A high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

Abstract

This invention discloses a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains. The kit includes HRM Master Mix, specific primer pairs HRM-F / HRM-R, ddH2O without positive nucleic acid, and standard quality particles for three different pathogenic IBDV serotypes. Experiments have demonstrated that this kit can rapidly and accurately identify highly virulent IBDV strains, novel variants, and attenuated vaccine strains. It features good versatility, high sensitivity, strong specificity, good reproducibility, simple operation, and rapid accuracy. Furthermore, the reaction process does not require opening the container, effectively avoiding contamination issues, and the amplified products can be used for gel electrophoresis analysis and subsequent sequencing. This invention provides a new technology and method for the rapid and accurate identification and detection of different pathogenic IBDV serotypes circulating clinically.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and aquaculture technology, specifically to a high-resolution melting curve kit for rapidly identifying highly virulent IBDV strains, novel variants, and attenuated vaccine strains. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, highly contagious viral disease of poultry caused by the infectious bursal disease virus (IBDV). It is one of the most economically destructive immunosuppressive viral diseases, seriously threatening poultry health worldwide and causing enormous economic losses. IBDV primarily targets B lymphocytes in the bursa of Fabricius, a central immune organ, leading to severe immunosuppression, which in turn increases susceptibility to other pathogens and reduces the effectiveness of vaccination. Currently, three main pathogenic strains exist clinically in my country: highly virulent strains, novel variants, and attenuated vaccine strains. The novel variants are newly discovered pathogenic types in China in recent years. Although large-scale IBD vaccination programs have been implemented in the poultry industry, different types of IBDV are increasingly being isolated from immunized and non-immunized flocks. Like other segmental RNA viruses, different pathogenic types of IBDV can co-circulate in the wild, and long-term mixed infections may provide conditions for viral evolution (such as gene rearrangement and recombination within genomic fragments). It has been confirmed that gene rearrangements and intra-segment recombination of different pathogenic IBDV strains can alter the virulence and antigenic properties of the virus strains, thereby helping them evade the host's immune system and overcome the protection provided by current commercial vaccines, posing a huge challenge to the prevention and control of the disease in poultry farming.

[0003] While several methods have been developed for identifying highly virulent and attenuated IBDV vaccine strains, such as conventional RT-PCR combined with cloning sequencing, these methods are cumbersome, lack automation, are time-consuming (5-7 days), and are costly. Restriction fragment length polymorphism reverse transcription polymerase chain reaction (RT-PCR-RFLP) is not only complex and extremely time-consuming (5-6 hours), but also suffers from unreliable restriction enzyme sites, making some results difficult to interpret. Furthermore, these methods can only identify single-infected samples, not different pathogenic strains in mixed-infection samples. Therefore, there is an urgent need to develop a kit that can rapidly and accurately identify clinically prevalent mixed-infection samples of different pathogenic IBDV strains to solve the long-standing technical challenge of rapidly and accurately identifying the pathogenic type of IBDV in infected chicken flocks. Summary of the Invention

[0004] One object of the present invention is to provide specific primer pairs for identifying highly virulent strains of IBDV, novel variants of IBDV, and attenuated vaccine strains of IBDV.

[0005] The specific primer pair provided by this invention for identifying highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains consists of primer HRM-F and primer HRM-R.

[0006] The primer HRM-F is either a1) or a2) as follows:

[0007] a1) The single-stranded DNA molecule shown in sequence 1 of the sequence listing;

[0008] a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1;

[0009] The HRM-R is as follows (a3) ​​or (a4):

[0010] a3) The single-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0011] a4) A single-stranded DNA molecule that has one or more nucleotides of sequence 2 replaced and / or deleted and / or added, and has the same function as sequence 2.

[0012] In the above-mentioned specific primer pairs, the molar ratio of primer HRM-F to primer HRM-R can be 1:1.

[0013] Another object of the present invention is to provide new uses for the above-mentioned specific primer pairs.

[0014] This invention provides the application of the above-described specific primer pairs in any of the following b1)-b10):

[0015] b1) Identify or assist in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains;

[0016] b2) To differentiate or assist in the differentiation of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains;

[0017] b3) Identify or assist in identifying whether the test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain.

[0018] b4) Detect or assist in detecting whether the sample to be tested is infected with highly virulent strains of IBDV, novel variants of IBDV, and / or attenuated vaccine strains of IBDV;

[0019] b5) Detect or assist in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested;

[0020] b6) Prepare products for identifying or assisting in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains;

[0021] b7) Prepare products that can differentiate or assist in differentiating IBDV super-virulent strains, novel IBDV variants, and IBDV attenuated vaccine strains;

[0022] b8) Prepare products for identification or auxiliary identification of the test strain as a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain.

[0023] b9) Prepare products for detecting or assisting in the detection of whether a sample to be tested is infected with a highly virulent strain of IBDV, a novel variant of IBDV, and / or an attenuated IBDV vaccine strain;

[0024] b10) Prepare products for detecting or assisting in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested.

