Detection primer and probe for H9 subtype AIV and IBV as well as detection method and application of detection primer and probe

Abstract

The invention provides a detection primer, a probe, a detection method and application of H9 subtype AIV and IBV, and belongs to the technical field of fluorescence detection. The detection method comprises a primer pair and a probe group for detecting H9 subtype AIV and IBV, and can detect and distinguish epidemic H9 viruses of different branches and IBV of different genotypes, so that specific, sensitive and rapid detection of H9 and IBV is realized, and the defects of an existing traditional detection technology are overcome. The method is very suitable for on-site rapid detection, the RT-RAA method can be used for reaction at constant temperature, the operation is simple, the constraint of complex instruments is eliminated, temperature change is not needed, and the reaction time is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, and in particular to a detection primer and probe for H9 subtype AIV and IBV, and a detection method and application thereof. Background Art

[0002] Avian influenza is an acute, highly contagious disease of poultry caused by influenza A virus of the Orthomyxoviridae family. Avian influenza virus (AIV) is a single-stranded negative-strand RNA virus, consisting of 8 independent RNA segments. One of its notable biological characteristics is that it has many subtypes and frequent mutations. In addition to infecting poultry, it can also infect humans, pigs, horses and other animals. According to the antigenic differences of the hemagglutinin (HA) on the surface of its virus particles, it can be divided into 16 HA subtypes, and the pathogenicity of different subtypes to the host varies significantly. According to the different pathogenicity, AIV can be divided into highly pathogenic AIV and low pathogenic AIV. Among all HA subtypes, only some strains of H5 and H7 subtypes are highly pathogenic to poultry, called highly pathogenic AIV, which can cause 100% death in chickens.

[0003] Among low pathogenic AIVs, the H9 subtype has a wide range of prevalence and a high prevalence rate. H9 subtype avian influenza outbreaks have occurred in many countries around the world. It can not only cause respiratory symptoms and reduced egg production, but also lead to immunosuppression in chickens, causing the spread of other diseases, causing continuous harm to the poultry industry and huge economic losses.

[0004] Infectious bronchitis is an acute, highly contagious respiratory disease caused by infectious bronchitis virus (IBV), and chickens of all ages are susceptible. It mainly harms the respiratory, urogenital and digestive systems of chickens, with the main clinical features being difficulty breathing, white or watery stools, high mortality in sick chicks, decreased egg production in adult chickens, and the production of deformed and soft-shelled eggs. It is currently one of the most serious viral infectious diseases that endangers my country's poultry industry. The disease exists in almost all poultry-raising countries around the world, and its prevalence in my country is on the rise.

[0005] There are many IBV serotypes, and more than 30 have been reported so far. Different serotypes have great differences in virulence, pathogenicity and tissue tropism, and there is no or only partial cross-immunity between them. In recent years, with the continuous mutation of the virus, new serotypes continue to emerge. However, since serotyping only describes the differences in IBV neutralizing antigenic sites and cannot reflect the level of the genome, it is limited in actual detection applications.

[0006] With the advancement of molecular biology technology, the phylogenetic classification of IBV isolates began to be divided into 9 genotypes and 38 lineages based on the S1 gene. Genotype I includes 30 different viral lineages (temporarily named GI-1 to GI-30), while the remaining genotypes are composed of one viral lineage. Genotype has become the most commonly used method for the classification of IBV isolates.

[0007] H9 subtype AIV and IBV are two common pathogens in chickens. After infection, both may cause respiratory symptoms and decreased egg production. The clinical symptoms of these two diseases are similar, and it is difficult to distinguish them directly from the clinical point of view. Laboratory technology is needed to identify and distinguish them. Virus isolation and serological tests are still the commonly used methods for diagnosing avian influenza and infectious bronchitis. Early rapid detection and diagnosis are undoubtedly the prerequisites for controlling these diseases. However, these methods are cumbersome and complicated to operate, and it is difficult to quickly diagnose the two pathogens, which is very unfavorable for the prevention and treatment of the two diseases.

