Fluorescent quantitative PCR (Polymerase Chain Reaction) primer, kit and method for detecting fowl adenovirus 4

Abstract

The invention provides fluorescent quantitative PCR primers, a kit and a method for detecting fowl adenovirus 4, and belongs to the technical field of biological detection. The nucleotide sequence of the fluorescent quantitative PCR primer pair for detecting the fowl adenovirus 4 provided by the invention is shown as SEQ ID NO.1 and SEQ ID NO.2 or a complementary sequence of the SEQ ID NO.1 and SEQ ID NO.2. The fluorescent quantitative PCR primer and the method for detecting the fowl adenovirus 4, provided by the invention, are high in sensitivity and strong in specificity, and are negative when being used for detecting fowl adenovirus 8, Newcastle disease virus, H9 subtype avian influenza virus, infectious bronchitis virus, infectious bursal disease virus, Marek's disease virus, egg drop syndrome virus and mycoplasma synoviae, and can be used for detecting fowl adenovirus 8, Newcastle disease virus, H9 subtype avian influenza virus, infectious bronchitis virus, infectious bursal disease virus, Marek's disease virus, egg drop syndrome virus and mycoplasma synoviae. And false positive can be effectively avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a fluorescent quantitative PCR primer and a method for detecting avian adenovirus type 4. Background Art

[0002] Adenovirus (FAdV) is a common infectious pathogen in poultry and wild birds. Avian adenovirus type 4 (FAdV-4) belongs to the genus Avian adenovirus of the family Adenoviridae and is a non-enveloped double-stranded DNA virus. FAdV-4 mainly causes pericardial effusion syndrome characterized by transparent effusion or yellowish jelly-like effusion in the pericardium. The capsid protein of this virus has three main structural proteins: Hexon, Penton and Fiber. Among them, the Fiber protein is encoded by two different Fiber genes, Fiber-1 and Fiber-2.

[0003] At present, for the detection of avian adenovirus, ordinary PCR can achieve rapid detection of pathogenic viruses, but the sensitivity is low and it cannot quantify pathogenic viruses; fluorescent quantitative PCR can quantitatively detect viruses, with high sensitivity and good specificity, and is suitable for high-throughput rapid detection and can be applied in production practice. However, for the primer design of fluorescent quantitative PCR of avian adenovirus, due to poor primer specificity, false positive results are easily caused, or the method is not sensitive enough, resulting in false negative results. Summary of the invention

[0004] In order to solve the above technical problems, an object of the present invention is to provide a fluorescent quantitative PCR primer and method for detecting avian adenovirus type 4.

[0005] Another object of the present invention is to provide a method for detecting avian adenovirus by using fluorescent quantitative PCR.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A fluorescent quantitative PCR primer pair for detecting avian adenovirus, wherein the nucleotide sequence of the primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences.

[0008] A kit for detecting avian adenovirus type 4 comprises a primer pair having nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences.

[0009] The kit as described above also includes 2×Taq Pro Universal AYBR qPCR Master Mix.

[0010] A fluorescent quantitative PCR method for detecting avian adenovirus type 4 comprises the following steps:

[0011] S1, sample processing and DNA extraction;

[0012] S2, using the primer pairs with nucleotide sequences such as SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences and the extracted DNA to set up a real-time fluorescence quantitative PCR reaction system;

[0013] S3, performing real-time fluorescence quantitative PCR amplification;

[0014] S4. Determine the result.

[0015] In the fluorescent quantitative PCR method as described above, preferably, the PCR reaction system includes 10.0-25 μL of 2×Taq Pro UniversalSYBR qPCR Master Mix, 0.4-1 μL of each primer pair as shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences at a concentration of 10 μM, 4.0-10 μL of template DNA, and ddH2O to make up the system to 20.0-50 μL.

[0016] In the fluorescence quantitative PCR method as described above, preferably, the program of real-time fluorescence quantitative PCR amplification is pre-denaturation at 95°C for 30s, 1 cycle; cyclic reaction: denaturation at 95°C for 5s, annealing at 55°C for 5s, extension at 72°C for 18s, 40 cycles.

[0017] In the fluorescent quantitative PCR method as described above, preferably, the result is judged as when there is an obvious S-shaped amplification curve and the CT value is ≤32.5, it indicates that the sample to be tested contains avian adenovirus type 4 nucleic acid, otherwise it is negative, that is, it does not contain avian adenovirus type 4 nucleic acid.