[0025] Another object of the present invention is to provide a kit containing the above-mentioned specific primer pairs; the kit functions as any one of c1)-c5) below:

[0026] c1) Identify or assist in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains;

[0027] c2) To differentiate or assist in the differentiation of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains;

[0028] c3) Detect or assist in the detection of whether the virus strain to be tested is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain;

[0029] c4) Detect or assist in detecting whether the sample to be tested is infected with a highly virulent strain of IBDV, a novel variant of IBDV, and / or an attenuated IBDV vaccine strain;

[0030] c5) Detect or assist in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested.

[0031] Furthermore, the kit also includes standard quality grains of IBDV super-virulent strain, IBDV novel variant strain, and IBDV attenuated vaccine strain.

[0032] The nucleotide sequence of the standard quality plasmid of the highly virulent IBDV strain is shown in Sequence 3 of the sequence listing.

[0033] The nucleotide sequence of the standard quality plasmid of the novel IBDV mutant is shown in Sequence 4 of the sequence listing.

[0034] The nucleotide sequence of the standard quality plasmid of the IBDV attenuated vaccine strain is shown in Sequence 5 of the sequence listing.

[0035] Furthermore, the kit may also include other reagents for PCR amplification, such as HRM MasterMix and ddH2O.

[0036] Another objective of this invention is to provide a method for detecting or assisting in the detection of whether a test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain.

[0037] The method for detecting or assisting in the detection of whether a test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain, provided by this invention, includes the following steps: extracting RNA from the test strain, then reverse transcribing the RNA into cDNA, and then performing PCR amplification using the cDNA as a template and the aforementioned specific primer pair. Simultaneously, standard quality spectra of the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain are used as control samples for PCR amplification under the same conditions, respectively. Finally, the method determines whether the test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain based on the high-resolution melting curves of the test strain and the control samples.

[0038] If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the control sample of the IBDV super-virulent strain, then the test strain is the IBDV super-virulent strain.

[0039] If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the control sample of the novel IBDV variant, then the test strain is the novel IBDV variant.

[0040] If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the IBDV attenuated vaccine strain control sample, then the test strain is the IBDV attenuated vaccine strain.

[0041] Another objective of this invention is to provide a method for detecting or assisting in the detection of whether a sample to be tested is infected with a highly virulent strain of IBDV, a novel variant of IBDV, and / or an attenuated IBDV vaccine strain.

[0042] The method for detecting or assisting in the detection of whether a test sample is infected with a highly virulent IBDV strain, a novel IBDV variant strain, and / or an attenuated IBDV vaccine strain, provided by this invention, includes the following steps: extracting RNA from the test sample, then reverse transcribing the RNA into cDNA, and then performing PCR amplification using the cDNA as a template and the aforementioned specific primer pair. Simultaneously, standard plasmids of the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain are used as control samples for PCR amplification under the same conditions. Finally, the method determines whether the test sample is infected with the highly virulent IBDV strain, the novel IBDV variant strain, and / or the attenuated IBDV vaccine strain based on the high-resolution melting curves of the test sample and the control samples.

[0043] If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the control sample of the highly virulent IBDV strain, then the test sample is infected with the highly virulent IBDV strain; otherwise, the test sample is not infected with the highly virulent IBDV strain.

[0044] If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the control sample of the novel IBDV variant, then the test sample is infected with the novel IBDV variant; otherwise, the test sample is not infected with the novel IBDV variant.

[0045] If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the control sample of the IBDV attenuated vaccine strain, then the test sample is infected with the IBDV attenuated vaccine strain; otherwise, the test sample is not infected with the IBDV attenuated vaccine strain.

[0046] The high-resolution melting curves described above can be peak-shaped melting curves and / or standardized melting curves. In practical applications, identification and detection can be performed solely based on peak-shaped melting curves, or simultaneously based on both peak-shaped melting curves and standardized melting curves.

[0047] The final objective of this invention is to provide a method for detecting or assisting in the detection of the content of highly virulent IBDV strains, novel IBDV variants, or attenuated IBDV vaccine strains in a sample to be tested.

[0048] The method for detecting or assisting in the detection of the content of highly virulent IBDV strains, novel IBDV variants, or attenuated IBDV vaccine strains in a test sample provided by the present invention includes the following steps: extracting RNA from the test sample, then reverse transcribing the RNA into cDNA, and then using the cDNA as a template, performing PCR amplification with the above-mentioned specific primer pair to obtain the Cq value of the test sample (the Cq value is the number of PCR cycles at which the sample reaction curve intersects the threshold line, and this value indicates the number of cycles required to detect a true positive signal from the sample), and substituting the Cq value of the test sample into the standard curve equation of the highly virulent IBDV strain, novel IBDV variant, or attenuated IBDV vaccine strain to obtain the content of the highly virulent IBDV strain, novel IBDV variant, or attenuated IBDV vaccine strain in the test sample;

[0049] The standard curve equation was obtained as follows: PCR amplification was performed on a series of standard plasmid solutions of IBDV super-virulent strains, novel IBDV variants, or attenuated IBDV vaccine strains at known concentrations using the above-mentioned specific primer pairs. The Cq values ​​corresponding to each concentration of the standard plasmid solution of IBDV super-virulent strains, novel IBDV variants, or attenuated IBDV vaccine strains were measured, thereby obtaining the standard curve equation between the concentration of the standard plasmid solution of IBDV super-virulent strains, novel IBDV variants, or attenuated IBDV vaccine strains and the Cq value.

