One-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus

Abstract

The present invention discloses a one-step TaqMan RT-PCR kit for differential diagnosis of a novel variant bursal disease virus. The kit comprises primers and probes with sequences shown in SEQ ID NOs. 1 to 4. The kit has high sensitivity and good specificity. The detection method is easy to operate, convenient, and rapid, significantly shortening the detection time. It can effectively screen a large number of samples and distinguish whether bursal atrophy is caused by infection with the novel variant bursal disease virus or by the vaccine strain.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and in particular relates to a one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus. Background Art

[0002] Infectious bursal disease (IBD) is a common viral disease affecting poultry worldwide. It was first discovered in Gumboro, USA in 1957. Infectious bursal disease virus (IBDV) is a bisegmented RNA virus (segments A and B). Currently, there are two serotypes of IBDV. Serotype 1 strains infect chickens and are classified according to their pathogenicity as avirulent, classical (also known as standard), American variant, very virulent (vv), and novel variant bursal disease virus (nVarIBDV). Serotype 2 strains are nonpathogenic to chickens. The VP2 protein, derived from genome segment A, is the primary immunogenic protein that induces neutralizing antibodies in the host. The vp2 gene contains a hypervariable region (HVR, nt616-1050) responsible for viral antigenic variation and virulence. Genome segment B encodes the RNA-dependent RNA polymerase VP1.

[0003] Classical and vvIBDV strains have different mortality rates, with vvIBDV strains causing higher mortality and morbidity than classical strains. vvIBDV strains cause 40-100% mortality in SPF chickens, 60% in laying hens, and 30% in broilers. Chickens infected with both classical and vvIBDV strains experience muscle hemorrhages, hemorrhages in the bursa of Fabricius, and a yellow, jelly-like appearance in the bursa of Fabricius. nVarIBDV strains (e.g., FJ2019-01 and SHG19) are novel pathogenic viruses that differ significantly from US variants. nVarIBDV strains cause atrophy of the bursa of Fabricius and decreased lymphocyte levels in SPF chickens, as well as intrafollicular macrophage infiltration, proliferation of perifollicular fibrous tissue, and severe follicular atrophy. Notably, no gross clinical signs or mortality were observed in chickens infected with the novel IBDV variants SHG19 or FJ2019-01. Currently available commercial IBDV vaccines offer no effective protection against infection with these nVarIBDV strains. nVarIBDVs can cause subclinical infection, increase susceptibility to infection with other pathogens, and interfere with the immune response of other vaccines.

[0004] Vaccination is the most effective method for controlling IBD. However, in chickens uninfected with nVarIBDV, chickens vaccinated with immune complex vaccines or live vaccines exhibit bursal atrophy compared to unvaccinated chickens. This makes it clinically difficult to distinguish between bursal atrophy caused by nVarIBDV infection and bursal atrophy caused by vaccination. Therefore, there is an urgent need to establish a method to distinguish nVarIBDV-infecting strains from live vaccine strains to effectively monitor vaccinated chickens and control nVarIBDV infection. Summary of the Invention

[0005] The purpose of the present invention is to provide a one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] A one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus (nVarIBDV), comprising primers and probes for differential diagnosis of novel variant bursal disease virus, the sequences of which are as follows:

[0008] Upstream primer F: 5'-CCTCCTTCTAYARYGCTRTCAT-3',

[0009] Downstream primer R: 5'-CGTATGAACGGAACAATCTG-3',

[0010] PnVar probe for novel variant bursal disease virus: 5'-TAGAGATCAGACGAACG-3',

[0011] Pnon-nVar probe for non-novel variant bursal disease virus: 5'-AGTAGAGATCAGACAAA-3'.

[0012] Furthermore, the 5' end of the PnVar probe is labeled with a VIC group, and the 3' end is labeled with an MGB group.

[0013] Furthermore, the 5' end of the Pnon-nVar probe is labeled with a FAM group, and the 3' end is labeled with a MGB group.