[0018] The fluorescent quantitative PCR method as described above preferably uses avian adenovirus DNA containing different concentrations as a standard, performs S1-S3 amplification, establishes a fluorescent quantitative standard curve, and performs quantitative analysis on the fluorescent quantitative PCR results.

[0019] Furthermore, the avian adenovirus DNA contains the sequence shown in SEQ ID NO.13.

[0020] The beneficial effects of the present invention are:

[0021] The fluorescent quantitative PCR primer pair for detecting fowl adenovirus type 4 provided by the present invention has good reaction performance and good specificity, and is negative when used for detecting fowl adenovirus type 8, Newcastle disease virus, H9 subtype avian influenza virus, infectious bronchitis virus, infectious bursal virus, Marek's virus, egg drop syndrome virus and Mycoplasma synoviae, and can effectively avoid false positives; the provided fluorescent quantitative PCR detection method for fowl adenovirus has the characteristics of simple and easy-to-use operation procedures, and the detection method has high sensitivity, effectively avoids false negative results, and greatly improves the detection efficiency and accuracy.

[0022] The detection method provided by the present invention uses specific primers with high sensitivity and specificity, and can detect a large number of tissue samples at a time with high throughput, and the time consumption is shorter than that of ordinary PCR; during the fluorescent quantitative PCR process, the detection process can be observed in real time, and the detection results can be obtained immediately after the reaction is completed, which greatly shortens the detection time and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Raw melting curve for primer pair 1 plotted against temperature.

[0024] Figure 2 This is the melting curve of primer pair 1 obtained by plotting the derivative of the change in temperature and fluorescence signal (-dRFU / dT).

[0025] Figure 3 Raw melting curves obtained by plotting fluorescence versus temperature for primer pair 2.

[0026] Figure 4 This is the melting curve of primer pair 2 obtained by plotting the derivative of the change in temperature and fluorescence signal (-dRFU / dT).

[0027] Figure 5 Raw melting curves obtained by plotting fluorescence versus temperature for primer pair 3.

[0028] Figure 6 This is the melting curve of primer pair 3 obtained by plotting the derivative of the change in temperature and fluorescence signal (-dRFU / dT).

[0029] Figure 7 Raw melting curves obtained by plotting fluorescence versus temperature for primer pair 4.

[0030] Figure 8 This is the melting curve of primer pair 4 obtained by plotting the derivative of the change in temperature and fluorescence signal (-dRFU / dT).

[0031] Fig. 9 Raw melting curves obtained by plotting fluorescence versus temperature for primer pair 5.

[0032] Fig.10 This is the melting curve of primer pair 5 obtained by plotting the derivative of the change in temperature and fluorescence signal (-dRFU / dT).

[0033] Fig.11 is the PCR fluorescence standard curve of primer pair 1.

[0034] Fig.12 is the standard curve of primer pair 1.

[0035] Fig.13 is the PCR fluorescence standard curve of primer pair 2.

[0036] Fig.14 is the standard curve of primer pair 2.

[0037] Fig.15 is the PCR fluorescence standard curve of primer pair 4.

[0038] Fig.16 is the standard curve of primer pair 4.

[0039] Fig.17 is the PCR fluorescence standard curve of primer pair 5.

[0040] Fig.18 is the standard curve of primer pair 5.

[0041] Fig.19 This is the amplification curve diagram of the specific detection results of primer pair 1 fluorescence quantitative PCR.

[0042] Fig. 20 This is the amplification curve diagram of the specific detection results of primer pair 2 fluorescence quantitative PCR.

[0043] Fig.21 This is the amplification curve of oral and cloaca detoxification detection in infected chickens. DETAILED DESCRIPTION

[0044] The existing avian adenovirus fluorescence quantitative PCR mostly designs primers with Hexon gene as the target gene. The present invention selects the highly conserved region of Fiber-2 gene to design primers. The primers have high sensitivity and specificity and are suitable for the rapid detection of avian adenovirus type 4. The method of the present invention has no non-specific amplification and high sensitivity for other 8 poultry pathogens, including avian adenovirus type 8 (FAdV-4), Newcastle disease virus (NDV), H9 subtype avian influenza virus (AIV H9), infectious bronchitis virus (IBV), infectious bursal virus (IBDV), egg drop syndrome virus (EDSV), Marek's virus (MDV), and Mycoplasma synoviae (MS). It can detect avian adenovirus type 4 with a concentration of 10 copies / μL. At present, the fluorescence quantitative PCR of avian adenovirus is mostly a probe method, and the probe method requires additional design and synthesis of probe sequences, which increases the detection cost. The present invention uses a fluorescent dye method, and the mix contains a dye. The dye method is economical and affordable, and the positive and negative results can be judged according to the CT value. The CT values ​​of all PCR products were analyzed, and a CT value greater than 32.5 was considered negative. The detection method provided by the present invention is suitable for large-scale rapid detection.

