attenuated vaccines, and viral histophagy. This invention is simple to operate, highly sensitive, specific, and reproducible, enabling rapid and accurate differential diagnosis and relative quantification. It can quantitatively determine IBV infectivity and replication efficiency, thereby implementing appropriate prevention or treatment plans and reducing economic losses in the poultry industry.

[0006] This invention establishes a detection method for IBV subgenomes, which can qualitatively and quantitatively analyze IBV infectivity and replication efficiency. It can be widely applied to basic and applied research on IBV and even coronaviruses, such as virus-host interaction, vaccine development, and pathogenic mechanisms.

[0007] This invention establishes a triple real-time fluorescence RT-PCR method to simultaneously detect IBV sgRNA, gRNA, and the internal reference gene β-actin, and evaluates the detection performance of the established method. This invention significantly improves detection efficiency by adding three fluorescent probes labeled with different fluorescent signal groups to the same quantitative fluorescence reaction system, enabling the simultaneous detection of three target genes—chicken internal reference β-actin, gRNA ORF1ab, and sgRNA-N—in a single reaction tube. The chicken internal reference gene introduced in this invention can serve as a quality control indicator for nucleic acid extraction and a relative quantitative internal reference. It can also detect the absolute and relative content of IBV subgenomes and the genome as important indicators for viral infectivity and replication efficiency. This method can replace chicken embryo inoculation experiments to evaluate the quality of inactivated and attenuated vaccines, replace chicken embryo growth curve determination to reflect viral replication efficiency, replace animal experiments to evaluate the risk of virulence reversion in attenuated vaccines, and replace animal experiments and histoimmunochemical methods to evaluate viral tissue phagocytosis. This invention enables simultaneous detection of both the IBV genome and IBV subgenome in a sample. It is simple to operate, highly sensitive, specific, and reproducible, allowing for rapid and accurate differential diagnosis and precise absolute and relative quantification. This enables the implementation of appropriate prevention or treatment plans, reducing economic losses in the poultry industry.

[0008] To address the problems existing in the prior art, the first aspect of the present invention provides a nucleic acid combination, wherein the nucleic acid combination is C1, C2, C3 or C4;

[0009] C1: First nucleic acid combination;

[0010] C2: A combination of the first nucleic acid combination and the second nucleic acid combination;

[0011] C3: A combination of the first and third nucleic acid combinations;

[0012] C4: A combination of the first, second, and third nucleic acid combinations;

[0013] The first nucleic acid combination is nucleic acid combination 1, nucleic acid combination 2, nucleic acid combination 3 or nucleic acid combination 4;

[0014] The nucleic acid combination 1 includes primer pairs with sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively;

[0015] The nucleic acid combination 2 includes primer pairs with sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively;

[0016] The nucleic acid combination 3 includes primer pairs with sequences as shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively;

[0017] The nucleic acid combination 4 includes primer pairs with sequences as shown in SEQ ID NO.11 and SEQ ID NO.12, respectively;

[0018] The second nucleic acid combination is nucleic acid combination a, nucleic acid combination b, nucleic acid combination c, or nucleic acid combination d;

[0019] The nucleic acid combination a includes primer pairs with sequences as shown in SEQ ID NO.14 and SEQ ID NO.15, respectively;

[0020] The nucleic acid combination b includes primer pairs with sequences as shown in SEQ ID NO.17 and SEQ ID NO.18, respectively;

[0021] The nucleic acid combination c includes primer pairs with sequences as shown in SEQ ID NO.20 and SEQ ID NO.21, respectively;

[0022] The nucleic acid combination d includes primer pairs with sequences as shown in SEQ ID NO.23 and SEQ ID NO.24, respectively;

[0023] The third nucleic acid combination includes primer pairs with sequences as shown in SEQ ID NO.26 and SEQ ID NO.27, respectively.

