Primer probe composition and kit for detecting H9 subtype AIV, NDV virulent strain and IBV and application

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a primer probe composition for detecting H9 subtype AIV, NDV virulent strains and IBV, a kit and application. The primer probe composition for detecting the H9 subtype AIV, the NDV virulent strain and the IBV comprises an upstream primer, a downstream primer and a probe which are designed aiming at the H9 subtype AIV virulent strain, and the sequences of the upstream primer, the downstream primer and the probe are shown as SEQ ID NO.1-3; the sequences of the upstream primer, the downstream primer and the probe are as shown in SEQ ID NO.4-6, and the probe is designed for the H9 subtype NDV virulent strain; the sequences of the upstream primer, the downstream primer and the probe designed aiming at the IBV are as shown in SEQ ID NO.7-9; according to the invention, H9 subtype AIV, NDV virulent strains and IBV with different branches or genotypes can be specifically detected, cross reaction with other common viruses is avoided, and the detection sensitivity is high.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and particularly relates to a primer and probe composition, kit, and application for detecting H9 subtype AIV, highly virulent NDV strains, and IBV. Background Technology

[0002] H9 subtype avian influenza virus (H9 subtype AIV), highly virulent Newcastle disease virus (NDV) strains, and infectious bronchitis virus (IBV) are three common and serious viruses that cause significant damage in poultry farming, widely spreading among various poultry populations such as chickens, ducks, and geese. Although H9 subtype AIV has a relatively low mortality rate, it easily causes respiratory symptoms and decreased egg production in poultry, and often results in co-infection with other pathogens, causing significant economic losses. Highly virulent NDV strains are extremely pathogenic and can cause acute illness and death in poultry, while attenuated strains are mostly used for vaccination. In clinical practice, it is necessary to quickly distinguish between virulent and attenuated strains to guide prevention and control. IBV has diverse genotypes and is prone to mutation. Different genotypes of IBV strains have different pathogenicity and transmission characteristics, posing a great challenge to diagnosis and control.

[0003] Currently, the main detection methods for the three viruses mentioned above include virus isolation and culture, conventional RT-PCR, and ordinary fluorescent RT-PCR. However, existing technologies have significant shortcomings: First, their specificity is poor, with some detection methods prone to cross-reaction with other avian viruses, leading to false positive results; second, their detection efficiency is low, with most methods only capable of detecting one virus at a time, failing to meet the needs of multi-virus joint screening in clinical settings; third, their sensitivity is limited, with high minimum detection limits, making it difficult to detect low-concentration virus-infected samples; fourth, their discriminatory power is insufficient, failing to effectively distinguish between different epidemic branches of H9 subtype AIV, different genotypes of IBV, and virulent strains of NDV, affecting the accurate implementation of prevention and control measures; and fifth, some methods are cumbersome and time-consuming, making them unsuitable for rapid detection in grassroots farming settings.

[0004] Previously, we applied for a patent: a primer and probe composition, kit, and application for detecting H9 subtype AIV and IBV (application number 202411297204.6). This invention can specifically detect H9 subtype avian influenza virus and chicken infectious bronchitis virus, as well as multiple branches of H9 subtype avian influenza virus and chicken infectious bronchitis virus, with a minimum detection limit of 1 copy for both viruses. However, this invention cannot detect and distinguish diseases caused by NDV that have similar clinical symptoms to H9 subtype AIV and IBV.

[0005] Therefore, developing a detection technology that can simultaneously and specifically detect H9 subtype AIV, NDV virulent strains and IBV, and possesses high sensitivity, strong distinguishing ability and simple operation, is of great significance for timely prevention and control of epidemics and reduction of economic losses, and is also an urgent need in the field of poultry disease detection. Summary of the Invention

