Primer probe combination, kit and method for detecting bearded dragon adenovirus type 1 based on real-time fluorescence quantitative PCR technology

Abstract

This invention provides a primer-probe combination, kit, and method for detecting benign adenovirus type 1 (BAA1) based on real-time quantitative PCR (qPCR), belonging to the field of biodetection technology. The primer-probe combination includes a forward primer, a reverse primer, and a probe, with nucleotide sequences shown in SEQ ID NO: 1-3. The primer-probe combination provided by this invention can specifically detect BAA1 in samples, featuring short detection time, high specificity, good repeatability, and high sensitivity. It fills the gap in the field of PCR detection of BAA1 both domestically and internationally, enabling rapid and accurate detection of BAA1.

Description

Technical Field

[0001] This invention pertains to the field of biological detection, and in particular relates to a primer and probe combination, kit, and method for detecting adenovirus type 1 of trichomoniasis based on real-time quantitative PCR technology. Background Technology

[0002] Bearded dragon adenovirus 1 (BDAdV-1), also known as Agamidadenovirus, belongs to the family Adenoviridae and the genus Atadenovirus. It can infect various bearded dragons, such as the Eastern Bearded Dragon, Central Bearded Dragon, Western Bearded Dragon, Nurabo Bearded Dragon, and Dresdale River Bearded Dragon. Adenoviruses are often referred to as the "AIDS of reptiles" because they are highly contagious DNA viruses. Bearded dragons infected with Bearded Dragon adenovirus 1 will exhibit symptoms such as loss of appetite, lethargy, weight loss, difficulty moving, diarrhea, and swelling of the jaw or abdomen. Furthermore, because the virus's effects on the nervous system can cause body convulsions or neck bending and gazing upwards, Bearded Dragon adenovirus 1 infection is also known as "wasting disease" or "stargazing disease." Younger bearded dragons are more susceptible to infection and disease, and most severely affected individuals will not live beyond three months.

[0003] Bearded lizards infected with adenovirus type 1 (AV1) are difficult to detect because: besides the potential for varying individual responses to adenovirus, the symptoms of AV1 infection are highly similar to many other diseases, making it easily confused with other illnesses. Furthermore, some AV1 carriers may be asymptomatic, further complicating detection. AV1 is highly contagious, spreading through direct, indirect, and vertical transmission among lizards. Even asymptomatic carriers can infect other lizards. There is no specific treatment for AV1 infection; only symptom management is possible. Therefore, prevention and timely detection of AV1 are crucial for the health of individual lizards and the aquaculture industry.

[0004] With the development of molecular biology techniques, PCR technology has been widely applied in the rapid detection of pathogens. Compared with conventional detection techniques, this method not only shortens the detection time but also saves manpower and resources, providing a reliable basis for the rapid diagnosis of pathogens. Real-time quantitative PCR is a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction. However, in the current technology, there are no products available for detecting adenovirus type 1 (ADV1) using real-time fluorescence PCR.

[0005] Therefore, there is an urgent need in this field for a detection reagent and method for beard adenovirus type 1 that can quickly, efficiently and accurately detect beard adenovirus type 1, and has the characteristics of short detection time, high specificity, good repeatability and high sensitivity. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a primer-probe combination for detecting adenovirus type 1 (ADV1) based on real-time quantitative PCR technology. The primer-probe combination comprises a forward primer, a reverse primer, and a probe, and the nucleotide sequences of the forward primer, reverse primer, and probe are as follows:

[0007] Forward primer: 5'-AAGGCCTCCGGCAGGCTT-3', as shown in SEQ ID NO:1;

[0008] Reverse primer: 5'-GACGTAGTCTCTGCACAGGCAT-3', as shown in SEQ ID NO:2;

[0009] Probe: 5'-AGTCAAAATCTTGGTCGGCAAC-3', as shown in SEQ ID NO:3, wherein the 5' end of the probe is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group.

[0010] Specifically, the fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED, or LCRED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3, or MGB.

[0011] Preferably, the fluorescent reporter group is FAM and the fluorescent quencher group is MGB.

[0012] In a second aspect, the present invention provides the application of the aforementioned primer-probe combination for detecting adenovirus type 1 based on real-time quantitative PCR technology in the preparation of a kit for detecting adenovirus type 1.