[0008] Currently, most PCR methods are for separate detection of H9 subtype AIV or IBV. Moreover, as the virus continues to mutate, existing methods are difficult to cover all strains or genotypes. It is urgent to establish an on-site rapid detection method that can detect all strains or genotypes and distinguish between the two diseases, providing a sensitive, rapid and practical method for early on-site rapid diagnosis of clinical diseases.

[0009] The key to the success of RT-RAA is to design reasonable primers and probes. Appropriate primers and probes are the key to determining the specificity and sensitivity of RT-RAA amplification. Multiplex RT-RAA has stricter requirements on primers so that multiple pathogens can be detected simultaneously.

[0010] In addition to each pair of primers having similar annealing temperatures and probes having similar Tm values, the primer and probe sequences must not form stable dimers, hairpin structures, and non-specific binding, so that the components in the system do not interfere with each other and affect the amplification specificity and sensitivity while ensuring the effective amplification of their respective target fragments.

[0011] Although there are many softwares that can help design primers and probes, the automatic search results are not ideal. In order to obtain primers with good effects, various softwares should be used in addition to automatic search. Although the software can help design primers and probes, its limitations are also very obvious: first, the primers and probes designed by the software are designed to find the optimal region on the full-length sequence, and due to the frequent genome mutations of RNA viruses, these regions are likely not the most conservative and most suitable regions; second, manual analysis and practical operation experience are essential in the design of primers and probes. A reliable multiplex RT-RAA detection system must be verified and screened by scientific and technological personnel through a large number of experiments. Summary of the invention

[0012] The purpose of the present invention is to provide a detection primer, probe and detection method and application of H9 subtype AIV and IBV, which have strong specificity and high sensitivity and can realize the diagnosis of these two diseases in a short time, thus buying time for the prevention and control of the above two diseases.

[0013] The objective of the present invention is achieved through the following technical solutions:

[0014] The present invention provides a primer and probe combination for detecting H9 subtype AIV and IBV. The primer and probe combination capable of detecting H9 subtype AIV and IBV comprises a primer pair and a probe for detecting H9 subtype AIV, and a primer pair and a probe for detecting IBV.

[0015] Furthermore, the sequences of the primers and probes include: forward primer H9-1 as shown in SEQ ID NO.1, reverse primer H9-2 as shown in SEQ ID NO.2, probe H9-P as shown in SEQ ID NO.3, forward primer IBV-1 as shown in SEQ ID NO.4, reverse primer IBV-2 as shown in SEQ ID NO.5 and probe IBV-P as shown in SEQ ID NO.6.

[0016] The present invention also provides a dual isothermal amplification detection method for H9 subtype AIV and IBV, and the dual isothermal amplification detection method comprises the steps of using the primers and probes for detection.

[0017] Furthermore, the reaction system of the dual isothermal amplification detection method is 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL of RNase Free dH2O, 2.5 μL of magnesium acetate, enzyme lyophilized powder, and 5.0 μL of template DNA.

[0018] Furthermore, the reaction conditions of the dual isothermal amplification detection method are isothermal amplification at 42° C. for 20 min.

[0019] The present invention also provides a kit for detecting H9 subtype AIV and IBV, and the kit comprises the primers and probes.

[0020] Furthermore, the specific components of the kit include: 2X reaction buffer 25 μL, 10 pmol / μL H9-1 and H9-2 2.0 μL each, 10 pmol / μL IBV-1 and IBV-2 1.8 μL each, 10 pmol / μL H9-P 0.6 μL, 10 pmol / μL IBV-P 0.8 μL, RNase Free dH2O 8.5 μL, magnesium acetate 2.5 μL, enzyme lyophilized powder, template 5.0 μL.

[0021] Furthermore, the reaction conditions of the kit are: isothermal amplification at 42°C for 20 minutes.

[0022] The present invention also provides an application of the primer and probe in preparing a product for detecting H9 subtype AIV and IBV. The product can be used for detecting H9 subtype AIV and IBV at the same time.