[0050] In any of the methods described above, the PCR amplification reaction procedure is as follows: pre-denaturation at 94℃ for 2 min; denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, repeated 40 times; the melting temperature of the HRM analyzer is set to collect fluorescence signals at a rate of 0.04℃ per step from 75℃ to 95℃.

[0051] The PCR amplification reaction system is as follows: 10 μL HRM Master Mix, 0.2 μL upstream primer HRM-F, 0.2 μL downstream primer HRM-R, 1 μL cDNA, and ddH2O to a final volume of 20 μL. The final concentration of both upstream primer HRM-F and downstream primer HRM-R in the reaction system is 0.2 μM.

[0052] The instrument used for PCR amplification is a fluorescence quantitative PCR instrument. 96 instruments.

[0053] In any of the above applications or methods, the sample to be tested may be a single infected sample or a mixed infected sample.

[0054] The single infected sample may be a sample infected with a highly virulent strain of IBDV, a novel variant of IBDV, or an attenuated IBDV vaccine strain.

[0055] The mixed infection sample may be a sample infected with at least two of the following strains: a highly virulent IBDV strain, a novel IBDV variant strain, and an attenuated IBDV vaccine strain, such as a double mixed infection sample or a triple mixed infection sample.

[0056] The aforementioned highly virulent IBDV strain is specifically the highly virulent IBDV strain NN1172.

[0057] The aforementioned novel IBDV variant is specifically the novel IBDV variant QZ191002.

[0058] The IBDV attenuated vaccine strain mentioned above is specifically IBDV attenuated vaccine strain B87.

[0059] The present invention has the following advantages:

[0060] 1. The high-resolution melting (HRM) technology used in this invention is a new technology for gene analysis. It is based on the principle that different melting temperatures of single nucleotides result in different melting curves, which can detect differences in individual bases and has extremely high sensitivity. In addition, it has the advantages of high automation, high throughput, closed-tube operation and low cost.

[0061] 2. This invention uses a one-step amplification method, with the entire amplification time being 60 minutes. This not only saves working time but also provides high sensitivity. In contrast, conventional methods such as ordinary PCR + enzyme digestion identification often require 5 to 6 hours, which are cumbersome and increase workload.

[0062] 3. The high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains provided by this invention requires only one pair of primers to identify three different pathogenic types of IBDV (highly virulent strains, novel variants, and attenuated vaccine strains). The melting peaks are concise and clear, and it can simultaneously qualitatively detect single and mixed infection samples (including various dual and triple mixed infections). Standard curves for the three different pathogenic strains have been established, and quantitative analysis of single infection samples can also be performed.

[0063] 4. Because the three pathogenic strains of IBDV are not significantly different in sequence, they are often difficult to distinguish directly using conventional PCR. Conventional PCR requires designing three different primers based on the sequences of the three different virus types. However, due to high sequence homology, it is difficult to design primer sequences that can accurately distinguish the three different strains. This invention differs from previous methods by designing primers based on extensive comparative screening of regions with different GC content in the genomes of highly virulent IBDV strains, novel variants, and attenuated vaccine strains. Only one pair of primers is needed to simultaneously distinguish the three different pathogenic strains of IBDV. Furthermore, this method is simple to operate, rapid, low-cost, and exhibits good sensitivity and specificity. The reaction process does not require opening the container, avoiding contamination issues. Additionally, the amplified products can be used for gel electrophoresis, subsequent sequencing, and sequence analysis, greatly improving the efficiency of IBDV identification and detection.

[0064] This invention establishes a high-resolution melting curve kit for rapidly and accurately identifying highly virulent IBDV strains, novel variants, and attenuated vaccine strains. This provides a new technology and means for the rapid and accurate identification and detection of different pathogenic IBDV strains circulating in clinical practice, greatly improving the efficiency of clinical identification and detection. Attached Figure Description

[0065] Figure 1 A schematic diagram of the standardized melting curve of a high-resolution melting curve kit for rapidly identifying highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0066] Figure 2 A schematic diagram of the peak shape of the high-resolution melting curve kit for rapidly identifying highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0067] Figure 3 This is a schematic diagram of the specificity assay for a high-resolution melting curve kit used to rapidly identify highly virulent IBDV strains, novel variants, and attenuated vaccine strains. A shows the specific peak-shaped melting curve of RT-qPCR-HRM. B shows the gel electrophoresis image of the specific amplification products of RT-qPCR-HRM. Wherein, M: DL 2000 Marker; 1-10 are the amplification products of avian leukosis virus (ALV), Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), adenovirus type 4 (FAdV-4), avian reticuloendotheliosis virus (REV), infectious bronchitis virus (IBV), avian influenza virus (AIV), avian metapneumovirus (aMPV), and avian reovirus (ARV), respectively; 11-13 are the amplification products of the highly virulent IBDV strain NN1172, the novel variant strain QZ191002, and the attenuated vaccine strain B87, respectively; 14: negative control.

