A probe method for fluorescent quantitative detection of psittacine beak and feather disease virus, a primer kit and application thereof

Abstract

The application discloses a parrot beak feather disease virus probe fluorescence quantitative detection primer, a kit and application thereof, and belongs to the technical field of virus detection. The detection primer comprises an upstream primer PBFDV-F with a nucleotide sequence as shown in SEQ ID NO. 3, a downstream primer PBFDV-R with a nucleotide sequence as shown in SEQ ID NO. 4 and a probe PBFDV-P with a nucleotide sequence as shown in SEQ ID NO. 5. The application designs parrot beak feather disease virus specific detection primers and Taq Man probes, constructs a recombinant standard plasmid, optimizes a reaction system and establishes a parrot beak feather disease virus detection method. The sensitivity, specificity and repeatability of the detection method are verified through experiments, and it is found that the detection method has good sensitivity, specificity and repeatability. The application provides reliable technical support for clinical diagnosis and epidemiological investigation of PBFD.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to a fluorescent quantitative detection primer and kit for parrot beak feather disease virus probe method and its application. Background Technology

[0002] Psittacine Beak and Feather Disease (PBFD) is an acute or chronic contagious disease caused by psittacine beak and feather disease virus (PBFDV). It primarily infects parrots and is characterized by abnormal feather loss, beak and claw deformities, and impaired immune function. Some affected birds may also experience growth retardation and diarrhea. The disease has a high mortality rate, causing significant economic losses and ecological threats to the global ornamental bird breeding industry and the conservation of wild parrot populations. PBFDV belongs to the genus Circovirus in the family Circviridae. The viral particles are non-enveloped, and the genome is a single-stranded circular negative-sense DNA. Its replication process does not involve RNA intermediates, and its gene structure is relatively simple but highly variable.

[0003] This virus has a wide range of transmission routes. It can be transmitted through direct contact with the feathers, feces, and secretions of infected birds, as well as indirectly through contaminated feed, water, and cages. The incubation period after infection is relatively long, and some virus-carrying birds show no obvious clinical symptoms, becoming potential sources of infection and increasing the difficulty of disease control. In recent years, with the increasing frequency of the ornamental bird trade, the prevalence of PBFD has continued to expand. PBFDV-positive cases have been detected in many breeding farms and wild bird rescue centers, and the genotypes of the circulating strains are also showing a trend of diversification.

[0004] Currently, clinical detection methods for PBFDV mainly include conventional PCR and enzyme-linked immunosorbent assay (ELISA). While conventional PCR has a certain degree of specificity, its sensitivity is low, making it difficult to detect low viral load samples. ELISA is susceptible to antibody cross-reactivity, resulting in unsatisfactory test results. Neither of these methods can meet the needs of rapid clinical diagnosis and epidemiological surveillance.

[0005] Quantitative real-time PCR (qPCR) technology, with its advantages of high sensitivity, high specificity, speed, and efficiency, has become the mainstream technique in the field of animal virus detection. Therefore, establishing a sensitive, specific, and stable qPCR detection method for PBFDV is of great significance for early diagnosis, epidemic tracing, and precise prevention and control of this disease. Summary of the Invention

[0006] The purpose of this invention is to provide primers, a kit, and their applications for the quantitative fluorescence detection of psittacosis beak feather disease virus (PBFD) using a probe method, thereby addressing the problems existing in the prior art. The detection method provided by this invention exhibits good sensitivity, specificity, and repeatability. This invention provides reliable technical support for the clinical diagnosis and epidemiological investigation of PBFD.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a fluorescent quantitative detection primer for parrot beak feather disease virus probe method. The detection primer includes an upstream primer PBFDV-F with a nucleotide sequence as shown in SEQ ID NO.3, a downstream primer PBFDV-R with a nucleotide sequence as shown in SEQ ID NO.4, and a probe PBFDV-P with a nucleotide sequence as shown in SEQ ID NO.5.