[0023] The beneficial effects of the present invention are:

[0024] The H9 subtype AIV and IBV dual fluorescence detection kit provided by the present invention searches for the H9 subtype AIV HA gene sequence in GISAID and GenBank, searches for the IBV 5' non-coding region gene sequence in GenBank, and designs specific primers and probes. After a large number of experiments and actual tests, two pairs of primers, probes and their reaction systems are screened and optimized. The results show that a dual isothermal amplification detection kit for simultaneously detecting H9 subtype AIV and IBV is prepared by optimizing the dual fluorescence RT-PCR reaction system and conditions. The kit has no cross reaction to common subtype avian influenza virus, infectious bursal virus of chickens, infectious laryngotracheitis virus of chickens, Newcastle disease virus of chickens, and normal tissues, and has strong specificity; it has high sensitivity for detecting H9 subtype AIV and IBV, and the minimum detection amount of the two viruses is 1 copy. Farms and grassroots laboratories can diagnose the two diseases in a short time, which buys time for the prevention and control of the two diseases, and can take measures in time to control the spread of the diseases within the smallest possible range. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1It is the result diagram of double RT-RAA amplification of H9 subtype AIV and IBV, among which, 1: H9 subtype AIV RT-RAA amplification curve; 2: IBV RT-RAA amplification curve; 3-15: avian influenza virus H1, H2, H3, H4, H5, H6, H7, H10, H11, H12, H13 subtype strains, chicken infectious bursal virus (IBDV) B87, chicken infectious laryngotracheitis virus (ILTV) vaccine strain, chicken Newcastle disease virus (NDV) Lasota strain, normal chicken tissue nucleic acid control;

[0027] Figure 2 The figure shows the sensitivity amplification results of the double RT-RAA test for H9 subtype AIV and chicken IBV. Among them, 1: is 10 4 Copy / reaction, 2:10 3 Copy / Reaction, 3:10 2 For copy / reaction, 4:10 1 Copy / reaction, 5:10 0 copy / react;

[0028] Figure 3 The results of the sensitivity test amplification of H9 subtype AIV and IBV double RT-RAA for IBV detection are shown in Figure 1. Among them, 1: is 10 4 Copy / reaction, 2:10 3 Copy / Reaction, 3:10 2 For copy / reaction, 4:10 1 Copy / reaction, 5:10 0 copy / react;

[0029] Figure 4 This is the detection spectrum of H9 subtype AIV and IBV dual RT-RAA against different branches of H9 virus;

[0030] Figure 5 This is the detection spectrum of H9 subtype AIV and IBV dual RT-RAA against different IBV genotypes. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below, and the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. If specific techniques or conditions are not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0032] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0033] The present invention prepares a primer and a probe for detecting H9 subtype AIV and IBV, which are as follows:

[0034] Primer H9-1 (SEQ ID NO. 1) 5'-CTTGCAGTTGGTCTRAGGAATGTKCCTT CT-3'

[0035] Primer H9-2 (SEQ ID NO. 2) 5'-GCCTTTTGRGTTGAKTCTCTRTCTGCTGC C-3'

[0036] Probe H9-P (SEQ ID NO.3)5'-GCAGGDTTTATAGARGGAGGTTGGYCAGGACTAGTTGCTGGTTGGTATGG-3'

[0037] The probe is labeled with a FAM luminescent group at the 32nd base T from the 5′ end, a base-free site of tetrahydrofuran (THF) is connected after the 32nd base, a BHQ1 quenching group is labeled at the 36th base T, and a C3-spacer blocking modification is performed at the 3′ end.

[0038] Primer IBV-1 (SEQ ID NO.4)5'-GCACCTGGCCACCTGTCAGGTTTTAGTT ATT-3'

[0039] Primer IBV-2 (SEQ ID NO. 5) 5'-TACATCCGTTGCTTGGGCTACCTAGTGC CAG-3'

[0040] Probe IBV-P (SEQ ID NO. 6) 5'-TTGTGTCTCACTTTATTACATCCGTTGCTTGGCTACCTAGTGCCAGCGT-3'

[0041] The probe is labeled with a ROX luminescent group at the 30th base T from the 5′ end, a base-free site of tetrahydrofuran (THF) is connected after the 31st base, a BHQ2 quenching group is labeled with a T at the 34th base, and a C3-spacer blocking modification is performed at the 3′ end.