[0006] The purpose of this invention is to provide a primer and probe composition, kit, and application for detecting H9 subtype AIV, NDV virulent strains, and IBV. This kit can specifically detect H9 subtype AIV, NDV virulent strains, and IBV of different branches or genotypes, without cross-reactivity with other common viruses, and has high detection sensitivity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a primer-probe composition for detecting H9 subtype AIV, NDV virulent strains, and IBV. The primer-probe composition includes an upstream primer designed for H9 subtype AIV virulent strains, a downstream primer designed for H9 subtype AIV virulent strains, a fluorescent probe designed for H9 subtype AIV virulent strains, an upstream primer designed for H9 subtype NDV virulent strains, a downstream primer designed for H9 subtype NDV virulent strains, a fluorescent probe designed for H9 subtype NDV virulent strains, an upstream primer designed for IBV, a downstream primer designed for IBV, and a fluorescent probe designed for IBV.

[0009] The upstream primer designed for the H9 subtype AIV virulent strain has the sequence shown in SEQ ID NO.1;

[0010] The downstream primers designed for the H9 subtype AIV virulent strain are shown in SEQ ID NO.2;

[0011] A fluorescent probe designed for the highly virulent H9 subtype AIV strain, the sequence of which is shown in SEQ ID NO.3;

[0012] The upstream primer designed for the H9 subtype NDV virulent strain has the sequence shown in SEQ ID NO.4;

[0013] The downstream primers designed for the H9 subtype NDV virulent strain are shown in SEQ ID NO.5.

[0014] A fluorescent probe designed for the highly virulent H9 subtype NDV strain has the sequence shown in SEQ ID NO.6;

[0015] The upstream primer designed for IBV has the sequence shown in SEQ ID NO.7;

[0016] The downstream primer designed for IBV has the sequence shown in SEQ ID NO.8;

[0017] A fluorescent probe designed for IBV has the sequence shown in SEQ ID NO.9;

[0018] Furthermore, the probe for detecting H9 subtype AIV is labeled with carboxyfluorescein FAM at the 5' end and with a quencher group BHQ1 at the 3' end.

[0019] Furthermore, the probe for detecting highly virulent NDV strains is labeled with carboxyfluorescein VIC at the 5' end and with a quencher group BHQ1 at the 3' end.

[0020] Furthermore, the probes for detecting IBV are all labeled with carboxyfluorescein CY5 at the 5' end and with quencher group BHQ3 at the 3' end.

[0021] This invention provides a triple fluorescence detection kit for H9 subtype AIV, highly virulent NDV strains and IBV, comprising a reaction buffer, a negative control, and a positive control; the reaction buffer comprises a primer-probe composition having nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.9.

[0022] Furthermore, the positive control is obtained by extracting the nucleic acid DNA / RNA of the H9 subtype AIV, NDV virulent strain and IBV to obtain the sample DNA / RNA template to be tested.

[0023] This invention provides an application of a triple fluorescence detection kit in the preparation of products for detecting H9 subtype AIV, highly virulent NDV strains, and IBV.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The primer and probe compositions (SEQ ID NO. 1-9) designed in this invention for detecting H9 subtype AIV, NDV virulent strains, and IBV can specifically detect H9 subtype AIV, NDV virulent strains, and IBV, as well as multiple branches of H9 subtype AIV, NDV virulent strains, and IBV, without cross-reactivity with other common viruses. Furthermore, they exhibit high detection sensitivity, with a minimum detection limit of 0.8 copies for each of the three viruses. Attached Figure Description

[0026] Figure 1The results of triple fluorescent RT-PCR amplification of H9 subtype AIV, virulent NDV strain, and IBV in Example 3 are shown below. 1: Fluorescent RT-PCR amplification curve of H9 subtype AIV; 2: Fluorescent RT-PCR amplification curve of virulent NDV strain; 3: Fluorescent RT-PCR amplification curve of IBV; 4-19: Nucleic acid control diagrams of AIV H1, H2, H3, H4, H5, H6, H7, H8, H10, H11, H12, H13, H14, H15, H16 subtype strains, infectious bursal virus (IBDV) B87, infectious laryngotracheitis virus (ILTV) vaccine strain, attenuated NDV strain, and normal chicken tissue.

