Infectious bronchitis virus detection primer combination and application thereof

Abstract

The invention discloses a detection primer combination for infectious bronchitis virus. The detection primer combination comprises a primer pair and a probe for detecting an infectious bronchitis virus subgenome-N, a primer pair and a probe for detecting an infectious bronchitis virus genome, and a primer pair and a probe for detecting a reference gene. The invention also discloses a non-diagnostic method for detecting the infectious bronchitis virus based on the primer combination. The primer combination is low in detection limit, good in sensitivity, good in specificity and good in repeatability when used for detecting the infectious bronchitis virus, can judge the infection period of the infectious bronchitis virus to a certain extent, and has an application prospect.

Description

Technical Field

[0001] The invention belongs to the field of virus detection and relates to a detection primer combination for infectious bronchitis virus and application thereof. Background Art

[0002] Infectious bronchitis (IB) is an acute, highly contagious viral respiratory disease of poultry caused by infectious bronchitis virus (IBV), characterized by sneezing, coughing, and tracheal rales in sick chickens. IBV was once listed by the UK as one of the main infectious pathogens of poultry that caused economic losses to the poultry industry. IBV can lead to a decrease in the feed conversion rate of chickens; mixed infection is the main reason for the elimination of broiler chickens; the kidneys of infected commercial chickens swell, forming "spotted kidneys"; the egg-laying performance and egg quality of infected laying hens decrease; and even lead to the death of infected chickens, causing huge economic losses to the world's poultry industry. For nearly half a century, the main measure for the prevention of infectious bronchitis has been vaccination. For a long time, vaccine strains such as H120, H52, Ma5, 28 / 86 and IBn have been used for prevention and control. In recent years, due to the continuous emergence of new variants, immune failure has occurred from time to time.

[0003] IBV belongs to the grama coronavirus of the Coronaviridae family and the Coronavirus genus. It is a single-stranded, non-segmented, positive-strand RNA virus (genomic RNA, gRNA). Coronavirus produces subgenomic RNA (sgRNA) during replication and transcription. The structures of gRNA and sgRNA are as follows Figure 1 As shown. Coronavirus sgRNA is produced by leader-to-body discontinuous transcription of the replication transcription complex (RTC). That is, the positive strand sgRNA contains a leader RNA (leader RNA) that is consistent with the 5' end sequence of the genome, and is connected to the sgRNA body (thebody of the sgRNA) that is identical to the 3' end sequence of the downstream genome. The coronavirus sgRNA-N content is high and has good specificity, and the subgenome can reflect the genetic period of viral replication. At present, the diagnostic method for IBV mainly uses molecular biological methods such as reverse transcription polymerase chain reaction (RT-PCR) to detect the genomic RNA (genomics RNA, gRNA) of the virus, but it cannot determine the infectivity of the virus and the state of viral replication.

[0004] There are currently no reports on the detection method of infectious bronchitis virus sgRNA, let alone reports on the combined detection of IBV genome and subgenome. Therefore, it is difficult to determine the infection period of IBV using existing technologies. Summary of the invention

[0005] The present invention establishes a triple fluorescence quantitative method for detecting genomic RNA (genomic RNA, gRNA), subgenomic RNA (subgenomic RNA, sgRNA) and chicken internal reference β-actin gene of avian infectious bronchitis virus (IBV). The present invention can realize accurate detection and relative quantification of sample IBV genome and IBV subgenome at the same time, and the detection result can be directly used as an important indicator for detecting virus infectivity and replication efficiency, and can be used for qualitative and quantitative evaluation of inactivated seedling quality, risk of attenuated vaccine toxicity reversion and virus tissue phagocytosis. The present invention is simple to operate, highly sensitive, specific, and highly repeatable, and can perform rapid and accurate differential diagnosis and relative quantification, and can quantitatively judge IBV infectivity and replication efficiency, thereby implementing a correct prevention or treatment plan and reducing economic losses in the poultry industry.

[0006] The detection method for IBV subgenome established by the present invention will be able to qualitatively and quantitatively analyze the infectivity and replication efficiency of IBV, and will be widely used in the basic and applied research fields of IBV and even coronavirus, such as research on virus-host interaction, vaccine development, pathogenic mechanism, etc.