[0042] The following examples are used to further illustrate the present invention, but are not intended to limit the present invention.

[0043] Example 1

[0044] In this embodiment, a dual RT-RAA kit for detecting H9 subtype AIV and IBV is prepared, which consists of an RNA reverse transcription system and an isothermal PCR reaction system in one tube, wherein the reaction system is: 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL of RNase Free dH2O, 2.5 μL of magnesium acetate, enzyme lyophilized powder (comprising recombinase, reverse transcriptase and polymerase), and 5.0 μL of template (H9 and IBV RNA).

[0045] The reaction conditions of the above kit are: constant temperature amplification at 42°C for 20 minutes.

[0046] Experimental Example 1 Specificity experiment of primer probe detection

[0047] The experimental materials selected were avian influenza virus H1, H2, H3, H4, H5, H6, H7, H9, H10, H11, H12, and H13 subtype strains, infectious bronchitis virus (IBV) H52, infectious bursal virus (IBDV) B87, infectious laryngotracheitis virus (ILTV) vaccine strain, and Newcastle disease virus (NDV) Lasota strain.

[0048] The nucleic acid DNA / RNA of the above viruses and normal chicken tissues were extracted using a commercial kit (DNA / RNA extraction kit, Xi'an Tianlong).

[0049] Prepare the reaction system: 50 μL total system, including 25 μL 2X reaction buffer, 2.0 μL each of 10 pmol / μL H9-1 and H9-2, 1.8 μL each of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL RNase Free dH2O, 2.5 μL magnesium acetate, enzyme lyophilized powder (including recombinase, reverse transcriptase and polymerase), and 5.0 μL sample RNA template to be tested. The templates are nucleic acids of avian influenza virus H1, H2, H3, H4, H5, H6, H7, H9, H10, H11, H12, and H13 subtype strains, infectious bronchitis virus (IBV) H52, infectious bursal virus (IBDV) B87, infectious laryngotracheitis virus (ILTV) vaccine strain, and Newcastle disease virus (NDV) Lasota strain.

[0050] The detection reaction conditions are: constant temperature amplification at 42°C for 20 minutes.

[0051] Test results see Figure 1 The results showed that H9 subtype AIV and IBV nucleic acids showed fluorescence amplification curves, while other common avian viruses and normal chicken tissue nucleic acid controls did not show amplification curves.

[0052] The results showed that the kit can specifically detect H9 subtype AIV and IBV and does not cross-react with other common viruses.

[0053] Experimental Example 2 Sensitivity experiment of primer probe detection

[0054] The amplified products of H9-1 and H9-2, IBV-1 and IBV-2 primers were connected to the T vector and then transcribed into cRNA in vitro. The nucleic acid concentration was determined, the copy number was calculated, and then diluted 10-fold to 2×10 5 , 2×10 4 , 2×10 3 , 2×102, 2×10 1 , 2×10 0 , 2×10 -1 copies / μL.

[0055] Prepare the reaction system: 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL of RNase Free dH2O, 2.5 μL of magnesium acetate, lyophilized enzyme powder (including recombinase, reverse transcriptase and polymerase), and take 5.0 μL of each of the above diluted nucleic acids as templates.

[0056] The detection reaction conditions are: constant temperature amplification at 42°C for 20 minutes.

[0057] Test results such as Figure 2 , 3 As shown, the results show that the primer and probe combination designed by the present invention has high sensitivity for detecting H9 subtype AIV and IBV, and the minimum detection amount of both viruses is 1 copy.

[0058] Test Example 3 Detection Spectrum

[0059] The nucleic acid DNA / RNA of the above viruses and normal chicken tissues were extracted using commercial kits, including one strain each of 10 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, and one strain each of 17 genotypes of infectious bronchitis virus 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.

[0060] Prepare the reaction system: 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL of RNase Free dH2O, 2.5 μL of magnesium acetate, lyophilized enzyme powder (including recombinase, reverse transcriptase and polymerase), and 5.0 μL of nucleic acid of each of the above-mentioned branch viruses or genotypes as templates.

[0061] The detection reaction conditions are: constant temperature amplification at 42°C for 20 minutes.

