Multiplex PCR (Polymerase Chain Reaction) primer group, kit and multiplex PCR detection method for detecting Jerseyvirus bacteriophage

Abstract

The invention belongs to the technical field of biotechnology and diagnostic detection, and discloses a multiple PCR (Polymerase Chain Reaction) primer group, a kit and a multiple PCR detection method for detecting Jerseyvirus bacteriophage. The primer group comprises a primer DpolF1 of which the nucleotide sequence is as shown in SEQ ID No.1, a primer DpolR1 of which the nucleotide sequence is as shown in SEQ ID No.2, a primer TlsF1 of which the nucleotide sequence is as shown in SEQ ID No.3, a primer TlsR2 of which the nucleotide sequence is as shown in SEQ ID No.4, a primer ImmF1 of which the nucleotide sequence is as shown in SEQ ID No.5 and a primer ImmR1 of which the nucleotide sequence is as shown in SEQ ID No.6, the multiplex PCR detection method of the Jerseyvirus bacteriophage comprises the following steps: carrying out PCR amplification on a to-be-detected bacteriophage sample by adopting the primer group, analyzing and judging according to an amplification result, and determining whether the to-be-detected bacteriophage is the Jerseyvirus bacteriophage or not. The detection method has the advantages of simple and rapid operation, strong specificity and high sensitivity, and can rapidly and accurately identify the Jerseyvirus bacteriophage.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and diagnostic detection, and particularly to a multiplex PCR primer set, a kit, and a multiplex PCR detection method for detecting phages of the genus Jerseyvirus. Background Art

[0002] Diseases caused by bacterial infections seriously endanger human public health safety and the healthy development of the livestock and poultry breeding industry. Currently, antibiotics are mainly used clinically for the prevention and treatment of bacterial diseases. However, the long-term and large-scale use of antibiotics has the following consequences: on the one hand, it leads to drug residues in agricultural products and environmental pollution, indirectly threatening human health; on the other hand, under the selective pressure of antibiotics, bacteria are prone to evolve into resistant bacteria, resulting in an increasingly serious problem of bacterial drug resistance. Therefore, there is an urgent need to develop new biological control agents for pathogenic bacteria.

[0003] Bacteriophages are viruses that specifically infect microorganisms such as bacteria and archaea, and have characteristics such as high specificity, wide distribution, low production cost, and high biosafety. As an alternative and complementary therapy to antibiotics, bacteriophages have great application potential in the prevention and treatment of bacterial diseases. Currently, the application of bacteriophages has been deeply studied and promoted in industries such as healthcare and livestock and poultry breeding, such as treating infections caused by superbugs, using them as feed additives to prevent and control bacterial infections, and improving the growth performance of livestock and poultry.

[0004] The application effect of bacteriophage products mainly depends on the stability of the activity of bacteriophages, and the activity of bacteriophages is affected by various environmental factors, among which temperature is one of the important influencing factors. In practice, at various stages such as the production process (such as spray drying, formulation), storage, transportation, and application of bacteriophages, higher temperatures will reduce the activity of bacteriophages, restricting the industrial development and application of bacteriophages. Therefore, isolating and identifying heat-resistant bacteriophages is of great significance for improving the application effect of bacteriophages and reducing production costs.

[0005] The current conventional method for screening heat-resistant bacteriophages is as follows: First, use the host bacteria to proliferate the bacteriophages, then place the bacteriophage proliferation solution at different temperatures for 30 min to 2 h, subsequently gradient dilute the bacteriophage suspension, and count the plaques by overnight culture using the double-layer plate method to measure the bacteriophage titer before and after different temperature treatments, so as to determine the temperature stability of the bacteriophages and screen out heat-resistant bacteriophages. This method is not only cumbersome to operate, but also has a large workload, consuming a lot of time and manpower. Currently, there is no method for quickly identifying and screening heat-resistant bacteriophages, and the existing technology needs to be further improved.