[0068] Figure 4 A schematic diagram of the sensitivity test for a high-resolution melting curve kit for rapidly identifying highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0069] Figure 5 A schematic diagram of the standard curve equations for three different pathogenic IBDV strains established using a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0070] Figure 6 This is a schematic diagram showing the results of detecting mixed plasmid samples using a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0071] Figure 7 This diagram illustrates the results of testing artificial mixed infection model samples using a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains. A: NN1172 single infection group; B: B87 single infection group; C: QZ191002 single infection group; D: NN1172+B87 double mixed infection group; E: NN1172+QZ191002 double mixed infection group; F: B87+QZ191002 double mixed infection group; G: NN1172+B87+QZ191002 triple mixed infection group. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0074] The highly virulent IBDV strain NN1172 described in the following examples is described in the literature “Wang W, Huang Y, Ji Z, Chen G, Zhang Y, Qiao Y, Shi M, Li M, Huang T, Wei T, Mo M, He X, Wei P. The Full Region of N-Terminal in Polymerase of IBDV Plays an Important Role in Viral Replication and Pathogenicity: Either Partial Region or Single Amino Acid V4I Substitution Does Not Completely Lead to the Virus Attenuation to Three-Yellow Chickens. Viruses. 2021 Jan 14;13(1):107. doi:10.3390 / v13010107.”

[0075] The novel IBDV variant strain QZ191002 and the attenuated vaccine strain B87 in the following examples are both described in the literature “Wang W, Huang Y, Zhang Y, Qiao Y, Deng Q, Chen R, Chen J, Huang T, Wei T, Mo M, He X, Wei P. The emerging naturally reassortant strain of IBDV (genotype A2dB3) having segment A from Chinese novel variant strain and segment B from HLJ 0504-like very virulent strain showed enhanced pathogenicity to three-yellow chickens. Transbound Emerg Dis. 2021 Sep 28. doi:10.1111 / tbed.14336.”

[0076] Example 1: A high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains, and its usage method.

[0077] I. High-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains

[0078] The high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains of the present invention includes: HRM Master Mix (Tiangen Biotech (Beijing) Co., Ltd.), a pair of specific primers HRM-F / HRM-R designed with conserved sequences on both sides of the region (2450-2603bp) in the VP3 gene of highly virulent IBDV strains, novel variants, and attenuated vaccine strains as the detection target, ddH2O without positive nucleic acid, and standard quality particles of three different pathogenic IBDV types.

[0079] The upstream primer of the specific primer pair, HRM-F: 5′-GCAGCAGCCAATGTGGAC-3′ (sequence 1);

[0080] The downstream primer of the specific primer pair is HRM-R: 5′-CCGCTTGTGGTGCGTTTG-3′ (sequence 2).

[0081] Three standard quality plasmids for IBDV with different pathogenicity are: pMD18-T-NN1172 (for the highly virulent IBDV strain NN1172), pMD18-T-QZ191002 (for the novel IBDV variant strain QZ191002), and pMD18-T-B87 (for the attenuated IBDV vaccine strain B87). The nucleotide sequence of pMD18-T-NN1172 is shown in Sequence 3. The nucleotide sequence of pMD18-T-QZ191002 is shown in Sequence 4. The nucleotide sequence of pMD18-T-B87 is shown in Sequence 5.

[0082] II. Instructions for using the high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0083] The method for detecting IBDV using the high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains (named the RT-qPCR-HRM method) in step one includes the following steps:

[0084] Using the three standard plasmids from step 1 as templates, PCR amplification was performed using the specific primer pairs from the high-resolution melting curve kit in step 1 to obtain PCR products.

[0085] PCR amplification instrument: Quantitative PCR 96 instruments.

[0086] PCR amplification reaction program: 94℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, 40 cycles; HRM analyzer (Roche, LightCycier 96) melting temperature setting was set to collect fluorescence signals from 75℃ to 95℃ at a rate of 0.04℃ per step.

[0087] PCR amplification reaction system: HRM Master Mix 10 μL, upstream primer HRM-F 0.2 μL, downstream primer HRM-R 0.2 μL, cDNA 1 μL, ddH2O to bring the system to 20 μL. The final concentration of both upstream primer HRM-F and downstream primer HRM-R in the reaction system is 0.2 μM.

[0088] Gel electrophoresis analysis of the PCR products revealed an amplified target fragment of 154 bp. After recovery and purification, the target fragment was ligated, transformed, and positive clones were selected for sequencing. The sequencing results were verified using NCBI BLAST, indicating that the amplified fragment was an IBDV sequence.