[0014] Furthermore, the above kit also contains 4×Fast one-Step Master, deionized water and positive plasmid standards, which are obtained as follows: referring to the reported primers AU / A1542L, AU: 5'-GGATACGATCGGTCTGACCCCGGGGGAGTC-3', A1542L: 5'-GTAGTCTACACCTTCCCCAATTGCAT--3'; extracting viral RNA of IBDV FJ2019-01 strain and IBDV BC6 / 85 strain, reverse transcribe it into cDNA as a template, and perform PCR amplification using primers AU / A1542L; cloning the purified PCR product into pCRTMⅡ-Blunt- The vector was inserted into the vector and transformed into Escherichia coli DH5α competent cells; plasmids pCR-FJ2019-01-A and pCR-BC6 / 85-A were extracted and used as standards.

[0015] Furthermore, the primers and probes were used at a concentration of 10 μM.

[0016] Furthermore, the PCR reaction procedure of the one-step TaqMan RT-PCR kit when used to detect samples is: reaction at 50°C for 10 minutes; insulation at 95°C for 20 seconds; denaturation at 95°C for 3 seconds, annealing / extension at 60°C for 30 seconds and collection of FAM and VIC fluorescence signals, for a total of 40 cycles.

[0017] The one-step TaqMan RT-PCR kit is used to prepare a product for diagnosing infectious bursal disease.

[0018] The one-step TaqMan RT-PCR kit is used to prepare a product for distinguishing new variant bursal disease virus from live vaccination strains.

[0019] The significant advantages of the present invention are:

[0020] In this study, we analyzed genetic polymorphisms in the overlapping region of the IBDV A segmental sequence, vp5 / vp2, and developed a one-step, real-time TaqMan-MGB assay using allele-specific probes to detect and differentiate nVarIBDV (novel variant bursal disease virus) from non-nVarIBDV (non-novel variant bursal disease virus), specifically targeting live vaccine strains. This method is rapid, sensitive, and specific, and can effectively screen large numbers of samples and distinguish whether bursal atrophy is caused by nVarIBDV infection or vaccine strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Feasibility of the one-step real-time TaqMan RT-PCR method. IBDV FJ2019-01 strain (a), positive plasmid pCR-FJ2019-01-A (b) and BC6 / 85 strain (c), positive plasmid pCR-BC6 / 85-A (d) demonstrate specific amplification, as indicated by VIC signals (dashed lines) and FAM signals (solid lines). No amplification signals were observed for IBDV-negative samples (e), AVE buffer (f), and H2O (g).

[0022] Figure 2 Specificity of the one-step real-time TaqMan RT-PCR assay. a: FJ2021 and B87 strains served as positive controls for VIC and FAM signals, respectively. Clinical samples negative for IBV, NDV, AIV, ILTV, FPV, MG, MS, and IBDV showed no VIC or FAM amplification signals during the assay. b: Six novel variants, BC6 / 85, and seven vaccine strains were analyzed, each specifically generating amplified VIC and FAM signals.

[0023] Figure 3 : Sensitivity of a one-step real-time TaqMan RT-PCR method. DETAILED DESCRIPTION

[0024] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0025] Materials and sources involved in this example:

[0026] Virus strains: IBDV FJ2019-01 strain (GenBank: MZ736578 or MZ044944) is stored in our laboratory. IBDV classic strain BC6 / 85 strain (GenBank: ON286951) was purchased from the China Veterinary Drug Administration and stored in our laboratory. The specificity of the kit was evaluated using live IBDV vaccine strains B87, D78, W2512, MB, K85, NF8, and CF; the classic strain BC6 / 85; and variants FJ2019-01, FJ2019-02 (GenBank: MZ044945), FJ2019-03 (GenBank: MZ044946), FJ2019-04 (GenBank: MZ044947), FJ2019-05 (GenBank: MZ044948), and FJ2021 (GenBank: MZ593902). The specificity of the kit was also evaluated using RNA or DNA extracted from Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and infectious bronchitis virus (IBV).