[0006] Phages of the genus Jerseyvirus belong to the order Caudovirales and have a typical icosahedral head and long tail structure; their genomes are all linear double-stranded DNA, about 40.7 - 43.7 kb in size, and do not contain antibiotic resistance genes and virulence genes. Research reports show that phages of this genus are strictly lytic phages, without a lysogenic stage, and mainly infect Salmonella, with high specificity. Therefore, Jerseyvirus phages have good application value in preventing and controlling Salmonella infections. Based on long-term and extensive phage research results, the inventors of this application unexpectedly found that phages of the genus Jerseyvirus generally have the characteristics of high heat resistance, high lysis activity, and a broad host spectrum. Phages of this genus are suitable as applied phages for industrial production and application of phages. However, how to quickly screen and identify phages of the genus Jerseyvirus is the primary problem faced.

[0007] Currently, the main method for identifying the biological classification of phages is through whole-genome sequencing. The sequencing results are compared with the existing phage sequences in the database, and combined with the measured biological characteristics to determine the biological classification of the phages. However, this existing method requires a large amount of manpower, material resources, and time. Therefore, developing a low-cost and rapid method for identifying phages of the genus Jerseyvirus helps to efficiently and conveniently screen out heat-resistant phages for subsequent further research or production. Summary of the Invention

[0008] In view of the above problems, the present invention provides a multiplex PCR primer set, a kit, and a multiplex PCR detection method for detecting phages of the genus Jerseyvirus. The multiplex PCR primer set can be used to quickly identify the phages to be tested by PCR, so as to quickly identify phages of the genus Jerseyvirus and use them as heat-resistant phages for research and application.

[0009] Due to the certain diversity of phages of the genus Jerseyvirus, there is currently no specific and rapid identification method for phages of the genus Jerseyvirus. In view of this problem, the present application provides the following technical solutions:

[0010] In a first aspect, the present application provides a multiplex PCR primer set for detecting phages of the genus Jerseyvirus, the primer set comprising: primer DpolF1 with a sequence as shown in SEQ ID No. 1, primer DpolR1 with a sequence as shown in SEQ ID No. 2, primer TlsF1 with a sequence as shown in SEQ ID No. 3, primer TlsR2 with a sequence as shown in SEQ ID No. 4, primer ImmF1 with a sequence as shown in SEQ ID No. 5, and primer ImmR1 with a sequence as shown in SEQ ID No. 6.

[0011] In the multiplex PCR primer set, the primer pair for detecting the phage Dpol gene is DpolF1 and DpolR1; the primer pair for detecting the phage Tls gene is TlsF1 and TlsR2; the primer pair for detecting the phage Imm gene is ImmF1 and ImmR1.

[0012] The present invention specifically selects three specific conserved regions (Dpol: 35660 - 38758, Tls: 21585 - 22856, and Imm: 7675 - 7973) of the gene sequence of the phage vB_SenS_SP8 strain (ON381768.1) of the genus Jerseyvirus, and correspondingly designs and synthesizes a set of primers DpolF1, DpolR1, TlsF1, TlsR2, ImmF1, and ImmR1 that cooperate with each other. This primer set can effectively distinguish phages of the genus Jerseyvirus from phages of other genera and can be used for the detection of phages of the genus Jerseyvirus.

[0013] When designing primers for the above three specific regions, the inventors designed multiple primers with different sites and lengths. Through a large amount of bioinformatics theoretical analysis and screening and actual sample detection, the primers described in the present invention were finally selected. The primer set provided by the present invention has good amplification specificity, a low mismatch rate, and few hairpin structures, can be used for the specific amplification and detection of phages of the genus Jerseyvirus, quickly and accurately identify phages of the genus Jerseyvirus, and provides a new method for the rapid screening of heat-resistant phages.

[0014] In a second aspect, the present application further provides a multiplex PCR detection kit for phages of the genus Jerseyvirus, which comprises the aforementioned multiplex PCR primer set.

[0015] Optionally, the multiplex PCR detection kit further comprises one or more of: a PCR amplification reaction solution, a positive control. The PCR amplification reaction solution includes a DNA polymerase, a buffer solution, etc.

[0016] Optionally, the amplification reaction solution is 2×PCR Master Mix, and the concentration of the primer is 10 μM.

[0017] In a third aspect, the present application also provides a multiplex PCR detection method for phages of the genus Jerseyvirus. The detection method is as follows: using the aforementioned multiplex PCR primer set to perform PCR amplification on the phage to be detected, and analyzing and judging according to the amplification results to determine whether the phage to be detected is a phage of the genus Jerseyvirus.