[0024] In some implementations, the following are selected: D1, D2, D3, D4, D5, D6, D7, D8, and D9;

[0025] D1: The nucleic acid combination 1 further includes probe 1; in probe 1, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:4, one end of which is connected to a first fluorescent group and the other end of which is connected to a first quenching group;

[0026] D2: The nucleic acid combination 2 further includes a probe 2; in the probe 2, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:7, one end of which is connected to a first fluorescent group, and the other end of which is connected to a first quenching group;

[0027] D3: The nucleic acid combination 3 further includes a probe 3; in the probe 3, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:10, one end of which is connected to a first fluorescent group, and the other end of which is connected to a first quenching group;

[0028] D4: The nucleic acid combination 4 further includes a probe 4; in the probe 4, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:13, one end of which is connected to a first fluorescent group and the other end of which is connected to a first quenching group;

[0029] D5: The nucleic acid combination a further includes probe a; in probe a, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:16, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group;

[0030] D6: The nucleic acid combination b further includes probe b; in probe b, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:19, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group;

[0031] D7: The nucleic acid combination c further includes probe c; in probe c, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:22, one end of which is connected to a second fluorescent group and the other end of which is connected to a second quenching group;

[0032] D8: The nucleic acid combination d further includes probe d; in probe d, the nucleic acid sequence or its complementary sequence as shown in SEQ ID NO:25 is connected to a second fluorescent group at one end and a second quenching group at the other end;

[0033] D9: The third nucleic acid combination also includes probe p; in probe p, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:28, one end of which is connected to a third fluorescent group and the other end of which is connected to a third quenching group.

[0034] In some embodiments, the first quenching group can quench the fluorescence emitted by the first fluorescent group, but the first quenching group cannot quench the fluorescence emitted by any one of the second fluorescent group, the second quenching group, the third fluorescent group, and the third quenching group.

[0035] The second quenching group can quench the fluorescence emitted by the second fluorescent group, but the second quenching group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the third fluorescent group, and the third quenching group.

[0036] The third quenching group can quench the fluorescence emitted by the third fluorescent group, but the third quenching group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the second fluorescent group, and the second quenching group.

[0037] The first fluorescent group cannot quench the fluorescence emitted by any one of the second fluorescent group, the second quenching group, the third fluorescent group, and the third quenching group;

[0038] The second fluorescent group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the third fluorescent group, and the third quenching group;

[0039] The third fluorescent group cannot quench the fluorescence emitted by any of the first fluorescent group, the first quenching group, the second fluorescent group, and the second quenching group.

[0040] In some implementations, the options are selected from E1, E2, E3, E4, E5, or E6;

[0041] E1: The first fluorescent group and the first quenching group are group 1 dyes; the second fluorescent group and the second quenching group are group 2 dyes; the third fluorescent group and the third quenching group are group 3 dyes;

[0042] E2: The first fluorescent group and the first quenching group are group 1 dyes; the second fluorescent group and the second quenching group are group 3 dyes; the third fluorescent group and the third quenching group are group 2 dyes;

[0043] E3: The first fluorescent group and the first quenching group are dyes of the second group; the second fluorescent group and the second quenching group are dyes of the first group; the third fluorescent group and the third quenching group are dyes of the third group;

[0044] E4: The first fluorescent group and the first quenching group are dyes of the second group; the second fluorescent group and the second quenching group are dyes of the third group; the third fluorescent group and the third quenching group are dyes of the first group;

[0045] E5: The first fluorescent group and the first quenching group are dyes of the third group; the second fluorescent group and the second quenching group are dyes of the first group; the third fluorescent group and the third quenching group are dyes of the second group;

[0046] E6: The first fluorescent group and the first quenching group are dyes of the third group; the second fluorescent group and the second quenching group are dyes of the second group; the third fluorescent group and the third quenching group are dyes of the first group;

[0047] The first group of dyes consists of FAM and TAMRA;

[0048] The second group of dyes consists of: VIC and BHQ1;

[0049] The third group of dyes consists of ROX and BHQ2.

[0050] In some implementations, the following are selected: F1, F2, F3, F4, F5, F6, F7, F8, and F9;

[0051] F1: In the probe 1, a first fluorescent group is attached to the 5' end and a first quenching group is attached to the 3' end;

[0052] F2: In the probe 2, a first fluorescent group is attached to the 5' end and a first quenching group is attached to the 3' end;

[0053] F3: In the probe 3, a first fluorescent group is attached to the 5' end, and a first quenching group is attached to the 3' end;

[0054] F4: In the probe 4, a first fluorescent group is attached to the 5' end and a first quenching group is attached to the 3' end;

[0055] F5: In the probe a, a second fluorescent group is attached to the 5' end and a second quenching group is attached to the 3' end;

[0056] F6: In the probe b, a second fluorescent group is attached to the 5' end and a second quenching group is attached to the 3' end;

[0057] F7: In the probe c, a second fluorescent group is attached to the 5' end and a second quenching group is attached to the 3' end;

[0058] F8: In the probe d, a second fluorescent group is attached to the 5' end and a second quenching group is attached to the 3' end;

[0059] F9: In the probe p, a third fluorescent group is attached to the 5' end and a third quenching group is attached to the 3' end.

