A multiplex PCR primer set for simultaneously detecting four pathogens, as well as a detection method and kit thereof

Abstract

The present invention provides a primer set for multiplex PCR for the simultaneous detection of four pathogens, a detection method, and a kit thereof, and relates to the field of biological detection technology. The present invention has developed and designed a rapid nucleic acid extraction method for four common pathogens in living minks and a primer set for multiplex PCR detection. The primer set includes upstream and downstream primers for detecting mink canine distemper virus, parvovirus, Aleutian virus, and Pseudomonas aeruginosa, respectively. The nucleic acid extraction method provided by the present invention is simple and rapid to operate, and has a good extraction effect; the primer set, detection system, and detection method for multiplex PCR detection are characterized by high efficiency, strong sensitivity, good specificity, and good repeatability, and overcome the cumbersome steps of single-plex PCR, are easy to operate, simple and efficient, and have pioneering significance.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a primer set for a multiplex PCR for simultaneously detecting four pathogens, a detection method thereof, and a kit. Background Art

[0002] Mink, a key fur-producing animal, has grown to over one million individuals since its introduction and expansion in the 1950s. Effective disease quarantine and timely disease prevention are crucial for ensuring efficient and healthy mink farming (Wang Bao et al., 2009). With advances in medical research, mink has become increasingly widely used as an animal model. Studies have found that establishing a vomiting model using mink can aid in the study of anti-vomiting mechanisms and the screening of new drugs (Yan Wenzhuo et al., 2020). Furthermore, mink is an ideal animal model for human influenza and embryo implantation-related diseases (Sun et al., 2021; Fenelon and Murphy, 2019). However, the use of mink in laboratory experiments is plagued by issues such as a lack of reliable sources and a lack of relevant disease testing systems. Controlling the pathogens carried by mink is essential for the successful use of mink as experimental animals. Therefore, promoting the establishment and improvement of a screening system for experimental mink and developing rapid and accurate detection methods for pathogens carried by mink is crucial for ensuring the safety of experimental personnel, the accuracy of experimental data, and the industrialization of experimental mink use.

[0003] Currently, there is an inadequate system for large-scale testing of common mink diseases. Summer and autumn are peak seasons for mink diseases, primarily acute diarrheal illnesses caused by canine distemper virus and enteritis parvovirus (Sun Shengnan et al., 2020; Zhu Xiangyu et al., 2020), and hemorrhagic pneumonia caused by Pseudomonas aeruginosa (Ma Xiaohui, 2019). These diseases typically develop rapidly and have high mortality rates. Furthermore, the Aleutian mink virus has a wide range of transmission pathways (horizontally and vertically), resulting in a high incidence rate, severely impacting mink reproduction and causing significant economic losses to the mink farming industry (Lu et al., 2021). Clinically, testing for canine distemper and parvovirus diseases often uses commercially available colloidal gold immunoassay strips. While this method is simple and rapid, it is costly and unsuitable for large-scale testing of animal populations. Convection immunoelectrophoresis (CIEP) is the internationally recognized gold standard for Aleutian virus detection (Dam-Tuxen et al., 2014) and is widely used in mink farms worldwide. However, the antigen preparation required by this method is complex and expensive, increasing mink farming costs. Pseudomonas aeruginosa requires laboratory isolation, nucleic acid extraction using kits, and PCR testing, which increases testing costs and is time-consuming. Therefore, it is crucial to develop a rapid, economical, and convenient disease detection system for common mink diseases.

[0004] Polymerase chain reaction (PCR) technology is an in vitro enzymatic method for synthesizing and amplifying specific DNA fragments. Using this method for detecting pathogens at the genomic level has proven to be the most effective detection method to date. Multiplex PCR, developed from conventional PCR, simultaneously performs specific amplification of multiple sites within a single reaction system, saving not only sample volume but also time and effort (Zhong Zecheng et al., 2020). Multiplex PCR detection methods can effectively improve detection efficiency and reduce testing costs. However, a multiplex PCR detection system is not simply the superposition of multiple single-plex PCR detection systems; instead, it requires repeated testing and optimization of multiple technical elements to achieve the optimal reaction system (Huang et al., 2018). The amplification template (sample nucleic acid) is particularly important. Currently, obtaining nucleic acid mostly relies on commercially available kits. Existing nucleic acid extraction kits are mainly divided into bacterial nucleic acid extraction kits and viral nucleic acid (DNA / RNA) extraction kits. For disease samples with mixed bacterial and viral infections, separate nucleic acid extraction kits must be used, increasing both the workload of testers and the cost of testing. Therefore, developing a new, simple and rapid method for the rapid extraction of bacterial and viral nucleic acids in the same reaction system for use in multiplex PCR detection will greatly improve its detection efficiency.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a primer set, nucleic acid extraction method, and detection method for multiplex PCR detection of canine distemper virus (CDV), Aleutian virus (AMDV), parvovirus (MEV), and Pseudomonas aeruginosa (PA) in mink. The present invention enables simultaneous extraction of nucleic acids from a mixed sample of canine distemper virus (CDV), Aleutian virus (AMDV), parvovirus (MEV), and Pseudomonas aeruginosa (PA) using a single reaction system. The resulting product can be directly used in a multiplex PCR reaction to detect these four pathogens. The method is simple, efficient, and has good specificity and sensitivity.