[0018] The method for analyzing and judging the amplification results is as follows:

[0019] If the length of the amplification product is one or more of the three bands of 193 bp, 570 bp, or 2355 / 1527 bp, it indicates that the phage sample to be detected is a phage of the genus Jerseyvirus; if there is no band, it indicates that the phage sample to be detected is not a phage of the genus Jerseyvirus.

[0020] Optionally, in the multiplex PCR detection method, the conditions for PCR amplification are as follows: pre-denaturation at 94°C for 5 min and then enter the cycle; denaturation at 94°C for 30 s, annealing at 50 - 58°C for 30 s, and extension at 72°C for 90 s; after 30 cycles, final extension at 72°C for 10 min.

[0021] Preferably, the optimal annealing temperature is 58°C.

[0022] Optionally, in the multiplex PCR detection method, a 25 μl PCR amplification reaction system includes the following components: 12.5 μL of the amplification reaction solution, 0.8 μL of primer DpolF1, 0.8 μL of primer DpolR1, 0.8 μL of primer TlsF1, 0.8 μL of primer TlsR2, 0.8 μL of primer ImmF1, 0.8 μL of primer ImmR1, 2.0 μL of the phage suspension template of the sample to be detected, and the rest is sterile deionized water.

[0023] In a fourth aspect, the present application also provides the application of the aforementioned multiplex PCR primer set in screening phage resources with excellent biological characteristics.

[0024] The phage resources with excellent biological characteristics refer to phages with characteristics such as strong lysis ability and high heat resistance, and can be applied to the preliminary screening or batch screening of target phages.

[0025] In a sixth aspect, the present application also provides the application of the above multiplex PCR primer set in detecting phages of the genus Jerseyvirus.

[0026] The present invention has the following beneficial effects:

[0027] 1. The present invention provides a multiplex PCR primer set for detecting phages of the genus Jerseyvirus. The phage suspension sample to be detected can be quickly and multiplexly PCR amplified and electrophoresed using the primer sets DpolF1, DpolR1, TlsF1, TlsR2, ImmF1 and ImmR1 to quickly identify phages of the genus Jerseyvirus.

[0028] 2. This application also provides a kit and a detection method for detecting phages of the genus Jerseyvirus using the above primer set. Compared with the existing analysis and identification of whole-genome sequencing and biological classification, this method for detecting phages of the genus Jerseyvirus is simple, rapid, highly specific, and highly sensitive, with the lowest phage titer being 1.0×10 4 PFU / mL (20 PFU / reaction).

[0029] 3. The identification method for phages of the genus Jerseyvirus provided by this application can be used to screen heat-resistant phage resources, providing a new path for the screening method of production-type phages. Description of the Drawings

[0030] Figure 1 It is the result diagram of the amplification of vB_SenS_SP8 by each group of primers;

[0031] Among them: Figure A is the result diagram of each group of primers for the Dpol region. Each lane: M: DL2000; 1: DpolF1 + TlsR1; 2: DpolF1 + DpolR2; 3: TlsF1 + TlsR3; 4 - 6: Sterilized deionized water; Figure B is the result diagram of each group of primers for the Tls region. Each lane: M: DL2000; 1: Tls-F1 + Tls-R1; 2: Tls-F1 + Tls-R2; 3 Tls-F1 + Tls-R3; 4: Tls-F2 + Tls-R1; 5: Tls-21 + Tls-R2; 6: Tls-F2 + Tls-R3; 7: Tls-F3 + Tls-R1; 8: Tls-F3 + Tls-R2; 9: Tls-F3 + Tls-R3; 10 - 18: Sterilized deionized water; Figure C is the result diagram of each group of primers for the Imm region. Each lane: M: DL2000; 1: imm-F1 + imm-R1; 2: imm-F1 + imm-R2; 3: imm-F1 + imm-R4; 4: imm-F2 + imm-R1; 5: imm-2 + imm-R2; 6: imm-F2 + imm-R4; 7: imm-F3 + imm-R1; 8: imm-F3 + imm-R2; 9: imm-F3 + imm-R4; 10 - 18: Sterilized deionized water;

