Dual NanoPCR Detection Kit for Porcine Reproductive and Respiratory Syndrome Virus and Pseudorabies Virus and Its Application
By designing a dual nanoPCR detection kit for pig breeding and respiratory syndrome virus and pseudorabies virus, the primer set and nanoPCR technology with strong specificity and high sensitivity are used to solve the problem of difficult to detect two virus infections at the same time in the existing technology, and efficient and low-cost virus detection is achieved.
Patent Information
- Application Number
- CN202111439874.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-11-30
- Publication Date
- 2025-07-25
- Estimated Expiration
- 2041-11-30
AI Technical Summary
The prior art is difficult to efficiently detect individual or mixed infections of pig breeding and respiratory syndrome virus and pseudorabies virus at the same time, and the detection cost is high and the time is long.
A dual nanoPCR detection kit for pig breeding and respiratory syndrome virus and pseudorabies virus was designed, which includes a primer set with strong specificity and high sensitivity and a positive control for recombinant plasmids. The detection was performed using nanoPCR technology, which can identify the infection status of the two viruses in a PCR reaction system at the same time.
It realizes the identification of individual or mixed infections of two viruses simultaneously in one detection operation, reducing detection cost and time, improving detection sensitivity, and no special instruments and equipment are required.
Smart Images
Figure CN114381549B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a dual nano-PCR detection kit for porcine reproductive and respiratory syndrome virus and pseudorabies virus and an application thereof. Background Art
[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral infectious disease characterized by reproductive disorders in pregnant sows, including abortion, mummification, stillbirth, and weak fetuses, as well as respiratory diseases in piglets and fattening pigs. The disease has spread globally, causing significant economic losses to the global pig industry. Pseudorabies (PRR) is caused by the porcine pseudorabies virus (PRV), a herpesvirus that causes fever, severe itching (except in pigs), and encephalopathy in a variety of domestic and wild animals. The disease can be transmitted to the reproductive system through breeding pigs. Once infected in pregnant sows, it can be transmitted vertically through the placenta to the fetus, resulting in severe abortion, stillbirth, weak fetuses, and mummification, leading to a decrease in the number and quality of newborn piglets.
[0003] The two diseases share many similar clinical symptoms: pregnant sows experience miscarriage, stillbirth, fetal mummification, and high neonatal mortality; fattening pigs experience loss of appetite, vomiting, diarrhea, fever, dyspnea, dermatitis, arthritis, neurological symptoms, growth retardation, and severely reduced immunity, making them highly susceptible to secondary infection with other pathogens. Adult pigs can carry and excrete the virus for long periods without clinical symptoms, and are also the primary cause of respiratory disease syndrome. Therefore, diagnosing PRRSV or PRV based solely on clinical symptoms is not advisable; laboratory testing is required, currently primarily through serology and molecular biology. Therefore, establishing detection methods for both PRRSV and PRV is essential for livestock production. Nano-PCR, as a new PCR technology, offers high sensitivity and specificity. Compared to fluorescent quantitative PCR, it requires less specialized laboratory equipment and is less expensive. Compared to conventional PCR, it also offers higher sensitivity, making it a new molecular diagnostic technology. Dual PCR can use one PCR reaction system to diagnose two diseases at the same time. It can not only detect single infection of two diseases, but also detect mixed infection of two diseases, which can save detection time and detection costs. In addition, nano-PCR has higher sensitivity than ordinary PCR and does not require special instruments and equipment. It is the current direction of reagent kit development and research. Summary of the Invention
[0004] The purpose of the present invention is to provide a dual nano-PCR detection kit for porcine reproductive and respiratory syndrome virus and pseudorabies virus and its application.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A dual nano-PCR detection kit for porcine reproductive and respiratory syndrome virus and pseudorabies virus, comprising a primer set for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and a primer set for detecting pseudorabies virus (PRV);
[0007] The primer set for detecting porcine reproductive and respiratory syndrome virus has the following sequence:
[0008] Upstream primer PRRSV-F: as shown in SEQ ID NO.1
[0009] Downstream primer PRRSV-R: as shown in SEQ ID NO.2;
[0010] The primer set for detecting pseudorabies virus has the following sequence:
[0011] Upstream primer PRV-F: as shown in SEQ ID NO.3
[0012] Downstream primer PRV-R: as shown in SEQ ID NO.4.