[0045] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0046] Unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are of analytical grade or above.

[0047] Example 1 Determination of melting curve of primers

[0048] 1. Primer design

[0049] The present invention selects the highly conserved region of the Fiber-2 gene to design primers, specifically the sequence of GeneBank accession number MN337322 to design specific primers, according to the known FAdV-4Fiber-2 gene sequence and fluorescent PCR primer design requirements: primer length 18 to 30 bases, GC content 40-60%, PCR amplification product length preferably between 100-200bp, avoid the secondary structure region of the template as much as possible, and the primer itself does not have a complementary sequence. The primers are synthesized by Beijing New Era Zhonghe Technology Co., Ltd., dissolved in double distilled water according to the instructions, to a final concentration of 10μM, and stored at -20°C. The designed primers are shown in Table 1 below.

[0050] Table 1 qPCR primers

[0051]

[0052]

[0053] 2. Extract DNA of avian adenovirus type 4 strain as a template, specifically isolated from Hebei, and extract DNA from the liver of diseased chickens as a qPCR template.

[0054] The primer melting curve determination qPCR reaction system was established according to the instructions of Novozyme Taq Pro Universal AYBR qPCR Master Mix. Four replicates were set for each primer concentration. The reaction system is shown in Table 2.

[0055] Table 2 qPCR reaction system

[0056]

[0057] The real-time fluorescence quantitative reaction program was 95°C pre-denaturation for 30 s, 1 cycle; 95°C denaturation for 5 s, 55°C annealing for 5 s, 72°C extension for 18 s, 40 cycles; after the PCR cycle, a heating and cooling program was added: 55-95°C, increasing by 0.5°C every 10 s at 520 nm until it was heated to 95°C, and the fluorescence was continuously measured to obtain the melting curve.

[0058] The melting curve of primer pair 1 is as follows Figure 1 , Figure 2 The melting curve of primer pair 2 is as follows Figure 3 , Figure 4 The melting curve of primer pair 3 is as follows Figure 5 , Figure 6 The melting curve of primer pair 4 is as follows Figure 7 , Figure 8 The melting curve of primer pair 5 is as follows Fig. 9 , Fig.10 The results showed that the melting curve of the product after PCR amplification of primer pair 3 was not a single melting point peak, indicating non-specific amplification. Therefore, primer pair 3 had poor specificity and was discarded. The best primer pair was subsequently selected from the other four primer pairs.

[0059] Example 2 Preparation of Fiber-2 gene qPCR standard curve

[0060] 1. Construction of PMD19-T-Fiber-2 plasmid

[0061]

[0062] Table 3 PCR amplification of F1iber-2 nucleic acid fragments

[0063]

[0064] PCR product gel recovery: using TIANGEN gel recovery kit. TA cloning of fiber-2 gene: using TAKARA's pMD TM 19-T Vector Cloning Kit, cloning system as shown in Table 4. Mix the reaction system evenly, react at 16℃ for 30min, then take out and cool on ice for 2min. Take 100μL DH5α competent cells, add 4μL ligation solution, and ice bath for 5min before blue-white screening and identification.

[0065] Blue-white spot screening and identification: Prepare LB agar medium containing 100 μg / mL Amp, apply 40 μL 20 mg / mL X-Gal (DMSO) and 4 μL 1M IPTG solution respectively, put it in a 37°C incubator for half an hour, then apply the transformed bacteria, culture in the dark for 16 hours, observe, and take the white colonies for expansion culture to extract the plasmid.

[0066] Plasmid extraction: Take a single white colony cultured by blue-white screening, inoculate it into 10 mL of LB liquid culture medium containing 100 μg / mL Amp, and culture it at 180 rpm and 37°C for 16 h before taking it out.

[0067] Table 4 TA cloning system of Fiber-2 gene

[0068]

[0069] The plasmid was extracted using the FastPure Plasmid Mini Kit from Vazyme. The plasmid concentration was detected using nanodrop. The plasmid was named PMD19T-Fiber-2 and stored at -20°C.

[0070] Nanodrop2000 was used to measure the plasmid concentration and calculate the plasmid copy number.