[0032] Figure 2 Results diagram of amplification of 15 phages (8 phages of the genus Jerseyvirus and 7 non-Jerseyvirus phages) with primer sets targeting the Dpol region;

[0033] Among them, M: DL2000; 1: vB_SenS_SP8; 2: SHY10; 3: SHY12; 4: SHY15; 5: SHY16; 6: S112; 7: FS77; 8: QE33; 9: DH15; 10: YH11; 11: YH15; 12: YH18; 13: KE88; 14: QS23; 15: GS22; 16: Sterilized deionized water. Figures A - C are the results diagrams of PCR amplification products of primer combinations DpolF1 + DpolR1, DpolF2 + DpolR1, and DpolF3 + DpolR1 respectively;

[0034] Figure 3 Results diagram of amplification of 15 phages (8 phages of the genus Jerseyvirus and 7 non-Jerseyvirus phages) with primer sets targeting the Tls region;

[0035] Among them, M: DL2000; 1: vB_SenS_SP8; 2: SHY10; 3: SHY12; 4: SHY15; 5: SHY16; 6: S112; 7: FS77; 8: QE33; 9: DH15; 10: YH11; 11: YH15; 12: YH18; 13: KE88; 14: QS23; 15: GS22; 16: Sterilized deionized water. Figures A - I are the results diagrams of PCR amplification products of primer combinations TlsF1 + TlsR1, TlsF1 + TlsR2, TlsF1 + TlsR3, TlsF2 + TlsR1, TlsF2 + TlsR2, TlsF2 + TlsR3, TlsF3 + TlsR1, TlsF3 + TlsR2, and TlsF3 + TlsR3 respectively;

[0036] Figure 4 Results diagram of amplification of 15 phages (8 phages of the genus Jerseyvirus and 7 non-Jerseyvirus phages) with primer sets targeting the Imm region;

[0037] Among them, M: DL2000; 1: vB_SenS_SP8; 2: SHY10; 3: SHY12; 4: SHY15; 5: SHY16; 6: S112; 7: FS77; 8: QE33; 9: DH15; 10: YH11; 11: YH15; 12: YH18; 13: KE88; 14: QS23; 15: GS22; 16: Sterilized deionized water. Figures A-I are respectively the results of PCR amplification products of primer combinations ImmF1+immR1, ImmF1+immR2, ImmF1+ImmR3, ImmF2+immR1, ImmF2+immR2, ImmF2+ImmR3, ImmF3+immR1, ImmF3+immR2, ImmF3+ImmR3;

[0038] Figure 5 It is the result diagram of the amplification coverage rate of 212 phages of the genus Jerseyvirus in the NCBI database by multiplex PCR;

[0039] Figure 6 It is the result diagram of the optimization of the annealing temperature for multiplex PCR amplification;

[0040] Among them, M: DL2000; 1: 50.0 °C; 2: 50.4 °C; 3: 51.3 °C; 4: 53.2 °C; 5: 54.8 °C; 6: 56.6 °C; 7: 57.6 °C; 8: 58.0 °C; 9: 50.0 °C; 10: 50.4 °C; 11: 51.3 °C; 12: 53.2 °C; 13: 54.8 °C; 14: 56.6 °C; 15: 57.6 °C; 16: 58.0 °C; 17: Sterilized deionized water; Lanes 1-8 are phage vB_SenS_SP8 of the genus Jerseyvirus, and 9-16 are phage YH15 of the genus Jerseyvirus.

[0041] Figure 7 It is the result diagram of the specificity test of multiplex PCR amplification;

[0042] Among them, M: DL2000; 1: vB_SenS_SP8; 2: SHY10; 3: SHY12; 4: SHY15; 5: SHY16; 6: S112; 7: FS77; 8: QE33; 9: DH15; 10: YH11; 11: YH15; 12: YH18; 13: KE88; 14: QS23; 15: GS22; 16: Sterilized deionized water;

[0043] Figure 8 It is the result diagram of the sensitivity test of multiplex PCR amplification; Among them, M: DL2000; 1: 2.0×10 10 PFU / ml; 2: 2.0×10 9PFU / ml; 3: 2.0×10 8 PFU / ml; 4: 2.0×10 7 PFU / ml; 5: 2.0×10 6 PFU / ml; 6: 2.0×10 5 PFU / ml; 7: 2.0×10 4 PFU / ml; 8: 1.0×10 4 PFU / ml; 9: 5.0×10 3 PFU / ml; 10: 5.0×10 2 PFU / ml; 11: 1.0×10 2 PFU / ml; 12: 5×10 1 PFU / ml; 13: Sterilized deionized water. Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained through commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment, and the "solution" involved is an aqueous solution unless otherwise specified.