[0007] The present invention establishes a triple real-time fluorescence RT-PCR method to simultaneously detect IBV sgRNA, gRNA and internal reference gene β-actin, and evaluates the detection performance of the established method. The present invention simultaneously detects chicken internal reference β-actin, gRNA ORF1ab and sgRNA-N three target genes in one tube reaction by adding three fluorescent probes labeled with different fluorescent signal groups in the same reaction fluorescence quantitative reaction system, greatly improving the detection efficiency. The chicken internal reference gene introduced in the present invention can be used as a nucleic acid extraction quality control indicator and a relative quantitative internal reference, and the absolute and relative content of the IBV subgenome and genome can be detected as an important indicator for virus infectivity and replication efficiency detection, which can replace the chicken embryo inoculation experiment to evaluate the quality of inactivated seedlings and attenuated seedlings, and can replace the chicken embryo growth curve determination to reflect the virus replication efficiency, and can replace animal experiments to evaluate whether there is a risk of virulence reversal in attenuated vaccines, and can replace animal experiments and tissue immunochemical methods to evaluate viral tissue phagocytosis. The present invention can detect the IBV genome and IBV subgenome of a sample at the same time, has simple operation, high sensitivity, good specificity, strong repeatability, and can perform rapid and accurate differential diagnosis, accurate absolute and relative quantification, thereby implementing the correct prevention or treatment plan and reducing the economic losses of the poultry industry.

[0008] In order to solve the problems existing in the prior art, the present invention provides a nucleic acid combination in a first aspect, 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 nucleic acid combination and the third nucleic acid combination;

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

[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 a primer pair whose sequences are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0015] The nucleic acid combination 2 includes a primer pair whose sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0016] The nucleic acid combination 3 includes a primer pair whose sequences are shown as SEQ ID NO.8 and SEQ ID NO.9 respectively;

[0017] The nucleic acid combination 4 includes a primer pair whose sequences are shown as 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 comprises a primer pair whose sequences are shown as SEQ ID NO.14 and SEQ ID NO.15 respectively;

[0020] The nucleic acid combination b includes a primer pair whose sequences are shown as SEQ ID NO.17 and SEQ ID NO.18 respectively;

[0021] The nucleic acid combination c includes a primer pair whose sequences are shown as SEQ ID NO.20 and SEQ ID NO.21 respectively;

[0022] The nucleic acid combination d comprises a primer pair whose sequences are shown as SEQ ID NO.23 and SEQ ID NO.24 respectively;

[0023] The third nucleic acid combination includes a primer pair whose sequences are shown as SEQ ID NO.26 and SEQ ID NO.27 respectively.

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

[0025] D1: The nucleic acid combination 1 further includes a probe 1; in the 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 comprises a probe a; in the 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 a probe b; in the 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 a probe c; in the 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 a probe d; in the probe d, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO: 25, one end of which is connected to a second fluorescent group, and the other end of which is connected to a second quenching group;

[0033] D9: The third nucleic acid combination further includes a probe p; in the 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 quencher group can quench the fluorescence emitted by the first fluorescent group, and the first quencher group cannot quench the fluorescence emitted by any of the second fluorescent group, the second quencher group, the third fluorescent group, and the third quencher group;

[0035] The second quenching group can quench the fluorescence emitted by the second fluorescent group, and 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, and 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 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 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 embodiments, selected from E1, E2, E3, E4, E5 or E6;

[0041] E1: the first fluorescent group and the first quenching group are the first group of dyes; the second fluorescent group and the second quenching group are the second group of dyes; the third fluorescent group and the third quenching group are the third group of dyes;

[0042] E2: the first fluorescent group and the first quenching group are the first group of dyes; the second fluorescent group and the second quenching group are the third group of dyes; the third fluorescent group and the third quenching group are the second group of 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 are: FAM and TAMRA;

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

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

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

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

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

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

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

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

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

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

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

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

[0060] The second aspect of the present invention provides a kit, wherein the kit contains the nucleic acid combination described in the first aspect of the present invention.

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

[0062] In some embodiments, the method comprises the steps of:

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

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

[0065] S3: performing thermal cycling on the reaction system and detecting infectious bronchitis virus through fluorescent signals.

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

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

[0068] The first step was reverse transcription at 50°C for 5 min;

[0069] The second step was pre-denaturation at 95 °C for 30 s, 1 cycle;

[0070] The third step was denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds, and cycled 35-40 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Schematic diagram of the IBVgRNA and sgRNA structures.

[0072] Figure 2 This is the amplification curve of triple TaqMan RT-PCR detection of avian infectious bronchitis attenuated vaccine IBV gRNA, IBV sgRNA, and β-actin gene.

[0073] Figure 3 This is the Ct value determination result of IBV gRNA detection.

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

[0075] Figure 5 This is the Ct value determination result of IBV sgRNA detection.

[0076] Figure 6 This is a standard curve graph for IBV sgRNA detection.