[0013] A third aspect of the present invention provides a kit for detecting adenovirus type 1 based on real-time quantitative PCR technology, the kit comprising PCR amplification reagents, a negative control and a positive control, wherein the PCR amplification reagents comprise the aforementioned primer-probe combination for detecting adenovirus type 1 based on real-time quantitative PCR technology.

[0014] Specifically, the PCR amplification reagents also include DNA polymerase, Mg + dNTPs, freeze-drying protectants.

[0015] Specifically, in the primer-probe combination, the concentrations of the forward primer, reverse primer, and probe are all 5-10 μM.

[0016] Specifically, the positive control is a positive sample of bearded adenovirus type 1, and the negative control is ultrapure water.

[0017] A fourth aspect of the present invention provides a method for detecting benign adenovirus type 1 (BV1) using real-time quantitative PCR for non-diagnostic purposes, comprising the steps of:

[0018] 1) Extract total DNA from the sample to be tested;

[0019] 2) Prepare the reaction system, wherein the reaction system includes the aforementioned PCR amplification reagents;

[0020] 3) Using the extracted total DNA from the sample to be tested as a template, and adding negative and positive controls as quality control templates, a real-time quantitative PCR reaction was performed;

[0021] 4) After the reaction is complete, determine the test result based on the Ct value.

[0022] Specifically, the procedure for the real-time quantitative PCR reaction is as follows:

[0023] Step 1: 94℃, 5 minutes;

[0024] Step2: 94℃, 30s, 60℃, 30s;

[0025] Step 2 executes 40 loops.

[0026] Beneficial effects:

[0027] The primer-probe combination, kit, and detection method provided in this application for real-time quantitative PCR detection can rapidly and accurately detect the adenovirus type 1 gene in the sample to be tested. It has no cross-reactivity with other pathogens, can effectively distinguish other adenovirus types from adenovirus type 1, and has high sensitivity, good specificity, and high practicality. The detection limit can reach 1000 copies / mL. It can perform specific detection in the early stage of pathogen infection and is well applied to the timely detection and prevention of adenovirus type 1. Attached Figure Description

[0028] Figure 1 This is a graph showing the results of plasmid linearity detection in Example 1.

[0029] Figure 2 This is a graph showing the results of testing a positive sample in Example 2. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Based on the nucleotide sequence accessed in GenBank, BDAdV-1 (accession number NC_076486.1) was selected for alignment analysis using bioinformatics software. Conserved regions were chosen as the target sequence for amplification, and a genovirus type 1 plasmid was synthesized. Multiple primer and probe pairs were designed using Primer Premier 5.0 and Oligo 7 software to amplify the plasmid, and experimental screening was performed. Finally, one primer pair and one probe were determined, including a forward primer, a reverse primer, and a probe. The nucleotide sequences are as follows:

[0033] Forward primer (BDAdV-1 F): 5'-AAGGCCTCCGGCAGGCTT-3', as shown in SEQ ID NO:1;

[0034] Reverse primer (BDAdV-1 R): 5'-GACGTAGTCTCTGCACAGGCAT-3', as shown in SEQ ID NO:2;

[0035] Probe (BDAdV-1 PF): 5'-AGTCAAAATCTTGGTCGGCAAC-3', as shown in SEQ ID NO:3.

[0036] The probe is labeled with a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group MGB at its 3' end. The amplification length of the forward primer, reverse primer, and probe is 168 bp.

[0037] This embodiment demonstrates a primer-probe combination and method for detecting adenovirus type 1 (ADV1) based on real-time quantitative PCR technology. Based on this method, plasmids are used to evaluate its analytical performance. The specific details are as follows:

[0038] 1) Linearity test

[0039] Table 1 PCR amplification system

[0040] Components concentration 1T dosage <![CDATA[ddH2O]]> / 7.5μL Buffer Mix 4× 5μL BDAdV-1 F 10μM 0.5μL BDAdV-1 R 10μM 0.5μL BDAdV-1 PF 10μM 0.5μL

[0041] The synthesized bearded adenovirus type 1 plasmid was diluted to a concentration of 1×10⁻⁶ after being fixed. 3 Copies / mL and 1×10 7 Copies / mL. Prepare PCR amplification reagents according to Table 1, and add 5 μL of each concentration of plasmid per T for amplification.