[0071] Plasmid copy number calculation formula:

[0072] (6.02×10 23 )×(ng / μl×10 -9 ) / (DNA length×660)=copies / μl

[0073] The PMD19T-Fiber-2 plasmid was diluted to 10 8 copies / μL, 107 copies / μL····10 1 copies / μL, each gradient was set in parallel, the primer concentration was 0.6μM, the reaction system was the same as before, the reaction procedure was 95℃ pre-denaturation for 30s, 1 cycle; 95℃ denaturation for 5s, 55℃ annealing for 5s, 72℃ extension for 18s, 40 cycles; real-time fluorescence measurement during annealing. After the reaction, the standard curve and slope parameters were measured: Ct value was used as the vertical coordinate, and the copy number was taken as the logarithm with the base of ten as the horizontal coordinate. The standard curve was plotted and the standard curve formula was calculated. The standard curve of primer pair 1 is shown in the figure. Fig.11 , Fig.12 The standard curve of primer pair 2 is shown in Fig.13 , Fig.14 The standard curve of primer pair 4 is shown in Fig.15 , Fig.16 The standard curve of primer pair 5 is shown in Fig.17 , Fig.18 The standard curve equation for primer pair 1 is Y = -3.354x + 35.622 (R 2 =0.997); the standard curve equation for primer pair 2 is Y=-2.9294x+32.878(R 2 =0.901); the standard curve equation for primer pair 4 is Y=-3.696x+20.991(R 2 =0.982); the standard curve equation for primer pair 5 is Y=-2.856x+22.195(R 2 =0.984); indicating that the qPCR reaction effectiveness of the four primer pairs and the good linear relationship between the different gradient logarithmic values ​​and the Ct values. However, when the plasmid was diluted to 10 4 When the plasmid was diluted to 10 copies / μL, no amplification curve was obtained. 1 When the detection limit of primer pairs 1 and 2 was 10 copies / μL, no amplification curve was obtained. Therefore, the sensitivity of fluorescence quantitative PCR using these two primer pairs was too low and it was not easy to use. The detection limit of primer pairs 1 and 2 was 10 copies / μL, indicating that the dual fluorescence quantitative PCR method established with these two primer pairs had good sensitivity.

[0074] Example 3 Specificity test

[0075] The cDNAs reverse transcribed from DNA or RNA of fowl adenovirus type 4 (FAdV-4), fowl adenovirus type 8 (FAdV-8), Newcastle disease virus (NDV), avian influenza virus H9 (AIV H9), infectious bronchitis virus (IBV), infectious bursal virus (IBDV), Marek's virus (MDV), egg drop syndrome virus (EDSV), and Mycoplasma synoviae (MS) were used as templates, and primer pairs 1 and 2 were used at a primer concentration of 0.6 μM, respectively. The reaction system was the same as in Table 2 above, and the reaction procedure was 95°C pre-denaturation for 30 s, 1 cycle; 95°C denaturation for 5 s, 55°C annealing for 5 s, 72°C extension for 18 s, 40 cycles; during annealing, real time fluorescence values ​​were measured to detect the specificity of the primers. The amplification curve of the specificity detection results of primer pair 1 fluorescence quantitative PCR is shown in the figure below. Fig.19 As shown, the amplification curve of the specific detection result of primer pair 2 fluorescence quantitative PCR is shown in Fig. 20 shown.

[0076] Among them, the total RNA of each virus was extracted by Trizol method. The specific operation is as follows: take 200μL of each sample to be tested in a 1.5mL centrifuge tube, add 600μL of Trizol and shake it in a vortex for 2-3min, add 480μL of chloroform, centrifuge, take the supernatant and transfer it to another 1.5mL centrifuge tube, add 200μL of isopropanol and centrifuge, take the precipitate and add 75% ethanol to wash the precipitate, dry it, and finally dissolve the precipitate with 40μLRNase-Free ddH2O, take 10μL for reverse transcription, and store the rest at -20℃. Reverse transcription is performed by one-step method. The reaction system is as follows: 4μL 5×ALL-in-one qPCR SuperMinx, 1μL Enzyme Mix, 1μg template RNA, add RNase-Free ddH2O to 20μL, and place the configured system in a PCR instrument at 50℃ for 15min and 85℃ for 5s. The reverse transcription product can be directly used for qPCR or stored at -80℃ for a long time.

[0077] Results (such as Fig.19 ) showed that except for the adenovirus type 4 sample, the primer pair 1 had a typical S-shaped amplification curve, but no amplification curve was obtained for the other 8 avian pathogens. Fig. 20 In addition to the typical S-shaped amplification curve of avian adenovirus type 8 (such as Fig. 20 The amplification curve of the samples with the number 2 was also shown, and the other samples were all negative (see Fig. 20 ), indicating that this pair of primers can detect not only adenovirus type 4, but also adenovirus type 8. Therefore, primer pair 1 has the best specificity.