[0007] The technical solutions provided by the present invention are as follows:

[0008] In one aspect, the present invention provides a primer set for multiplex PCR for simultaneously detecting four pathogens, wherein the pathogens are mink canine distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa, and the primer set includes: upstream and downstream primers for canine distemper virus detection, whose sequences are shown in SEQ ID No.1 and SEQ ID No.2; upstream and downstream primers for parvovirus detection, whose sequences are shown in SEQ ID No.3 and SEQ ID No.4; upstream and downstream primers for Aleutian virus detection, whose sequences are shown in SEQ ID No.5 and SEQ ID No.6; and upstream and downstream primers for Pseudomonas aeruginosa detection, whose sequences are shown in SEQ ID No.7 and SEQ ID No.8.

[0009] The present invention designs four pairs of specific primers for multiplex PCR reactions targeting conserved sequences in the genes of four common mink pathogens. Simultaneously detecting multiple different pathogens in a single reaction places high demands on primer design, not only considering the amplification effect of a single primer pair, but also considering and avoiding interactions and cross-reactions between different primers / probes to prevent false positives and other problems. The above-mentioned primer combination of the present invention was selected after trying a large number of primer combinations and found to be the most effective primer combination. Experimental confirmation shows that these primers do not cross-react with each other and have high detection sensitivity and strong specificity.

[0010] In one aspect, the present invention provides a kit for multiplex PCR detection of mink canine distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa, comprising the aforementioned primer set.

[0011] In one embodiment, the kit further comprises a PCR detection reagent; preferably, the PCR detection reagent comprises a PCR reaction buffer, a reaction enzyme, dNTPs, and RNase-free H2O.

[0012] In one embodiment, specific primers for the same pathogen can be packaged in the same tube within the kit, or different primers can be packaged together. In one embodiment, the kit can also contain other components, including, but not limited to, positive controls and negative controls. In one embodiment, the kit also includes reagents for reverse transcription, or can be used in conjunction with a commercially available reverse transcription kit. The kit can be stored at -20°C.

[0013] In one embodiment, the present invention provides an integrated nucleic acid extraction method for a mink sample co-infected with canine distemper virus, parvovirus, Aleutian virus, and Pseudomonas aeruginosa. The method comprises mixing the mixed sample of mink canine distemper virus, parvovirus, Aleutian virus, and Pseudomonas aeruginosa with a lysis buffer, followed by incubation in a water bath. The lysis buffer comprises 90-110 mmol / L Tris-HCl, 10-15 mmol / L EDTA, 0.1-1.0% by volume NP-40, and 0.1-1.0% by volume Tween 20. The present invention also provides an extraction method for simultaneously extracting CDV, AMDV, MEV, and PA nucleic acids in a single reaction system.

[0014] In another aspect, the present invention provides a multiplex PCR detection method for mink distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa for non-disease diagnosis purposes, the method comprising: performing a multiplex PCR amplification reaction on the PCR template using the aforementioned primer set or the aforementioned kit.

[0015] In one embodiment, the method further comprises simultaneously extracting total nucleic acid from the sample to be tested within a single reaction system and performing reverse transcription to obtain the PCR template. In one embodiment, the extraction comprises mixing a mixed sample of mink distemper virus, parvovirus, Aleutian virus, and Pseudomonas aeruginosa with a lysis buffer, followed by simultaneous incubation in a water bath. The lysis buffer comprises 90-110 mmoL / L Tris-HCl; 10-15 mmoL / L EDTA; 0.1-1.0% by volume NP-40; and 0.1-1.0% by volume Tween.

[0016] In a specific embodiment, the lysate includes 1M Tris HCl (pH 8.0) at a final concentration of 100 mM, 0.5M EDTA (pH 8.0) at a final concentration of 10 mM, Tween 80 or Tween 20, 0.5% (v / v); NP-40, 0.5% (v / v); and the remainder is ddH2O.

[0017] In a specific embodiment, proteinase K is added to the above lysate for concentration (20 mg / ml), and the final concentration of proteinase K is 0.6 mg / mL.

[0018] In one embodiment, the water bath incubation condition is 55-60° C. for 25-35 min, followed by 90-95° C. for more than 10 min to inactivate the protease.

