Primer probe group for detecting and distinguishing infectious pancreatic necrosis viruses 1 and 5 and application of primer probe group

Abstract

The invention discloses a primer probe group for detecting and distinguishing infectious pancreatic necrosis viruses 1 and 5 and application of the primer probe group. The invention provides a primer probe group for detecting and distinguishing infectious pancreatic necrosis virus type 1 and infectious pancreatic necrosis virus type 5. The primer probe group consists of a primer pair (SEQ ID No.1 and SEQ ID No.2) and a probe (SEQ ID No.3) for detecting the infectious pancreatic necrosis virus, a primer pair (SEQ ID No.4 and SEQ ID No.5) for identifying the infectious pancreatic necrosis virus type 1 and a primer pair (SEQ ID No.6 and SEQ ID No.7) for identifying the infectious pancreatic necrosis virus type 5. The primer probe group and the method provided by the invention can specifically, quickly and sensitively detect the IPNV and carry out typing of type 1 or type 5, and can be used for detecting the IPNV in samples of Atlantic salmon, rainbow trout and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer probe set for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5 and an application thereof. Background Art

[0002] Infectious pancreatic necrosis (IPN) is an acute, highly contagious disease that mainly harms salmon and trout. It is caused by infectious pancreatic necrosis virus (IPNV) and has a mortality rate of generally 10%-90%.

[0003] According to the phylogenetic tree analysis of the VP2 / NS protein gene sequence, IPNV can be divided into 7 genotypes (genotype 1-7) (Blake S, Ma JY, D CaPorale, et al. Phylogenetic relationships of aquaticbirnaviruses based on deduced amino acid sequences of genome segment A cDNA[J]. Diseases of Aquatic Organisms, 2001, 45(2): 89-102.; Nishizawa, T. An approach for genogrouping of Ja Panese isolates of aquabirnaviruses in a new genotype, VII, based on the VP2 / NS junction region[J]. Journal of General Virology, 2005, 86 (7): 1973-1978.), VP2 protein is the main protective antigen of IPNV, which is often used for pathogen detection and vaccine development. However, the VP2 protein of IPNV of different genotypes not only has large sequence differences, but also has different protein sizes. For example, the VP2 gene sequence of infectious pancreatic necrosis virus type 1 strain is usually 1326bp, while the VP2 gene sequence of infectious pancreatic necrosis virus type 5 strain can be 1536 or 1539bp. This situation leads to poor conservatism of the IPNV strain, which belongs to different genotypes and serotypes (references: Duan, K.; Zhao, J.; Ren, G.; Shao, Y.; Lu, T.; Xu, L.; Tang, X.; Zhao, W.; Xu, L. Molecular Evolution of Infectious Pancreatic Necrosis Virus in China. Viruses, 2021, 13, 488.), which increases the difficulty of pathogen detection and vaccine development. The main prevalent genome types in my country are type 1 and type 5, among which type 1 is dominant (Duan, K.; Zhao, J.; Ren, G.; Shao, Y.; Lu, T.; Xu, L.; Tang, X.; Zhao, W.; Xu, L. Molecular Evolution of Infectious Pancreatic Necrosis Virus in China. Viruses, 2021, 13, 488.), which is listed as a Category III animal epidemic in my country.

[0004] At present, there is no systematic technical means in China to quickly identify and differentiate between type 1 and type 5 IPNV. Summary of the invention

[0005] The purpose of the present invention is to provide a primer probe set for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5 and an application thereof.

[0006] In a first aspect, the present invention claims a primer probe set for detecting and distinguishing between infectious pancreatic necrosis virus type 1 and type 5.

[0007] The primer probe set for detecting and distinguishing infectious pancreatic necrosis virus type 1 (genotype I) and type 5 (genotype V) claimed in the present invention consists of the following:

[0008] (A1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2;

[0009] (A2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end;

[0010] (A3) A primer pair for identifying infectious pancreatic necrosis virus type 1: consisting of two primers shown in SEQ ID No. 4 and SEQ ID No. 5;

[0011] (A4) Primer pair for identifying infectious pancreatic necrosis virus type 5: consisting of two primers shown in SEQ ID No. 6 and SEQ ID No. 7.

[0012] In one embodiment of the present invention, in (A2), the fluorescent group is labeled at the 5' end, and the quenching group is labeled at the 3' end. The fluorescent group is specifically FAM, and the quenching group is specifically MGB.