[0089] 4. High-resolution melting curve analysis (HRM analysis)

[0090] 1) Standardized melting curve

[0091] A schematic diagram of the standardized melting curve is shown below. Figure 1 As shown in the figure, the red curve represents the highly virulent IBDV strain NN1172, the light blue curve represents the novel IBDV variant QZ191002, and the orange curve represents the attenuated IBDV vaccine strain B87. The figure reveals significant differences in the melting curves of the three different pathogenic IBDV strains due to variations in GC content.

[0092] 2) Peak-shaped melting curve

[0093] A schematic diagram of the peak-shaped melting curve is shown below. Figure 2 As shown in the figure, the red curve represents the highly virulent IBDV strain NN1172, the light blue curve represents the novel IBDV variant QZ191002, and the orange curve represents the attenuated IBDV vaccine strain B87. The figure shows that the three different pathogenic IBDV strains exhibit different melting peaks and different Tm values ​​due to variations in GC content.

[0094] Example 2: Specificity detection of a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0095] Tested viruses: Avian leukosis virus (ALV), Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), adenovirus type 4 (FAdV-4), avian reticuloendotheliosis virus (REV), infectious bronchitis virus (IBV), avian influenza virus (AIV), avian metapneumovirus (aMPV), avian reovirus (ARV), IBDV super-virulent strain NN1172, novel IBDV variant strain QZ191002, and IBDV attenuated vaccine strain B87.

[0096] 1. RNA extraction

[0097] Viral nucleic acid (RNA) was extracted from the tested viruses.

[0098] 2. Reverse transcription

[0099] The viral RNA obtained in step 1 was reverse transcribed into cDNA.

[0100] 3. RT-qPCR

[0101] The RT-qPCR-HRM method described in step two of Example 1 was used to detect the cDNA of the tested virus to verify the specificity of the method. Meanwhile, ddH2O without positive nucleic acid was used as a negative control.

[0102] The results are as follows Figure 3 As shown in the figure, the results indicated that only three different pathogenic IBDV serotypes showed specific peak-shaped melting curves and specific bands in the gel electrophoresis images of the amplification products detected by RT-qPCR-HRM, while the detection results for other common viruses were negative. This demonstrates that the method has good specificity.

[0103] Example 3: Sensitivity test of a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0104] Test samples: The standard plasmids pMD18-T-NN1172 (IBDV supervirulent strain NN1172), pMD18-T-QZ191002 (IBDV novel mutant strain QZ191002), and pMD18-T-B87 (IBDV attenuated vaccine strain B87) were serially diluted 10-fold to obtain 10 samples each. -1 Dilute gradient standard plasmid solutions of different strains, 10 -2 Dilute gradient standard plasmid solutions of different strains, 10 -3 Dilute gradient standard plasmid solutions of different strains, 10 -4 Dilute gradient standard plasmid solutions of different strains, 10 -5 Dilute gradient standard plasmid solutions of different strains, 10 -6Dilute gradient standard plasmid solutions of different strains, 10 -7 Dilute gradient standard plasmid solutions of different strains, 10 -8 Dilute gradient standard plasmid solutions of different strains, 10 -9 Dilute gradient standard plasmid solutions of different strains and 10 -10 Standard granule solutions of different strains in dilute gradients.

[0105] 10 -1 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 9 copies / μL;

[0106] 10 -1 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 9 copies / μL;

[0107] 10 -1 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 9 copies / μL;

[0108] 10 -2 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 8 copies / μL;

[0109] 10 -2 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 8 copies / μL;

[0110] 10 -2 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 8 copies / μL;

[0111] 10 -3 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 7 copies / μL;

[0112] 10 -3 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 7 copies / μL;

[0113] 10 -3The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 7 copies / μL;

[0114] 10 -4 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 6 copies / μL;

[0115] 10 -4 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 6 copies / μL;

[0116] 10 -4 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 6 copies / μL;

[0117] 10 -5 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 5 copies / μL;

[0118] 10 -5 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 5 copies / μL;

[0119] 10 -5 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 5 copies / μL;

[0120] 10 -6 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 4 copies / μL;

[0121] 10 -6 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 4 copies / μL;

[0122] 10 -6 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 4 copies / μL;

[0123] 10 -7The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 3 copies / μL;

[0124] 10 -7 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 3 copies / μL;

[0125] 10 -7 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 3 copies / μL;

[0126] 10 -8 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 2 copies / μL;

[0127] 10 -8 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 2 copies / μL;

[0128] 10 -8 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 2 copies / μL;

[0129] 10 -9 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172, obtained by dilution gradient, was 6.12 × 10⁻⁶. 1 copies / μL;

[0130] 10 -9 The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002, obtained by dilute gradient assay, was 6.11 × 10⁻⁶. 1 copies / μL;

[0131] 10 -9 The concentration of the dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution was 6.75 × 10⁻⁶. 1 copies / μL;

[0132] 10 -10 The concentration of the standard plasmid solution for the highly virulent IBDV strain NN1172 with dilute gradients was 6.12 copies / μL.