[0077] Figure 7 This is the Ct value determination result of β-actin gene detection.

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

[0079] Fig. 9 These are the sensitivity test results for IBV gRNA detection.

[0080] Fig.10 These are the sensitivity test results for IBV sgRNA detection.

[0081] Fig.11 This is the sensitivity test result of β-actin gene detection.

[0082] Fig.12 This is the specific detection amplification curve of IBV gRNA and IBV sgRNA. DETAILED DESCRIPTION

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

[0084] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0085] The application scenarios of the method for detecting infectious bronchitis virus for non-disease diagnosis purposes include but are not limited to: studying the interaction between IBV and the host; studying the ability of IBV to protect against virus attack after vaccination; and environmental assessment of whether there is infectious bronchitis virus in chicken farms.

[0086] Example 1: Preparation of standard plasmid

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

[0088] In the application tests of the 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] Shanghai Shenggong Bioengineering Technology Service Co., Ltd. was commissioned to construct a synthetic gene fragment, including IBVgRNAORF1ab (corresponding to the sequence between primers gRNA-F1 and gRNA-R1), sgRNA-N (corresponding to the sequence between primers sgRNA-F1 and sgRNA-R1), and a partial sequence of the chicken β-actin gene (corresponding to the sequence between primers β-actin-F and β-actin-R). The sequences of the three parts are shown in Table 2. Restriction enzyme sites were added to the ends of the synthetic fragments, and the fragments and vectors were cut separately. The three fragments were inserted into the pUC57 cloning vector in sequence to form a standard plasmid (named pUC57-gIBV-sgIBV-βactin), which will be used in subsequent experiments. The plasmid contains the three gene fragments in series, and the concentration of the standard plasmid is measured using a Nanodrop spectrophotometer. The standard plasmid was diluted 10 times with DNase / RNase-free distilled water to establish a concentration gradient range of 1×10 0 -1×10 10 The standards were stored at -20 °C for subsequent experiments.

[0095] Table 2. Synthetic gene fragment information

[0096]

[0097] Example 2: Optimization of triple TaqMan RT-PCR system

[0098] Dilute the standard plasmid to a final concentration of 1×10 7The real-time quantitative primer, probe concentration and annealing temperature were optimized by matrix method on QuantStudio 5 fluorescence quantitative PCR instrument with 1000 copies / μL as template. The final concentration of each primer was between 0.15-0.35μmol / L, and the final concentration of the probe was between 0.25-0.4μmol / L. Fluorescence quantitative PCR was performed to select the optimal concentration of primers and probes. The annealing temperature was optimized at 56-60℃. Reaction system: template 3μL, 2×Premix Ex Taq 10μL, gIBV / sgIBV / β-actin primer (10μM) was 0.3-0.7μL for ratio combination, and gIBV / sgIBV / β-actin probe (10μM) was 0.5-0.8μL for ratio combination. Enzyme-free water was added to 20μL. Water was used as negative control and parallel treatment was performed.

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

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

[0101] Components Volume (20 μL) 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 triple TaqMan RT-PCR standard curve

[0103] According to the optimized reaction conditions and procedures in Example 2, 1×10 3 -1×10 10The standard plasmid with 1000 copies / μL was used as a template to establish a triple TaqMan RT-PCR standard curve. The Ct value and logarithm of the plasmid copy number were subjected to linear regression analysis. Standard curves were generated for IBV gRNA, IBV sgRNA, and chicken internal reference β-actin, and water was used as a negative control.

[0104] Results: Figure 3 , 4 As shown in the figure, the IBV gRNA standard curve was successfully established, and the correlation coefficient (R 2 ), equation slope and amplification efficiency (E%) are 0.999, -3.453, 94.791 respectively; Figure 5 , 6 As shown in Figure 2, the IBV sgRNA standard curve was successfully established, and the correlation coefficient (R 2 ), equation slope and amplification efficiency (E%) are 0.999, -3.496, 93.23 respectively; Figure 7 , 8 As shown in the figure, the chicken β-actin standard curve was successfully established, and the correlation coefficient (R 2 ), equation slope and amplification efficiency (E%) were 0.999, -3.496 and 93.23, respectively. 2 Both the E% and E% values ​​indicate that there is 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 diluted 10-fold (the diluent was water) with a concentration gradient ranging from 1×10 0 -1×10 6 The standard sample with 100 copies / μL was used as a template, and the optimized reaction system and procedure were run according to the reaction conditions and procedures optimized in Example 2 to determine the sensitivity of the triple TaqMan RT-PCR.