[0008] Optionally, the 5′ end of the probe PBFDV-P is modified with FAM, and the 3′ end is modified with BHQ1.

[0009] The present invention also provides the application of the detection primers in the preparation of a detection kit for parrot beak feather disease virus.

[0010] The present invention also provides a parrot beak feather disease virus detection kit, comprising the aforementioned detection primers.

[0011] Optionally, the steps for detecting parrot beak feather disease virus using the kit are as follows: Using the DNA of the sample to be tested as a template, a Taq Man qPCR reaction was performed using the aforementioned detection primers. Fluorescence signals were collected to obtain the Ct value of the sample to be tested.

[0012] Optionally, if the Ct value of the sample to be tested is ≤40, the sample to be tested is determined to contain psittacosis virus; if the Ct value is >40 and the amplification line shows a peak, the sample to be tested is determined to be suspected of carrying psittacosis virus; if the Ct value is >40 and the amplification line does not show a peak, the sample to be tested is determined not to contain psittacosis virus.

[0013] Optionally, the reaction system for the TaqMan qPCR reaction is as follows: 2 μL template DNA, 0.2 μL upstream primer PBFDV-F, 0.2 μL downstream primer PBFDV-R, 0.1 μL probe PBFDV-P, 10 μL 2× Taq Pro U+ M μL step ProbeqPCR Mix, and 7.5 μL RNase-free ddH2O.

[0014] Optionally, the reaction conditions for the Taq Man qPCR reaction are: 37℃ for 2 min; 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, 45 cycles, with corresponding fluorescence collected at 60℃.

[0015] The present invention discloses the following technical effects: This invention designs primers and a Taq Man probe for the specific detection of psittacosis beak feather disease virus (PBFD) using a fluorescent quantitative PCR method. By constructing a recombinant standard plasmid and optimizing the reaction system, a Taq Man fluorescent quantitative PCR method for PBFD detection was established. Experiments verified the sensitivity, specificity, and repeatability of the detection method, demonstrating that the method provided by this invention exhibits good sensitivity, specificity, and repeatability. Therefore, this invention provides reliable technical support for the clinical diagnosis and epidemiological investigation of PBFD. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 PCR identification of standard plasmids; M: DL2000 DNA relative molecular mass standard; 1: standard plasmid; Figure 2 Electrophoresis results of bacterial PCR; M: relative molecular mass standard of DL2000 DNA; 1-3: amplification products of bacterial culture in tubes 1, 2, and 3, respectively; 4: negative control; Figure 3 Taq Man qPCR amplification curve of the PBFDV gene; Figure 4 Taq Man qPCR standard curve for the PBFDV gene; Figure 5 The results are for the specific detection of the PBFDV gene using the Taq Man qPCR method; 1: PBFDV; 2: Hepatitis A virus; 3: ALV; 4: FADV; 5: AMPV; 6: AIV; 7: IBV; 8: NDV; 9: Negative control; Figure 6 This is a sensitivity test for standard PCR; M: relative molecular mass of DL2000 DNA; 1~9: template concentrations of 1.35×10⁻⁹ respectively. 8 ~1.35×10 0 copies / μL; 10: negative control; Figure 7 For sensitivity detection by qPCR; 1-6: template concentrations were 1.35×10⁻⁶ respectively. 7 ~1.35×10 2 copies / μL; 7: Negative control; Figure 8 Taq Man qPCR detection for clinical samples. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 1. Materials, Reagents and Main Instruments 1.1 Materials Parrot disease feed containing PBFDV, hepatitis A virus, avian leukosis virus (ALV), avian adenovirus (FADV), avian metapneumovirus (AMPV), avian influenza virus (AIV), avian infectious bronchitis virus (IBV), and Newcastle disease virus (NDV). Provided by the Preventive Veterinary Laboratory of Xinyang Agricultural and Forestry College.