[0019] In a specific embodiment, the water bath incubation condition is 55° C. water bath incubation for 30 min, followed by 95° C. water bath incubation for 10 min to inactivate proteinase K.

[0020] In one embodiment, the lysate after lysis with a lysis buffer is reverse transcribed using a reverse transcription kit; preferably, the PerfectStart Uni RT Kit (Cat. No.: AUQ-01) is used.

[0021] For multiplex PCR testing, consistent amplification conditions for all four pathogens are essential, making primer design even more challenging. The designed primer combination, combined with nucleic acid extraction, reverse transcription, and optimized reaction conditions, allows for a better amplification curve. Therefore, optimizing the reaction conditions for multiplex PCR is crucial.

[0022] In one embodiment, when performing the multiplex PCR, the final concentration of each primer in the amplification system is 0.1-1 μM; preferably 0.5-0.8 μM.

[0023] In one embodiment, the strip annealing temperature of the multiplex PCR amplification reaction is 50-62°C.

[0024] In one embodiment, the conditions of the multiplex PCR amplification reaction are: pre-denaturation at 94-95°C for 8-10 min, denaturation at 94-95°C for 10-20 s, 50-62°C for 15-30 s, extension at 72°C for 25-30 s, and 35-45 cycles.

[0025] In a specific embodiment, the optimal reaction system conditions for the multiplex PCR amplification reaction are: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, storage at 4°C, and 35 cycles.

[0026] In one embodiment, the multiplex PCR detection method includes the steps of (1) primer design and synthesis, (2) DNA / RNA extraction and reverse transcription of the test sample, (3) multiplex PCR amplification in a single reaction system, and (4) determination of the results.

[0027] The present invention adopts an improved lysis method to simultaneously extract nucleic acids (DNA / RNA) from the sample to be tested, so that the lysis products undergo a reverse transcription process at the same time, and the cDNA obtained by reverse transcription is used as a template for multiple PCR reactions.

[0028] In one embodiment, the present invention uses 2% agarose gel to determine the results; mink distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa show specific amplification bands at 917bp, 515bp, 366bp and 232bp, respectively.

[0029] In one embodiment, the multiplex PCR method established above can be used to test clinical samples to verify the feasibility of the method and to determine whether the unknown sample is infected with a single virus or a mixed virus.

[0030] In the present invention, four pathogens can be detected simultaneously by testing samples of living animals (oral and nasal secretions, blood, and anal secretions).

[0031] In the present invention, the multiplex PCR detection method is not intended for disease detection and / or treatment, but can be used in other applications, such as laboratory research and analysis of pathogens.

[0032] Beneficial effects:

[0033] The primers and kit provided by the present invention have the advantages of strong sensitivity and good specificity. The detection method of the present invention is highly efficient and can realize the rapid detection of four pathogens simultaneously by a single PCR analysis of samples infected with mixed diseases, and the method has good repeatability and stability.

[0034] The optimized lysis method used in the present invention can lyse viruses and bacteria in the sample within 30 minutes, releasing DNA / RNA. The obtained nucleic acid product can be directly used for reverse transcription. This not only simplifies the nucleic acid extraction step but also greatly reduces the workload and cost of nucleic acid extraction. The nucleic acid obtained by the lysis method has no significant difference in the subsequent PCR detection effect compared with the kit.

[0035] The optimized real-time quantitative PCR system of the present invention ensures that different amplified target fragments have similar amplification efficiencies under the same PCR reaction conditions; the detection method is simple, time-saving and labor-saving.

[0036] The method of the present invention can achieve rapid detection, providing technical support for the purification and research of pathogens in mink populations. At the same time, it also lays a good foundation for the purification of mink diseases in experiments, fills the gap in related technologies, and has pioneering significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1Agarose gel electrophoresis diagram for establishing a multiplex PCR detection method for nucleic acids extracted by the lysis method provided in an embodiment of the present invention; M: Marker; 1: Pseudomonas aeruginosa (PA); 2: Mesovirus (MEV); 3: Aleutian AMDV (AMDV); 4: Canine distemper virus (CDV); 5: Single-tube multiplex PCR electrophoresis diagram for four pathogens;

[0039] Figure 2 A comparison of the effects of nucleic acid extraction using the lysis method and the kit provided in an embodiment of the present invention; wherein, A is an electrophoresis diagram of nucleic acids extracted using the kit: M: Marker; 1: Pseudomonas aeruginosa (PA); 2: Parvovirus (MEV); 3: Mink Aleutian virus (AMDV); 4: Canine distemper virus (CDV); 5: Multiplex PCR electrophoresis diagram of four pathogens; B is an electrophoresis diagram of nucleic acids extracted using the lysis method: the order is the same as above; C is a comparison of multiplex PCR electrophoresis diagrams using two nucleic acid extraction methods: M: Marker; 1: Multiplex PCR electrophoresis diagram of nucleic acids extracted using the kit; 2: Multiplex PCR electrophoresis diagram of nucleic acids extracted using the lysis method;