[0107] Results: Within 40 cycles, 1×10 IBV gRNA, sgRNA, and chicken β-actin gene were expressed. 1 -1×10 6 The copies / μL standards all had amplification curves, and the negative control (water) did not have any amplification. The sensitivity was determined to be 1×10 1 copies / μL.

[0108] Results: The sensitivity of IBV gRNA, IBV sgRNA and chicken β-actin can reach 1×10 1 copies / μL, such as Fig. 9 ,10 , as shown in 11.

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

[0110] Extract nucleic acids (DNA / cDNA) of pathogens such as avian infectious bronchitis virus M41 strain, Newcastle disease virus La Sota strain, avian leukosis virus J subtype HPRS103 strain, avian influenza virus H9N2 Re-2 vaccine strain, chicken astrovirus GXJL815 strain, avian infectious laryngotracheitis virus K317 strain and avian reticuloendotheliosis virus HA9901 strain, prepare an optimized system, run the program for detection, and use water as a negative control. The specificity of the IBV gRNA, IBV sgRNA and chicken β-actin triple TaqMan RT-PCR method established in Example 2 was verified.

[0111] Results: Fig.12 As shown in the figure, only IBV gRNA and IBV sgRNA amplified "S-shaped" curves with Ct values ​​<36, while other pathogens Newcastle disease virus, avian leukosis virus, avian influenza virus, chicken astrovirus, avian infectious laryngotracheitis, avian reticuloendotheliosis virus and negative controls had no obvious amplification curves. This shows that the triple TaqMan RT-PCR method of the present invention has good specificity.

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

[0113] Four 10-fold dilutions of the standard plasmid (1×10 3 -1×10 6 1000 copies / μL) was used as a template, and repeatability was tested according to the optimized reaction system and operation procedure of Example 2. Each gradient of plasmid was repeated 3 times in each test, divided into 3 batches (1d, 3d and 5d) to verify the repeatability of the intra-group and inter-group data of this method.

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

[0115] Table 4. Intra-group and inter-group repeatability

[0116]

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

[0118] 89 clinical samples were tested using the IBV gRNA, IBV sgRNA and chicken β-actin triple TaqMan RT-PCR detection method established in Example 2 of the present invention, and these samples were used to inoculate chicken embryo allantoic fluid for virus detection. The samples mainly consisted of throat swabs, trachea, kidney and other tissue and organ samples collected from different regions such as Jilin, Liaoning and Heilongjiang. The supernatant of the sample was aseptically inoculated into the allantoic cavity of 10-day-old SPF chicken embryos, and the chicken embryos that died within 24 hours of inoculation were discarded, and the allantoic fluid of chicken embryos that survived 3 days after inoculation was collected. Total RNA was extracted from the chicken embryo allantoic fluid for subsequent IBV fluorescence quantitative detection.

[0119] Results: RNA test results were positive in 61 of 89 throat swabs and tissue samples of chickens suspected of being infected with infectious bronchitis virus. The sgRNA test results of 28 gRNA-negative samples were all negative. 53 of 61 gRNA-positive samples 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 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 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 rate of sgRNA was highly consistent with the detection rate of inoculated chicken embryo allantoic fluid. And for the same sample, the Ct value of gRNA is less than the Ct of sgRNA. For samples with positive sgRNA detection, the difference in Ct values ​​between gRNA and sgRNA is compared, Ct(sgRNA)-Ct(gRNA)=5.21±2.82, and the 95% confidence interval is (4.72, 5.70). IBV subgenome detection has high sensitivity and specificity, and is highly consistent with the results of chicken embryo isolation and culture of IBV. The triple TaqMan RT-PCR method established in the present invention can qualitatively and quantitatively analyze the viral replication status of IBV in vivo, thereby implementing the correct prevention or treatment plan and reducing the economic losses of the poultry industry.

[0120] According to common sense, the gRNA content is low in the early stage of IBV infection, and there is no or very little sgRNA. As the infection time increases, the gRNA and sgRNA content both increase, and the sgRNA increases faster. The smaller the difference between the Ct value of gRNA and the Ct value of sgRNA in the early and middle stages of infection, the more sgRNA is expressed, which in turn indicates that the infection time is longer. The triple TaqMan RT-PCR method of the present invention can determine the infection period of IBV to a certain extent.