[0062] Test results see Figure 4 , 5 The results showed that the nucleic acids of all 10 branches of H9 subtype AIV and 17 genotypes of IBV showed fluorescence amplification curves.

[0063] The results showed that the kit can detect the H9 subtype AIV and common IBV genotypes that are currently prevalent in China.

[0064] Test Example 4 Clinical Testing Application

[0065] A total of 182 throat swab samples were collected from various poultry in a live poultry market in a certain province, and the swabs were placed in 1.0 mL of PBS solution (pH 7.0-7.4, 0.01 mol / L).

[0066] The nucleic acid RNA of the above samples was extracted using a commercial kit.

[0067] Prepare the reaction system: 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, 8.5 μL of RNase Free dH2O, 2.5 μL of magnesium acetate, lyophilized enzyme powder (including recombinase, reverse transcriptase and polymerase), and 5.0 μL of the nucleic acid of the above samples as templates.

[0068] The detection reaction conditions are: constant temperature amplification at 42°C for 20 minutes.

[0069] The results showed that among the 182 samples, 80 samples were positive for H9 subtype AIV nucleic acid, 76 samples were positive for IBV nucleic acid, and 32 samples were positive for H9 subtype AIV and IBV nucleic acid, indicating a mixed infection.

[0070] The results showed that the kit can quickly and accurately identify H9 subtype AIV and IBV and has good application prospects.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A primer and probe combination for detecting H9 subtype AIV and IBV, It is characterized in that The primer and probe combination capable of detecting H9 subtype AIV and IBV comprises a primer pair and a probe for detecting H9 subtype AIV, and a primer pair and a probe for detecting IBV.

2. The primer and probe composition for detecting H9 subtype AIV and IBV according to claim 1, It is characterized in that The sequences of the primers and probes include: forward primer H9-1 as shown in SEQ ID NO.1, reverse primer H9-2 as shown in SEQ ID NO.2, probe H9-P as shown in SEQ ID NO.3, forward primer IBV-1 as shown in SEQ ID NO.4, reverse primer IBV-2 as shown in SEQ ID NO.5 and probe IBV-P as shown in SEQ ID NO.

6.

3. A dual isothermal amplification detection method for H9 subtype AIV and IBV, It is characterized in that The dual isothermal amplification detection method comprises the step of using the primers and probes described in any one of claims 1-2 for detection.

4. The dual isothermal amplification detection method according to claim 3, It is characterized in that The reaction system of the dual isothermal amplification detection method is 25 μL of 2X reaction buffer, 2.0 μL of 10 pmol / μL H9-1 and H9-2, 1.8 μL of 10 pmol / μL IBV-1 and IBV-2, 0.6 μL of 10 pmol / μL H9-P, 0.8 μL of 10 pmol / μL IBV-P, RNase Free dH 2 HO 8.5μL, magnesium acetate 2.5μL, enzyme lyophilized powder, template DNA 5.0μL.

5. The dual isothermal amplification detection method according to claim 3, It is characterized in that The reaction conditions of the dual isothermal amplification detection method are isothermal amplification at 42° C. for 20 min.

6. A kit for detecting H9 subtype AIV and IBV, It is characterized in that The kit comprises the primers and probes according to any one of claims 1 to 2.

7. The kit according to claim 6, It is characterized in that The specific components of the kit include: 2X reaction buffer 25 μL, 10 pmol / μL H9-1 and H9-2 2.0 μL each, 10 pmol / μL IBV-1 and IBV-2 1.8 μL each, 10 pmol / μL H9-P 0.6 μL, 10 pmol / μL IBV-P 0.8 μL, RNase Free dH 2 O 8.5μL, magnesium acetate 2.5μL, enzyme lyophilized powder, template 5.0μL.

8. The kit according to claim 6, It is characterized in that The reaction condition of the kit is isothermal amplification at 42°C for 20 min.

9. Use of the primers and probes according to any one of claims 1 to 2 in the preparation of products for detecting H9 subtype AIV and IBV, It is characterized in that The product can be used for the detection of H9 subtype AIV and IBV at the same time.

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