[0040] Figure 3 Electrophoresis results of reverse transcription template volume optimization provided in an embodiment of the present invention; M: Marker; 1-6: reverse transcription template volumes are 1, 3, 5, 7, and 9 from left to right;

[0041] Figure 4 Electrophoresis results of multiplex PCR template volume optimization provided in an embodiment of the present invention; M: Marker; 1-6: PCR template volumes are 0.5, 1, 2, 3, 4, 5, and 6 μL, respectively;

[0042] Figure 5 Electrophoresis results of optimized multiplex PCR primer concentrations provided in an embodiment of the present invention; M: Marker; 1-7: Reverse transcription template volumes from left to right: 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0 μL, respectively;

[0043] Figure 6 The electrophoresis results of the multiplex PCR annealing temperature optimization provided in the embodiment of the present invention; M: Marker; 1-7: annealing temperatures are 50°C, 53°C, 56°C, 59°C, 62°C, 65°C, and 68°C, respectively;

[0044] Figure 7 Electrophoresis results of a specificity test of the multiplex PCR system provided in an embodiment of the present invention; M: DNA; 1-9: PA, MEV, AMDV, CDV, CAV, CPIV, BVDV, IBRV, E. coli, in that order;

[0045] Figure 8Electrophoresis results of the multiplex PCR sensitivity experiment provided by the embodiment of the present invention; M: DNA Marker DL2000; from top to bottom are CDV, ADV, MEV, PA; 1-9: CDV concentration 1.38×10 10 ~1.38×10 2 copies / μL, and the MEV concentration was 0.87×10 10 ~0.87×10 2 copies / μL, AMDV concentration 0.84×10 10 ~0.84×10 2 copies / μL, PA concentration was 2.12×10 3 ~2.12×10 -6 cfu / μL;

[0046] Figure 9 These are the electrophoresis diagrams of multiplex PCR detection of 32 clinical samples. A-1: ​​represents the electrophoresis diagram of the positive sample, A-(2-17): all are sample electrophoresis diagrams, B-1: represents the electrophoresis diagram of the positive sample, B-(2-17): all are sample electrophoresis diagrams.

[0047] Figure 10 The electrophoresis results of multiplex PCR with different primer pair combinations are shown in Figure 5. M: DNA Marker. 1-4: From top to bottom, the amplification products of CDV, PA, ADV, and MEV, respectively. 5-6: The amplification product of CDV.

[0048] Figure 11 The electrophoresis results of multiplex PCR with different primer pair combinations are shown in Figure 4. M: DNA Marker. 1-4: From top to bottom, the amplified products of CDV, ADV, MEV, and PA, respectively.

[0049] Figure 12 The electrophoresis results of multiplex PCR with different primer pair combinations are shown in Figure 4. M: DNA Marker. 1-4: From top to bottom, the amplified products of CDV, PA, ADV, and MEV are shown.

[0050] Figure 13 : Multiplex PCR electrophoresis results of nucleic acid extracted by conventional alkaline lysis method; M: DNA Marker; 1-2: From top to bottom, the amplification products of CDV, PA, ADV, and MEV are respectively;

[0051] Figure 14 : Multiplex PCR electrophoresis results of nucleic acid extracted by other alkaline lysis methods; M: DNA Marker; 1-4: From top to bottom, they are CDV, ADV, MEV, and PA amplification products;

[0052] Figure 15: Multiplex PCR electrophoresis results of different reverse transcription products; M: DNA Marker; 1-4: From top to bottom, the amplification products of CDV, ADV, MEV, and PA, respectively. DETAILED DESCRIPTION

[0053] 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.

[0054] Example

[0055] 1. Materials and Methods

[0056] 1.1 Sample

[0057] Canine distemper virus and parvovirus were purchased from Petway (canine distemper and parvovirus bivalent vaccine); mink Aleutian virus was kindly provided by Professor Shao Xiqun of the Institute of Specialty Products, Chinese Academy of Agricultural Sciences; and Pseudomonas aeruginosa (PA) was kindly provided by Professor Bai Xue of the Institute of Specialty Products, Chinese Academy of Agricultural Sciences. Clinical samples were collected from affected mink farms near Changli, Hebei Province.