[0013] In a second aspect, the present invention claims a primer probe set for detecting infectious pancreatic necrosis virus type 1.

[0014] The primer probe set for detecting infectious pancreatic necrosis virus type 1 (genotype I) claimed in the present invention consists of the following:

[0015] (B1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2;

[0016] (B2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end;

[0017] (B3) Primer pair for identifying infectious pancreatic necrosis virus type 1: consisting of two primers shown by SEQ ID No.4 and SEQ ID No.5.

[0018] In one embodiment of the present invention, in (B2), the fluorescent group is labeled at the 5' end, and the quenching group is labeled at the 3' end. The fluorescent group is specifically FAM, and the quenching group is specifically MGB.

[0019] In a third aspect, the present invention claims a primer probe set for detecting infectious pancreatic necrosis virus type 5.

[0020] The primer probe set for detecting infectious pancreatic necrosis virus type 5 (genome type V) claimed in the present invention is composed of the following:

[0021] (C1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2;

[0022] (C2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end;

[0023] (C3) Primer pair for identifying infectious pancreatic necrosis virus type 5: consisting of two primers shown by SEQ ID No.6 and SEQ ID No.7.

[0024] In one embodiment of the present invention, in (C2), the fluorescent group is labeled at the 5' end, and the quenching group is labeled at the 3' end. The fluorescent group is specifically FAM, and the quenching group is specifically MGB.

[0025] In a fourth aspect, the present invention claims a kit comprising the primer-probe set described in any one of the first to third aspects above.

[0026] Furthermore, in the kit containing the primer probe set described in the first aspect above, standard plasmid 1 and standard plasmid 2 may also be contained. In the kit containing the primer probe set described in the second aspect above, standard plasmid 1 may also be contained. In the kit containing the primer probe set described in the third aspect above, standard plasmid 2 may also be contained. Standard plasmid 1 (standard plasmid corresponding to the VP2 gene of IPNV 1) is a plasmid carrying the DNA fragment shown in SEQ ID No. 8; standard plasmid 2 (standard plasmid corresponding to the VP2 gene of IPNV 5) is a plasmid carrying the DNA fragment shown in SEQ ID No. 9.

[0027] In a fifth aspect, the present invention claims protection for any of the following applications:

[0028] (D1) Use of the primer probe set described in the first aspect above in the preparation of a product for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5;

[0029] (D2) Use of the primer probe set described in the second aspect above in the preparation of a product for detecting infectious pancreatic necrosis virus type 1;

[0030] (D3) Use of the primer probe set described in the third aspect above in the preparation of a product for detecting infectious pancreatic necrosis virus type 5.

[0031] Among them, the detection and differentiation of infectious pancreatic necrosis virus type 1 and type 5 includes: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 1 and / or infectious pancreatic necrosis virus type 5.

[0032] The detecting of infectious pancreatic necrosis virus type 1 comprises: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 1.

[0033] The detection of infectious pancreatic necrosis virus type 5 includes: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 5.

[0034] In a sixth aspect, the present invention claims a non-disease diagnostic method for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5. The detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5 comprises: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 1 and / or infectious pancreatic necrosis virus type 5.

[0035] The non-disease diagnostic method for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5 claimed in the present invention may include the following steps:

[0036] (E1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA;

[0037] (E2) using the cDNA as a template, performing fluorescent quantitative PCR amplification using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus according to the Ct value and the amplification curve as follows:

[0038] If the Ct value is ≤35 and there is a typical amplification curve, the sample to be tested is determined to be positive, that is, it contains infectious pancreatic necrosis virus;

[0039] If 1) the Ct value is greater than 40, or 2) there is no Ct value and a typical amplification curve, the sample to be tested is determined to be negative, i.e., it does not contain infectious pancreatic necrosis virus;

[0040] If 35<Ct value≤40, repeat the test, if there is still a Ct value (Ct value≤40) and a typical amplification curve, the sample to be tested is determined to be positive, otherwise it is negative.

