Primer-probe combinations for detecting avian group I adenovirus and their applications

Abstract

This invention discloses a primer-probe combination for detecting avian group I adenovirus and its applications. Firstly, this invention discloses a primer-probe composition for detecting avian group I adenovirus, comprising specific tailed primer pairs, universal primer pairs, and double-labeled probes, with sequences shown in SEQ ID NO. 1-6. This invention also discloses a kit containing the primer-probe composition, and a method for detecting avian group I adenovirus based on the primer-probe composition. The method of this invention is sensitive and specific, applicable not only to the risk monitoring of avian group I adenovirus in imported and exported poultry feces, whole blood, and diseased tissues, but also to the rapid screening of large batches of avian group I adenovirus in domestic poultry farming enterprises.

Description

Technical Field

[0001] This invention relates to the field of animal quarantine. More specifically, it relates to a primer-probe combination for detecting avian group I adenovirus and its application. Background Technology

[0002] Avian adenoviruses (FAdVs) belong to the genus Avian adenovirus in the family Adenoviridae. They are divided into three groups: Group I, Group II, and Group III. Group II adenoviruses are the pathogens of hemorrhagic enteritis in turkeys, while Group III adenoviruses are the pathogens of egg production reduction syndrome. Group I adenoviruses are relatively complex, currently believed to include five species (A, B, C, D, and E), which can be classified into 12 serotypes based on cross-neutralization tests and 12 genotypes based on hexazosome gene sequences. Some strains can cause inclusion body hepatitis, pericardial effusion syndrome, gizzard erosion, tenosynovitis, pancreatic necrosis in poultry, and bronchitis in quail. The severity of these conditions is exacerbated when co-infected with chicken infectious anemia virus (CIAV) or infectious bursal disease virus (IBDV). Avian group I adenovirus infection has been reported worldwide, exhibiting endemic characteristics. Infected hosts include chickens, turkeys, ducks, geese, pigeons, guinea fowl, and ostriches. It can cause harm such as decreased egg production in chickens and turkeys, slow growth of young birds, reduced feed efficiency, and increased mortality in chicks.

[0003] Avian group I adenovirus infection can be initially diagnosed through pathological dissection and histopathological examination of intranuclear inclusion bodies. Confirmation can be achieved through direct observation of virus particle morphology under electron microscopy and virus isolation and identification, but these methods are cumbersome, have long testing cycles, and low detection rates. Reported nucleic acid detection methods include conventional PCR and real-time quantitative PCR based on fluorescent dyes. These methods are generally designed to target different regions of the hexagonal gene or the 52K gene. Due to the lack of specific probes, result interpretation is often affected by non-specific amplification, making accurate result determination through electrophoresis or melting curves difficult. Because avian group I adenovirus strains are complex, there is currently no rapid, sensitive, universal real-time quantitative PCR detection method for all strains of avian group I adenovirus. Summary of the Invention

[0004] In view of the above-mentioned problems, one object of the present invention is to provide primer and probe compositions, kits and their applications for detecting avian group I adenovirus.

[0005] Another objective of this invention is to provide a method for detecting avian group I adenovirus using the above-mentioned primer-probe composition or kit. This method is sensitive and specific, and is applicable not only to the risk monitoring of avian group I adenovirus in imported and exported poultry feces, whole blood, and lesion tissues, but also to the rapid screening of avian group I adenovirus in large batches of samples from domestic poultry farming enterprises.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a primer-probe composition for detecting avian group I adenovirus, the composition comprising a specific tailed primer pair, a universal primer pair, and a dual-labeled probe;

[0008] The specific tailing primer pair has the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2:

[0009] SEQ ID NO.1: 5'-AACTTCCACGACCACATGGCKCAGATGGCYAAGG-3', (positive primer);

[0010] SEQ ID NO.2: 5'-GAGCACGGTATCCACGCGCCTGGGTCAAACCGA-3', (antisense primer);

[0011] The universal primer pair consists of the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4:

[0012] SEQ ID NO.3: 5'-TT+CCAC+GAC+CAC-3', (positive primer);

[0013] SEQ ID NO.4: 5'-CAC+GGTAT+C+CAC-3', (antisense primer);

[0014] In this case, the nucleotide following the "+" sign represents a LAN-modified nucleotide;

[0015] The dual-labeled probe has the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6:

[0016] SEQ ID NO.5: 5'-ATTCCGCAGATGACTGACGCCGAGTAC-3', (probe 1);

[0017] SEQ ID NO.6: 5'-ATCCCTCAGATGTCTGACGCGGACTAC-3', (probe 2).