[0027] Example 1

[0028] 1 Test method

[0029] 1.1 Design of primers and probes

[0030] Approximately 80 IBDV genomes were retrieved from the GenBank database and multiple sequence alignments were performed using MEGA7 software. Primers and probes were designed based on the highly conserved regions observed in the multiple sequence alignment, along with the 5' untranslated region of the A segment and the vp5 / vp2 overlap region. The upstream primer sequence F: 5'-CCTCCTTCTAYARYGCTRTCAT-3' (Y represents T and C, R represents A and G) and the downstream primer sequence R: 5'-CGTATGAACGGAACAATCTG-3' were used to amplify a 105-bp region containing the SNPs. The a / G shift maximizes the Tm difference between allele-specific probes. Therefore, the probe sequences used in this study were: PnVar: 5'-TAGAGATCAGACGAACG-3' (5' and 3' ends were labeled with VIC and MGB groups, respectively, for nVarIBDV), and Pnon-nVar: 5'-AGTAGAGATCAGACAAA-3' (5' and 3' ends were labeled with FAM and MGB groups, respectively, for non-nVarIBDV).

[0031] Viral RNA Extraction and Reverse Transcription: Viral RNA was extracted from samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the kit instructions. Commercially available lyophilized vaccine was resuspended in PBS (ThermoFisher) and RNA was extracted. The extracted RNA was reverse transcribed (RT) into cDNA using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Lithuania, USA).

[0032] 1.2 Construction of positive plasmid standards

[0033] Referring to the reported specific primers AU (10 μM; 5'-GGATACGATCGGTCTGACCCCGGGGGAGTC-3') and A1542L (10 μM; 5'-GTAGTCTACACCTTCCCCAATTGCAT-3') and methods for amplifying the A segment sequence of the IBDV genome, the genomic RNA of the IBDV FJ2019-01 strain and the IBDV BC6 / 85 strain were used as templates, and the RevertAid RT kit (Thermo, Lithuania, Vilnius) and specific primers AU / A1542L were used to amplify the partial A segment of the FJ2019-01 strain and the BC6 / 85 strain. The purified PCR product was cloned into pCRTMⅡ-Blunt- The vector was transfected into competent Escherichia coli DH5α cells (TIANGEN, Beijing, China). Plasmid DNA was extracted using the Qiagen plasmid Mini kit (Qiagen, Hilden, Germany), and plasmid concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo, Rockford, IL, USA). The target copy number of the positive plasmid was calculated as follows: copies / μL = (concentration in ng × 6.023 × 10 23 ) / (genome length × 1 × 10 9 ×660 dalton / bp). The constructed plasmids were named pCR-BC6 / 85-A and pCR-FJ2019-01-A, respectively, and served as positive controls and sensitivity evaluation for IBDV detection.

[0034] 1.3 Preparation of one-step real-time TaqMan RT-PCR kit

[0035] A one-step real-time TaqMan RT-PCR assay was performed on a LightCycler 96 (Roche, Basel, Switzerland) to distinguish between nVar IBDV strains and non-nVar IBDV strains (including classical strains, vv IBDV, US variants, and live vaccine strains). The total reaction volume was 20 μL and contained: 5 μL of 4× Fast one-Step Master mix (Thermo, Vilnius, Lithuania), 0.50 μL of each 10 μM upstream and downstream primers, 0.25 μL of 10 μM PnVar probe or 10 μM Pnon-nVar probe, 5 μL of template RNA or plasmid DNA, and 8.5 μL of deionized water. Reaction conditions were: 50°C for 10 min; 95°C for 20 s; cycles of 95°C for 3 s and 60°C for 30 s. Fluorescence intensity of the specific reporter fluorophore was measured during the 60°C step of each cycle and at the end of the run. All reactions and amplifications were performed using The data were analyzed using 96SW 1.1 software.

[0036] 1.3 Determination of primer and probe set specificity:

[0037] The specificity of primer pairs and probes was determined using NCBI BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) (Bethesda, Maryland, USA). BLAST searches were performed to predict the specificity of primer and probe sequences and to assess nonspecific homology between the sequences and the IBDV or chicken genomes. The specificity of the established method was validated using RNA from B87, D78, W2512, MB, K85, NF8, CF, BC6 / 85, FJ2019-01, FJ2019-02, FJ2019-03, FJ2019-04, FJ2019-05, and FJ2021, as well as RNA or DNA samples from NDV, IBV, AIV, ILTV, FPV, MG, and MS strains.

