Detection method for detecting infectious bronchitis virus

Abstract

The invention discloses a primer pair and a fluorescent probe for detecting an infectious bronchitis virus as well as application and a detection method of the primer pair and the fluorescent probe, and relates to the technical field of biological virus detection. Wherein the nucleotide sequence of a forward primer in the primer pair is as shown in SEQ ID No.1, the nucleotide sequence of a reverse primer is as shown in SEQ ID No.2, and the nucleotide sequence of the fluorescent probe is as shown in SEQ ID No.3; the 5'end of the fluorescent probe is modified by FAM, and the 3 'end of the fluorescent probe is modified by TAMRA. By implementing the kit, IBV of various genotypes can be accurately detected, and a good basis is provided for early diagnosis of IBV.

Description

[0001] This invention is a divisional application. The name of the original application is "Primer pairs for detecting infectious bronchitis virus of avian chickens, their applications, and detection methods"; the application number is 202211384708.2, and the application date is 20221107. Technical Field

[0002] The present invention relates to the technical field of biological virus detection, in particular to a primer pair for detecting avian infectious bronchitis virus, an application thereof and a detection method thereof. Background Art

[0003] Infectious bronchitis (IB) is a highly contagious disease caused by the infectious bronchitis virus (IBV) that severely harms the poultry industry. IBV can cause chick mortality, kidney disease, and permanent oviduct degeneration, leading to reduced feed conversion rates and decreased egg production and quality in laying hens, severely impacting the poultry industry. IBV is prone to mutation and has numerous genotypes. Therefore, existing detection methods often struggle to effectively detect all genotypes, especially newly emerging ones. For example, existing literature reports (Fraga et al., 2016; Marandino et al., 2016; Laconi et al., 2020; Okino et al., 2018) suggest that IBV detection is primarily based on single- or multiplex fluorescence quantitative assays, lacking a universal detection method.

[0004] In addition, in addition to single infection, IBV is often mixed with bacteria and viruses. Among the mixed viral infections, mixed infection with avian influenza virus (AIV) and / or Newcastle disease virus (NDV) is the main case, which places high demands on the detection specificity of IBV. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a primer pair and a fluorescent probe for avian infectious bronchitis virus, which can realize the detection of avian infectious bronchitis virus of multiple genotypes and distinguish it from other common viruses (AIV, NDV, etc.).

[0006] The technical problem that the present invention also aims to solve is to provide the use of the above primer pair and fluorescent probe for avian infectious bronchitis virus in a kit for detecting IBV.

[0007] Another technical problem to be solved by the present invention is to provide a test kit.

[0008] The technical problem that the present invention also aims to solve is to provide a method for detecting avian infectious bronchitis virus.

[0009] In order to solve the above technical problems, the present invention provides a primer pair and a fluorescent probe for detecting avian infectious bronchitis virus, characterized in that the nucleotide sequence of the forward primer is shown in SEQ ID No. 1, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID No. 3;

[0010] The 5' end of the fluorescent probe is modified with FAM, and the 3' end of the fluorescent probe is modified with TAMRA.

[0011] Correspondingly, the present invention also discloses the use of the above-mentioned primer pair and fluorescent probe for detecting avian infectious bronchitis virus in (1) or (2):

[0012] (1) Detection of avian infectious bronchitis virus;

[0013] (2) A kit for detecting avian infectious bronchitis virus.

[0014] Correspondingly, the present invention also discloses a kit for detecting avian infectious bronchitis virus, which comprises the above-mentioned primer pair and fluorescent probe.

[0015] As an improvement of the above technical solution, it also includes PCR reaction solution, standard negative control substance and standard positive control substance;

[0016] Wherein, the PCR reaction solution includes amplification buffer, dNTP, DNA polymerase and water;

[0017] The standard negative control substance is water;

[0018] The standard positive control substance is a standard positive plasmid, and the standard positive plasmid is a pIBVcDNA-5E plasmid containing an avian infectious bronchitis virus sequence.