[0045] Microbial materials for experiments

[0046] 1. Bacteriophage

[0047] The test bacteriophage vB_SenS_SP8 of the genus Jerseyvirus (NCBI GenBank accession number of the genome: ON381768.1) was isolated and preserved by Qingdao Nuoan Biotech Co., Ltd.

[0048] The test bacteriophages SHY10, S112, FS77, YH15, YH18, KE88, QS23 of the genus Jerseyvirus were isolated and preserved by Qingdao Nuoan Biotech Co., Ltd.

[0049] The test control bacteriophage strains are: SHY12, SHY15, SHY16, QE33, DH15, YH11, GS22, all of which were isolated and preserved by Qingdao Nuoan Biotech Co., Ltd.

[0050] 2. Reagents

[0051] 2×Taq PCR MasterMix and DNA molecular weight standard DL2000 DNA Marker, etc. were all purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0052] Example 1 Primer Design and Screening

[0053] 1.1 Primer Design

[0054] Using the phage genome sequence gene sequences of the genus Jerseyvirus in the National Center of Biotechnology Information (NCBI) database in the United States, three specific conserved regions of the phage vB_SenS_SP8 (GenBank: ON381768.1) of the genus Jerseyvirus were found through BLASTN alignment, which were: the regions of 35660 - 38758 bp (Dpol), 21585 - 22856 bp (Tls), and 7675 - 7973 bp (Imm). ClustalW was used to perform nucleotide sequence alignment and analysis on these three conserved sequences of the phage of the genus Jerseyvirus. The software genefisher2 was used to design multiple degenerate primer sequences for the above three conserved regions respectively, and they were synthesized by Shanghai Sangon Biotech Co., Ltd. The degenerate primer sequences are shown in Table 1 below.

[0055] Table 1 Primer Sequence Information

[0056]

[0057] Based on the above multiple different upstream and downstream primer combinations, multiple different primer sets are shown in Table 2 below.

[0058] Table 2 Primer Combinations

[0059]

[0060]

[0061] 1.2 Screening of Primer Combinations

[0062] 1.2.1 Experimental Method

[0063] 1.2.1.1 Preparation of Phage Suspension

[0064] The phage vB_SenS_SP8 of the genus Jerseyvirus was mixed with its exponential-phase host bacteria according to the optimal MOI until the culture medium became clear. After centrifuging the culture medium, the supernatant was collected and filtered through a 0.22-μm filter membrane to obtain the phage proliferation solution, which was stored at 4°C for later use.

[0065] Phage suspensions of 7 phage strains of the genus Jerseyvirus, namely SHY10, S112, FS77, YH15, YH18, KE88, QS23, and 7 other non-Jerseyvirus phage strains as controls, namely SHY12, SHY15, SHY16, QE33, DH15, YH11, GS22, were all prepared according to the above method and stored at 4°C for later use.

[0066] 1.2.1.2 Construction of the PCR system

[0067] It was configured according to the 25-μL system recommended by the 2×Taq PCR MasterMix kit. Among them, 12.5 μL of 2×Taq PCR MasterMix, 1.0 μL of the upstream and downstream primers of each primer combination (both at a concentration of 10 μM), and 2.0 μL of the bacterial suspension sample were used, and the volume of the reaction system was made up to 25 μL with sterile deionized water. After mixing, PCR amplification was carried out.

[0068] The amplification reaction conditions were as follows: pre-denaturation at 94°C for 5 min and then enter the cycle; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 90 s; after 30 cycles, final extension at 72°C for 10 min. Finally, agarose nucleic acid gel electrophoresis was used to identify the length of the PCR products.