[0133] 10 -10The concentration of the standard plasmid solution for the novel IBDV mutant strain QZ191002 with dilute gradient was 6.11 copies / μL;

[0134] 10 -10 The concentration of the standard plasmid solution for the dilute gradient IBDV attenuated vaccine strain B87 was 6.75 copies / μL.

[0135] The test samples were tested using the RT-qPCR-HRM method described in step two of Example 1 to verify the sensitivity of the method.

[0136] The results are as follows Figure 4 As shown. The results revealed that the three different pathogenic IBDV strains ranged from 10 -1 Dilute to 10 -9 Specific melting peaks were observed in all samples. Compared to conventional PCR detection, which requires at least 100 copies / μL, the detection limits of the super-virulent strain NN1172, the novel mutant strain QZ191002, and the attenuated vaccine strain B87 in this invention are 61.2 copies / μL, 61.1 copies / μL, and 67.5 copies / μL, respectively. This indicates that the sensitivity of this invention is high and significantly superior to conventional PCR detection methods.

[0137] Example 4: Establishment of standard curves for highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0138] Test sample: 10 from Example 3 -2 Dilute gradient standard plasmid solutions of different strains, 10 -3 Dilute gradient standard plasmid solutions of different strains, 10 -4 Dilute gradient standard plasmid solutions of different strains, 10 -5 Dilute gradient standard plasmid solutions of different strains, 10 -6 Standard granule solutions of different strains in dilute gradients.

[0139] The test samples were tested using the RT-qPCR-HRM method described in step two of Example 1. Each dilution was replicated in triplicate. Standard curves were then constructed with the logarithm of the standard plasmid copy number on the x-axis and the Cq value measured by the HRM analyzer (Roche, LightCycier96) on the y-axis for the highly virulent IBDV strain, the novel variant strain, and the attenuated vaccine strain, respectively.

[0140] The results are as follows Figure 5 As shown. The results show that this invention selected 10 amplification curves with relatively stable results. -2 ~10 -6These five dilutions were used to establish standard curve equations for three different pathogenic IBDV serotypes, which can be used to perform accurate quantitative analysis of the three different pathogenic IBDV serotypes.

[0141] Example 5: Repeatability test of a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains.

[0142] Test samples: 10 samples from different batches (within-batch and between-batch) of Example 3 were selected. -2 Dilute gradient standard plasmid solutions of different strains, 10 -3 Dilute gradient standard plasmid solutions of different strains, 10 -4 Dilute gradient standard plasmid solutions of different strains, 10 -5 Dilute gradient standard plasmid solutions of different strains, 10 -6 Standard granule solutions of different strains in dilute gradients.

[0143] The RT-qPCR-HRM method described in step two of Example 1 was used to perform repeatability tests on the test samples under the same reaction conditions.

[0144] The results are shown in Tables 1-3. The results show that the coefficients of variation for intra-batch replicates of the highly virulent IBDV strain NN1172, the novel variant strain QZ191002, and the attenuated vaccine strain B87 all did not exceed 1%, and the coefficients of variation for inter-batch replicates all did not exceed 1.5%. This indicates that the RT-qPCR-HRM method established in this invention for three different pathogenic IBDV serotypes has good reproducibility.

[0145] Table 1. Intra-batch and inter-batch repeatability tests of pMD18-T-NN1172

[0146]

[0147] Note: 1-5 represent 10 respectively. -2 -10 -6 Dilute gradient IBDV super-virulent strain NN1172 standard plasmid solution.

[0148] Table 2. Intra-batch and inter-batch repeatability tests of pMD18-T-QZ191002

[0149]

[0150]

[0151] Note: 1-5 represent 10 respectively. -2 -10 -6 Dilute gradient standard plasmid solutions of the novel IBDV mutant strain QZ191002.

[0152] Table 3. Intra-batch and inter-batch repeatability tests of pMD18-T-B87

[0153]

[0154] Note: 1-5 represent 10 respectively. -2 -10 -6 Dilute gradient IBDV attenuated vaccine strain B87 standard plasmid solution.

[0155] Example 6: The effectiveness of a high-resolution melting curve kit for rapid identification of highly virulent IBDV strains, novel variants, and attenuated vaccine strains on mixed samples.

[0156] I. Test Sample

[0157] 1. Mixed plasmid sample

[0158] B87+NN1172: Mix equal volumes of standard plasmid solution of IBDV attenuated vaccine strain B87 (50 ng / μL) and standard plasmid solution of IBDV super-virulent strain NN1172 (50 ng / μL) to obtain sample B87+NN1172.

[0159] QZ191002+NN1172: The standard plasmid solution of the novel IBDV mutant strain QZ191002 with a concentration of 50 ng / μL was mixed in equal volumes with the standard plasmid solution of the highly virulent IBDV strain NN1172 with a concentration of 50 ng / μL to obtain the QZ191002+NN1172 sample.