[0027] Figure 2 The results of the sensitivity amplification experiment for the detection of H9 subtype AIV by triple fluorescent RT-PCR of H9 subtype AIV, NDV virulent strain, and IBV in Example 4 are shown; where 1:8×10 3 Copy / reaction, 2: 8 × 10 2 Copy / reaction, 3: 8 × 10 1 Copy / reaction, 4: 8 × 10 0 Copy / reaction, 5: 8 × 10 -1 Copy / reaction diagram.

[0028] Figure 3 The results of the sensitivity test of triple fluorescent RT-PCR for detecting virulent NDV strains (H9 subtype AIV, NDV, and IBV) in Example 4 are shown; where 1: 8 × 10³ copies / reaction, 2: 8 × 10³ copies / reaction. 2 Copy / reaction, 3: 8 × 10 1 Copy / reaction, 4: 8 × 10 0 Copy / reaction, 5: 8 × 10 -1 Copy / reaction diagram.

[0029] Figure 4 The results of the sensitivity amplification experiment of triple fluorescent RT-PCR for IBV detection of H9 subtype AIV, NDV virulent strain, and IBV in Example 4 are shown; where 1:8×10 3 Copy / reaction, 2: 8 × 10 2 Copy / reaction, 3: 8 × 10 1 Copy / reaction, 4: 8 × 10 0 Copy / reaction, 5: 8 × 10 -1 Copy / reaction diagram.

[0030] Figure 5This is a spectrum of triple fluorescent RT-PCR detection of different branches of H9 virus using H9 subtype AIV, NDV virulent strains, and IBV in Example 5. The curves in the figure represent the viruses from 10 branches: h9.4.2.5a, h9.4.2.2, h9.4.2.5d, h9.4.2.5b, h9.4.2.1, h9.4.2.3, h9.4.2.5c, h9.4.2.4, h9.4.2.6, and h9.4.2.5e, from left to right.

[0031] Figure 6 This is a spectrum of triple fluorescent RT-PCR detection of different genotypes of the virulent NDV strain (H9 subtype AIV, NDV virulent strain, and IBV) in Example 5. Wherein, 1: genotype VI; 2: genotype VII; 3: genotype XII.

[0032] Figure 7 This is a spectrum of detection of common IBV genotypes in China by triple fluorescent RT-PCR of H9 subtype AIV, NDV virulent strain, and IBV in Example 5. The curves in the figure represent IBV genotypes GI-19, GI-22, GVI-1, GI-1, GI-2, GI-28, GI-3, GI-13, GI-4, GI-5, GI-29, GI-6, GI-18, GI-7, GI-16, GI-25, and GVII-1 from left to right. Detailed Implementation

[0033] Example 1

[0034] Primer and probe sequences were designed based on the specific sequences of H9 subtype AIV, highly virulent NDV strains, and IBV.

[0035] The primer pair for detecting H9 subtype AIV is as follows:

[0036] H9-1 (SEQ ID NO.1): 5'-AAGTGAAGAGGGCGTTGG-3';

[0037] H9-2 (SEQ ID NO.2): 5'-CTGTTGTAGGTCCCGTTCC-3';

[0038] The probe used to detect H9 subtype AIV is:

[0039] H9-P (SEQ ID NO.3): 5'-CGAAACATCCTTTCCCATCTTCCACCGC-3', the 5' end of the probe is labeled with carboxyfluorescein FAM, and the 3' end is labeled with the quencher group BHQ1.

[0040] The primer pair for detecting virulent NDV strains is as follows:

[0041] NDV-1 (SEQ ID NO.4): 5'-AGACAGGGTCAATCATAGT-3';

[0042] NDV-2 (SEQ ID NO.5): 5'-AACCCCAAGAGCTACA-3';

[0043] The probe for detecting highly virulent NDV strains is:

[0044] NDV-P (SEQ ID NO.6): 5'-ATAAAGCGTTTYTGTCTCCTTCCTCC-3', the 5' end of the probe is labeled with carboxyfluorescein VIC, and the 3' end is labeled with the quencher group BHQ1.