[0060] A second aspect of the present invention provides a kit containing the nucleic acid combination described in the first aspect of the present invention.

[0061] The third aspect of the present invention discloses a method for detecting infectious bronchitis virus for non-disease diagnostic purposes, wherein the method comprises: performing qPCR detection on nucleic acids extracted from a target test sample using the nucleic acid combination described in the first aspect of the present invention.

[0062] In some embodiments, the method includes the following steps:

[0063] S1: Extract RNA from the target sample and prepare it into cDNA;

[0064] S2: Mix the cDNA, the nucleic acid combination, and the qPCR reaction reagent to obtain the reaction system;

[0065] S3: The reaction system is subjected to thermal cycling, and the infectious bronchitis virus is detected by fluorescence signal.

[0066] In some embodiments, the GenBank number of the infectious bronchitis virus described is DQ834384.1.

[0067] In some embodiments, the thermal cycle includes the following steps:

[0068] Step 1: Reverse transcription at 50℃ for 5 minutes;

[0069] The second step is to pre-denature at 95°C for 30 seconds, followed by one cycle.

[0070] The third step is to denature at 95℃ for 5 seconds, anneal at 60℃ for 30 seconds, and extend the heat for 35-40 times. Attached Figure Description

[0071] Figure 1 This is a schematic diagram of the structures of IBVgRNA and sgRNA.

[0072] Figure 2 The graph shows the amplification curves of IBV gRNA, IBV sgRNA, and β-actin genes in avian infectious bronchitis attenuated vaccine detected by triple TaqMan RT-PCR.

[0073] Figure 3 The Ct value was determined for IBV gRNA detection.

[0074] Figure 4 This is a standard curve for IBV gRNA detection.

[0075] Figure 5 The Ct value was determined for IBV sgRNA detection.

[0076] Figure 6 The standard curve for IBV sgRNA detection.

[0077] Figure 7 The Ct value was determined for β-actin gene detection.

[0078] Figure 8 This is a standard curve for β-actin gene detection.

[0079] Figure 9 The results of a sensitivity assay for IBV gRNA detection.

[0080] Figure 10 The results of a sensitivity assay for IBV sgRNA detection.

[0081] Figure 11 The results are from a sensitivity test for β-actin gene detection.

[0082] Figure 12 Amplification curves for specific detection of IBV gRNA and IBV sgRNA. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0084] Materials and instruments not described in this invention are conventional materials and instruments in the art. Operational details not described in this invention are conventional operations in the art. The software used in this invention is operated by conventional methods in accordance with the software provider's instructions. The reagent kits used in this invention are operated by conventional methods in accordance with the reagent kit's instruction manual.

[0085] Applications of methods for detecting infectious bronchitis virus (IBV) for non-disease diagnostic purposes include, but are not limited to: studying the interaction between IBV and the host; studying the protective ability against IBV after vaccination; and environmental assessment of whether infectious bronchitis virus exists in chicken farms.

[0086] Example 1: Preparation of standard plasmids

[0087] This invention utilizes gRNA ORF1 (genomic RNA ORF1, also known as ORF1ab, positions 530-12391, 12466-20424 of GenBank:DQ834384.1), sgRNA-N (subgenomic RNA-N, sequence shown in SEQ ID NO.1 below, where positions 1-64 are from the gRNA 5'UTR), and the chicken β-actin gene (GenBank:L08165.1) of the avian infectious bronchitis virus M41 strain (GenBank:DQ834384.1) to analyze the genetic sequence using Primer Express. The 3.0.1 software was used to design multiple pairs of highly conserved primers and probes, as shown in Table 1. In each group of nucleic acids, F after - represents the upstream primer, R after - represents the downstream primer, and P after - represents the probe. For probes targeting sgRNA, the 5' end is connected to the FAM group and the 3' end is connected to the TAMRA group; for probes targeting gRNA, the 5' end is connected to the VIC group and the 3' end is connected to the BHQ1 group; for probes targeting β-actin, the 5' end is connected to the ROX group and the 3' end is connected to the BHQ2 group.