[0018] Furthermore, the 5' end of the dual-labeled TaqMan probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0019] According to a specific embodiment of the present invention, the 5' end of the dual-labeled TaqMan probes shown in SEQ ID NO.5 and SEQ ID NO.6 is labeled with a fluorescent reporter group of FAM and the 3' end is labeled with a fluorescent quencher group of BHQ1.

[0020] Secondly, the present invention provides the application of the above-mentioned primer-probe composition in the preparation of reagents or kits for detecting avian group I adenovirus.

[0021] Thirdly, the present invention provides a kit for detecting avian group I adenovirus, the kit containing the above-described primer and probe composition for detecting avian group I adenovirus.

[0022] Furthermore, the kit also includes reagents required for the real-time PCR reaction, such as at least one of the following: real-time PCR reaction solution, negative control, positive control, water, etc. The real-time PCR reaction solution may include PCR buffer, MgCl2, dNTP, BSA, and Taq DNA polymerase, etc.

[0023] Furthermore, the negative control can be a chicken tissue sample, prepared as a 5% suspension using 0.01 mol / L pH 7.2 PBS buffered saline.

[0024] Furthermore, the positive control may be plasmid DNA containing the target amplification gene;

[0025] For example, a 920bp fragment of the 52K gene of avian group I adenovirus, as shown in SEQ ID NO.7, was cloned into the Sac II / Not I site of the pBluescript II SK+ vector to obtain pBSK-Fade-1-GROUP-920.

[0026] The present invention also protects the method for preparing the kit, including the step of individually packaging each primer and TaqMan probe.

[0027] Fourthly, the present invention provides a method for detecting avian group I adenovirus (TaqMan probe-based quantitative PCR detection method) comprising the following steps:

[0028] 1) Extract DNA from the sample to be tested;

[0029] 2) Using the DNA extracted in step 1) as a template, perform a real-time PCR amplification reaction using the primer and probe combination or kit described above;

[0030] 3) Result judgment.

[0031] Furthermore, in the fluorescence quantitative PCR amplification reaction, the molar ratio of the specific tailed primer pair, the universal primer pair, and the dual-labeled probe is 4:2:1.

[0032] Furthermore, the PCR amplification system was 25 μL, and the reaction conditions were: 95℃ / 3 min; 95℃ / 15 s, 58℃ / 35 s, 42 cycles; fluorescence collection was set at 58℃ during the annealing and extension phase.

[0033] The beneficial effects of this invention are as follows:

[0034] This invention uses the 52K gene of avian group I adenovirus as the target region, designs composite primers (including specific tailed primers and universal primers) and dual TaqMan probes to establish and optimize a detection method for avian group I adenovirus, achieving excellent technical results: 1) Rapid: Compared with current conventional PCR methods based on gel electrophoresis and real-time quantitative PCR methods based on dyes, this method does not require electrophoresis or melting curve determination, thus being faster. 2) More sensitive: The detection method is more sensitive than using a single pair of primers; probability regression analysis was used to detect serially diluted plasmid DNA. The results showed that the limit of detection was 4.11 copies. 3) High specificity: The primers and probes target three different regions of the target sequence, resulting in higher specificity. 4) Good versatility: The dual-probe design ensures coverage of all five species of avian group I adenovirus, but cannot detect other avian adenoviruses. Attached Figure Description

[0035] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0036] Figure 1 This demonstrates the principle of using a composite primer in conjunction with a TaqMan dual probe to detect avian group I adenovirus.

[0037] Figure 2 The design region of the specific primer probe for avian group I adenovirus detection is shown.