[0160] B87+QZ191002: Mix equal volumes of standard plasmid solution of IBDV attenuated vaccine strain B87 (50 ng / μL) and standard plasmid solution of novel IBDV mutant strain QZ191002 (50 ng / μL) to obtain sample B87+QZ191002.

[0161] B87+QZ191002+NN1172: Equal volumes of standard plasmid solutions of IBDV attenuated vaccine strain B87 (50 ng / μL), novel IBDV variant strain QZ191002 (50 ng / μL), and highly virulent IBDV strain NN1172 (50 ng / μL) were mixed to obtain the B87+QZ191002+NN1172 sample.

[0162] 2. Artificial mixed infection model samples

[0163] Forty unvaccinated Sanhuang chickens aged 1 day were raised to 28 days old and tested negative for antibodies. The chickens in each group were then treated with an IBDV strain at a dose of 10... 5 TCID50 Eight groups of five Sanhuang chickens were challenged orally with a dose of 0.5 mL. The treatment method for each group was as follows:

[0164] Group A (NN1172-only infection group): challenged with the highly virulent IBDV strain NN1172;

[0165] Group B (B87 isolated infection group): challenged with IBDV attenuated vaccine strain B87;

[0166] Group C (QZ191002-only infection group): challenged with the novel IBDV mutant strain QZ191002;

[0167] Group D (NN1172+B87 dual infection group): challenged with IBDV super-virulent strain NN1172 and attenuated vaccine strain B87;

[0168] Group E (NN1172+QZ191002 double mixed infection group): challenged with the highly virulent IBDV strain NN1172 and the novel mutant strain QZ191002;

[0169] Group F (B87+QZ191002 dual mixed infection group): challenged with IBDV attenuated vaccine strain B87 and novel mutant strain QZ191002;

[0170] Group G (NN1172+B87+QZ191002 triple mixed infection group): challenged with IBDV super-virulent strain NN1172, attenuated vaccine strain B87 and novel mutant strain QZ191002;

[0171] Group H (blank control group): Orally administered 0.5 mL PBS;

[0172] In each of the above mixed infection groups, the challenge dose for each strain in each group was 10. 5 TCID 50 / 0.5mL.

[0173] All chickens in the above experimental groups were euthanized at 35 days of age (i.e. 7 days after challenge with the virus), and the bursa of Fabricius tissue was collected and homogenized to obtain samples infected with different strains of the virus.

[0174] II. Detection using the RT-qPCR-HRM method

[0175] The RT-qPCR-HRM method described in step two of Example 1 was used to detect the test samples from step one.

[0176] The detection results of the mixed plasmid sample are as follows: Figure 6As shown in the figure. The results show that all mixed samples can exhibit melting peaks corresponding to the positive samples within them. After RT-qPCR-HRM reaction, the binary or triple mixed samples showed melting curves with two or three peaks, respectively.

[0177] The detection results of samples from the artificial mixed infection model are as follows: Figure 7 As shown in the figure. The results show that the kit of the present invention can effectively identify infected samples of different single strains and mixed strains, with a detection rate and accuracy of 100%.

[0178] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A specific primer pair for identifying highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains, wherein the specific primer pair consists of primer HRM-F and primer HRM-R; The primer HRM-F is a single-stranded DNA molecule as shown in sequence 1 of the sequence listing; The primer HRM-R is a single-stranded DNA molecule as shown in sequence 2 of the sequence listing.

2. The specific primer pair according to claim 1, characterized in that: The molar ratio of primer HRM-F to primer HRM-R is 1:

1.

3. The use of the specific primer pair according to claim 1 or 2 for non-disease diagnostic and therapeutic purposes in any of the following b1)-b10): b1) Identify or assist in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains; b2) To differentiate or assist in the differentiation of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains; b3) Identify or assist in identifying whether the test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain. b4) Detect or assist in detecting whether the sample to be tested is infected with highly virulent strains of IBDV, novel variants of IBDV, and / or attenuated vaccine strains of IBDV; b5) Detect or assist in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested; b6) Prepare products for identifying or assisting in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains; b7) Prepare products that can differentiate or assist in differentiating IBDV super-virulent strains, novel IBDV variants, and IBDV attenuated vaccine strains; b8) Prepare products for identification or auxiliary identification of the test strain as a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain. b9) Prepare products for detecting or assisting in the detection of whether a sample to be tested is infected with a highly virulent strain of IBDV, a novel variant of IBDV, and / or an attenuated IBDV vaccine strain; b10) Prepare products for detecting or assisting in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested. The highly virulent IBDV strain is IBDV strain NN1172; The novel IBDV variant strain is IBDV variant strain QZ191002; The IBDV attenuated vaccine strain is IBDV attenuated vaccine strain B87.

4. A kit containing the specific primer pair as described in claim 1 or 2; The kit has the function of any one of the following c1)-c5): c1) Identify or assist in the identification of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains; c2) To differentiate or assist in the differentiation of highly virulent IBDV strains, novel IBDV variants, and attenuated IBDV vaccine strains; c3) Detect or assist in the detection of whether the virus strain to be tested is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain; c4) Detect or assist in detecting whether the sample to be tested is infected with a highly virulent strain of IBDV, a novel variant of IBDV, and / or an attenuated IBDV vaccine strain; c5) Detect or assist in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the sample to be tested.