[0078] Determination of results: The Ct values ​​of the eight dilutions of primer pair 1 for each sample were all ≤32.5. Therefore, if the Ct value of the amplified product was ≤32.5 and had a typical S curve, the sample to be tested was positive for avian adenovirus type 4, otherwise it was negative.

[0079] Example 4 Oral and cloaca excretion detection of infected chickens

[0080] SPF chickens were challenged with FAdV-4. Oral and anal swabs were collected from the test chickens at 1, 3, 5, and 7 days after the challenge. The cotton swab DNA was extracted: a 1.5 ml EP tube was taken, and a sterile cotton swab was placed in each tube and soaked with 500 μL PBS buffer solution. The oral cavity and anus of the test chicken were sampled, and the sampled cotton swab was returned to the 1.5 mL EP tube, broken and marked, and placed at 4 ° C for full soaking for 24 hours. The cotton swab was taken out, 200 μL of sample was taken from each sample, 200 μL buffer AL and 20 μL Proteinase K were added, and the sample was immediately vortexed for 15 seconds to mix thoroughly. DNA was used as a template for qPCR detection, and the nucleotide sequence of primer pair 1 used was SEQ ID NO.1 (qPCR-Fiber2-F): 5'-GATCAATCCTAACATGTTCACGGTC-3'; SEQ ID NO.2 (qPCR-Fiber2-R): 5'-GTTGTTGACAATCTGTAGGCTTTCA-3'. The reaction procedure was 95°C pre-denaturation for 30s, 1 cycle; 95°C denaturation for 5s, 55°C annealing for 5s, 72°C extension for 18s, 40 cycles; real time fluorescence value was measured during annealing.

[0081] Test results such as Fig.21 As shown, the Ct value of the amplified product is ≤32.5 and the amplified product presents a typical S curve, indicating that toxins are excreted in the oral cavity and cloaca of the test chickens 1, 3, 5, and 7 days after the infection. This also shows that the primer pair 1 provided by the present invention has good specificity and sensitivity for detecting a pair of samples.

Claims

1. A fluorescent quantitative PCR primer pair for detecting avian adenovirus, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences.

2. A kit for detecting avian adenovirus type 4, characterized in that: It includes a primer pair having nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences.

3. The kit according to claim 2, characterized in that Also included is 2× Taq Pro Universal AYBR qPCR Master Mix.

4. A fluorescent quantitative PCR method for detecting avian adenovirus type 4, characterized in that: It includes the following steps: S1, sample processing and DNA extraction; S2, using the primer pairs with nucleotide sequences such as SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences and the extracted DNA to set up a real-time fluorescence quantitative PCR reaction system; S3, performing real-time fluorescence quantitative PCR amplification; S4. Determine the result.

5. The fluorescent quantitative PCR method according to claim 4, characterized in that: The PCR reaction system includes 10.0-25 μL of 2×Taq ProUniversal AYBR qPCR Master Mix, 0.4-1 μL of each primer pair with a concentration of 10 μM as shown in SEQ ID NO.1 and SEQ ID NO.2 or their complementary sequences, 4.0-10 μL of template DNA, and ddH2O to make up the system to 20.0-50 μL.

6. The fluorescent quantitative PCR method according to claim 4, characterized in that: The program of real-time fluorescence quantitative PCR amplification was pre-denaturation at 95°C for 30s, 1 cycle; cycle reaction: denaturation at 95°C for 5s, annealing at 55°C for 5s, extension at 72°C for 18s, 40 cycles.

7. The fluorescent quantitative PCR method according to claim 4, characterized in that: The result is judged as follows: when there is an obvious amplification curve and the CT value of the PCR product is ≤32.5, it means that the sample to be tested contains avian adenovirus type 4 nucleic acid, otherwise it is negative.

8. The fluorescent quantitative PCR method according to claim 4, characterized in that: Avian adenovirus DNA with different copy number concentrations was used as the standard for S1-S3 amplification. The Ct value was used as the ordinate and the logarithm of the copy number to the base of ten was used as the abscissa. A fluorescence quantitative standard curve was constructed to quantitatively analyze the results of fluorescence quantitative PCR.

9. The fluorescent quantitative PCR method according to claim 8, characterized in that: The avian adenovirus DNA contains the sequence shown in SEQ ID NO.13.

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