[0038] 1.4 Kit sensitivity determination

[0039] Two positive plasmids were serially diluted (2 × 10 6 copies / μL~2×10 1 10 copies / μL) were added to the respective reaction mixtures to evaluate the sensitivity of primers and probes. 7 to 10 2) and log10, a standard curve can be obtained. 96SW 1.1 software was used to establish the standard curve and determine the efficiency. 7 ,1:10 6 1:10 5 ,1:10 4 ,1:10 3 ,1:10 2 ,1:0,10 7 :1,10 6 :1,10 5 :1,10 4 :1,10 3 :1,10 2 :1,0:1;1=10 5 The two plasmids of different copies / reaction were mixed to verify the interference between different probes.

[0040] 1.5 Clinical sample testing

[0041] Eighty-four clinical bursal specimens were collected from 20- to 40-day-old chickens immunized with live vaccines against W2512, B87, D78, mb, K85, and CF strains. RNA was extracted from all specimens and analyzed using a developed one-step real-time TaqMan RT-PCR kit. The vp5 gene (for all IBDV genotypes) was detected by RT-PCR using an IBDV fluorescent reverse transcription-PCR (RT-PCR) detection kit (BIOTECHSY, Beijing, China). The HVRs of the vp2 gene were detected by PCR using the vp2-f (5'-CCTCAGCTTACCCACATC-3') and vp2-r (5'-CCTTCCCCAATTGCATGG-3') primers. PCR products were sequenced and analyzed according to published methods to determine the IBDV genotypes in the samples.

[0042] 2 Test results

[0043] 2.1 Feasibility of One-Step Real-Time TaqMan RT-PCR Kit

[0044] This study exploited two single nucleotide polymorphisms (SNPs) in the vp5 / vp2 overlap region to distinguish nVarIBDV from non-nVarIBDV strains. The C103A and G117A SNPs were selected to distinguish nVarIBDV from non-nVarIBDV strains (including cIBDV, vvIBDV, US variants, and live vaccine strains). Because the A / G shift maximizes the Tm difference between allele-specific probes, two TaqMan-MGB probes were designed to distinguish between nVarIBDV and non-nVarIBDV genotypes, respectively. The PnVar probe is labeled with the VIC fluorescent dye and is specific for nVarIBDV strains. The Pnon-nVar probe is labeled with the FAM fluorescent dye and is specific for non-nVarIBDV strains, particularly live vaccine strains.

[0045] The established one-step real-time TaqMan RT-PCR kit can simultaneously identify and quantify nVarIBDV and non-nVarIBDV genotype strains. The results showed that only nVarIBDV positive plasmids were identified on the VIC channel, and only non-nVarIBDV positive plasmids were identified on the FAM channel ( Figure 1 ). It is noteworthy that nonspecific cross-reactivity between nVarIBDV-positive plasmids and non-nVarIBDV-positive plasmids was not observed ( Figure 1 Only the matching primer and probe combination could detect the amplification of nVarIBDV (FJ2019-01) and non-nVarIBDV (BC6 / 86), showing a typical "S" amplification curve. The Cq values ​​were all less than 35. No specific and nonspecific amplification was detected from the mismatched probe combination. No amplification curve was observed in the negative control ( Figure 1 These results indicate that the established one-step real-time TaqMan RT-PCR method is highly feasible and suitable for the simultaneous identification of nVarIBDV and non-nVarIBDV viruses.

[0046] 2.2 Specificity of the One-Step Real-Time TaqMan RT-PCR Kit

[0047] Nucleotide BLAST searches for each primer and probe revealed only homology to the expected IBDV genome. No amplification was observed in IBDV-negative samples and non-IBDV pathogens (IBV, NDV, AIV, ILTV, FPV, MG, and MS), nor in the negative or blank controls used in the assay. Figure 2 a). Detection and analysis of 6 nVarIBDV and 7 vaccine strains showed that the method had a 100% diagnostic accuracy for all strains, and a 100% sensitivity and effectiveness ( Figure 2b) This indicates that the two probes have high specificity and feasibility, are suitable for targeting IBDV genogroups, and have no cross-reactivity between nVarIBDV and non-nVarIBDV strains.