[0041] If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following tests (E3) and (E4); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further tests are performed;

[0042] (E3) using the cDNA as a template, performing PCR amplification using the primer pair shown by SEQ ID No.4 and SEQ ID No.5, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 1 as follows: if the amplified product contains a target fragment of 502 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 1; if the amplified product does not contain the target fragment of 502 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 1;

[0043] (E4) using the cDNA as a template and using the primer pair shown by SEQ ID No.6 and SEQ ID No.7 to perform PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 5 as follows: if the amplified product contains a target fragment of 543 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 5; if the amplified product does not contain the target fragment of 543 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 5.

[0044] In a seventh aspect, the present invention claims a non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 1. The detection of infectious pancreatic necrosis virus type 1 comprises: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 1.

[0045] The non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 1 claimed in the present invention comprises the following steps:

[0046] (F1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA;

[0047] (F2) using the cDNA as a template, performing fluorescent quantitative PCR amplification using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus according to the Ct value and the amplification curve as follows:

[0048] If the Ct value is ≤35 and there is a typical amplification curve, the sample to be tested is determined to be positive, that is, it contains infectious pancreatic necrosis virus;

[0049] If 1) the Ct value is greater than 40, or 2) there is no Ct value and a typical amplification curve, the sample to be tested is determined to be negative, i.e., it does not contain infectious pancreatic necrosis virus;

[0050] If 35<Ct value≤40, repeat the test, if there is still a Ct value (Ct value≤40) and a typical amplification curve, the sample to be tested is determined to be positive, otherwise it is negative.

[0051] If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following test (F3); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further test is performed;

[0052] (F3) using the cDNA as a template and using the primer pair shown by SEQ ID No.4 and SEQ ID No.5 to perform PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 1 as follows: if the amplified product contains a target fragment of 502 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 1; if the amplified product does not contain the target fragment of 502 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 1.

[0053] In an eighth aspect, the present invention claims a non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 5. The detection of infectious pancreatic necrosis virus type 5 comprises: detecting whether the sample to be tested contains infectious pancreatic necrosis virus, and if so, further detecting whether the infectious pancreatic necrosis virus contained is infectious pancreatic necrosis virus type 5.

[0054] The non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 5 claimed in the present invention may include the following steps:

[0055] (G1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA;

[0056] (G2) Using the cDNA as a template, using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3 to perform fluorescent quantitative PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus according to the Ct value and the amplification curve as follows:

[0057] If the Ct value is ≤35 and there is a typical amplification curve, the sample to be tested is determined to be positive, that is, it contains infectious pancreatic necrosis virus;

[0058] If 1) the Ct value is greater than 40, or 2) there is no Ct value and a typical amplification curve, the sample to be tested is determined to be negative, i.e., it does not contain infectious pancreatic necrosis virus;

[0059] If 35<Ct value≤40, repeat the test, if there is still a Ct value (Ct value≤40) and a typical amplification curve, the sample to be tested is determined to be positive, otherwise it is negative.

[0060] If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following test (G3); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further testing is performed;

[0061] (G3) Using the cDNA as a template, PCR amplification is performed using the primer pair shown by SEQ ID No.6 and SEQ ID No.7, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 5 as follows: if the amplified product contains a target fragment of 543 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 5; if the amplified product does not contain the target fragment of 543 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 5.

[0062] In the sixth to eighth aspects above, the non-disease diagnosis method may specifically be simple virus identification and genotyping research, etc.

[0063] In the sixth to eighth aspects above, when performing the fluorescent quantitative PCR amplification, the final concentrations of the upstream and downstream primers and probes in the reaction system can both be 5 μM.

[0064] In the sixth to eighth aspects, when performing the fluorescent quantitative PCR amplification, the annealing temperature may be 55° C. Specifically, when performing the fluorescent quantitative PCR amplification, the reaction procedure is: 94° C. for 30 s; 94° C. for 5 s, 55° C. for 20 s (40 cycles).

[0065] In one embodiment of the present invention, the sample to be tested is selected from: infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus type 1, and infectious pancreatic necrosis virus type 5.

[0066] Furthermore, the sample to be tested may be a tissue sample from fish such as Atlantic salmon and rainbow trout.

[0067] In a specific implementation case of the present invention, the infectious pancreatic necrosis virus type 1 is selected from the following: GS2017-1 strain, HLJ2019-5 strain, HLJ2019-4 strain, GS2019-2 strain, GS2017-2 strain, XZ2019-2 strain, LN2018-1 strain, HLJ2019-2 strain, JL2019-1 strain, QH2019-1 strain, HLJ2019-1 strain, GS2019-1 strain, HLJ2019-3 strain, LN2019-2 strain, GS2018-1 strain, LN2019-1 strain, ChRtm213 strain, XZ2019-1 strain, GS2019-3 strain. The infectious pancreatic necrosis virus type 5 is selected from the following: GS2020-1 strain, GS2020-2 strain, BJ2020-1 strain.