[0013] The above-mentioned kit also includes: a positive control, a negative control, colloidal gold nanoparticles and PCR amplification solution.
[0014] Furthermore, the positive control is a recombinant plasmid containing porcine reproductive and respiratory syndrome virus PRRSV and pseudorabies virus PRV; the negative control is double distilled water.
[0015] The use of the above-mentioned kit in detecting porcine reproductive and respiratory syndrome virus and pseudorabies virus is for non-disease diagnosis purposes.
[0016] A dual nano-PCR detection method for porcine reproductive and respiratory syndrome virus and pseudorabies virus comprises performing nano-duplex PCR on a sample to be tested using the above-mentioned kit to determine whether the sample contains porcine reproductive and respiratory syndrome virus and / or pseudorabies virus.
[0017] Furthermore, the reaction conditions of the nano-duplex PCR are: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, 35 cycles; and extension at 72°C for 10 min.
[0018] Compared with the prior art, the present invention has the following characteristics:
[0019] (1) The designed primer sequences have strong specificity, good reproducibility and high sensitivity;
[0020] (2) This method can simultaneously identify two viruses that are infected alone or together in one test, thus reducing the testing cost;
[0021] (3) The same PCR amplification procedure can simultaneously amplify PRRSV and PRV target fragments, saving detection time;
[0022] (4) By constructing a recombinant plasmid, it can be used as a positive control for each test, which is stable and convenient;
[0023] (5) Nano-PCR technology is used, which has higher sensitivity than conventional PCR and does not require special equipment compared to fluorescent PCR, making the method simple;
[0024] (6) Provide technical support for disease prevention and control in pig farms. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0026] Figure 1 The results of PRRSV single-plex PCR amplification are shown. M: DL5000 Marker, 1: negative control, 2: 52°C, 3: 54°C, 4: 56°C, 5: 58°C, 6: 60°C.
[0027] Figure 2 The results of PRV singleplex PCR amplification are shown in Figure 1. M: DL5000 Marker, 1: negative control, 2: 52°C, 3: 54°C, 4: 56°C, 5: 58°C, and 6: 60°C.
[0028] Figure 3 The amplification results of PRRSV and PRV recombinant plasmids are shown in Figure 1. M: DL5000 Marker, 1: negative control, 2: PRRSV, 3: PRV.
[0029] Figure 4 The figure shows the nano-PCR amplification results of the recombinant plasmid standard. M: DL5000 Marker, 1: negative control, 2: PRRSV and PRV nano-duplex PCR amplification bands.
[0030] Figure 5The following are the specific amplification results of the recombinant plasmid standard using nanoPCR. 1: Negative control; 2: PRRSV and PRV nanoduplex PCR amplification bands; 3-7: cDNA or DNA amplification bands for CSFV, PEDV, TGEV, JEV, and PCV, respectively.
[0031] Figure 6 The results of dual PCR amplification of PRRSV and PRV at different dilutions are shown. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 .
[0032] Figure 7 The results of nano-duplex PCR amplification of PRRSV and PRV at different dilutions are shown. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 .
[0033] Figure 8 The results of nano-duplex PCR amplification of 10 suspected PRRSV and PRV-infected samples are shown. M: DL5000 Marker, 1: negative control, 2: positive control, 3-12: amplified bands of the 10 suspected samples. DETAILED DESCRIPTION
[0034] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0035] The test reagents and samples used in the present invention are as follows:
[0036] Reagents: Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; Tiangen 2× Taq PCR Mastermix; Abcam reverse transcription kit; RNA and DNA virus extraction kits, plasmid mini-extraction kits, and DNA purification and recovery kits were all purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; DL5000 DNA Marker was purchased from Dalian Baosheng Biotechnology; and colloidal gold nanoparticles were purchased from Cytodiagnostics.
[0037] Samples: PRRSV and PRV positive samples retained by the applicant's laboratory; PRRSV and PRV suspected samples provided by a large-scale breeding farm.
[0038] Example 1
[0039] Establishment of a dual nano-PCR detection method
[0040] 1. Primer Screening
[0041] The PRRSV ORF3 and pseudorabies virus gE gene sequences were downloaded from the NCBI website, and one pair of primers each for specific amplification of PRV and PRRSV were designed using Primer 5.0 primer design software. The primer sequences and the sizes of the target amplified fragments are shown in Table 1. The primer sequences were synthesized by Shanghai Bioengineering.