[0038] Figure 3 The results of fluorescence PCR detection of avian group I adenovirus on a series of diluted standard plasmid DNA are shown.

[0039] Figure 4 The standard curve for the fluorescent PCR detection method of avian group I adenovirus is shown.

[0040] Figure 5 The results of the probability regression assay for the lowest detection limit are shown.

[0041] Figure 6 The results of the specific tests are shown.

[0042] Figure 7The comparison shows the amplification results with and without universal primers. Detailed Implementation

[0043] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1: Establishment of a rapid detection method for avian group I adenovirus

[0045] I. Primer and probe composition for detecting avian group I adenovirus

[0046] This invention selects a specific sequence of the 52K gene of avian group I adenovirus as the target region. Based on multiple sequence alignment, it designs and screens composite primers (including specific tailing primers and universal primers) and TaqMan probes. The design principle is described in [link to design principles]. Figure 1 Primer and probe design target regions are shown in [link / reference]. Figure 2 The specific tailing primers are 34 nt in length, do not form stable secondary structures within the primers, and have a melting temperature (Tm) difference of less than 2 °C between the primers; the universal primers are LNA-modified short primers, 12 nt in length. The dual-labeled TaqMan probe has a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end.

[0047] The specific primer pairs and TaqMan probe nucleotide sequences are as follows:

[0048] 1) 5'-AACTTCCACGACCACATGGCKCAGATGGCYAAGG-3' (SEQ ID NO: 1);

[0049] 2) 5'-GAGCACGGTATCCACGGCGCCTGGGTCAAACCGA-3' (SEQ ID NO: 2);

[0050] 3) 5'-TT+CCAC+GAC+CAC-3' (SEQ ID NO: 3);

[0051] 4)5'-CAC+GGTAT+C+CAC-3' (SEQ ID NO: 4);

[0052] 5) 5'-[FAM]-ATTCCGCAGATGACTGACGCCGAGTAC-[BHQ1]-3' (SEQ ID NO: 5);

[0053] 6) 5'-[FAM]-ATCCCTCAGATGTCTGACGCGGACTAC-[BHQ1]-3' (SEQ ID NO: 6).

[0054] SEQ ID NO:1 and SEQ ID NO:2 are the specific tailed positive and antisense primers for detecting avian group I adenovirus, respectively; SEQ ID NO:3 and SEQ ID NO:4 are the universal positive and antisense primers for detecting avian group I adenovirus, respectively; SEQ ID NO:5 and SEQ ID NO:6 are TaqMan double-labeled probes for detecting avian group I adenovirus, with the 5' end labeled with the reporter fluorescent group FAM and the 3' end labeled with the quencher group BHQ1, and the nucleotides after the "+" represent LAN-modified nucleotides.

[0055] II. Methods for detecting avian group I adenovirus

[0056] A detection method for avian group I adenovirus was established using TaqMan probe-based quantitative real-time PCR technology as follows: DNA was extracted from the sample to be tested; using the extracted DNA as a template, quantitative real-time PCR amplification was performed using the primer and probe combination described above; and the results were interpreted. Specifically, this includes:

[0057] 1. Sample pretreatment and DNA extraction

[0058] It can be used for nucleic acid detection in avian cloacal swabs, EDTA-anticoagulated whole blood, and diseased tissues.

[0059] When collecting a cloacal swab, insert the swab deep into the cloaca, rotate it once, and collect a small amount of feces. Place the swab into a 1.5 mL centrifuge tube containing 1.0 mL of PBS. After thoroughly mixing on a shaker, collect the supernatant for DNA extraction. EDTA-anticoagulated whole blood can be directly aspirated for DNA extraction. For pathological tissue, after thorough grinding, prepare a 20% tissue suspension with PBS, centrifuge at 3000 rpm for 5 min, and collect the supernatant for DNA extraction.

[0060] Effective nucleic acid (DNA) extraction methods can be used, as well as various commercially available viral DNA extraction kits or fully automated nucleic acid extractors and matching reagents. The extracted DNA must be tested as soon as possible or stored at -20°C or below for later use.

[0061] 2. Using the DNA from the sample to be tested in step 1 as a template, perform real-time PCR amplification to obtain the amplification curve.