[0019] Correspondingly, the present invention also discloses a method for detecting avian infectious bronchitis virus, which comprises the following steps:

[0020] Extract the sample to be tested and prepare the nucleic acid template through reverse transcription;

[0021] Amplifying the nucleic acid template using the above primer pair and fluorescent probe, separating the amplified product, and reading the fluorescence value;

[0022] The sample to be tested is specifically identified according to the obtained fluorescence value.

[0023] As an improvement to the above technical solution, the step of performing specific identification on the sample to be tested according to the obtained Ct value includes:

[0024] If the Ct value is less than 38 and there is a complete amplification curve, it means that the sample to be tested contains avian infectious bronchitis virus;

[0025] If the Ct value is greater than 38, it indicates that the sample to be tested is suspected to contain avian infectious bronchitis virus;

[0026] If the Ct value is Undetermined, it means that the sample to be tested does not contain avian infectious bronchitis virus or contains other sources of infection.

[0027] As an improvement of the above technical solution, the other infection sources include avian influenza virus and / or Newcastle disease virus.

[0028] As an improvement of the above technical solution, the infectious bronchitis virus is H120 infectious bronchitis virus, M41 infectious bronchitis virus, QX infectious bronchitis virus, and / or Beaudette infectious bronchitis virus.

[0029] As an improvement of the above technical solution, the amplification reaction system includes: 2×AceQ Universal U+ProbeMaster Mix V210μL, forward primer 0.4μL, reverse primer 0.4μL, fluorescent probe 0.2μL, nucleic acid template 4μL, and water 5μL.

[0030] As an improvement of the above technical solution, the amplification reaction procedure includes: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and 45 cycles.

[0031] The implementation of the present invention has the following beneficial effects:

[0032] The primer pairs and fluorescent probes designed in the present invention can accurately detect various genotypes of IBV, providing a good foundation for IBV detection. The detection method of the present invention has the characteristics of strong specificity and high sensitivity, providing a solid foundation for the early detection of IBV virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a structural map of the pBR-322-r5E-KC plasmid;

[0034] Figure 2 2 is a graph showing the test results of various samples in Example 2;

[0035] Figure 3 This is a diagram showing the detection results of the pIBVcDNA-5E plasmid in Example 3;

[0036] Figure 4 This is a sensitivity test result diagram in Example 3;

[0037] Figure 5 It is a standard curve diagram obtained from the sensitivity experiment in Example 3. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0039] Traditional PCR detection methods for IBV are difficult to detect across multiple genotypes of IBV and difficult to accurately detect in the early stages of infection, when the viral load is low. To this end, the present invention provides a primer pair and fluorescent probe for detecting infectious bronchitis virus (IBDV). The nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID No. 1, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID No. 3. The 5' end of the fluorescent probe is modified with FAM, and the 3' end of the fluorescent probe is modified with TAMRA. The RT-PCR detection method based on this primer combination and fluorescent probe can detect multiple genotypes of IBV in the early stages of infection.

[0040] The present invention also provides the use of the above primer pair and fluorescent probe in any of the following:

[0041] (1) Detection of avian infectious bronchitis virus;

[0042] (2) A kit for detecting avian infectious bronchitis virus.

[0043] Accordingly, the present invention also provides a kit for detecting avian infectious bronchitis virus, which includes the above-mentioned primer pair, a fluorescent probe, and a PCR reaction solution. Furthermore, in order to improve the accuracy of the detection, the kit also includes a standard positive control and a standard negative control. The PCR reaction solution includes an amplification buffer, dNTPs, DNA polymerase, and water. In one embodiment of the present invention, the PCR reaction solution can be selected from 2×AceQ UniversalU+Probe MasterMix, but is not limited thereto.

[0044] Accordingly, the present invention also provides a method for detecting avian infectious bronchitis virus, which comprises the following steps:

[0045] S1: Extract the sample to be tested and prepare it as a nucleic acid template;

[0046] Specifically, in one embodiment of the present invention, total RNA is extracted from the sample to be tested, reverse transcribed into cDNA, and diluted 100-fold to serve as a nucleic acid template. Total RNA extraction can be performed using the TRZOL hand-held method or kit method commonly used in the art, but is not limited thereto. Preferably, total RNA is extracted using an RNA extraction kit from Axygen.