[0042] Table 1 Primer sequences
[0043]
[0044] 2. PCR Amplification
[0045] 2.1 Amplification template preparation
[0046] RNA was extracted from PRRSV-positive samples using the Tiangen RNA virus extraction kit, and the reverse transcription system was prepared according to the Abcam reverse transcription kit:
[0047] RNA template 6 μL
[0048] ACCURT Reaction Mix 2 μL.
[0049] After mixing, let it stand at room temperature for 5 minutes, then add the following reagents:
[0050] ACCURT Reaction Stooper 2μL
[0051] 5XAll-In-One RT MasterMix 4μL
[0052] Nuclease-free H2O 6μL.
[0053] The prepared system was centrifuged briefly to mix, and reverse transcribed on a PCR instrument. The reaction procedure was: 25°C for 10 min, 42°C for 50 min, 85°C for 5 min, and 4°C for 5 min. The reverse-transcribed cDNA was stored in a -20°C refrigerator for later use.
[0054] DNA was extracted from PRV-positive samples using the Tiangen DNA virus extraction kit, and the extracted DNA was stored in a -20°C refrigerator for later use.
[0055] 2.2 Singleplex PCR amplification system
[0056] The singleplex PCR reaction system for PRRSV and PRV was 20 μL: 12.5 μL of 2× Taq PCR Mastermix, 1.0 μL each of upstream and downstream primers (25 pmol / L), and ddH2O to 20 μL. The PCR protocol was as follows: 95°C for 5 min; 35 cycles of 95°C for 45 s, annealing at 52°C, 54°C, 56°C, 58°C, and 60°C for 35 s, and 72°C for 30 s; and 72°C for 10 min. After PCR, 5 μL of the amplified product was analyzed by electrophoresis on a 2% agarose gel.
[0057] Amplification results such as Figure 1 and Figure 2 As shown, both PRRSV and PRV primers can amplify target fragments of 395 bp and 221 bp respectively. The optimal annealing temperature of PRRSV is 56°C, and the optimal annealing temperature of PRV is 54°C. The target fragments are clear without any mixed bands.
[0058] 2.3 Construction and identification of recombinant plasmid standards
[0059] 50 μL of PRRSV and PRV were amplified by ordinary PCR, and the amplified products were purified by gel recovery kit and connected to Peasy-T1 vector. Then 50 μL of Trans1-T1 competent cells were taken from -80℃, 5 μL of connection product was added, ice bathed for 30 minutes, heat shocked at 42 for 90 seconds, ice bathed for 2 minutes, 500 μL of LB liquid culture medium without antibiotics was added, and cultured at 37℃ and 250 r / min for 1 hour. After that, 30 μL was evenly spread on the LB solid plate containing 50 μg / mL ampicillin with a pipette, and cultured at 37℃ for 12 hours. A single white colony was picked from the above-mentioned transformation culture plate, inoculated into 15 mL of LB liquid culture medium containing ampicillin, and cultured at 37℃ and 250 r / min overnight (not more than 16 hours), centrifuged to remove the supernatant, collected the bacteria, and extracted the plasmid. The extraction steps refer to the kit. The recombinant plasmid was identified by the PCR method in step 2.2, and the target fragment was run out. The results are as follows Figure 3 The constructed plasmid was stored in a -80℃ refrigerator under the action of bacterial protection agent until use.
[0060] 2.4 Nanoparticle Duplex PCR Setup and Optimization
[0061] Using the constructed standards, the optimal nanoPCR reaction system was determined by optimizing the PCR reaction conditions: 1 μL of 10-fold diluted nanoparticles (20 nm), 1 μL of upstream and downstream primers (10 mmol / L), 1 μL of template, 12.5 μL of 2× Taq PCR Mastermix, and ddH2O to 25 μL. The optimal reaction conditions were: 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, followed by a 10-minute extension at 72°C. The amplified product was analyzed by gel electrophoresis. Figure 4 As shown, the target gene sizes of the constructed PRRSV and PRV standards amplified by the nano-duplex PCR method were 395 bp and 221 bp, respectively, which are consistent with the amplification value size in step 2.2.
