Avian pathogen primer compositions, kits and uses
By combining RAA and CRISPR/Cas13a detection technologies and using specific primer combinations for single-tube isothermal amplification of nucleic acids, the problem of insufficient sensitivity and specificity in IBV detection has been solved, achieving rapid and accurate detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-02
Smart Images
Figure CN122128472A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to microbial detection technology, and more particularly to a primer composition, kit, and application of avian pathogens. Background Technology
[0002] Avian infectious bronchitis virus (IBV) belongs to the genus Gamma-coronavirus. Due to its tendency to mutate and recombine, resulting in numerous serotypes, it is one of the most important pathogens affecting poultry farming. This virus mainly causes respiratory symptoms, decreased egg production, and kidney disease in chickens. It spreads rapidly and is difficult to control, therefore, there is an urgent need to establish rapid, accurate, and field-applicable detection methods.
[0003] Currently, IBV detection technologies mainly include quantitative real-time RT-PCR, conventional reverse transcription-recombinase-mediated strand displacement amplification (RAA amplification), and CRISPR / Cas13a targeted detection. While quantitative real-time RT-PCR offers high sensitivity, it relies on precise temperature control instruments and is cumbersome to operate, making it difficult to promote at the grassroots level. Existing RAA isothermal amplification technology, while eliminating the need for thermal cyclers, lacks specificity when used alone, easily leading to false positives. Although CRISPR / Cas13a technology offers high specificity, traditional methods require separate nucleic acid amplification and cleavage reactions; opening the lid during these steps can easily cause aerosol contamination, resulting in inaccurate result interpretation.
[0004] To address the aforementioned shortcomings, detection technologies combining RAA with CRISPR / Cas13a are becoming a research hotspot. However, similar technologies used for IBV detection still suffer from issues such as reaction step separation and reliance on specific equipment or consumables. Furthermore, existing primer designs are mostly targeted at the 3'UTR or specific genotype regions, and there is currently no detection scheme that can achieve a single-tube isothermal reaction and is easy to interpret. Summary of the Invention
[0005] Objectives of the invention: The first objective is to provide a primer composition for avian pathogens targeting the highly conserved NP region of avian infectious bronchitis virus; the second objective is to provide a kit containing the primer composition for avian pathogens; and the third objective is to provide the application of the primer composition for avian pathogens and the kit in single-tube detection of avian infectious bronchitis virus.
[0006] Technical solution: The avian pathogen primer composition of the present invention consists of crRNA with the sequence shown in SEQ ID NO: 1, an upstream RT-RAA primer with the sequence shown in SEQ ID NO: 2, and a downstream RT-RAA primer with the nucleotide sequence shown in SEQ ID NO: 3.
[0007] Preferably, the molar ratio of the crRNA, the upstream primer of RT-RAA, and the downstream primer of RT-RAA is (0.5~0.9):1:1.
[0008] Preferably, the primer composition further contains a fluorescent probe with the sequence TTTTT.
[0009] Preferably, the molar ratio of the fluorescent probe to the upstream primer and the downstream primer of RT-RAA is (0.2~0.6):1.
[0010] Preferably, the fluorescent probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end; more preferably, the fluorescent group is selected from any one of 6-FAM, HEX, Cy5, Cy3, ROX, and TAMRA, and the quenching group is selected from any one of BHQ1, BHQ2, and Dabcyl.
[0011] The kit described in this invention contains the aforementioned primer composition for avian pathogens.
[0012] Preferably, the kit further contains reaction buffer, Cas13a protein, T7 RNA polymerase, NTP mixture, magnesium acetate, and reverse transcription-recombinase-mediated strand displacement amplification (RT-RAA) enzyme system.
[0013] Application of the avian pathogen primer composition or kit described in this invention in the detection of avian pathogens Preferably, the pathogen is avian infectious bronchitis virus.
[0014] Preferably, the steps of the application include: collecting and extracting RNA from poultry samples, detecting and interpreting the results using the aforementioned avian pathogen primer composition or kit, wherein the reaction temperature of the detection does not exceed 40°C and the reaction time does not exceed 20 min.