5. The reagent kit according to claim 4, characterized in that: The kit also includes standard quality grains of IBDV super-virulent strain, IBDV novel variant strain, and IBDV attenuated vaccine strain.

6. The reagent kit according to claim 5, characterized in that: The nucleotide sequence of the standard quality plasmid of the highly virulent IBDV strain is shown in sequence 3 of the sequence listing. The nucleotide sequence of the standard quality plasmid of the novel IBDV mutant is shown in sequence 4 of the sequence listing. The nucleotide sequence of the standard quality plasmid of the IBDV attenuated vaccine strain is shown in Sequence 5 of the sequence listing.

7. A method for detecting or assisting in the detection of a test strain as a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain for non-disease diagnosis and treatment purposes, comprising the following steps: extracting RNA from the test strain, then reverse transcribing the RNA into cDNA, and then performing PCR amplification using the cDNA as a template and the specific primer pair described in claim 1; simultaneously using standard quality spectra of the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain as control samples for the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain, respectively, and performing PCR amplification under the same conditions; finally, determining whether the test strain is a highly virulent IBDV strain, a novel IBDV variant strain, or an attenuated IBDV vaccine strain based on the high-resolution melting curves of the test strain and the control samples. If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the control sample of the IBDV super-virulent strain, then the test strain is the IBDV super-virulent strain. If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the control sample of the novel IBDV variant, then the test strain is the novel IBDV variant. If the high-resolution melting curve of the test strain is consistent with the high-resolution melting curve of the IBDV attenuated vaccine strain control sample, then the test strain is the IBDV attenuated vaccine strain. The highly virulent IBDV strain is IBDV strain NN1172; The novel IBDV variant strain is IBDV variant strain QZ191002; The IBDV attenuated vaccine strain is IBDV attenuated vaccine strain B87.

8. A method for detecting or assisting in the detection of whether a test sample is infected with a highly virulent IBDV strain, a novel IBDV variant strain, and / or an attenuated IBDV vaccine strain for purposes other than disease diagnosis and treatment, comprising the following steps: extracting RNA from the test sample, then reverse transcribing the RNA into cDNA, and then performing PCR amplification using the cDNA as a template and the specific primer pair described in claim 1; simultaneously using standard plasmids of the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain as control samples for the highly virulent IBDV strain, the novel IBDV variant strain, and the attenuated IBDV vaccine strain, respectively, and performing PCR amplification under the same conditions; finally, determining whether the test sample is infected with the highly virulent IBDV strain, the novel IBDV variant strain, and / or the attenuated IBDV vaccine strain based on the high-resolution melting curves of the test sample and the control samples. If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the control sample of the highly virulent IBDV strain, then the test sample is infected with the highly virulent IBDV strain; otherwise, the test sample is not infected with the highly virulent IBDV strain. If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the control sample of the novel IBDV variant, then the test sample is infected with the novel IBDV variant; otherwise, the test sample is not infected with the novel IBDV variant. If the high-resolution melting curve of the test sample is consistent with the high-resolution melting curve of the IBDV attenuated vaccine strain control sample, then the test sample is infected with the IBDV attenuated vaccine strain; otherwise, the test sample is not infected with the IBDV attenuated vaccine strain. The highly virulent IBDV strain is IBDV strain NN1172; The novel IBDV variant strain is IBDV variant strain QZ191002; The IBDV attenuated vaccine strain is IBDV attenuated vaccine strain B87.

9. A method for detecting or assisting in the detection of the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in a test sample for non-disease diagnosis and treatment purposes, comprising the following steps: extracting RNA from the test sample, then reverse transcribing the RNA into cDNA, and then using the cDNA as a template to perform PCR amplification using the specific primer pair described in claim 1 to obtain the Cq value of the test sample, and substituting the Cq value of the test sample into the standard curve equation of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV to obtain the content of highly virulent IBDV strains, novel variants of IBDV, or attenuated vaccine strains of IBDV in the test sample; The standard curve equation was obtained as follows: PCR amplification was performed on a series of standard plasmid solutions of IBDV super-virulent strains, novel IBDV variants, and attenuated IBDV vaccine strains at known concentrations using the specific primer pair described in claim 1. The Cq values ​​corresponding to each concentration of the standard plasmid solutions of IBDV super-virulent strains, novel IBDV variants, and attenuated IBDV vaccine strains were measured, thereby obtaining the standard curve equation between the concentration of the standard plasmid solutions of IBDV super-virulent strains, novel IBDV variants, and attenuated IBDV vaccine strains and the Cq value. The highly virulent IBDV strain is IBDV strain NN1172; The novel IBDV variant strain is IBDV variant strain QZ191002; The IBDV attenuated vaccine strain is IBDV attenuated vaccine strain B87.

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