[0042] Table 2 Linearity Test Results

[0043]

[0044] Test results as follows Figure 1 As shown in Table 2, the primer-probe combination and detection method provided in this application have an amplification efficiency of 103%, and the amplification rate is between 90% and 110%, indicating good amplification.

[0045] 2) Specificity test

[0046] Bearded dragon adenovirus type 1 belongs to the family Adenoviridae, genus Atadenovirus. This genus includes bovine adenovirus, snake adenovirus, parrot adenovirus, and lizard adenovirus type 2, among others. Clinical samples of these adenoviruses were collected for specificity testing. PCR amplification reagents were prepared according to Table 1, with 5 μL of sample added per T for amplification, repeated three times.

[0047] Table 3 Specificity test results

[0048]

[0049] As shown in Table 3, the primer-probe combination and detection method provided in this application have good specificity and have not detected other adenoviruses in the same genus.

[0050] 3) Repeatability test

[0051] The synthesized bearded adenovirus type 1 plasmid was diluted to a concentration of 1×10⁻⁶ after being fixed. 4Copies / mL and 1×10 6 Copies / mL. Prepare PCR amplification reagents according to Table 1. Pipette 5 μL of each concentration into the PCR amplification reagent for amplification, and repeat 10 times for each concentration.

[0052] Table 4 Repeatability Test Results

[0053]

[0054] As shown in Table 4, the primer-probe combination and detection method provided in this application are within 1×10 4 Copies / mL and 1×10 6 The CV values ​​of the two plasmid concentrations (Copies / mL) were 0.8% and 1.1%, respectively, with CV < 5%, indicating good reproducibility.

[0055] 4) Sensitivity test

[0056] The synthesized bearded adenovirus type 1 plasmid was diluted to a concentration of 1×10⁻⁶ after being fixed. 3 Copies / mL and 5×10 2 Copies / mL. Prepare PCR amplification reagents according to Table 1, and add 5 μL of each concentration to the PCR amplification reagent for amplification, repeating 20 times.

[0057] Table 5 Sensitivity Test Results

[0058]

[0059] As shown in Table 5, the primer-probe combination and detection method provided in this application are within 1×10⁻⁶. 3 The detection rate of Copies / mL concentration was 100%, at 5×10 2 The detection rate was 65% for copies / mL. Therefore, the sensitivity for beard dragon adenovirus type 1 was 1×10⁻⁶. 3 Copies / mL.

[0060] 4) Summary

[0061] Test results as follows Figure 1 As shown in Tables 2-4, it can be seen that the primer-probe combination and detection method provided in this application have a detection limit of up to 1000 copies / mL, and have the characteristics of short detection time, high detection specificity, good repeatability and high sensitivity.

[0062] Example 2

[0063] This embodiment illustrates a kit for detecting beard dragon adenovirus type 1 based on real-time quantitative PCR technology, including PCR amplification reagents, a negative control, and a positive control. The PCR amplification reagents include primer and probe compositions for detecting beard dragon adenovirus type 1 as shown in SEQ ID NO: 1-3, and 4×Buffer Mix (DNA polymerase, Mg...). + The reagents used included dNTPs and lyophilization protectant; the positive control was a synthetic plasmid containing the target fragment of adenovirus type 1 (Adenovirus 1); and the negative control was ultrapure water. The concentrations of the forward primer, reverse primer, and probe were all 10 μM. The PCR amplification reagents are listed in Table 1. The specific operating steps are as follows:

[0064] 1) Extract nucleic acid from the sample to be tested; 200 μL of fecal sample was aspirated into the lysis buffer of Gangzhu Medical Nucleic Acid Extraction Reagent Consumable (Cartridge), and nucleic acid was extracted from the fecal sample of the diseased bearded dragon using Gangzhu Medical Nucleic Acid Extractor.

[0065] 2) Prepare the reaction system. The total volume of the reaction system is 20 μL. The reaction components are shown in Table 1.