[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The primer composition for avian pathogens can be used for the detection of avian infectious bronchitis virus, with high specificity. It shows no cross-reactivity with common avian virus samples such as avian influenza virus, Newcastle disease virus, and infectious laryngotracheitis virus. At the same time, the primer composition has high sensitivity, with a detection limit as low as 4.25 × 10⁻⁶. 2 1. The sample size is 1000 copies / μL, and the repeatability and results are stable. 2. This avian pathogen primer composition is suitable for single-tube isothermal amplification detection of nucleic acids. The reaction conditions are mild and the detection time is greatly shortened, which has excellent industrial application value. Attached Figure Description
[0016] Figure 1 The graph shows the sensitivity test results of the primer composition for detecting avian infectious bronchitis virus, where A is the fluorescence amplification curve and B is the observation result graph. Figure 2The figure shows the specific experimental results of the primer composition for avian pathogen detection of avian infectious bronchitis virus, where A is the fluorescence amplification curve and B is the observation result figure. Figure 3 Figure 1 shows the experimental results of using primer sets of different avian pathogen sequences for the detection of avian infectious bronchitis virus. Figure 2 shows the fluorescence amplification curve and Figure 3 shows the observation results. Detailed Implementation
[0017] The technical solution of the present invention will be further described below.
[0018] Example 1: Preparation of primer composition for avian pathogens and construction of detection system 1. Preparation of primer compositions for avian pathogens The crRNA, RT-RAA upstream primer (F1), RT-RAA downstream primer (R1), and fluorescent probe were synthesized by General Biotechnology (Anhui) Co., Ltd., and the specific sequences are shown in Table 1. Table 1. Nucleotide sequence of primer compositions
[0019] As shown in Table 1, the 5' end of the fluorescent probe is modified with a 6-FAM fluorescent group, and the 3' end is modified with a BHQ1 quencher group.
[0020] Example 2: Sensitivity verification of avian pathogen primer composition for the detection of avian infectious bronchitis virus We commissioned General Biotechnology (Anhui) Co., Ltd. to synthesize a plasmid based on the pTC57-T vector for preparing the avian infectious bronchitis virus nucleocapsid protein gene (IBV NP) fragment. Using this DNA plasmid as a template, we then used HiScribe... ® The T7 Rapid and Efficient RNA Synthesis Kit (purchased from New England Biolabs, catalog number E2050S) was used for in vitro transcription to obtain IBV NP RNA fragments, the sequence of which is shown in SEQ ID NO: 4. The obtained IBV NP RNA was then serially diluted 10-fold to prepare a series of concentration gradient samples, with each concentration diluted to 8.5 × 10⁻⁶. 6 Copies / μL, 8.6×10 5 Copies / μL, 8.5×10 4 Copies / μL, 8.5×10 3 Copies / μL, 8.5×10 2 Copies / μL, 8.5×10 1 Copies / μL, 8.5×10 0Copies / μL, with RNase-free sterile water used as a negative control.
[0021] The following system was constructed for detection: 25 μL RT-RAA buffer, 2 μL RT-RAA upstream primer (10 μmol / L), 2 μL RT-RAA downstream primer (10 μmol / L), 1.5 μL crRNA (10 μmol / L), 2 μL Cas13a protein (Tolo Biotech Co., Ltd., catalog number 32117), 1 μL T7 RNA polymerase (Nanjing Novizan Biotechnology Co., Ltd., catalog number TR101-01), 1 μL fluorescent probe (10 μmol / L), 4 μL NTP Mixture (Sangon Biotech Co., Ltd., catalog number B600057), and 3 μL magnesium acetate (280 mM) were prepared from an RNA isothermal nucleic acid amplification kit (purchased from Wuxi Leshang Biotechnology Co., Ltd., catalog number RNA-LS01). Add μL of the aforementioned RNA sample to be tested or the lyophilized powder of the enzyme composition in the RNA isothermal nucleic acid amplification kit resuspended in RNase-free sterile water, and finally add RNase-free sterile water to a final volume of 50 μL. Gently mix the prepared reaction system, centrifuge, seal, and incubate at 39°C in a metal bath for 20 min.
[0022] Two methods were used to interpret the results: 1. Precise interpretation: The reaction tube was placed in the Bori FQD-16A real-time quantitative PCR analyzer, and the fluorescence value was read once per minute during the reaction to record the fluorescence amplification curve; 2. Rapid interpretation: After the reaction was completed, the reaction tube was placed under a blue light gel cutter to directly observe the generation of fluorescence signals.