[0058] 1.2 Main reagents and instruments

[0059] TIANamp Bacteria DNA Kit (Cat. No. DP302) and TIANamp Virus DNA / RNA Kit (Cat. No. DP315) were purchased from Tiangen Biochemical Technology Co., Ltd.; the reverse transcription kit PerfectStart Uni RT Kit (Cat. No. AUQ-01) was purchased from Beijing Quanshijin Biotechnology; PrimerStar SH Premix (Cat. No. R040A) was purchased from Takara; the pUC-TAQuick Ligation Kit (Cat. No. CW2592S) was purchased from Kangwei Century; competent E. coli Trans-T1 cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; all conventional reagents used in the experiment were of analytical grade and purchased from Sigma. A gradient PCR instrument (ETC-811) was purchased from Beijing Dongsheng; a nucleic acid electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; and a UV analyzer (JY02S) was purchased from Beijing Junyi.

[0060] 1.3 Experimental methods

[0061] 1.3.1 Primer design and synthesis

[0062] Four pairs of specific primers were designed with reference to the CDV, MEV, AMDV, and PA gene sequences included in GenBank and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 1-1.

[0063] Table 1. Primer sequences and product lengths

[0064]

[0065] 1.3.2 Extraction of viral and bacterial DNA / RNA by lysis method

[0066] (1) Lysis solution formula

[0067] The lysis buffer formula is shown in Table 2.

[0068] Table 2. Lysis buffer formula

[0069]

[0070] Note: Lysis buffer formula (prepared immediately before use): Add 30 μL of proteinase K concentrate (20 mg / ml) to 1 ml of the above lysate, and the final proteinase K concentration is 0.6 mg / ml.

[0071] (2) Lysis and extraction of nucleic acid DNA / RNA

[0072] During the laboratory optimization phase, live vaccines and cultured bacterial cultures were used to obtain DNA / RNA. Specifically, canine distemper, parvovirus, and Aleutian virus cultures were mixed with overnight Pseudomonas aeruginosa cultures. 10 μL of the mixed sample was then added to 30 μL of lysis buffer (Table 2) and suspended. The mixture was then incubated in a 55°C waterbath for 30 minutes, followed by a 10-minute incubation at 95°C to inactivate proteinase K.

[0073] (3) Reverse transcription

[0074] The obtained cleavage product was reverse transcribed using a reverse transcription kit. The reverse transcription system is shown in Table 3.

[0075] Reaction conditions: After thoroughly mixing the liquid, incubate at 50°C for 5 minutes; heat at 85°C for 5 seconds, and store the product at -20°C for subsequent testing. The cDNA obtained by this method is labeled as a cleavage group.

[0076] Table 3. Reverse transcription reaction system (20 μL)

[0077]

[0078] 1.3.3 Establishment of multiplex PCR detection method

[0079] The PCR reaction system is shown in Table 4.

[0080] PCR reaction conditions: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, final extension at 72°C for 10 min, storage at 4°C, for 35 cycles.

[0081] The PCR products were subjected to agarose gel electrophoresis at 2% agarose gel electrophoresis at 120 V, 130 mA, for 40 min, and the results were observed using a gel imaging system.

[0082] Table 4. PCR reaction system

[0083]

[0084] 1.3.4 Identification of multiplex PCR amplification products

[0085] The PCR products containing CDV, MEV, AMDV and PA gene sequences were purified and recovered, and connected to the pUC-TA vector; the ligation products were transformed into competent cells E. coli Trans-T1 using the heat shock method and plated on Amp + LB plates were plated and cultured at 37°C for 16 h. Monoclonal colonies were picked and cultured overnight. Plasmids were extracted according to the instructions of the plasmid extraction kit and PCR identification was performed. The extracted positive plasmids were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the results were compared with the sequences registered in GenBank.

[0086] 1.4. Comparison of kit extraction effects

[0087] Equal volumes of viral and bacterial samples were taken and CDV RNA, MEV, and AMDV DNA were extracted according to the instructions in the viral DNA / RNA extraction kit. DNA from the bacterial group was extracted using a bacterial genomic DNA extraction kit, and the resulting DNA / RNA was labeled the kit group. Finally, nucleic acid products from the lysis group and the kit extraction group were amplified by multiplex PCR under the same conditions, and the products were compared by 2% agarose gel electrophoresis.

[0088] 1.5. Optimization of multiplex PCR reaction conditions

[0089] (1) Reverse transcription template concentration: The nucleic acid product obtained by the lysis method was reverse transcribed, and the template addition volume was set to 1, 3, 5, 7, 9, and 11 μL. The reverse transcription products were subjected to multiple PCR amplification, and the results were observed by 2% agarose gel electrophoresis to determine the optimal reverse transcription template addition amount.

[0090] (2) Multiplex PCR template concentration: The multiplex PCR template volumes were set to 0.5, 1, 2, 3, 4, and 5 μL, respectively. The results were observed after 2% agarose gel electrophoresis to determine the optimal template addition amount for the multiplex PCR reaction.