[0069] 1.2.1.3 Bioinformatics analysis of each primer combination

[0070] After retrieving through NCBI, a total of 212 phages of the genus Jerseyvirus were uploaded to the NCBI database. The amplification specificity of each primer combination was analyzed and verified by bioinformatics using these genomic sequences, and a multiplex PCR system for phages of the genus Jerseyvirus was constructed. And the constructed multiplex PCR system was verified by alignment using the software Snapgene to verify its coverage.

[0071] 1.2.2 Experimental results and analysis

[0072] (1) Using each group of primer combinations designed in Table 2 to amplify the phage strain VB_SENS_SP8 of the genus Jerseyvirus, the amplification results are shown in Table 1. This result shows that each group of primer combinations can obtain the target fragment with the expected size.

[0073] (2) For the Tls, Dpol, and Imm regions, the above three regions of 15 phages (the above-mentioned 8 phages of the genus Jerseyvirus and 7 non-Jerseyvirus phages) were amplified using the respective primer combinations described above. The results are shown in Figure 2 、 Figure 3 、 Figure 4 。

[0074] Figure 2 From the results of Figure 3 H, it can be seen that there are no bands for 2 phages of the genus Jerseyvirus in the combination of DpolF3 + DpolR1 (see lanes 13 and 14);

[0075] (3) The amplification specificity of each primer combination was verified by bioinformatics analysis using the genomic sequences of 212 phages of the genus Jerseyvirus on NCBI. The analysis and verification results of the amplification coverage rate are shown in Table 3 below.

[0076] From the results of Table 3, it can be seen that the amplification ratios of the target sequences of each Dpol primer combination are relatively high. The primer combination with the longest product length, namely the combination of DpolF1 and DpolR1, was selected for multiplex PCR construction.

[0077] Since the product lengths of the primer combinations TlsF2 and TlsR2 (222 bp), TlsF3 and TlsR2 (186 bp), and TlsF3 and TlsR3 (369 bp) are similar to the product length of the Imm primer combination (162 - 248 bp), the agarose gel electrophoresis bands will overlap. These three primer combinations were excluded; based on the PCR amplification results (see Figure 2 ), as well as the theoretical amplification ratio results of each primer combination with the phages of the genus Jerseyvirus in the NCNI database (see Table 3), the primer combination of TlsF1 and TlsR2 with a relatively high theoretical amplification number was selected to construct a multiplex PCR system.

[0078] The genomic size range of phages of the genus Jerseyvirus in the NCBI database is generally 40.7 to 43.6 kb, but the genome length of a phage, Salmonella phage L13, is relatively short (21248 bp). This phage only paired successfully with the sequence in the Imm primer combination (using immR1 for the downstream sequence); and since the products of the Imm primers are all relatively short, to prevent experimental deviation caused by non-specific amplification, the primer combination with a longer product length (immF1 and immR1) was selected to construct a multiplex PCR system.

[0079] Therefore, the primers for the multiplex PCR system of phages belonging to the genus Jerseyvirus to be constructed are: DpolF1 + DpolR1, TlsF1 + TlsR2, and ImmF1 + ImmR1.

[0080] Table 3 Amplification coverage of each primer combination for 212 phages belonging to the genus Jerseyvirus in the NCBI database

[0081]

[0082] (4) Use the software Snapgene to perform alignment verification on the above-mentioned multiplex PCR system to be constructed, and the results are shown in Figure 5 , and this result shows that for 212 phages belonging to the genus Jerseyvirus, the above-established multiplex PCR system can successfully match 207 phages, and only 5 phages cannot be successfully matched (Salmonella phage Jersey (NCBI GenBank accession number: NC021777.1), Salmonella phage LSPA1 (NCBI GenBank accession number: NC_026017.1), Salmonellaphage S4lw (NCBI GenBank accession number: OQ660438.1), Salmonella phage L223 (NCBI GenBank accession number: PP034127.1), Salmonella phage Ayanbimpe (NCBI GenBank accession number: PQ139365.1)), and the coverage rate reaches 97.65%.