[0088] In the application tests of subsequent embodiments of the present invention, gRNA-F1, gRNA-R1, and gRNA-P1 were selected for IBV gRNA, sgRNA-F1, sgRNA-R1, and sgRNA-P1 were selected for sgRNA-N, and β-actin-F, β-actin-R, and β-actin-P were selected for chicken internal reference β-actin.

[0089] The sgRNA-N sequence is as follows (SEQ ID NO.1, where U is represented by T):

[0090]

[0091] Table 1. Primer and probe information

[0092]

[0093]

[0094] A synthetic gene fragment was constructed by Shanghai Sangon Biotech Co., Ltd., comprising IBVgRNAORF1ab (the sequence between primers gRNA-F1 and gRNA-R1), sgRNA-N (the sequence between primers sgRNA-F1 and sgRNA-R1), and a partial sequence of the chicken β-actin gene (the sequence between primers β-actin-F and β-actin-R). The sequences of these three parts are shown in Table 2. Restriction enzyme sites were added to the ends of each synthetic fragment, and the fragments and vectors were digested separately. The three fragments were then sequentially inserted into the pUC57 cloning vector to construct a standard plasmid (named pUC57-gIBV-sgIBV-βactin), which will be used in subsequent experiments. This plasmid contains the three gene fragments in tandem. The concentration of the standard plasmid was measured using a Nanodrop spectrophotometer. The standard plasmid was serially diluted 10-fold with DNase / RNase-free distilled water to establish a concentration gradient range of 1×10⁻⁶. 0 -1×10 10 Copy / μL of the standard and then store it at -20°C for subsequent experiments.

[0095] Table 2. Information on synthesized gene fragments

[0096]

[0097] Example 2: Optimization of Triple TaqMan RT-PCR System

[0098] The standard plasmid was diluted to a final concentration of 1×10⁻⁶ with DNase / RNase-free distilled water. 7Using a template of 3 μL / μL, the matrix method was applied to optimize the real-time quantitative PCR conditions, including primer and probe concentrations and annealing temperature, on a QuantStudio 5 real-time PCR instrument. Each primer was performed at a final concentration between 0.15-0.35 μmol / L, and the probe at a final concentration between 0.25-0.4 μmol / L to select the optimal primer and probe concentrations. The annealing temperature was optimized at 56-60℃. The reaction system consisted of 3 μL template, 10 μL 2×Premix Ex Taq, 0.3-0.7 μL of gIBV / sgIBV / β-actin primer (10 μM), and 0.5-0.8 μL of gIBV / sgIBV / β-actin probe (10 μM). Enzyme-free water was added to a final volume of 20 μL. Water was used as a negative control for parallel processing.

[0099] Results: In the final optimized 20 μL triplet TaqMan RT-PCR reaction system, the final F / R volumes of the gIBV / sgIBV / β-actin upstream and downstream primers were 0.6 μL (0.3 μmol / L), 0.6 μL (0.3 μmol / L), and 0.3 μL (0.15 μmol / L), respectively; the final volumes of the gIBV / sgIBV / β-actin probe were 0.8 μL (0.4 μmol / L), 0.8 μL (0.4 μmol / L), and 0.6 μL (0.3 μmol / L), as shown in Table 3; the optimal annealing temperature was 60℃. The triplet TaqMan RT-PCR reaction program was as follows: Step 1, reverse transcription at 50℃ for 5 min; Step 2, pre-denaturation at 95℃ for 30 s, 1 cycle; Step 3, denaturation at 95℃ for 5 s, annealing and extension at 60℃ for 30 s; 40 cycles. Figure 2 As shown, the triple TaqMan RT-PCR optimization system can successfully amplify the genome, subgenome, and chicken β-actin gene of the attenuated live attenuated avian infectious bronchitis vaccine IBV.