[0091] (3) Annealing temperature: The annealing temperature gradient of the multiplex PCR reaction was set at 50°C, 53°C, 56°C, 59°C, 62°C, and 65°C. The reaction products were observed by agarose gel electrophoresis to screen out the optimal annealing temperature.

[0092] (4) Primer concentration: Premix the four primer pairs to 10 μM, and add 1, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 μL, respectively, for multiplex PCR amplification. Observe the results by agarose gel electrophoresis, screen out the optimal primer concentration, and optimize the multiplex PCR reaction conditions.

[0093] Specificity experiments

[0094] The nucleic acid extraction method described in 1.3.2 was used to extract nucleic acids such as canine parainfluenza virus (CPIV), canine adenovirus (CAV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and Escherichia coli (E. coli), and amplified using a multiplex PCR system. A positive control was set up at the same time, and specific bands were detected by 2% agarose gel electrophoresis.

[0095] 1.7. Sensitivity test

[0096] Positive plasmids were constructed by ligating CDV, MEV, and AMDV amplification products (fragment sizes of 917 bp, 515 bp, and 366 bp, respectively) with the pUC-TA vector. Concentrations were measured using a UV spectrophotometer and converted to plasmid copy number. Simultaneously, Pseudomonas aeruginosa cultured in LB liquid medium was diluted 10-fold in a gradient, and colonies were counted on LB solid plates. Genomic DNA was extracted from the enumerated bacterial suspension using a lysis method. The extracted DNA was thoroughly mixed with the three positive plasmids described above in a 1:1:1:1 ratio. The mixture was then diluted 10-fold in a gradient with sterile ultrapure water. Multiplex PCR amplification was performed at nine dilutions, and the results were observed by agarose gel electrophoresis to test the sensitivity of the multiplex PCR system established in this experiment.

[0097] 1.8. Clinical sample testing

[0098] Viral and bacterial DNA / RNA were extracted from diseased samples. First, ocular, nasal, and anal secretions were collected from 11 suspected mink samples sent to a clinic in Changli, Hebei Province. A small amount of sterile water was added, the samples were shaken, and then allowed to stand for 5 minutes. The supernatant was aspirated and centrifuged at 12,000 rpm for 3 minutes. The supernatant was discarded, and the pellet was suspended and mixed with 30 μL of lysis buffer (detailed formula see Table 2). After incubation at 55°C for 30 minutes, proteinase K was inactivated in a 95°C water bath for 10 minutes. The products were reverse transcribed and detected using the established multiplex PCR method.

[0099] 2. Results Analysis

[0100] 2.1 Establishment of multiplex PCR detection method and identification of amplification products

[0101] The nucleic acids of canine distemper and parvovirus vaccine, mink Aleutian virus culture fluid and Pseudomonas aeruginosa culture fluid were extracted by lysis method. After reverse transcription, the established multiplex PCR was used to amplify the specific sequences of CDV, AMDV, MEV and PA with the expected sizes. The amplified target fragments were 917 bp, 515 bp, 366 bp and 232 bp respectively. Figure 1 The recovered product was sequenced and showed 100% homology to the GenBank accession sequence. These results demonstrate that the nucleic acid extraction and multiplex PCR pathogen detection methods established in this study are feasible, rapid, specific, and simple.

[0102] 2.2 Comparison with the test kit

[0103] CDV, MEV, AMDV virus solution and overnight cultured PA were extracted using different methods for multiplex PCR verification. The results showed that there was no significant difference in the amplification effect of nucleic acid extracted from lysate and nucleic acid extracted from kit ( Figure 2 ). Therefore, the nucleic acid obtained by treating the sample with the lysis method adopted in the present invention can meet the requirements of multiplex PCR amplification for the concentration of nucleic acid in the sample.

[0104] 2.3 Optimization of multiplex PCR reaction conditions

[0105] The reverse transcription template volume optimization experiment showed that specific bands could be amplified when the template volume was between 5-11 μL ( Figure 3 ); Multiplex PCR template volume optimization experiments showed that specific bands could be amplified when the template volume was between 0.5-5 μL ( Figure 4 ); Optimization experiments on annealing temperature and primer concentration showed that when the annealing temperature was 50-62℃ ( Figure 5 ), when the primer concentration is 1-0.2mmol ( Figure 4 ), multiplex PCR could amplify specific bands.

[0106] 2.4 Specificity test

[0107] The method established by the present invention was used to extract DNA / RNA from cell culture samples of canine distemper virus and parvovirus, Aleutian virus, Pseudomonas aeruginosa, canine parainfluenza virus, and canine adenovirus, which are common in minks. The results of multiple PCR amplification showed that canine distemper virus and parvovirus, Aleutian virus, and Pseudomonas aeruginosa amplified specific bands of expected sizes, respectively, while canine adenovirus (CAV), canine parainfluenza virus (CPIV), bovine viral diarrhea virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), and Escherichia coli (E. coli) were all negative ( Figure 7 ). The experimental results show that the multiplex PCR detection method established by the present invention has good specificity.