[0066] 3) Take 5 μL of the nucleic acid sample to be tested, the positive control, and the negative control into the PCR amplification reagent, respectively. Use the Gangzhu 8PLUS fluorescence PCR amplification instrument, select the Beard Dragon Adenovirus Type I test, and perform real-time fluorescence quantitative PCR reaction;

[0067] 4) After the reaction is complete, determine the test result based on the Ct value.

[0068] When making a judgment, the fluorescence curve above the threshold should have a clear S-shaped curve; otherwise, the experiment is considered invalid, and errors in instruments, reagents, amplification conditions, etc., should be checked.

[0069] When using the method provided by this invention to detect beard adenovirus type 1, the judgment criteria are as follows:

[0070] Under the conditions that the negative control should show no amplification and the positive control Ct value should be less than 32, samples with Ct ≤ 36.0 are considered positive, and samples with Ct > 38.0 are considered negative. If the Ct value of the sample is 36.0 < Ct ≤ 38.0, retesting is recommended. The retest result with Ct ≤ 38.0 is considered positive, and the result with Ct > 38.0 is considered negative.

[0071] Test results as follows Figure 2 As shown in Table 6, it can be seen that the primer-probe combination, detection kit and detection method provided in this application can efficiently and accurately detect beard dragon adenovirus type 1 in the sample to be tested.

[0072] Table 6 Sample Detection Results

[0073]

[0074] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting beard dermal adenovirus type 1 based on real-time quantitative PCR technology, characterized in that, The primer-probe combination includes a forward primer, a reverse primer, and a probe, and the nucleotide sequences of the forward primer, reverse primer, and probe are as follows: Forward primer: 5'-AAGGCCTCCGGCAGGCTT-3', as shown in SEQ ID NO:1; Reverse primer: 5'-GACGTAGTCTCTGCACAGGCAT-3', as shown in SEQ ID NO:2; Probe: 5'-AGTCAAAATCTTGGTCGGCAAC-3', as shown in SEQ ID NO:3, wherein the 5' end of the probe is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group.

2. The primer and probe combination for detecting beard dermal adenovirus type 1 based on real-time quantitative PCR technology according to claim 1, characterized in that, The fluorescent reporter group is selected from any one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED, or LC RED460, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2, BHQ3, or MGB.

3. The use of the primer and probe combination for detecting adenovirus type 1 based on real-time fluorescence quantitative PCR technology as described in any one of claims 1 or 2 in the preparation of a kit for detecting adenovirus type 1.

4. A kit for detecting beard dermal adenovirus type 1 based on real-time quantitative PCR technology, characterized in that, The kit includes PCR amplification reagents, a negative control, and a positive control. The PCR amplification reagents include the primer and probe combination for detecting adenovirus type 1 based on real-time quantitative PCR technology as described in any one of claims 1 or 2.

5. The kit for detecting beard dragon adenovirus type 1 based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that, The PCR amplification reagents also include DNA polymerase, Mg + dNTPs, freeze-drying protectants.

6. The kit for detecting beard dragon adenovirus type 1 based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that, In the primer-probe combination, the concentrations of the forward primer, reverse primer, and probe are all 5-10 μM.

7. The kit for detecting beard dragon adenovirus type 1 based on real-time fluorescence quantitative PCR technology according to claim 4, characterized in that, The positive control is a plasmid containing a fragment of beard adenovirus type 1, and the negative control is ultrapure water.

8. A method for detecting benign adenovirus type 1 based on real-time quantitative PCR technology for non-diagnostic purposes, characterized in that, Including the following steps: 1) Extract total DNA from the sample to be tested; 2) Prepare a reaction system, wherein the reaction system comprises the PCR amplification reagent according to any one of claims 4-7; 3) Using the extracted total DNA from the sample to be tested as a template, and adding negative and positive controls as quality control templates, a real-time quantitative PCR reaction was performed; 4) After the reaction is complete, determine the test result based on the Ct value.

9. The method for detecting beard dermal adenovirus type 1 based on real-time quantitative PCR technology for non-diagnostic purposes according to claim 8, characterized in that, The procedure for the real-time quantitative PCR reaction is as follows: Step 1: 94℃, 5 minutes; Step2: 94℃, 30s, 60℃, 30s; Step 2 executes 40 loops.

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