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

[0122]

[0123] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A nucleic acid combination, wherein the nucleic acid combination is C1, C2, C3 or C4; C1: first nucleic acid combination; C2: a combination of the first nucleic acid combination and the second nucleic acid combination; C3: a combination of the first nucleic acid combination and the third nucleic acid combination; C4: a combination of the first nucleic acid combination, the second nucleic acid combination, and the third nucleic acid combination; The first nucleic acid combination is nucleic acid combination 1, nucleic acid combination 2, nucleic acid combination 3 or nucleic acid combination 4; The nucleic acid combination 1 includes a primer pair whose sequences are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively; The nucleic acid combination 2 includes a primer pair whose sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively; The nucleic acid combination 3 includes a primer pair whose sequences are shown as SEQ ID NO.8 and SEQ ID NO.9 respectively; The nucleic acid combination 4 includes a primer pair whose sequences are shown as SEQ ID NO.11 and SEQ ID NO.12 respectively; The second nucleic acid combination is nucleic acid combination a, nucleic acid combination b, nucleic acid combination c or nucleic acid combination d; The nucleic acid combination a comprises a primer pair whose sequences are shown as SEQ ID NO.14 and SEQ ID NO.15 respectively; The nucleic acid combination b includes a primer pair whose sequences are shown as SEQ ID NO.17 and SEQ ID NO.18 respectively; The nucleic acid combination c includes a primer pair whose sequences are shown as SEQ ID NO.20 and SEQ ID NO.21 respectively; The nucleic acid combination d comprises a primer pair whose sequences are shown as SEQ ID NO.23 and SEQ ID NO.24 respectively; The third nucleic acid combination includes a primer pair whose sequences are shown as SEQ ID NO.26 and SEQ ID NO.27 respectively.

2. The nucleic acid combination according to claim 1, characterized in that Selected from the following D1, D2, D3, D4, D5, D6, D7, D8 and D9; D1: The nucleic acid combination 1 further includes a probe 1; in the 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; 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; 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; 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; D5: The nucleic acid combination a further comprises a probe a; in the 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; D6: The nucleic acid combination b further includes a probe b; in the 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; D7: The nucleic acid combination c further includes a probe c; in the 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; D8: The nucleic acid combination d further includes a probe d; in the probe d, the nucleic acid sequence or its complementary sequence is as shown in SEQ ID NO: 25, 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 further includes a probe p; in the 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 nucleic acid combination according to claim 1 or 2, characterized in that The first quenching group can quench the fluorescence emitted by the first fluorescent group, and 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, and 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, and 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 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 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 nucleic acid combination according to 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 the first group of dyes; the second fluorescent group and the second quenching group are the second group of dyes; the third fluorescent group and the third quenching group are the third group of dyes; E2: the first fluorescent group and the first quenching group are the first group of dyes; the second fluorescent group and the second quenching group are the third group of dyes; the third fluorescent group and the third quenching group are the second group of 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 are: FAM and TAMRA; The second group of dyes are: VIC and BHQ1; The third group of dyes includes: ROX and BHQ2.

5. The nucleic acid combination according to claim 2, characterized in that Selected from the following F1, F2, F3, F4, F5, F6, F7, F8 and F9; F1: In the probe 1, the 5' end is connected to a first fluorescent group, and the 3' end is connected to a first quenching group; F2: In the probe 2, the 5' end is connected to a first fluorescent group, and the 3' end is connected to a first quenching group; F3: In the probe 3, the 5' end is connected to the first fluorescent group, and the 3' end is connected to the first quenching group; F4: In the probe 4, the 5' end is connected to a first fluorescent group, and the 3' end is connected to a first quenching group; F5: In the probe a, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group; F6: In the probe b, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group; F7: In the probe c, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group; F8: In the probe d, the 5' end is connected to a second fluorescent group, and the 3' end is connected to a second quenching group; F9: In the probe p, the 5' end is connected to a third fluorescent group, and the 3' end is connected to a third quenching group.

6. A kit comprising the nucleic acid combination according to any one of claims 1 to 5.

7. A method for detecting infectious bronchitis virus for non-disease diagnosis purposes, the method comprising: performing qPCR detection on nucleic acid extracted from a target detection sample using the nucleic acid combination described in any one of claims 1 to 5.

8. The method according to claim 7, characterized in that The method comprises the following steps: S1: Extract RNA from the target test sample and prepare it into cDNA; S2: mixing the cDNA, the nucleic acid combination, and the qPCR reaction reagent to obtain a reaction system; S3: performing thermal cycling on the reaction system and detecting infectious bronchitis virus through fluorescent signals.

9. The method according to claim 8, characterized in that The GenBank number of the infectious bronchitis virus is DQ834384.

1.

10. The method according to claim 8, characterized in that The thermal cycle comprises the following steps: The first step was reverse transcription at 50°C for 5 min; The second step was pre-denaturation at 95 °C for 30 s, 1 cycle; The third step was denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds, and cycled 35-40 times.

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