[0083] Therefore, based on the above results, the primers used for the multiplex PCR system of phages belonging to the genus Jerseyvirus are finally determined as: the combination of DpolF1 (sequence shown in SEQ ID No. 1) and DpolR1 (sequence shown in SEQ ID No. 2), TlsF1 (sequence shown in SEQID No. 3) and TlsR2 (sequence shown in SEQ ID No. 4), and ImmF1 (sequence shown in SEQ ID No. 5) and ImmR1 (sequence shown in SEQ ID No. 6).

[0084] Example 2 Establishment of the multiplex PCR method

[0085] 2.1 Preparation of phage suspension

[0086] The phage vB_SenS_SP8 of the genus Jerseyvirus for the experiment was mixed and cultured with its exponential-phase host bacteria according to the optimal MOI until the culture medium became clear. After centrifuging the culture medium, the supernatant was collected and filtered through a 0.22-μm filter membrane to obtain the phage proliferation solution, which was stored at 4°C for later use.

[0087] The phages of the genus Jerseyvirus for the experiment (SHY10, S112, FS77, YH15, YH18, KE88, QS23) and the control phage strains (SHY12, SHY15, SHY16, QE33, DH15, YH11, GS22) were all prepared into phage suspensions according to the above method and stored at 4°C for later use.

[0088] 2.2. Preparation of reaction solution and optimization of annealing temperature

[0089] It was prepared according to the 25-μL system recommended by the 2×Taq PCR MasterMix kit, including 12.5 μL of 2×Taq PCR MasterMix, 1.0 μL of each of the upstream and downstream primers of each primer combination (6 primers, namely DpolF1 and DpolR1, TlsF1 and TlsR2, and ImmF1 and ImmR1, with each concentration being 10 μM), 2.0 μL of the phage suspension sample, and the volume of the reaction system was made up to 25 μL with sterile deionized water. After mixing, PCR amplification was carried out.

[0090] The amplification reaction conditions were as follows: pre-denaturation at 94°C for 5 min and then entering the cycle; denaturation at 94°C for 30 s,

[0091] annealing at △T (50 - 58°C) for 30 s, extension at 72°C for 90 s; after 30 cycles, final extension at 72°C for 10 min. The length of the PCR product was identified by agarose nucleic acid gel electrophoresis. △T (50 - 58°C) indicates that the annealing temperature was optimized within this range.

[0092] Sixteen experimental groups were set up, using the above amplification system and amplification conditions. The difference was that in experimental groups 1 - 8, phage vB_SenS_SP8 was used as the template (all three primer pairs could amplify), and in experimental groups 9 - 16, phage YH15 was used as the template (DpolF1 - DpolR1 and TlsF1 - TlsR2 could amplify, and the amplified fragment length of DpolF1 - DpolR1 was 1527 bp). The annealing temperatures were set as: 50.0°C, 50.4°C, 51.3°C, 53.2°C, 54.8°C, 56.6°C, 57.6°C, 58.0°C.

[0093] The experimental results are shown in Figure 6This result shows that the target band has relatively high brightness at 50 - 58°C. Therefore, the single PCR annealing temperature of 53°C is continued as the multiple PCR annealing temperature.

[0094] Example 3 Specificity Test of the Multiple PCR System

[0095] 1. Experimental Method

[0096] According to the optimized multiple PCR system and amplification conditions in Example 2, eight phage strains belonging to the genus Jerseyvirus (vB_SenS_SP8, SHY10, S112, FS77, YH15, YH18, KE88, QS23), seven phages not belonging to the genus Jerseyvirus (SHY12, SHY15, SHY16, QE33, DH15, YH11, GS22), and a sterilized deionized water control group were amplified respectively.

[0097] 2. Experimental Results and Analysis

[0098] The experimental results are shown in Figure 7 . This result shows that the amplification results of only the phages belonging to the genus Jerseyvirus are positive, and the amplification results of the remaining phages not belonging to the genus Jerseyvirus are negative. This result indicates that the multiple PCR system and detection method constructed in this application have high specificity.