[0100] Table 3. Triple TaqMan RT-PCR reaction system

[0101] 2×Premix Ex Taq 10 β-actin-F 0.3 β-actin-R 0.3 β-actin-P 0.6 sgRNA-F1 0.6 sgRNA-R1 0.6 sgRNA-P1 0.8 gRNA-F1 0.6 gRNA-R1 0.6 gRNA-P1 0.8 <![CDATA[ddH2O]]> 1.8 template 3

[0102] Example 3: Establishment of a triple TaqMan RT-PCR standard curve

[0103] Based on the optimized reaction conditions and procedures in Example 2, 1×10⁻⁶ serially diluted 10-fold was used. 3 -1×10 10A triplet TaqMan RT-PCR standard curve was constructed using a standard plasmid of copy number / μL as a template. Linear regression analysis was performed on the Ct value and logarithm of the plasmid copy number. Standard curves were also generated for IBV gRNA, IBV sgRNA, and chicken internal control β-actin, with water used as a negative control.

[0104] Result: As Figure 3 , 4 As shown, a standard curve for IBV gRNA was successfully established, with a correlation coefficient (R²). 2 The equation slope and amplification efficiency (E%) were 0.999, -3.453, and 94.791, respectively; Figure 5 , 6 As shown, a standard curve for IBV sgRNA was successfully established, with a correlation coefficient (R²). 2 The equation slope and amplification efficiency (E%) were 0.999, -3.496, and 93.23, respectively; Figure 7 , 8 As shown, a standard curve for chicken β-actin was successfully established, with a correlation coefficient (R²). 2 The equation slope and amplification efficiency (E%) were 0.999, -3.496, and 93.23, respectively. 2 Both the E% and Ct values ​​indicate a strong linear relationship between the initial template and the Ct value, thus demonstrating the reliability of the triple TaqMan RT-PCR standard curve.

[0105] Example 4: Sensitivity test of triple TaqMan RT-PCR method

[0106] The standard plasmid was serially diluted 10-fold (using water as the diluent), with a concentration gradient ranging from 1×10⁻⁶. 0 -1×10 6 Using a standard of 1 copy / μL as a template, the optimized reaction system and program were run according to the optimized reaction conditions and procedures in Example 2 to determine the sensitivity of the triplet TaqMan RT-PCR.

[0107] Results: Within 40 cycles, IBV gRNA, sgRNA, and chicken β-actin gene levels were 1×10⁻⁶. 1 -1×10 6 All copies / μL standards showed amplification curves, while the negative control (water) showed no amplification. The sensitivity was determined to be 1×10⁻⁶. 1 Copy / μL.

[0108] Results: The sensitivity to IBV gRNA, IBV sgRNA, and chicken β-actin all reached 1×10⁻⁶. 1 Copy / μL, such as Figure 9 ,10 As shown in Figure 11.

[0109] Example 5: Specificity test of triple TaqMan RT-PCR method

[0110] Nucleic acid (DNA / cDNA) was extracted from the following pathogens: avian infectious bronchitis virus M41 strain, Newcastle disease virus La Sota strain, avian leukosis virus J subtype HPRS103 strain, avian influenza virus H9N2 type Re-2 vaccine strain, chicken astrovirus GXJL815 strain, avian infectious laryngotracheitis virus K317 strain, and avian reticuloendothelial proliferator-associated virus HA9901 strain. An optimized system was prepared, and the detection program was run, with water as a negative control. The specificity of the IBV gRNA, IBV sgRNA, and chicken β-actin triple TaqMan RT-PCR methods established in Example 2 was verified.

[0111] Result: As Figure 12 As shown, only IBV gRNA and IBV sgRNA amplified with "S-shaped" curves and Ct values ​​<36. Other pathogens, including Newcastle disease virus, avian leukosis virus, avian influenza virus, chicken astrovirus, avian infectious laryngotracheitis virus, avian reticuloendothelial proliferator-associated virus, and the negative control, showed no obvious amplification curves. This indicates that the triplet TaqMan RT-PCR method of this invention has good specificity.