[0024] 1.2 Main Reagents Nucleic acid extraction kits were purchased from Guangzhou Cybex Biotechnology Co., Ltd.; 2×Phanta Max Master Mix (Dye Plus), 5×MLtra-Universal TOPO Cloning Mix, 2×Taq Pro U+ MμLtiple ProbeqPCR Mix, and RNase-free ddH2O were purchased from Nanjing Novizan Biotechnology Co., Ltd.; M5 DL2000 DNA Marker, gel extraction kit, 2×M5 Hiper plus Taq HiFi PCR mix (with blue dye), and small-plasmid extraction kit were purchased from Beijing Polymer Biotechnology Co., Ltd.; competent cells were purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0025] 1.3 Main Instruments The Tanon-2500 fully automated digital gel imaging system was purchased from Guangzhou Yuwei Biotechnology Instruments Co., Ltd., and the quantitative real-time polymerase chain reaction (PCR) detection system was purchased from Hangzhou Borui Technology Co., Ltd.

[0026] 2. Test methods 2.1 Design and Synthesis of Primers and Probes The complete genome sequence of PBFDV (accession numbers: AF311299 and AF311301) was downloaded from NCBI. Primers and probes were designed using Primer 5.0 software based on the conserved PBFDV gene sequence, and this was accomplished by Jiangsu Saisofe Biotechnology Co., Ltd. The gene sequences of the primers and probes and the expected sizes of the target fragments are shown in Table 1.

[0027] Table 1 Primer and probe gene sequences 2.2 Preparation of standard plasmids Viral DNA was extracted according to the instructions of the DNA extraction kit, and the extracted DNA was used as a template for conventional PCR amplification (PBFDV Rep gene).

[0028] PCR reaction system: 2× Phanta Max Master Mix 25μL, RNase-free ddH2O 11μL, DNA 10μL, PFDVrepF 2μL and PFDVrepR 2μL.

[0029] PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 60 s, 35 cycles; 72℃ final extension for 5 min.

[0030] The PCR product was recovered and purified using a DNA gel recovery kit. The purified target gene was ligated into the CE3Blunt vector (Nanjing Novizan Biotechnology Co., Ltd.) for transformation. Three colonies were picked from the transformed plate, and 500 μL of antibiotic-free LB and 0.5 μL of ampicillin were added. The plates were shaken for 4 hours, and the resulting three bacterial cultures were used for PCR.

[0031] PCR reaction system: 10 μL of 2× M5 Hiper plus Taq HiFi PCR mix, 5 μL of RNase-free ddH2O, 4 μL of bacterial culture, 0.5 μL of PFDVrepF and 0.5 μL of PFDVrepR.

[0032] PCR reaction program: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 58℃ annealing for 25 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0033] The gel image after electrophoresis showed that tube 3 was the brightest, so tube 3 was selected for culture shaking. The culture medium was 5 μL A. + 50 μL of bacterial culture and 5 mL of broth were added to tube #3 and cultured for 18 hours. Plasmids were then extracted according to the instructions of the small-sample plasmid extraction kit. The extracted plasmids were identified by PCR. Positive plasmids were sent to Henan Shangya Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the nucleotide sequence of the reference strain on NCBI for homology. The copy number was calculated using formula (1).

[0034] Plasmid copy number (copies / μL) = 6.02 × 10⁻⁶ 23 ×10 -9 × plasmid concentration ÷ molecular weight (1).

[0035] 2.3 Establishment of TaqMan qPCR reaction system The established TaqMan qPCR reaction system consisted of 20 μL: 2 μL template DNA, 0.2 μL each of forward and reverse primers (PBFDV-F and PBFDV-R), 0.1 μL probe PBFDV-P, 10 μL of 2× Taq Pro U+ M, 10 μL of step Probe qPCR Mix, and 7.5 μL of RNase-free ddH2O.

[0036] Reaction conditions: 37℃ for 2 min; 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing extension for 30 s; 45 cycles; corresponding fluorescence was collected at 60℃.