[0048] 2.3. Sensitivity of the One-Step Real-Time TaqMan RT-PCR Kit

[0049] The sensitivity was determined by a one-step real-time TaqMan RT-PCR method using a series of plasmid standards with known concentrations. Plasmid pCR-FJ2019-01-A was used as a template at 10 2 to 10 7 There was a linear relationship between the copy number / reaction, R 2 was 0.99, and the amplification efficiency was 93% ( Figure 3 a and 3c). Using pCR-BC6 / 85-A plasmid as template, the 2 to 10 7 There was a linear relationship between the copy number / reaction, R 2 is 1.00, and the amplification efficiency is 94% ( Figure 3 b and 3c). The results of the cross-interference test of the two probes showed that low copy number (less than 10 3 When a 100-copy / reaction) template is mixed with another high-copy number template, no FAM or VIC amplification signal is generated.

[0050] 2.4. Detection of clinical samples using a one-step real-time TaqMan RT-PCR kit

[0051] A total of 84 clinical bursal samples were collected from six chicken flocks inoculated with strains W2512, B87, D78, mb, K85, and CF. The results showed that the one-step real-time TaqMan RT-PCR assay detected 84 (84 / 84, 100%) positive samples for IBDV. RT-PCR of the vp5 gene using a commercially available kit (targeting all genotypes of IBDV strains) yielded positive results for all 84 clinical bursal samples, with a 100% concordance rate. This demonstrates that the established one-step real-time TaqMan RT-PCR assay is suitable for the detection of IBDV. For the detection of nVarIBDV and non-nVarIBDV strains, the one-step real-time TaqMan RT-PCR assay revealed that 19 (22.62%) samples were positive for nVarIBDV, 67 (79.76%) samples were positive for non-nVarIBDV, and 2 (2.38%) samples were positive for both IBDV and nVarIBDV. Conventional amplification and sequencing of the HVRs of the vp2 gene and sequence analysis revealed that IBDV-positive samples with successfully amplified and analyzed HVRs could be correctly distinguished between nVarIBDV and non-nVarIBDV using the one-step real-time TaqMan RT-PCR assay. This result demonstrates the potential genotyping capabilities and high validity of the established one-step real-time TaqMan RT-PCR assay. Failure to amplify the HVRs in some IBDV-positive samples may be due to low viral load or low template copy number, resulting in PCR amplification failure and failure to analyze the HVRs. Compared with conventional sequence analysis of the HVRs of the vp2 gene, the one-step real-time TaqMan RT-PCR assay demonstrated a specificity and sensitivity of 100% and 100%, respectively. Distinguishing between bursal atrophy caused by vaccine strains and nVarIBDV strains is crucial for clinical vaccine efficacy assessment and nVarIBDV prevention and control. Compared with conventional RT-PCR and sequence analysis, the one-step real-time TaqMan RT-PCR assay allows for rapid differentiation of IBDV strains.

Claims

1. A one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus, characterized by: The primers and probes for differential diagnosis of novel variant bursal disease virus contain the following sequences: Upstream primer F: 5'-CCTCCTTCTAYARYGCTRTCAT-3', Downstream primer R: 5'-CGTATGAACGGAACAATCTG-3', The PnVar probe for novel variant bursal disease virus is: 5'-TAGAGATCAGACGAACG-3' and the Pnon-nVar probe for non-novel variant bursal disease virus is: 5'-AGTAGAGATCAGACAAA-3'.

2. The one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus according to claim 1, characterized in that: The 5' end of the PnVar probe is labeled with a VIC group, and the 3' end is labeled with an MGB group.

3. The one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus according to claim 1, characterized in that: The 5' end of the Pnon-nVar probe is labeled with a FAM group, and the 3' end is labeled with a MGB group.

4. The one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus according to claim 1, characterized in that: The primers and probes were used at a concentration of 10 μM.

5. The one-step TaqMan RT-PCR kit for differential diagnosis of novel variant bursal disease virus according to claim 1, characterized in that: The PCR reaction procedure of the one-step TaqMan RT-PCR kit for detecting samples is as follows: reaction at 50°C for 10 min; insulation at 95°C for 20 s; denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s and collection of FAM and VIC fluorescence signals, for a total of 40 cycles.

6. Use of the one-step TaqMan RT-PCR kit according to claim 1 in the preparation of a product for diagnosing infectious bursal disease.

7. Use of the one-step TaqMan RT-PCR kit according to claim 1 in preparing a product for distinguishing novel variant bursal disease virus from non-novel variant bursal disease virus.

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