[0112] Example 6: Repeatability test of triple TaqMan RT-PCR method

[0113] The standard plasmids were diluted 10-fold at four different concentration gradients (1×10⁻⁶). 3 -1×10 6 Using a copy / μL) as a template, repeatability testing was performed according to the optimized reaction system and operating procedure of Example 2. Each gradient of plasmid was repeated three times in each experiment, divided into three batches (1d, 3d, and 5d) to verify the repeatability of intra- and inter-group data of the method.

[0114] Results: The coefficient of variation (CV) for within-group replicates was 0.16-0.44%, and the CV for between-group replicates was 0.11-0.59%. Both CV values ​​were below 1%, indicating that the established triple TaqMan RT-PCR method for IBV gRNA, IBV sgRNA, and chicken β-actin has good stability and repeatability, as shown in Table 4.

[0115] Table 4. Within-group and between-group repeatability

[0116]

[0117] Example 7: Clinical sample detection using the triple TaqMan RT-PCR method

[0118] Eighty-nine clinical samples were tested using the triple TaqMan RT-PCR detection method for IBV gRNA, IBV sgRNA, and chicken β-actin established in Example 2 of this invention. Simultaneously, these samples were inoculated into chicken embryo allantoic fluid for virus detection. The samples mainly consisted of throat swabs, tracheal swabs, and kidney tissue samples collected from different regions including Jilin, Liaoning, and Heilongjiang. The supernatant of the tested samples was aseptically inoculated into the allantoic cavity of 10-day-old SPF chicken embryos. Chicken embryos that died within 24 hours of inoculation were discarded, and allantoic fluid from surviving chicken embryos was collected 3 days after inoculation. Total RNA was extracted from the chicken embryo allantoic fluid for subsequent quantitative IBV detection.

[0119] Results: Of the 89 suspected infectious bronchitis virus (IBC) infected chickens, 61 samples from throat swabs and tissue / organ samples tested positive for RNA. All 28 gRNA-negative samples tested negative for sgRNA. Of the 61 gRNA-positive samples, 53 were gRNA-positive, with a positive rate of 86.89%. As shown in Table 5, when the Ct value of gRNA was <30, the sgRNA positive rate was 100.00%, and the positive rate of inoculated chicken embryo allantoic fluid was also 100.00%. When the Ct value of gRNA was in the range of 30–32, the detection rate of sgRNA was 75.00%, and the positive rate of inoculated chicken embryo allantoic fluid was also 75.00%. When the Ct value of gRNA was in the range of 32–35, the detection rate of sgRNA was 44.44%, and the positive rate of inoculated chicken embryo allantoic fluid was 33.33%. The detection rates of sgRNA and inoculated chicken embryo allantoic fluid showed a high degree of consistency. Furthermore, for the same sample, the Ct value of gRNA was consistently lower than that of sgRNA. For samples positive for sgRNA, comparing the Ct values ​​of gRNA and sgRNA, Ct(sgRNA) - Ct(gRNA) = 5.21 ± 2.82, with a 95% confidence interval of (4.72, 5.70). IBV subgenomic detection exhibits high sensitivity and specificity, showing strong consistency with IBV isolation and culture results from chicken embryos. This invention establishes a triple TaqMan RT-PCR method for qualitative and quantitative analysis of IBV replication status in vivo, enabling the implementation of appropriate prevention or treatment strategies and reducing economic losses in the poultry industry.

[0120] Common sense dictates that in the early stages of IBV infection, gRNA levels are low, and sgRNA is absent or minimal. As infection progresses, both gRNA and sgRNA levels increase, with sgRNA increasing more rapidly. In the early and middle stages of infection, a smaller difference between the Ct values ​​of gRNA and sgRNA indicates a higher relative expression level of sgRNA, thus suggesting a longer infection duration. The triplet TaqMan RT-PCR method of this invention can, to some extent, determine the stage of IBV infection.

[0121] Table 5. IBV gRNA positive samples and detection rate of IBV sgRNA in inoculated chicken embryos

[0122]

[0123] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method for detecting infectious bronchitis virus for non-disease diagnostic purposes, the method comprising: performing qPCR detection on nucleic acids extracted from a target test sample using a nucleic acid combination; The GenBank number for the infectious bronchitis virus is DQ834384.1; The nucleic acid combination is C2 or C4; C2: A combination of the first nucleic acid combination and the second nucleic acid combination; C4: A combination of the first, second, and third nucleic acid combinations; The first nucleic acid combination includes primer pairs with sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; The second nucleic acid combination comprises primer pairs with sequences as shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; The third nucleic acid combination includes primer pairs with sequences as shown in SEQ ID NO.26 and SEQ ID NO.27, respectively.

