Primer probe composition and kit for detecting H9 subtype AIV (avian influenza virus), NDV (Newcastle Disease Virus) and IBV (Infectious Bursal Virus) and application

Abstract

The invention discloses a primer probe composition for detecting H9 subtype AIV, NDV and IBV, a kit and application, and belongs to the technical field of fluorescence detection. The sequence of the primer probe combination is shown as SEQ ID NO.1-SEQ ID NO.9, the primer probe combination comprises a primer pair and a probe for detecting the H9 subtype avian influenza virus, the Newcastle disease virus and the infectious bronchitis virus, can detect and distinguish epidemic H9 viruses of different branches, NDV of different genotypes and IBV of different genotypes, does not have cross reaction with other common viruses, and can be used for detecting and distinguishing the H9 viruses of different branches, the NDV of different genotypes and the IBV of different genotypes. The detection sensitivity is high, and the minimum detectable quantity of the three viruses is 0.8 copy, so that the specific, sensitive and rapid detection of H9, NDV and IBV can be realized, and the defects of the existing traditional detection technology are overcome. The method has important practical significance on timely blocking epidemic situation propagation, reducing breeding loss, guaranteeing public health safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to a primer and probe composition, kit, and application for detecting H9 subtype AIV, NDV, and IBV. Background Technology

[0002] H9 subtype avian influenza virus (H9 subtype AIV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) are three core pathogenic viruses that threaten the poultry industry. They are widespread in various poultry populations, including chickens, ducks, geese, and pigeons, and mixed infections are common, causing serious economic losses to the poultry industry. H9 subtype AIV is characterized by its wide spread and high infection rate. Although the mortality rate of single infection is low, it easily causes respiratory symptoms and decreased egg production in poultry, and the disease can be aggravated when it works in synergy with other pathogens. NDV has diverse genotypes, and the pathogenicity of different genotypes varies significantly, ranging from asymptomatic infection to acute death. Its spread directly threatens the survival of poultry populations. IBV has a high degree of genetic variation and has evolved into multiple genotype branches. Different genotypes have different tissue tropisms and pathogenic spectra, making it difficult for routine testing to fully cover all cases, thus increasing the difficulty of prevention and control.

[0003] Current detection technologies for the three viruses mentioned above mainly include virus isolation and culture, conventional RT-PCR, and ordinary fluorescent RT-PCR. However, existing technologies have many limitations: First, the detection efficiency is low, as most methods can only detect a single virus, failing to meet the needs of multi-virus joint screening in clinical practice, increasing detection costs and time. Second, specificity is insufficient, as the primer and probe designs of some detection methods lack specificity, easily leading to cross-reactions with other avian viruses (such as other subtypes of AIV, infectious bursal virus, etc.), resulting in false positive results and interfering with epidemic assessment. Third, sensitivity is limited, as conventional detection methods have high minimum detection limits, making it difficult to capture early samples of low-concentration viral infections, easily leading to missed detections. Fourth, the ability to differentiate is weak, failing to effectively distinguish different epidemic branches of H9 subtype AIV, different genotypes of NDV, and multiple genotypes of IBV, hindering accurate tracing of the epidemic source and the development of targeted prevention and control measures. Fifth, the operation procedures are cumbersome, with some methods relying on complex experimental conditions and specialized equipment, unsuitable for the needs of grassroots farming scenarios or rapid on-site testing.