[0045] The primers for detecting IBV are:

[0046] IBV-1 (SEQ ID NO.7): 5'-TGTTGCTGGTATCACTGCTTGT-3';

[0047] IBV-2 (SEQ ID NO.8): 5'-CAGAGGTCCTTCGCACTCG-3';

[0048] The probe used to detect IBV is:

[0049] IBV-P (SEQ ID NO.9): 5'-CCAGCGTCCTACGGGCGCCGT-3', the 5' end of the probe is labeled with carboxyfluorescein CY5, and the 3' end is labeled with quencher group BHQ3.

[0050] Among them, the primer sequences SEQ ID NO.4-SEQ ID NO.6 are modified based on the primer and probe sequences in the national standard GB / T 16550-2020 "Newcastle Disease Diagnostic Techniques".

[0051] Example 2

[0052] A method for preparing a detection kit for H9 subtype AIV, NDV virulent strains and IBV.

[0053] 1. Negative control: Nuclease-free water.

[0054] 2. Positive control: Nucleic acid DNA / RNA was extracted from the above-mentioned H9 subtype AIV, NDV virulent strains and IBV using a commercial kit (Fine Pure Virus DNA / RNA Extraction Kit, Jifan Biotechnology) to obtain the sample DNA / RNA template to be tested;

[0055] The following reaction systems were used for testing:

[0056] 10.0 μL of 2× fluorescent RT-PCR buffer, 0.4 μL of reverse transcriptase and reverse transcriptase inhibitor, 0.4 μL of DNA polymerase, 0.9 μL each of primers H9-1 and H9-2 (final concentration 0.45 μM), 0.8 μL each of primers NDV-1 and NDV-2 (final concentration 0.4 μM), 0.8 μL each of primers IBV-1 and IBV-2 (final concentration 0.4 μM), 0.6 μL of probe H9-P (final concentration 0.3 μM), 0.8 μL of probe NDV-P (final concentration 0.4 μM), 0.8 μL of probe IBV-P (final concentration 0.4 μM), and 2.0 μL of RNA template from the sample to be tested;

[0057] The reaction procedure is as follows:

[0058] First stage, 42℃ / 5min; second stage, 95℃ / 30s; third stage, 95℃ / 5s, 59℃ / 30s (collect fluorescence), 40 cycles;

[0059] The primer amplification products of H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-2 were obtained;

[0060] The amplification products of primers H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-2 were inserted into the pMD19-T vector (TaKaRa), and then amplified, identified, and extracted by plasmids.

[0061] The artificially synthesized plasmids H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-2 were quantified to 10⁻⁶. 7 After copy number / mL, a positive control was prepared by mixing the samples at a volume ratio of 1:1:1. In the obtained positive control, the concentrations of the synthetic plasmids H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-2 were 3.33 × 10⁻⁶. 6 copies / mL;

[0062] 3. Select the reaction system and reaction procedure from step 2, and then assemble the detection kit. The detection kit includes a reaction buffer, a negative control, a positive control, and an external reference. Aliquot 100 μL into each tube for the negative and positive controls. Store the nucleic acid detection kit at -20°C.

[0063] Example 3

[0064] Specific detection

[0065] The specific steps for detecting the specificity of primer-probe combinations for H9 subtype AIV, highly virulent NDV strains, and IBV are as follows:

[0066] Avian influenza virus subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16, IBV H52, infectious bursal virus (IBDV) B87, infectious laryngotracheitis virus (ILTV) vaccine strain, virulent NDV strain, and attenuated NDV strain were extracted from the above viruses and normal chicken tissues using a commercially available kit (Fine Pure Virus DNA / RNA Extraction Kit, Jifan Biotechnology) to obtain the DNA / RNA templates for the samples to be tested.