[0023] Accurate interpretation results such as Figure 1 As shown in A, the quick interpretation result is as follows: Figure 1 As shown in B, at 8.5 × 10 6 ~8.5×10 1 Within the range of copies / μL concentration, the fluorescence signal intensity gradually weakened as the IBV NP RNA sample concentration decreased, but all gradient samples could still be collected with obvious fluorescence signals and specific amplification curves during the reaction, which were judged as positive results; when the sample concentration decreased to 8.5×10 0 When the concentration of primers was 100 μL, the fluorescence signal was not significantly different from the negative control, and no specific amplification curve was observed, indicating a negative result. Therefore, the detection limit of the avian infectious bronchitis virus-specific primer composition of this invention is 425 × 10⁻⁶. 2 Copies / reaction, with high detection sensitivity, enable accurate detection of low-load IBV samples.
[0024] Example 3: Specificity verification of avian pathogen primer composition for avian infectious bronchitis virus detection The avian influenza virus was strain H7N9, and viral RNA samples were provided by Jiangsu Vocational College of Agriculture and Forestry. Newcastle disease virus attenuated vaccine (Harbin Pharmaceutical Group vaccine, Clone30 strain) and infectious laryngotracheitis virus attenuated vaccine (Laryngbituo, K317 strain) were purchased from agricultural and forestry veterinary drug stores. Viral RNA was extracted from these attenuated vaccines using the following steps: 100 μL of vaccine solution was taken, and total RNA was extracted using a viral genomic DNA / RNA extraction kit (purchased from Tiangen Biotech Co., Ltd., catalog number DP315). After extraction, the RNA concentration and purity were measured, and the RNA concentration was adjusted to 50 ng / μL to obtain Newcastle disease virus RNA samples and infectious laryngotracheitis virus RNA samples.
[0025] The following reaction system was constructed for detection: 25 μL RT-RAA buffer, 2 μL RT-RAA upstream primer (10 μmol / L), 2 μL RT-RAA downstream primer (10 μmol / L), 1.5 μL crRNA (10 μmol / L), 2 μL Cas13a protein, 1 μL T7 RNA polymerase, 1 μL fluorescent probe (10 μmol / L), 4 μL NTP mixture, and 3 μL magnesium acetate (280 mM). Then, 5 μL of the aforementioned RNA sample to be tested, or the IBV NP RNA (50 ng / μL) obtained in Example 1, or the lyophilized enzyme composition from the RNA isothermal nucleic acid amplification kit resuspended in RNase-free sterile water was added. Finally, the volume was brought to 50 μL with RNase-free sterile water. The constructed reaction system was gently mixed, centrifuged, and sealed, and incubated in a 39°C metal bath for 20 min.
[0026] Two methods were used to interpret the results: 1. Precise interpretation: The reaction tube was placed in the Bori FQD-16A real-time quantitative PCR analyzer, and the fluorescence value was read once per minute during the reaction to record the fluorescence amplification curve; 2. Rapid interpretation: After the reaction was completed, the reaction tube was placed under a blue light gel cutter to directly observe the generation of fluorescence signals.
[0027] Accurate interpretation results such as Figure 2 As shown in A, the quick interpretation result is as follows: Figure 2As shown in B, only the avian infectious bronchitis virus (IBV) positive control sample showed positive results in both interpretation methods, that is, a clear fluorescence amplification curve was detected by the real-time fluorescence quantitative PCR analyzer, and significant green fluorescence was visible under the blue light gel cutter; no fluorescence amplification curve was detected in avian influenza virus, Newcastle disease virus, infectious laryngotracheitis virus, healthy poultry tissue samples and blank control, and no fluorescence signal was observed under the blue light gel cutter, and they were judged as negative.
[0028] Example 4: Repeatability validation of avian pathogen primer composition for avian infectious bronchitis virus detection The following system was constructed for detection: 25 μL RT-RAA buffer, 2 μL RT-RAA upstream primer (10 μmol / L), 2 μL RT-RAA downstream primer (10 μmol / L), 1.5 μL crRNA (10 μmol / L), 2 μL Cas13a protein, 1 μL T7 RNA polymerase, 1 μL fluorescent probe (10 μmol / L), 4 μL NTP mixture, 3 μL magnesium acetate (280 mM), followed by 5 μL of IBV NP RNA (8.5 × 10⁻⁶) obtained in Example 1. 3 8.5×10 4 8.5×10 5 The enzyme composition lyophilized powder from the RNA isothermal nucleic acid amplification kit (copies / μL) or RNase-free sterile water resuspended in washer-dried water was added, and the volume was brought to 50 μL with RNase-free sterile water. The prepared reaction system was gently mixed, centrifuged, and sealed, and reacted in a 39°C metal bath for 20 min. Three replicate assays were performed, with three parallel samples in each assay.