[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] With the large-scale and intensive development of poultry farming, the speed of virus transmission has accelerated, making the demand for rapid, accurate, and efficient multi-virus joint detection technologies increasingly urgent. Therefore, developing a detection technology capable of simultaneously and specifically detecting H9 subtype AIV, NDV, and IBV, with advantages such as high sensitivity, broad detection spectrum, and ease of operation, is of significant practical importance for timely blocking of epidemic transmission, reducing farming losses, and ensuring industry safety. It is also a pressing technical challenge that needs to be addressed in the field of poultry disease detection. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a primer and probe composition, kit and application for detecting H9 subtype AIV, NDV and IBV, which can specifically detect H9 subtype AIV, NDV and IBV of different branches or genotypes, without cross-reaction with other common viruses, and with high detection sensitivity.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a primer-probe combination comprising primer pairs and probes for detecting H9 subtype AIV, primer pairs and probes for detecting NDV, and primer pairs and probes for detecting IBV; wherein: The primer pair for detecting H9 subtype AIV is H9-1 and H9-2, with sequences shown in SEQ ID NO.1~SEQ ID NO.2; the probe is H9-P, with a sequence shown in SEQ ID NO.3. The primer pair for detecting NDV is NDV-1 and NDV-2, with sequences shown in SEQ ID NO.4~SEQ ID NO.5; the probe is NDV-P, with a sequence shown in SEQ ID NO.6. The primer pair for detecting IBV is IBV-1 and IBV-2, with sequences shown in SEQ ID NO.7~SEQ ID NO.8; the probe is IBV-P, with a sequence shown in SEQ ID NO.9.

[0008] Preferably, 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.

[0009] Preferably, the probe for detecting NDV is labeled with carboxyfluorescein VIC at the 5' end and with a quencher group BHQ1 at the 3' end.

[0010] Preferably, the probes for detecting IBV are all labeled with carboxyfluorescein CY5 at the 5' end and with quencher group BHQ3 at the 3' end.

[0011] A second objective of this invention is to provide the application of the primer-probe combination in the preparation of products for detecting H9 subtype AIV, NDV, and / or IBV.

[0012] Preferably, the application includes a method for detecting AIV, NDV, and IBV using the primer-probe combination.

[0013] Preferably, the reaction system of the method is as follows: 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.

[0014] Preferably, the reaction procedure of the method is as follows: first stage, 42℃ / 5min; second stage, 95℃ / 30s; third stage, 95℃ / 5s, 58℃ / 30s for fluorescence collection, 40 cycles.

[0015] Preferably, the product includes a reagent kit.

[0016] The third objective of this invention is to provide a triple fluorescence detection kit, wherein the triple fluorescence detection kit includes the primer-probe combination.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The primer-probe combination designed in this invention contains primer pairs and probes for detecting H9 subtype avian influenza virus, Newcastle disease virus, and infectious bronchitis virus (IBV). It can detect and differentiate between different branches of H9 virus, different genotypes of NDV, and different genotypes of IBV, without cross-reactivity with other common viruses. It exhibits high detection sensitivity, with a minimum detection limit of 0.8 copies for all three viruses. Therefore, it enables specific, sensitive, and rapid detection of H9, NDV, and IBV, thus overcoming the shortcomings of existing traditional detection technologies. This invention has significant practical implications for timely prevention of disease transmission, reduction of livestock losses, and protection of public health and safety. Attached Figure Description

[0018] Figure 1 The results of triple fluorescent RT-PCR amplification of H9 subtype AIV, NDV, and IBV in Example 2 of this invention are shown below. 1: Fluorescent RT-PCR amplification curve of H9 subtype AIV; 2: Fluorescent RT-PCR amplification curve of NDV; 3: Fluorescent RT-PCR amplification curve of IBV; 4-18: AIV H1, H2, H3, H4, H5, H6, H7, H8, H10, H11, H12, H13, H14, H15, H16 subtype strains, Infectious Bursal Disease Virus (IBDV) B87, Infectious Laryngotracheitis Virus (ILTV) vaccine strain, and normal chicken tissue nucleic acid control.

[0019] Figure 2 This refers to the sensitivity amplification results of triple fluorescent RT-PCR for the detection of H9 subtype AIV, NDV, and IBV in Example 3 of the present invention; wherein, 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.

[0020] Figure 3 The results of the sensitivity amplification experiment of triple fluorescent RT-PCR for NDV detection of H9 subtype AIV, NDV, and IBV in Example 3 of this invention are shown; wherein, 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.