[0067] The following reaction systems were used for testing:

[0068] 10.0 μL of 2× fluorescent RT-PCR buffer, 0.4 μL of reverse transcriptase and reverse transcriptase inhibitor, 0.4 μL of DNA polymerase, 0.9 μL each of primers H9-1 and H9-2, 0.8 μL each of primers NDV-1 and NDV-2, 0.8 μL each of primers IBV-1 and IBV-2, 0.6 μL of probe H9-P, 0.8 μL of probe NDV-P, 0.8 μL of probe IBV-P, and 2.0 μL of RNA template from the sample to be tested;

[0069] The reaction procedure is as follows:

[0070] First stage, 42℃ / 5min; second stage, 95℃ / 30s; third stage, 95℃ / 5s, 59℃ / 30s (collect fluorescence), 40 cycles.

[0071] Figure 1 The results showed that H9 subtype AIV, virulent NDV strains, and IBV nucleic acids exhibited fluorescence amplification curves, while other common avian viruses (including attenuated NDV strains) and normal chicken tissue nucleic acids did not show amplification curves. This indicates that the primer and probe composition of the present invention for detecting H9 subtype AIV, virulent NDV strains, and IBV can specifically detect H9 subtype AIV, virulent NDV strains, and IBV without cross-reacting with other common viruses.

[0072] Example 4

[0073] Sensitivity detection

[0074] The primer amplification products of H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-2 obtained in step 2 of Example 2 were ligated into the pMD19-T vector (TaKaRa), then transcribed into cRNA in vitro, the nucleic acid concentration was measured, the copy number was calculated, and then serially diluted 10-fold to 4 × 10⁻⁶. 3 4×10 2 4×10 1 4×10 0 4×10 -1 Copy / μL.

[0075] Following the reaction system of Example 3, using 2 μL of each diluted nucleic acid as a template, the reaction was carried out according to the reaction procedure of Example 3 and the results were detected. Figures 2-4 As shown.

[0076] Figure 2 , Figure 3 and Figure 4 The results show that the primer and probe combination designed in this invention has high sensitivity for detecting H9 subtype AIV, NDV virulent strains and IBV, with a minimum detection limit of 0.8 copies for all three viruses.

[0077] Example 5

[0078] The detection spectra of primer-probe combinations for detecting H9 subtype AIV, highly virulent NDV strains, and IBV are determined by the following steps:

[0079] One strain each of the ten branches of H9 subtype avian influenza virus (h9.4.2.1, h9.4.2.2, h9.4.2.3, h9.4.2.4, h9.4.2.6, h9.4.2.5a, h9.4.2.5b, h9.4.2.5c, h9.4.2.5d, and h9.4.2.5e) identified and preserved by the China Animal Health and Epidemiology Center was used. One virulent NDV strain each of genotypes VI, VII, and XII was also used. One strain each of IBV genotypes GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-13, GI-16, GI-18, GI-19, GI-22, GI-25, GI-28, GI-29, GVI-1, and GVII-1 was also used. A commercially available kit (Fine) was used for analysis. Pure Virus DNA / RNA Extraction Kit (Jifan Biotechnology) was used to extract nucleic acid RNA from the above-mentioned virus and normal chicken tissue to obtain the sample DNA / RNA template to be tested. The reaction was carried out according to the reaction system and procedure described in Example 3, and the results are as follows: Figures 5-7 As shown.

[0080] Figure 5 , Figure 6 and Figure 7The present invention provides a primer and probe composition for detecting H9 subtype AIV, highly virulent NDV strains, and IBV. This composition can detect currently prevalent H9 subtype AIV (h9.4.2.1, h9.4.2.2, h9.4.2.3, h9.4.2.4, h9.4.2.6, h9.4.2.5a, h9.4.2.5b, h9.4.2.5c, h9.4.2.5d, h9.4.2.5e), highly virulent NDV strains (genotypes VI, VII, and XII), and common IBV genotypes (GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-13, GI-16, GI-18, GI-19, GI-22, GI-25, GI-28, GI-29, GVI-1, and GVII-1).