[0029] Two methods were used to interpret the results: 1. Precise interpretation: The reaction tube was placed in the Bori FQD-16A real-time quantitative PCR analyzer, and the fluorescence value was read once per minute during the reaction to record the fluorescence amplification curve; 2. Rapid interpretation: After the reaction was completed, the reaction tube was placed under a blue light gel cutter to directly observe the generation of fluorescence signals.
[0030] Table 2. Fluorescence signal measurement values
[0031] The results are shown in Table 2. All detections showed stable fluorescence signals. The detection results of the blue light gel excimer and the real-time PCR instrument were consistent, with a small coefficient of variation, which proves that the detection method has good repeatability and stable results.
[0032] Comparative Example 1: Preparation of primer sets for avian pathogens based on different sequences and detection of avian infectious bronchitis virus The upstream primer-2 (F2) and downstream primer-2 (R2) of RT-RAA were synthesized by General Biotechnology (Anhui) Co., Ltd., and their specific sequences are shown in Table 3. Table 3. Nucleotide sequence list of primers for avian pathogens with different sequences
[0033] The following system was constructed for detection: 25 μL RT-RAA buffer, 2 μL RT-RAA upstream primer (10 μmol / L), 2 μL RT-RAA downstream primer (10 μmol / L), 1.5 μL crRNA (10 μmol / L), 2 μL Cas13a protein, 1 μL T7 RNA polymerase, 1 μL fluorescent probe (10 μmol / L), 4 μL NTP mixture, 3 μL magnesium acetate (280 mM), followed by 5 μL of IBV NP RNA (8.5 × 10⁻⁶) obtained in Example 1. 1 The enzyme composition lyophilized powder from the RNA isothermal nucleic acid amplification kit (copies / μL) or RNase-free sterile water resuspended in isothermal water was added to a final volume of 50 μL. The prepared reaction system was gently mixed, centrifuged, and sealed, and then incubated in a 39°C metal bath for 20 min.
[0034] Two methods were used to interpret the results: 1. Precise interpretation: The reaction tube was placed in the Bori FQD-16A real-time quantitative PCR analyzer, and the fluorescence value was read once per minute during the reaction to record the fluorescence amplification curve; 2. Rapid interpretation: After the reaction was completed, the reaction tube was placed under a blue light gel cutter to directly observe the generation of fluorescence signals.
[0035] The results are as follows Figure 3 As shown, the detection performance based on F2 and R2 is significantly worse than that based on the avian pathogen primer combination.
Claims
1. A primer composition for avian pathogens, characterized in that, The primer composition consists of crRNA with the sequence shown in SEQ ID NO: 1, an upstream RT-RAA primer with the sequence shown in SEQ ID NO: 2, and a downstream RT-RAA primer with the nucleotide sequence shown in SEQ ID NO:
3.
2. The avian pathogen primer composition according to claim 1, characterized in that, The molar ratio of crRNA, RT-RAA upstream primer, and RT-RAA downstream primer is (0.5~0.9):1:
1.
3. The avian pathogen primer composition according to claim 1, characterized in that, The primer composition also contains a fluorescent probe with the sequence TTTTT.
4. The avian pathogen primer composition according to claim 3, characterized in that, The molar ratio of the fluorescent probe to the upstream and downstream primers of RT-RAA is (0.2~0.6):
1.
5. The avian pathogen primer composition according to claim 3, characterized in that, The fluorescent probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end.
6. A reagent kit, characterized in that, The kit contains the avian pathogen primer composition according to any one of claims 1 to 5.
7. The reagent kit according to claim 6, characterized in that, Reaction buffer, Cas13a protein, T7 RNA polymerase, NTP mixture, magnesium acetate, and reverse transcription-recombinase-mediated strand displacement nucleic acid amplification enzyme system.
8. The use of a primer composition for avian pathogens according to any one of claims 1 to 5 or a kit according to claims 6 to 7 in the detection of avian pathogens.
9. The application according to claim 8, characterized in that, The pathogen is avian infectious bronchitis virus.
10. The application according to claim 8, characterized in that, The steps of the application include: collecting and extracting RNA from poultry samples, detecting and interpreting the results using the avian pathogen primer composition according to any one of claims 1 to 5 or the kit according to claims 6 to 7, wherein the reaction temperature of the detection does not exceed 40°C and the reaction time does not exceed 20 min.