[0062] 2.5 nm Duplex PCR Specificity Verification
[0063] The established PRRSV and PRV nano-duplex PCR method was used to amplify the cDNA or DNA of CSFV, PEDV, TGEV, JEV, and PCV, which can cause abortion in breeding sows. The verification results showed that except for the two clear bands of PRRSV and PRV, no other bands were amplified. The amplification results are shown in Figure 5 This indicates that the established nano-duplex PCR method has strong specificity.
[0064] 2.6 nm Duplex PCR Sensitivity Validation
[0065] The initial concentrations of the plasmid standards were measured by photometer and were 5.17×10 9 copies / μL and 4.64×10 9 Copies / μL. Two plasmid standards were diluted 10-fold respectively, and used as templates, and then diluted 10-fold, with the dilution ratios of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Conventional duplex PCR and nano-PCR were performed. The minimum copy numbers of PRRSV and PRV plasmid standards that could be detected by conventional duplex PCR were 5.17×10 4 copies / μL and 4.64×10 4 copies / μL, while the minimum copy numbers of the two plasmid standards that could be detected by nanoPCR were 5.17×10 2 copies / μL and 4.64×10 2 copies / μL (such as Figure 6 and Figure 7As shown in Figure 3, the sensitivity of the nano-PCR detection method is 100 times higher than that of conventional PCR. This shows that the nano-PCR detection method for PRRSV and PRV established in this experiment has high sensitivity.
[0066] Example 2
[0067] The method of Example 1 was used to perform nano-duplex PCR verification on 10 samples positive for both PRRSV and PRV and 20 samples positive for PRRSV and PRV alone retained by the applicant's laboratory. The constructed verification results showed that the detection results of the nano-duplex PCR were consistent with the detection results of the previous sample registration, with a compliance rate of 100%. The results are shown in Table 2.
[0068] Table 2 Single-plex duplex PCR verification results
[0069]
[0070] Ten suspicious samples were then verified by single-plex and nano-duplex PCR. The results were as follows: Figure 8 The results showed that 2 of the 10 samples were infected with PRRSV and PRV together, 1 was infected with PRRSV, and 2 were infected with PRV. This indicates that the primers for PRRSV and PRV designed in the present invention can be well amplified by nano-duplex PCR, and a single detection operation can simultaneously identify single or co-infection of the two viruses.
[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Fujian Aonong Biotechnology Group Co., Ltd. <120> Dual nano-PCR detection kit for porcine reproductive and respiratory syndrome virus and pseudorabies virus and its application <130> 20211129 <141> 2021-11-30 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ttacacggta tgcccgcttt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ccaggtgaaa ccaattgccg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gatcgtcgtc ctcctgatct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 attgttcttc tgcgatggtg 20
Claims
1. A dual nano-PCR detection kit for porcine reproductive and respiratory syndrome virus and pseudorabies virus, characterized in that: It includes a primer set for detecting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and a primer set for detecting Pseudorabies Virus (PRV). The primer set for detecting Porcine Reproductive and Respiratory Syndrome Virus has the following sequences: Forward primer PRRSV-F: as shown in SEQ ID NO.1 Reverse primer PRRSV-R: as shown in SEQ ID NO.2; The primer set for detecting Pseudorabies Virus has the following sequences: Forward primer PRV-F: as shown in SEQ ID NO.3 Reverse primer PRV-R: as shown in SEQ ID NO.4; It also includes: a positive control, a negative control, colloidal gold nanoparticles, and a PCR amplification solution; The positive control is a recombinant plasmid containing Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Pseudorabies Virus (PRV); the negative control is double-distilled water.
2. Use of the kit according to claim 1 in detecting Porcine Reproductive and Respiratory Syndrome Virus and Pseudorabies Virus, and the use is for non-disease diagnosis purposes.
3. A dual nano-PCR detection method for porcine reproductive and respiratory syndrome virus and pseudorabies virus, characterized in that: The method is to perform nano-dual PCR on a test sample using the kit according to claim 1 to determine whether the test sample contains Porcine Reproductive and Respiratory Syndrome Virus and / or Pseudorabies Virus; the method is for non-disease diagnosis purposes.
4. The method according to claim 3, wherein: The reaction conditions for the nano-dual PCR are: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for extension for 10 min.