[0047] The samples to be tested include, but are not limited to, trachea, lung, kidney, pharyngeal swab, and allantoic fluid. Specifically, when the sample to be tested is trachea, lung, or kidney, the tissue is minced and further processed in a mortar. An appropriate amount of PBS is added and mixed before extraction. When the sample to be tested is a pharyngeal swab or allantoic fluid, extraction can be performed directly after centrifugation.

[0048] S2: amplifying the nucleic acid template using the above primer pair and fluorescent probe, separating the amplified product, and reading the fluorescence value;

[0049] The amplification reaction system includes an amplification buffer, dNTPs, a DNA polymerase, a nucleic acid template, and water. Preferably, in one embodiment of the present invention, the amplification reaction system includes: 10 μL of 2×AceQ Universal U+ProbeMasterMix V2, 0.4 μL of forward primer, 0.4 μL of reverse primer, 0.2 μL of fluorescent probe, 4 μL of nucleic acid template (after 100-fold dilution), and 5 μL of water.

[0050] The amplification reaction procedure includes: pre-denaturation at 95° C. for 5 min, denaturation at 95° C. for 10 s, annealing at 60° C. for 30 s, and 45 cycles. S3: performing specific identification on the sample to be tested based on the obtained separation product.

[0051] If the Ct value is less than 38, it means that the sample contains avian infectious bronchitis virus;

[0052] If the Ct value is Undetermined, it means that the sample to be tested does not contain avian infectious bronchitis virus or contains other sources of infection.

[0053] If the Ct value is greater than 38, it indicates that the sample to be tested is suspected of containing avian infectious bronchitis virus and needs to be retested; if the Ct value after retest is ≤38 and there is an amplification curve, it indicates that the sample to be tested contains avian infectious bronchitis virus; if the Ct value after retest is greater than 38 and there is no obvious amplification curve, or the Ct value is Undertermined, it indicates that the sample to be tested does not contain avian infectious bronchitis virus or contains other sources of infection.

[0054] The present invention will be further described below with specific embodiments:

[0055] Example 1 Establishment of detection method

[0056] 1. Preparation of test samples and extraction of nucleic acid templates

[0057] (1) Collection and pretreatment of the sample to be tested: The sample to be tested can be trachea, lung, kidney, throat swab, or allantoic fluid. When the sample to be tested is trachea, lung, or kidney, mince the tissue, further process it in a mortar, add an appropriate amount of PBS, mix well, and then extract. When the sample to be tested is a throat swab or allantoic fluid, directly centrifuge and extract.

[0058] (2) Extraction of nucleic acid template: Take the pretreated tissue sample to be tested and use TRZOL or Axygen RNA to extract total RNA. Use Axygen's viral total RNA / DNA extraction kit to extract viral nucleic acid from the allantoic fluid sample for later use.

[0059] 2. Testing

[0060] (1) Sequence analysis and primer design

[0061] SnapGene software was used to design IBV universal detection primers and probes, as shown in the following table:

[0062]

[0063] According to the above results, primers and fluorescent probes were synthesized by Invitrogen.

[0064] (2) Construction of standard positive plasmid

[0065] The primers with nucleotide sequences as shown in SEQ ID No. 1-2 were used to amplify the 5E fragment using the Beaudette-p65 viral nucleic acid reverse transcription product as a template, and then ligated with the pGEM-T easy plasmid vector to obtain the standard positive plasmid (pIBVcDNA-5E plasmid). Figure 1 shown.

[0066] (3) RT-PCR amplification

[0067] The specific reaction system is shown in the following table:

[0068]

[0069] Mix the reaction system evenly and then perform amplification on a PCR instrument. The specific amplification procedure is as follows:

[0070] (4) Analysis and judgment:

[0071] The determination is made based on the band fragments and fluorescence values ​​(Ct values) of the amplified products, as follows:

[0072] If the Ct value is less than 38, it means that the sample contains avian infectious bronchitis virus;

[0073] If the Ct value is >38, the sample is suspected of containing IBDV and retesting is recommended. If the Ct value after retest is ≤38 and an amplification curve is present, the sample contains IBDV. If the Ct value after retest is >38 and there is no obvious amplification curve, or if the Ct value is "Undertermined," the sample does not contain IBDV or may contain other sources of infection.