[0081] Example 6

[0082] Clinical testing was conducted using the primer and probe composition provided by this invention for detecting H9 subtype AIV, NDV virulent strains, and IBV. The specific steps are as follows: 182 pharyngeal swab samples were collected from chickens, ducks, geese, and pigeons at the Qingxiu District Agricultural Products Trading Market in Nanning City. The swabs were placed in 1.0 mL of PBS solution (pH 7.0~7.4, 0.01 mol / L), and the nucleic acid RNA of the above samples was extracted using a commercial kit (Fine Pure Viral DNA / RNA Extraction Kit, Jifan Biotechnology) to obtain the sample DNA / RNA template to be tested.

[0083] The reaction was carried out according to the reaction system and reaction procedure described in Example 3, and the results were detected. The results are shown in Table 1.

[0084] Table 1. Results of clinical testing using the primer and probe composition provided by this invention.

[0085]

[0086] The results showed that among the 182 samples, 80 samples were positive for H9 subtype AIV nucleic acid, 2 samples were positive for virulent NDV, and 76 samples were positive for IBV nucleic acid; 1 sample was positive for both H9 subtype AIV and virulent NDV nucleic acid, indicating a mixed infection; and 32 samples were positive for both H9 subtype AIV and IBV nucleic acid, also indicating a mixed infection. These results demonstrate that the primer-probe composition provided by this invention can rapidly and accurately identify H9 subtype AIV, virulent NDV strains, and IBV, showing promising application prospects.

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer-probe composition for detecting H9 subtype AIV, highly virulent NDV strains, and IBV, characterized in that, The primer-probe composition includes an upstream primer designed for a virulent strain of H9 subtype AIV, a downstream primer designed for a virulent strain of H9 subtype AIV, a fluorescent probe designed for a virulent strain of H9 subtype AIV, an upstream primer designed for a virulent strain of H9 subtype NDV, a downstream primer designed for a virulent strain of H9 subtype NDV, a fluorescent probe designed for a virulent strain of H9 subtype NDV, an upstream primer designed for IBV, a downstream primer designed for IBV, and a fluorescent probe designed for IBV. The upstream primer designed for the H9 subtype AIV virulent strain has the sequence shown in SEQ ID NO.1; The downstream primers designed for the H9 subtype AIV virulent strain are shown in SEQ ID NO.2; A fluorescent probe designed for the highly virulent H9 subtype AIV strain, the sequence of which is shown in SEQ ID NO.3; The upstream primer designed for the H9 subtype NDV virulent strain has the sequence shown in SEQ ID NO.4; The downstream primers designed for the H9 subtype NDV virulent strain are shown in SEQ ID NO.

5. A fluorescent probe designed for the highly virulent H9 subtype NDV strain has the sequence shown in SEQ ID NO.6; The upstream primer designed for IBV has the sequence shown in SEQ ID NO.7; The downstream primer designed for IBV has the sequence shown in SEQ ID NO.8; The fluorescent probe designed for IBV has the sequence shown in SEQ ID NO.

9.

2. The primer-probe composition according to claim 1, characterized in that, The probe for detecting H9 subtype AIV is labeled with carboxyfluorescein FAM at the 5' end and with quencher group BHQ1 at the 3' end.

3. The primer-probe composition according to claim 1, characterized in that, The probe for detecting highly virulent NDV strains was labeled with a carboxyfluorescein (VIC) at the 5' end and with a quencher group (BHQ1) at the 3' end.

4. The primer-probe composition according to claim 1, characterized in that, The probes used to detect IBV were all labeled with carboxyfluorescein CY5 at the 5' end and with quencher group BHQ3 at the 3' end.

5. A triple fluorescence detection kit for H9 subtype AIV, highly virulent NDV strains, and IBV, characterized in that, It includes a reaction buffer, a negative control, and a positive control; the reaction buffer includes a primer-probe composition having nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.

9.

6. The triple fluorescence detection kit according to claim 5, characterized in that, The positive control is obtained by extracting the nucleic acid DNA / RNA of the H9 subtype AIV, NDV virulent strain and IBV to obtain the sample DNA / RNA template to be tested.

7. The application of the triple fluorescence detection kit as described in claim 5 or 6 in the preparation of products for detecting H9 subtype AIV, highly virulent NDV strains and IBV.

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