[0062] The reaction system for the real-time PCR amplification reaction is shown in Table 1.

[0063] Table 1 Formulation of the reaction system

[0064] Components Final concentration 5× buffer solution 1× buffer solution <![CDATA[25mmol / L MgCl2]]> 4.5 mmol / L 2.5 mmol / L dNTP 0.4 mmol / L Specific tailed positive primer 0.8 μmol / L Specific tailed antisense primers 0.8 μmol / L Universal positive primer 0.4 μmol / L Universal antisense primer 0.4 μmol / L TaqMan Probe 1 (FAM Marker) 0.2 μmol / L TaqMan Probe 2 (FAM Marker) 0.2 μmol / L Taq DNA polymerase (5 U / μL) 0.25μL DNA template 5μL BSA 0.15％ Add DEPC water to the total volume 25μL

[0065] Among them, dNTP was purchased from TaKaRa, primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd., and the composition of 5×PCR buffer was: 400 mmol / L KCl, 250 mmol / L Tris-HCl (pH 9.0, 25°C), 15 mmol / L MgCl2, 50 mmol / L DTT (pH 8.9, 25°C); it was purchased from Beijing Xikai Innovation Technology Co., Ltd.

[0066] Taq DNA polymerase at 5 U / μL was purchased from TaKaRa.

[0067] BSA was purchased from Beijing Xikai Innovation Technology Co., Ltd.

[0068] DEPC water: After double-distilling pure water, add DEPC to a final concentration of 0.1%, stir at 37°C for 12 hr, and autoclave at 15 lbf / in2 (1.034×10

[0074] ,

[0073] ,

[0072] , , Pa) for 15 minutes.

[0069] The negative control was a chicken tissue sample, made into a 5% suspension with 0.01 mol / L pH 7.2 PBS buffer saline.

[0070] The positive control was pBSK-Fade-1-GROUP-920 obtained by cloning the 920-bp fragment of the avian group I adenovirus 52K gene shown in SEQ ID NO.7 into the Sac II / Not I site of the pBluescript II SK+ vector. [[ID=​​​​​​​​​Example 2: Sensitivity, specificity, and repeatability tests of the detection method and the detection of submitted samples.

[0075] 1. Establishment of standard curve and evaluation of method amplification efficiency

[0076] The plasmid pBSK-Fade-1-GROUP-920, containing the 52K gene fragment of avian group I adenovirus, was serially diluted 10-fold starting at 0.004 ng / μL. Fluorescent PCR reaction systems were prepared according to the formulations in Table 1 for detection. Results showed that detection was possible for DNA samples ranging from 20 pg to 0.002 pg. The amplification curves are shown in the figure below. Figure 3 The standard curve is shown below. Figure 4 The results showed that the slope of the standard curve was -3.39, the intercept was 40.92, and R0 was [value missing]. 2 The value was 0.999, and the amplification efficiency was 97.10%, indicating that the established system had a high amplification efficiency.

[0077] 2. Determination of the minimum detection limit

[0078] To further determine the detection limit of the method, repeated detections were performed on plasmid DNA containing the target gene sequence in the range of 0.5–20 copies, with 8 tubes per gradient. Probability regression was used to determine the analytical sensitivity within the 95% confidence interval. The results showed that, at a 95% probability, the detection limit of the established method was 4.11 copies, and the upper and lower confidence limits were 3.16 and 6.90 copies, respectively. (See attached table). Figure 5 .

[0079] 3. Specificity test

[0080] The results of the specificity test on the avian pathogen nucleic acid (see Table 2) preserved in our laboratory using the method established in Example 1 of this invention showed that the established method had no cross-reactivity with other avian pathogen nucleic acids and was highly specific.

[0081] Table 2. Viral nucleic acids used in specificity testing studies.

[0082]

[0083]

[0084] 4. Repeatability test

[0085] Repeatability tests were performed on pBSK-Fade-1-GROUP-920 plasmid DNA at concentrations of 0.4 fg / μL, 4 fg / μL, and 40 fg / μL, and the results are shown in Table 3.