[0021] Figure 4The results of the sensitivity amplification experiment of triple fluorescent RT-PCR for IBV detection of H9 subtype AIV, NDV, and IBV in Example 3 of this invention are shown; wherein, 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.

[0022] Figure 5 This is the detection spectrum of different branches of H9 virus by triple fluorescent RT-PCR of H9 subtypes AIV, NDV and IBV in Example 4 of the present invention.

[0023] Figure 6 This is the detection spectrum of different NDV genotypes by triple fluorescent RT-PCR of H9 subtype AIV, NDV and IBV in Example 4 of the present invention.

[0024] Figure 7 This is the detection spectrum of common IBV genotypes in China by triple fluorescent RT-PCR of H9 subtype AIV, NDV and IBV in Example 4 of the present invention. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0026] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0027] Example 1 This embodiment provides a primer-probe composition for detecting H9 subtype AIV, NDV, and IBV. Wherein: 1. Primer pairs for detecting H9 subtype AIV include: H9-1 (SEQ ID NO.1): 5'-AAGTGAAGAGGGCGTTGG-3'; H9-2 (SEQ ID NO.2): 5'-CTGTTGTAGGTCCCGTTCC-3'; The probe used to detect H9 subtype AIV is: 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.

[0028] 2. Primer pairs for detecting NDV include: NDV-1 (SEQ ID NO.4): 5'-GACTCGGTATTCATCACCACCT-3'; NDV-2 (SEQ ID NO.5): 5'-TCCGTCATTCGGGACACTT-3'; The probe used to detect NDV is: NDV-P (SEQ ID NO.6): 5'-CACACCGGCTGTGATTTCCTCATTCCC-3', the 5' end of the probe is labeled with carboxyfluorescein VIC, and the 3' end is labeled with the quencher group BHQ1.

[0029] 3. Primers for detecting IBV include: IBV-1 (SEQ ID NO.7): 5'-TGTTGCTGGTATCACTGCTTGT-3'; IBV-2 (SEQ ID NO.8): 5'-CAGAGGTCCTTCGCACTCG-3'; The probe used to detect IBV is: 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.

[0030] Example 2 This embodiment is used to detect the specificity of the primer-probe composition, and the specific steps are as follows: 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, and NDV were identified and preserved by the China Animal Health and Epidemiology Center. Nucleic acid DNA / RNA was 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 sample DNA / RNA templates for testing.

[0031] The following reaction systems were used for testing: 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; The reaction procedure is as follows: First stage, 42℃ / 5min; second stage, 95℃ / 30s; third stage, 95℃ / 5s, 58℃ / 30s (collect fluorescence), 40 cycles.

[0032] Figure 1 The results showed that H9 subtype AIV, NDV, and IBV nucleic acids exhibited fluorescence amplification curves, while other common avian viruses 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, NDV, and IBV can specifically detect H9 subtype AIV, NDV, and IBV without cross-reacting with other common viruses.

[0033] Example 3 This embodiment is used to detect the sensitivity of the primer-probe composition, and the specific steps are as follows: The amplification products of primers H9-1 and H9-2, NDV-1 and NDV-2, and IBV-1 and IBV-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.

[0034] Following the reaction system of Example 2, using 2 μL of each diluted nucleic acid as a template, the reaction was carried out according to the reaction procedure of Example 1, and the results were detected. Figures 2-4 As shown.

[0035] 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 and IBV, with a minimum detection limit of 0.8 copies for all three viruses.

[0036] Example 4 This embodiment is used to detect the detection spectrum of the primer-probe composition, and the specific steps are as follows: 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, one strain each of seven different genotypes of NDV (including Class I genotypes 1.1 and 1.2, and Class II genotypes I, II, VI, VII, and XII), and IBV were collected. One strain each of 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 used to extract nucleic acid RNA from the above viruses and normal chicken tissues using a commercially available kit (Fine Pure Virus DNA / RNA Extraction Kit, Jifan Biotechnology) to obtain sample DNA / RNA templates for testing.