[0099] Example 4 Sensitivity Test of the Multiple PCR System

[0100] 1. Experimental Method

[0101] The suspension of phage vB_SenS_SP8 was diluted to 2.0×10 10 PFU / ml, 2.0×10 9 PFU / ml, 2.0×10 8 PFU / ml, 2.0×10 7 PFU / ml, 2.0×10 6 PFU / ml, 2.0×10 5 PFU / ml, 2.0×10 4 PFU / ml, 1.0×10 4 PFU / ml, 5.0×10 3 PFU / ml, 5.0×10 2 PFU / ml, 1.0×10 2 PFU / ml, 5×10 1 PFU / ml. Then, using them as templates respectively, amplification was carried out according to the PCR system and amplification conditions in Example 2 to determine the sensitivity of this multiple PCR.

[0102] 2. Experimental Results and Analysis

[0103] The experimental results are shown in Figure 8 , and the results show that the lowest detectable phage titer is 1.0×10 4 PFU / mL (20 PFU / reaction), indicating that the system and method have good sensitivity.

[0104] Example 5 Clinical Application

[0105] 1. Experimental method

[0106] The multiplex PCR detection was performed on 121 Salmonella phage samples (specifically shown in Table 4) isolated and preserved by the applicant using the PCR system and amplification conditions of Example 2.

[0107] 2. Experimental results and analysis

[0108] The experimental results are shown in Table 4. The results show that 41 phages were positive. Randomly select 10 positive samples and send them to Guangdong Megagenomics Technology Co., Ltd. for whole-genome sequencing analysis. After sequence alignment and analysis of the sequencing results, it was found that the 10 samples sent were all phages of the genus Jerseyvirus.

[0109] Table 4 Clinical application results

[0110]

[0111]

[0112]

[0113] Note: Among them, "-" indicates negative; "+" indicates positive.

[0114] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. It can be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and the concept of the present invention, and all such changes or substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A multiplex PCR primer set for detecting bacteriophages of the genus Jerseyvirus, characterized in that: The primer set includes: primer DpolF1 with a nucleotide sequence as shown in SEQ ID No.1, primer DpolR1 with a nucleotide sequence as shown in SEQ ID No.2, primer TlsF1 with a nucleotide sequence as shown in SEQ ID No.3, primer TlsR2 with a nucleotide sequence as shown in SEQ ID No.4, primer ImmF1 with a nucleotide sequence as shown in SEQ ID No.5 and primer ImmR1 with a nucleotide sequence as shown in SEQ ID No.

6.

2. A multiplex PCR detection kit for Jerseyvirus bacteriophage, characterized in that: Comprising the multiplex PCR primer set as claimed in claim 1.

3. The multiplex PCR detection kit according to claim 2, characterized in that: Also includes: One or more of PCR amplification reaction solution and positive control.

4. A multiplex PCR detection method for Jerseyvirus bacteriophage, characterized in that: The detection method comprises: performing PCR amplification on the phage sample to be detected using the multiplex PCR primer set as claimed in claim 1, and analyzing and judging according to the amplification result to determine whether the phage to be detected is a phage of the genus Jerseyvirus.

5. The multiplex PCR detection method according to claim 4, characterized in that: The analysis and judgment method of amplification results is: If the length of the amplified product is one or more of the three bands of 193 bp, 570 bp or 2355 / 1527 bp, it indicates that the phage sample to be tested is a phage of the genus Jerseyvirus; if there is no band, it indicates that the phage sample to be tested is not a phage of the genus Jerseyvirus.

6. The multiplex PCR detection method according to claim 4, characterized in that: The conditions for PCR amplification were as follows: pre-denaturation at 94°C for 5 min before entering the cycle; denaturation at 94°C for 30 s, annealing at 50-58°C for 30 s, and extension at 72°C for 90 s; after 30 cycles, final extension at 72°C for 10 min.

7. The multiplex PCR detection method according to claim 4, characterized in that: The 25ul PCR amplification reaction system includes the following components: 12.5μL of amplification reaction solution, 0.8μL of primer DpolF1, 0.8μL of primer DpolR1, 0.8μL of primer TlsF1, 0.8μL of primer TlsR2, 0.8μL of primer ImmF1 and 0.8μL of primer ImmR1, 2.0μL of phage suspension template of the sample to be tested, and the rest is sterile deionized water.

8. Use of the multiplex PCR primer set as claimed in claim 1 in screening phage resources with excellent biological properties.

9. Use of the multiplex PCR primer set as claimed in claim 1 in detecting bacteriophages of the genus Jerseyvirus.

Images