[0037] Result interpretation: If the Ct value of the sample is ≤40, it is positive, and the sample is determined to contain psittacosis virus; if the Ct value is >40 and the amplification line shows a peak, the sample is suspected of carrying psittacosis virus; if the Ct value is >40 and the amplification line does not show a peak, the sample is determined not to contain psittacosis virus.

[0038] 2.4 Plotting the Standard Curve The standard plasmid prepared in 2.2 was serially diluted 10-fold, and the 1.35 × 10⁻⁶ plasmids were analyzed using the TaqMan qPCR method established in 2.3. 7 ~1.35×10 3 Five concentration gradients (copies / μL) were used for detection. A standard curve was plotted with the logarithm of the standard plasmid copy number on the x-axis and the CT value on the y-axis.

[0039] 2.5 Specificity test The established TaqMan qPCR method was used to amplify DNA extracted from parrot disease samples, hepatitis A disease samples, avian leukosis virus (ALV), avian adenovirus (FADV), avian metapneumovirus (AMPV), avian influenza virus (AIV), avian infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and negative samples (ddH2O) to determine the specificity of the method.

[0040] 2.6 Sensitivity Experiment For 1.35×10 7 ~1.35×10 2 Six gradients of standard plasmids (copies / μL) and a negative control (ddH2O) were analyzed by TaqMan qPCR to determine the lowest detection limit of this method for PBFDV standard plasmids. Simultaneously, the same batch of standard plasmids (concentration 1.35 × 10⁻⁶) were detected using the conventional PCR method described in section 2.2. 8 ~1.35×10 0 (copies / μL), comparing the differences in sensitivity between the two.

[0041] 2.7 Repeatability Experiment Continuous 3 days for 1.35×10 7 ~1.35×10 4 TaqMan qPCR was performed using four gradients of standard plasmids (copies / μL), with three replicates within each gradient. The mean CT value, standard deviation, and coefficient of variation were calculated to determine the intra- and inter-group repeatability of the method.

[0042] 2.8 Clinical Sample Testing Thirty-two PBFDV-positive samples were collected, and PBFDV DNA was extracted using a nucleic acid extraction kit. The samples were then amplified by Taq Man qPCR according to the method established in this invention to verify its effectiveness.

[0043] 3. Results and Analysis 3.1 Preparation of standard plasmids Electrophoresis after bacterial PCR showed that the amplification product in tube 3 was the brightest, with the target band visible at approximately 873 bp, consistent with the expected fragment size. Figure 1 The band was recovered, purified, and ligated into a vector for transformation. Three different colonies were picked to obtain three sets of bacterial culture samples. These three sets of bacterial cultures were identified by bacterial culture PCR. The electrophoresis results showed that the target band was visible at approximately 873 bp, consistent with the expected fragment size. Figure 2 This preliminarily confirms that the PBFDV target gene fragment was successfully ligated into the CE3Blunt vector. The plasmid concentration, measured using a micro-ultraviolet spectrophotometer, was 39.6 ng / μL, and its copy number was calculated to be 1.35 × 10⁻⁶. 10 copies / μL.

[0044] 3.2 Plotting the Standard Curve Using a copy number of 1.35 × 10 7 ~1.35×10 3 TaqMan qPCR was performed using copies / μL of the standard, with three replicates per group and a negative control group (ddH2O template). The results showed that the amplification curves of this gene all exhibited a relatively typical S-shaped pattern. Figure 3 The amplification curves at different dilutions were evenly spaced, and the negative control showed no amplification. The standard curve intercept was 42.68, the slope was -3.4, the regression equation was Y=3.4X+42.68, the correlation coefficient was 0.999, and the amplification efficiency was 96.82. A standard curve was plotted with the logarithm of the initial template copy number as the X-axis and the CT value as the Y-value. Figure 4 As can be seen, a good linear relationship is observed at different dilutions.