2. The method as described in claim 1, characterized in that, Selected from D1, D5, and D9 below; D1: The first nucleic acid combination further includes probe 1; in probe 1, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:4, one end is connected to a first fluorescent group, and the other end is connected to a first quenching group; D5: The second nucleic acid combination further includes probe a; in probe a, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:16, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group; D9: The third nucleic acid combination also includes probe p; in probe p, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO:28, one end of which is connected to a third fluorescent group and the other end of which is connected to a third quenching group.

3. The method as described in claim 2, characterized in that, The first quenching group can quench the fluorescence emitted by the first fluorescent group, but the first quenching group cannot quench the fluorescence emitted by any one of the second fluorescent group, the second quenching group, the third fluorescent group, and the third quenching group. The second quenching group can quench the fluorescence emitted by the second fluorescent group, but the second quenching group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the third fluorescent group, and the third quenching group. The third quenching group can quench the fluorescence emitted by the third fluorescent group, but the third quenching group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the second fluorescent group, and the second quenching group. The first fluorescent group cannot quench the fluorescence emitted by any one of the second fluorescent group, the second quenching group, the third fluorescent group, and the third quenching group; The second fluorescent group cannot quench the fluorescence emitted by any one of the first fluorescent group, the first quenching group, the third fluorescent group, and the third quenching group; The third fluorescent group cannot quench the fluorescence emitted by any of the first fluorescent group, the first quenching group, the second fluorescent group, and the second quenching group.

4. The method as described in claim 2 or 3, characterized in that, Selected from E1, E2, E3, E4, E5, or E6; E1: The first fluorescent group and the first quenching group are group 1 dyes; the second fluorescent group and the second quenching group are group 2 dyes; the third fluorescent group and the third quenching group are group 3 dyes; E2: The first fluorescent group and the first quenching group are group 1 dyes; the second fluorescent group and the second quenching group are group 3 dyes; the third fluorescent group and the third quenching group are group 2 dyes; E3: The first fluorescent group and the first quenching group are dyes of the second group; the second fluorescent group and the second quenching group are dyes of the first group; the third fluorescent group and the third quenching group are dyes of the third group; E4: The first fluorescent group and the first quenching group are dyes of the second group; the second fluorescent group and the second quenching group are dyes of the third group; the third fluorescent group and the third quenching group are dyes of the first group; E5: The first fluorescent group and the first quenching group are dyes of the third group; the second fluorescent group and the second quenching group are dyes of the first group; the third fluorescent group and the third quenching group are dyes of the second group; E6: The first fluorescent group and the first quenching group are dyes of the third group; the second fluorescent group and the second quenching group are dyes of the second group; the third fluorescent group and the third quenching group are dyes of the first group; The first group of dyes consists of FAM and TAMRA; The second group of dyes consists of: VIC and BHQ1; The third group of dyes consists of ROX and BHQ2.

5. The method as described in claim 2, characterized in that, Select from F1, F5, and F9 below; F1: In the probe 1, a first fluorescent group is attached to the 5' end and a first quenching group is attached to the 3' end; F5: In the probe a, a second fluorescent group is attached to the 5' end and a second quenching group is attached to the 3' end; F9: In the probe p, a third fluorescent group is attached to the 5' end and a third quenching group is attached to the 3' end.

6. The method as described in claim 1, characterized in that, The method includes the following steps: S1: Extract RNA from the target sample and prepare it into cDNA; S2: Mix the cDNA, the nucleic acid combination, and the qPCR reaction reagent to obtain the reaction system; S3: The reaction system is subjected to thermal cycling, and the infectious bronchitis virus is detected by fluorescence signal.

7. The method as described in claim 6, characterized in that, The thermal cycle includes the following steps: Step 1: Reverse transcription at 50℃ for 5 minutes; The second step is to pre-denature at 95°C for 30 seconds, followed by one cycle. The third step is to denature at 95℃ for 5 seconds, anneal at 60℃ for 30 seconds, and extend the heat for 35-40 times.

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