[0086] Table 3 Results of Repeatability Tests

[0087]

[0088] The data in the table above show that the established quantitative real-time PCR detection method has good repeatability and stability.

[0089] 5. Testing of collected and submitted samples

[0090] The method established in this invention and the SYBR Green I-based quantitative PCR method were used to detect and compare 286 laboratory-collected samples, verifying the applicability of the method in actual samples. The results are shown in Table 4. In 6 organ samples from a chicken farm, 5 were positive using both methods, indicating that the established method can be applied to the detection of submitted samples.

[0091] Table 4. Results of avian group I adenovirus nucleic acid detection in collected samples.

[0092]

[0093] Example 3: Effectiveness test of universal primers in avian group I adenovirus real-time quantitative PCR system

[0094] According to the reaction system ratios in Table 1, systems containing universal primers and systems without universal primers were prepared respectively. The plasmid DNA of 40 pg / μL pBSK-Fade-1-GROUP-920 was detected in 3 replicates in each system, and the Ct values ​​were statistically analyzed.

[0095] The plasmid DNA of pBSK-Fade-1-GROUP-920 at a concentration of 40 pg / μL was detected, and the results are shown in the figure. Figure 7 It can be seen that, under the same amplification conditions, the amplification effect of the system containing universal primers is significantly better than that of the system without universal primers.

[0096] Statistical analysis was performed on the Ct values ​​of amplification results with and without universal primers. The results are shown in Table 5. The t-test results showed that the difference between the two was significant.

[0097] Table 5. Statistical analysis of amplification results for systems with and without universal primers.

[0098]

[0099] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A primer probe composition for detecting avian group I adenovirus, characterized by, The composition comprises a specific tailing primer pair, a universal primer pair and a dual-labeled probe; The specific tailing primer pair is the nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2: SEQ ID NO. 1: 5'-AACTTCCACGACCACATGGCKCAGATGGCYAAGG-3'; SEQ ID NO. 2: 5'-GAGCACGGTATCCACGCGCCTGGGTCAAACCGA-3'; The universal primer pair is the nucleotide sequence shown in SEQ ID NO. 3 and SEQ ID NO. 4: SEQ ID NO. 3: 5'-TT+CCAC+GAC+CAC-3'; SEQ ID NO. 4: 5'-CAC+GGTAT+C+CAC-3'; The nucleotide after "+" represents a LAN modified nucleotide; The dual-labeled probe is the nucleotide sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6: SEQ ID NO. 5: 5'-ATTCCGCAGATGACTGACGCCGAGTAC-3'; SEQ ID NO. 6: 5'-ATCCCTCAGATGTCTGACGCGGACTAC-3'; The 5' end of the dual-labeled probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

2. The primer probe composition of claim 1, wherein, The fluorescent reporter group labeled at the 5' end of the dual-labeled probe shown in SEQ ID NO. 5 and SEQ ID NO. 6 is FAM, and the fluorescent quencher group labeled at the 3' end is BHQ1.

3. Use of the primer probe composition of any one of claims 1-2 in the preparation of a reagent or kit for detecting avian group I adenovirus.

4. A kit for detecting avian group I adenovirus, characterized in that, The kit contains the primer probe composition of any one of claims 1-2.

5. The kit of claim 4, wherein The kit further comprises reagents required for fluorescent quantitative PCR reaction.

6. The kit of claim 4, wherein The kit further comprises a positive control.

7. The kit of claim 6, wherein The positive control is a plasmid DNA containing the 52K gene fragment of avian group I adenovirus shown in SEQ ID NO.

7.

8. A method of detecting avian group I adenovirus for non-diagnostic purposes characterised in that, The method comprises the following steps: 1) Extracting DNA from the sample to be tested; 2) Using the primer probe composition of any one of claims 1-2 or the kit of any one of claims 4-7 to perform a fluorescent quantitative PCR amplification reaction using the DNA extracted in step 1) as a template; 3) Result judgment.

9. The method of claim 8, wherein, In the fluorescent quantitative PCR amplification reaction, the molar ratio of the specific tailing primer pair, the universal primer pair and the dual-labeled probe is 4:2:1.

Images