[0074] If the Ct value is Undetermined, it means that the sample to be tested does not contain avian infectious bronchitis virus or contains other sources of infection.

[0075] Example 2 Testing of Several Samples

[0076] IBV strains (H120, M41, Beaudette, and three QX strains), H9 and H5 subtype avian influenza A viruses (AIV), and NDV strains were collected from the laboratory. Viral nucleic acid was extracted using an Axygen Total RNA / DNA Extraction Kit and transcribed into cDNA using Tiangen Biotechnology's RNA Reverse Transcription Kit (KP118-02). Using the cDNA as the nucleic acid template, the method established in Example 1 was used for testing.

[0077] Among them, the IBV strain originated from the clinical isolation strain of the testing center of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.

[0078] The AIV strain originated from the clinical isolation strain of the testing center of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.

[0079] The NDV strain was derived from the clinical isolation strain of the testing center of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.

[0080] Specific results such as Figure 2 The results showed that this assay can simultaneously detect the common QX-like and Mass genotypes of IBV and has no cross-reactivity with other viruses, such as AIV and NDV. This indicates that the assay is highly specific.

[0081] Example 3 Methodology Verification

[0082] (1) Sensitivity test

[0083] The pIBVcDNA-5E (pGEM-T easy) positive plasmid was transformed into TOP10 competent cells and amplified in Amp+ resistant LB liquid medium at 37°C. After 16 hours, the plasmid was extracted with a concentration of 538.2 ng / μL and a copy number of 5.0×1010. The plasmid was identified by BsaI digestion, and three bands of 6744 bp, 1617 bp, and 1421 bp were obtained ( Figure 3 ), correct.

[0084] The concentration of the extracted and correctly identified plasmid pIBVcDNA-5E (pGEM-T easy) was determined. The positive standard plasmid was diluted 10-fold with RNase-free water to 1010-101 copies / μL and used as a nucleic acid template. The detection was performed according to the method established in Example 1. The determination was repeated 3 times for each concentration. The detection limit of the detection method was 10.

[0085] After the test, the standard curve of the established RT-qPCR detection method was drawn with the common logarithm of the plasmid copy number LgC (copy number) as the horizontal axis and the Ct value as the vertical axis. The fitting curve was y = -3.3208x + 40.263 (R 2 = 0.9983), and the detection limit was < 4000 copies / mL ( Figure 4 、 Figure 5 ), with ideal sensitivity, and can be used as a follow-up test application and has the potential to be developed into an IBV nucleic acid detection kit.

[0086] (2) Accuracy test

[0087] The positive standard plasmid was diluted with RNase-free water to Log10(Copies) values ​​of 2.60206, 3.60206, 4.60206, 5.60206, 6.60206, 7.60206, 8.60206, and 9.60206, respectively. Each concentration was repeated three times, and three consecutive experiments were performed at different times. The inter-assay and intra-assay coefficients of variation were calculated.

[0088] Coefficient of variation (%) = [mean value of SD / (1.414×mean binding rate)]×100%, intra-assay and inter-assay precision analysis were performed, and SD is standard deviation.

[0089] The specific test results are shown in the table below. As can be seen from the table, the average intra-assay and inter-assay coefficients of variation of RT-qPCR were 0.79% and 3.99%, respectively, both less than 4%, indicating that the method is highly precise.

[0090]

[0091]

[0092] The above description is only a preferred embodiment of the present invention and cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for detecting avian infectious bronchitis virus, characterized in that: The following steps are involved: Extract the sample to be tested and prepare the nucleic acid template through reverse transcription; Amplifying the nucleic acid template using the primer pair and fluorescent probe according to claim 1, separating the amplified product, and reading the fluorescence value; The sample to be tested is specifically identified according to the obtained fluorescence value: If the Ct value is less than 38 and there is a complete amplification curve, it means that the sample to be tested contains avian infectious bronchitis virus; If the Ct value is greater than 38, it indicates that the sample to be tested is suspected to contain avian infectious bronchitis virus; If the Ct value is Undetermined, it means that the sample to be tested does not contain avian infectious bronchitis virus or contains other sources of infection.

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