[0108] 2.5 Sensitivity test

[0109] The concentration was measured by UV spectrophotometer and converted into plasmid copy number. The result was CDV of 1.38×10 per microliter. 10 copies, MEV is 0.87×10 per microliter 10 copies, ADV is 0.84×10 per microliter 10 The results of colony counting showed that the number of PA bacterial solution was 2.12×10 per microliter. 3 CFU, and the mixed template was serially diluted 10-fold for multiplex PCR verification.

[0110] The results showed that the minimum detection amount of CDV nucleic acid was 1.38×10 2 copies; the minimum nucleic acid detection amount of MEV was 0.87×10 3 The minimum nucleic acid detection amount of AMDV was 0.84×10 4 copies; PA is 2.12×10 per microliter -1 CFU( Figure 8 ).

[0111] 2.6 Clinical Sample Test Results

[0112] Clinical mink samples were collected and nucleic acid was extracted according to the method established in this study. Multiplex PCR detection was performed. The results showed that the CDV infection rate was 44% (14 / 32), the AMDV infection rate was 6% (2 / 32), and the MEV infection rate was 100% (32 / 32). No target band was amplified by PA ( Figure 9 ).

[0113] Comparative Example 1

[0114] The present invention involves the detection of four different pathogens. Simultaneously screening for the most optimal primer combination for multiple pathogen detection requires adapting different reaction systems and procedures to achieve the desired multiplex PCR detection results. Therefore, primer design and screening are time-consuming and labor-intensive. During the screening process, the present invention attempted various primer combinations, but in many cases, they were unsuitable and yielded suboptimal results. The following are some illustrative examples.

[0115] Table 5. Control primer sequences

[0116]

[0117] Multiplex PCR was performed using the control primer sequences. The results showed that the amplified product of PA primer pair No. 9 and No. 10 was 711 bp in size. When multiplex PCR was performed with CDV, AMDV, and MEV primers, the PA, AMDV, and MEV bands became clear and bright as the template volume increased, but the CDV band was absent. When CDV primer pair No. 1 and No. 2 were used alone for PCR amplification, a clear target band was visible ( Figure 10 ).

[0118] The size of the amplified product of PA primer pair No.11 and No.12 was 263 bp. When multiple PCR was performed with CDV, AMDV, and MEV primers, as the template volume continued to decrease, the target bands of CDV (917 bp), AMDV (515 bp), MEV (366 bp), and PA (263 bp) became weaker, and there was a miscellaneous band at 100 bp ( Figure 11 ).

[0119] The amplified product of PA primer pair No.13 and No.14 was 747 bp in size. When multiplex PCR was performed with CDV, AMDV, and MEV primers, the PA, AMDV, and MEV bands became clear and bright as the template volume increased, but the CDV target band disappeared. In addition, the CDV and PA bands in this primer combination were close in size, making it difficult to distinguish the presence or absence of the bands. Figure 12 ).

[0120] Comparative Example 2

[0121] In the present invention, CDV, AMDV, MEV, and PA nucleic acids (DNA / RNA) are simultaneously extracted in a single reaction system using a lysis method. The resulting products are then reverse transcribed for PCR detection. The lysis buffer used in the present invention is the first lysis buffer combination designed for the simultaneous extraction of CDV, AMDV, MEV, and PA nucleic acids. During the research and development process, the present invention also experimented with various types of lysis buffers, but the results were unsatisfactory. The following are illustrative examples.

[0122] When using a conventional lysis buffer (components include 1% SDS, 200mM NaCl, 5mM EDTA, 20mM Tris-HCl (pH 8.0)) for the extraction of CDV, AMDV, MEV, and PA nucleic acids, the specific experimental process is as follows:

[0123] An overnight PA bacterial culture was centrifuged at 10,000 rpm / min for 1 minute, and the supernatant discarded. CDV, AMDV, and MEV viral suspensions were added and mixed thoroughly. 5 mg / ml lysozyme and 20 μl proteinase K were added, and the mixture was allowed to stand at room temperature for 1 minute. An equal volume of lysis buffer was added, and the mixture was incubated at 56°C for 20 minutes. An equal volume of phenol-chloroform-isopropanol (25:24:1) was added, and the mixture was gently shaken for 2-5 minutes. The mixture was centrifuged at 10,000 rpm / min for 5 minutes, and the supernatant was transferred to a new centrifuge tube. Two volumes of anhydrous ethanol were added, and the mixture was precipitated at -20°C for 10 minutes. The mixture was centrifuged at 10,000 rpm / min at 4°C for 15 minutes, and the supernatant discarded. 0.5 ml of 70% ethanol was added, and the mixture was centrifuged at 4°C for 12 minutes, and the supernatant discarded. The ethanol was removed by centrifugation, and the mixture was dried at room temperature. 50 μl of RNase-free water was added. The nucleic acid products were reverse transcribed and analyzed by multiplex PCR.