[0045] 3.3 Specificity test The results showed that only PBFDV exhibited an amplification curve, while hepatitis A virus, ALV, FADV, AMPV, AIV, IBV, NDV, and the negative control (ddH2O) did not show an amplification curve. Figure 5 This indicates that the method has no cross-reactivity with hepatitis A virus, ALV, FADV, AMPV, AIV, IBV, and NDV, and can specifically detect PBFDV.

[0046] 3.4 Sensitivity Test The results showed that the detection limit of conventional PCR was 1.35 × 10⁻⁶. 4 copies / μL ( Figure 6 qPCR at 1.35 × 10⁻⁶ 7 ~1.35×10 2 Bands were observed in both copies / μL ( Figure 7 That is, the detection limit of this method is 1.35 × 10⁻⁶. 2 The copies / μL indicates that the sensitivity of the qPCR method constructed in this experiment is 100 times that of the conventional PCR method.

[0047] 3.5 Repeatability Experiment The results are shown in Tables 2 and 3. The coefficient of variation within groups was 0.21%–1.83%, and the coefficient of variation between groups was 0.33%–1.83%, indicating that the method has good repeatability.

[0048] Table 2. Intra-batch repeatability experiments of quantitative real-time PCR Table 3. Inter-batch repeatability of quantitative real-time PCR experiments 3.6 Clinical Sample Testing The results showed that all 32 PBFDV-positive samples had amplification curves. Figure 8 Of the 30 samples tested, 30 had a Ct value ≤ 38, indicating a positive result and the presence of psittacosis virus (PBFDV). One sample had a Ct value between 38 and 40, indicating a weak positive result. One sample had a Ct value > 40 and showed an amplification peak, suggesting it might be carrying PBFDV. The positive rate was 96.88% (31 / 32). Based on the clinical sample test results, this method can be used for PBFDV detection in practice.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer for quantitative real-time detection of parrot beak feather disease virus using a probe method, characterized in that, The detection primers include the upstream primer PBFDV-F with the nucleotide sequence shown in SEQ ID NO.3, the downstream primer PBFDV-R with the nucleotide sequence shown in SEQ ID NO.4, and the probe PBFDV-P with the nucleotide sequence shown in SEQ ID NO.

5.

2. The detection primer as described in claim 1, characterized in that, The probe PBFDV-P is modified with FAM at its 5′ end and with BHQ1 at its 3′ end.

3. The use of the detection primers as described in claim 1 or 2 in the preparation of a psittacosis virus detection kit.

4. A test kit for parrot beak feather disease virus, characterized in that, It contains the detection primers as described in claim 1 or 2.

5. The kit according to claim 4, characterized in that, The steps for detecting parrot beak feather disease virus using the kit are as follows: Using the DNA of the sample to be tested as a template, a Taq Man qPCR reaction is performed using the detection primers described in claim 1 or 2, and the fluorescence signal is collected to obtain the Ct value of the sample to be tested.

6. The reagent kit as described in claim 5, characterized in that, If the Ct value of the sample to be tested is ≤40, the sample is determined to contain psittacosis virus; if the Ct value is >40 and the amplification line shows a peak, the sample is determined to be suspected of carrying psittacosis virus; if the Ct value is >40 and the amplification line does not show a peak, the sample is determined not to contain psittacosis virus.

7. The kit according to claim 5, characterized in that, The reaction system for the TaqMan qPCR reaction was as follows: 2 μL template DNA, 0.2 μL upstream primer PBFDV-F, 0.2 μL downstream primer PBFDV-R, 0.1 μL probe PBFDV-P, 10 μL 2×Taq Pro U+ M μL step Probe qPCR Mix, and 7.5 μL RNase-free ddH2O.

8. The reagent kit as described in claim 5, characterized in that, The reaction conditions for the Taq Man qPCR reaction were as follows: 37℃ for 2 min; 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing and extension for 30 s; 45 cycles; and corresponding fluorescence was collected at 60℃.

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