[0037] The reaction was carried out according to the reaction system and reaction procedure described in Example 2, and the results were detected. Figure 5 , Figure 6 and Figure 7 As shown.

[0038] Figure 5 , Figure 6 and Figure 7 The invention demonstrates that the primer and probe composition for detecting H9 subtype AIV, NDV, and IBV 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) and NDV (Class I gene 1.1 and gene 1.2 subtypes, Class I gene 1.1 and IBV, Class I gene 1.2 ... IBV genotypes (genotypes I, II, 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).

[0039] Example 5 The purpose of this embodiment is to use the primer-probe composition provided by the present invention for clinical testing, and to compare its effectiveness with other technologies. The specific steps are as follows: 182 throat swab samples were collected from chickens, ducks, geese, and pigeons at the Qingxiu District Agricultural Products Trading Market in Nanning City, Guangxi. 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.

[0040] The methods in this invention and those in the previous patent application were used for testing, and the results are shown in Table 1.

[0041] The results showed that, using the method of this invention, out of 182 samples, 80 samples were positive for H9 subtype AIV nucleic acid, 5 samples were positive for NDV, 76 samples were positive for IBV nucleic acid, and 56 samples were negative for all three pathogens (56 = 182 - [80 + 5 + 76 - (3 + 32)]). Meanwhile, among the positive samples, 3 samples were positive for both H9 subtype AIV and NDV nucleic acid, indicating mixed infection; and 32 samples were positive for both H9 subtype AIV and IBV nucleic acid, also indicating mixed infection. However, using the method in the previously filed patent application, out of 182 samples, only 79 samples were positive for H9 subtype AIV nucleic acid, 75 samples were positive for IBV nucleic acid, and NDV-positive samples and samples with mixed NDV infection were undetectable.

[0042] Table 1. Results of clinical testing using the primer and probe composition provided by this invention.

[0043] In summary, the above results demonstrate that the primer-probe composition provided by this invention can rapidly and accurately identify H9 subtypes AIV, NDV, and IBV, and has promising application prospects.

[0044] 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 combination, characterized in that, This includes primer pairs and probes for detecting H9 subtype AIV, primer pairs and probes for detecting NDV, and primer pairs and probes for detecting IBV; among which: The primer pair for detecting H9 subtype AIV is H9-1 and H9-2, with sequences shown in SEQ ID NO.1~SEQ ID NO.2; the probe is H9-P, with a sequence shown in SEQ ID NO.

3. The primer pair for detecting NDV is NDV-1 and NDV-2, with sequences shown in SEQ ID NO.4~SEQ ID NO.5; the probe is NDV-P, with a sequence shown in SEQ ID NO.

6. The primer pair for detecting IBV is IBV-1 and IBV-2, with sequences shown in SEQ ID NO.7~SEQ ID NO.8; the probe is IBV-P, with a sequence shown in SEQ ID NO.

9.

2. The primer-probe combination 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 combination according to claim 2, characterized in that, The probe for detecting NDV is labeled with carboxyfluorescein VIC at the 5' end and with a quencher group BHQ1 at the 3' end.

4. The primer-probe combination according to claim 3, 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. The use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of products for detecting H9 subtype AIV, NDV and / or IBV.

6. The application according to claim 5, characterized in that, The application includes a method for detecting AIV, NDV and IBV using the primer-probe combination according to any one of claims 1 to 4.

7. The application according to claim 6, characterized in that, The reaction system of the method is as follows: 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.

8. The application according to claim 7, characterized in that, The reaction procedure of the method is as follows: first stage, 42℃ / 5min; second stage, 95℃ / 30s; third stage, 95℃ / 5s, 58℃ / 30s for fluorescence collection, 40 cycles.

9. The application according to claim 8, characterized in that, The product includes a reagent kit.

10. A triple fluorescence detection kit, characterized in that, This includes the primer-probe combination as described in any one of claims 1 to 4.

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