[0124] The results are as follows Figure 13 As shown: After multiplex PCR detection, the nucleic acid obtained by this lysis method showed poor multiplex effect, the PA target band was missing, and the CDV band was weak.

[0125] In addition, the present invention also tried other lysis methods: the components include (50mM NaOH, 1MTris-HCl pH8.0), the specific method is as follows: add 200μl 50mM sodium hydroxide to the mixed solution of CDV, AMDV, MEV, and PA, lyse in a 95°C water bath for 30 minutes, shake thoroughly, add 20μl 1M Tris-HCl (pH 8.0) to each tube, shake to mix, and centrifuge at 13000rpm / min for 5 minutes. The nucleic acid obtained in the above steps is reverse transcribed, and after multiple PCR amplification reaction, the nucleic acid extraction effect is identified by agarose gel electrophoresis.

[0126] The results showed that only AMDV and PA bands were amplified after the nucleic acid was extracted by lysis method, and no CDV and MEV target bands were found. The nucleic acid extraction effect was poor ( Figure 14 ).

[0127] Comparative Example 3

[0128] The present invention has also found through experiments that, compared with other reverse transcription kits or reverse transcription products, after obtaining the lysis product with the lysis buffer, when it is combined with the commercial reverse transcription kit PerfectStart Uni RT Kit (Cat. No.: AUQ-01) purchased from Beijing Quanshijin Biotechnology for reverse transcription and then performing multiple PCR reaction, the effect is better ( Figure 15 shown).

[0129] 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.

Claims

1. A multiplex PCR kit for simultaneous detection of four pathogens, characterized in that: The pathogens are mink distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa. The kit includes a primer set, which includes: Upstream and downstream primers for canine distemper virus detection, the sequences of which are shown in SEQ ID No. 1 and SEQ ID No. 2; The upstream and downstream primers for parvovirus detection are shown in SEQ ID No. 3 and SEQ ID No. 4; Upstream and downstream primers for Aleutian virus detection, the sequences of which are shown in SEQ ID No. 5 and SEQ ID No. 6; and Upstream and downstream primers for detecting Pseudomonas aeruginosa, the sequences of which are shown in SEQ ID No. 7 and SEQ ID No. 8; The kit also includes a lysis solution, which comprises: 90-110 mmoL / L Tris-HCl, 10-15 mmoL / L EDTA, 0.1-1.0 volume % NP-40, 0.1-1.0 volume % Tween 20, 0.6 mg / mL proteinase K, and the remainder is ddH2O.

2. The kit according to claim 1, wherein The kit also includes PCR detection reagents.

3. The kit according to claim 1, wherein The PCR detection reagent includes PCR reaction buffer, reaction enzyme, dNTPs, and RNase-free H2O.

4. A multiplex PCR method for detecting canine distemper virus, parvovirus, Aleutian virus, and Pseudomonas aeruginosa in mink for non-disease diagnosis purposes, characterized in that: include: The total nucleic acid of the sample to be tested is extracted simultaneously in one reaction system, and reverse transcription is performed to obtain the PCR template; performing a multiplex PCR amplification reaction on the PCR template using the primer set of claim 1 or the kit of any one of claims 1 to 3; The extraction comprises mixing a mixed sample of mink distemper virus, parvovirus, Aleutian virus and Pseudomonas aeruginosa with a lysis solution and then incubating in a water bath. The components of the lysis solution are: 90-110 mmoL / L Tris-HCl, 10-15 mmoL / L EDTA, 0.1-1.0 volume% NP-40, 0.1-1.0 volume% Tween 20, 0.6 mg / mL proteinase K, and the rest is ddH2O.

5. The method according to claim 4, characterized in that The water bath incubation conditions are 55-60°C for 25-35 minutes, followed by 95-100°C for more than 10 minutes to inactivate proteinase K.

6. The method according to claim 4, characterized in that When performing the multiplex PCR, the final concentration of each primer in the amplification system is 0.1-1 μM.

7. The method according to claim 6, characterized in that When performing the multiplex PCR, the final concentration of each primer in the amplification system is 0.5-0.8 μM.

8. The method according to claim 4, characterized in that The annealing temperature of the multiplex PCR amplification reaction is 50-62°C.

9. The method according to any one of claims 6 to 8, characterized in that: The conditions of the multiplex PCR amplification reaction are: pre-denaturation at 94-95°C for 8-10 min, denaturation at 94-95°C for 10-20 s, 50-62°C for 15-30 s, extension at 72°C for 25-30 s, and 35-50 cycles.

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