[0068] The primer probe set and method provided by the present invention can detect IPNV specifically, quickly and sensitively, and can perform type 1 or type 5 typing and construct a virus phylogenetic tree, and can be used for IPNV detection in samples such as Atlantic salmon and rainbow trout. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The number of cycles for detecting positive signals under different primer and probe concentrations. Different lowercase letters indicate significant differences (P<0.05).

[0070] Figure 2 The numbers of cycles at which positive signals were detected were different. In the figure, * indicates significant difference (P<0.05).

[0071] Figure 3 This is a specific analysis of the detection of IPNV by fluorescence quantitative PCR. In the figure, NTC represents the negative control, and DEPC water is used as the template.

[0072] Figure 4 This is the standard curve for detecting IPNV by fluorescence quantitative PCR.

[0073] Figure 5 This is the sensitivity test of fluorescent quantitative PCR detection of IPNV. In the figure, 1-9 represents the standard plasmid concentration of 10 1 -10 9 copies / μL; NTC represents negative control, using DEPC water as template.

[0074] Figure 6 This is the gel electrophoresis diagram of IPNV 1 / 5 typing primer PCR identification. The lanes are as follows: 1. GS2017-1; 2. HLJ2019-5; 3. GS2020-1; 4. HLJ2019-4; 5. GS2019-2; 6. GS2017-2; 7. XZ2019-2; 8. LN2018-1; 9. GS2020-2; 10. HLJ2019-2; 11. JL2019-1; 12.QH2019-1; 13.HLJ2019-1; 14.GS2019-1; 15.HLJ2019-3; 16.LN2019-2; 17.BJ 2020-1; 18.GS2018-1; 19.LN2019-1; 20.ChRtm213; 21.XZ2019-1; 22.GS2019-3. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0076] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0077] Type 1 IPNV (IPNV 1) ChRtm213 and type 5 IPNV (IPNV 5) BJ2020-1: recorded in the article "Duan, K.; Zhao, J.; Ren, G.; Shao, Y.; Lu, T.; Xu, L.; Tang, X.; Zhao, W.; Xu, L. Molecular Evolution of Infectious Pancreatic Necrosis Virus in China. Viruses, 2021, 13, 488.", which can be obtained by the public from the applicant and can only be used to repeat the experiments of the present invention and may not be used for other purposes.

[0078] Infectious hematopoietic necrosis virus (IHNV) LN15: recorded in “Lin Y, Ren G, Zhao J, et al. Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss). Microbiol Spectr. 2022; 10(6): e0324522.”, which is available to the public from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes.

[0079] Example 1: Design of a primer probe set for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5

[0080] The following primers were designed based on the VP2 gene sequence characteristics of infectious pancreatic necrosis virus type 1 and type 5 IPNV isolates: SEQ ID No.1-3 are primer probe sets for fluorescent probe PCR detection of IPNV, SEQ ID No.4-5 are specific PCR detection primers for type 1 IPNV, and SEQ ID No.6-7 are specific PCR detection primers for type 5 IPNV.

[0081] SEQ ID No. 1: 5'-AAGTAYGACCCYGARGGYCT-3' (Y is C or T, R is A or G).

[0082] SEQ ID No. 2: 5'-ATGTCCAGCTCCTCYCTGTG-3' (Y is C or T).

[0083] SEQ ID No. 3: 5'FAM-AACTATGCCAAGATGATCCTGTC-3'MGB.

[0084] SEQ ID No. 4: 5'-CGCTGGTCTGTATGCACTCAATG-3'.

[0085] SEQ ID No. 5: 5'-CGTGGAGCTTGTCACGGTGA-3'.

[0086] SEQ ID No. 6: 5'-CGACATTCAAAGCTCCACACTACCG-3'.

[0087] SEQ ID No. 7: 5'-TTGTCGTTTTGTGGCCAGCACG-3'.

[0088] The theoretical amplification product size of the specific PCR detection primers SEQ ID No. 4 and SEQ ID No. 5 for type 1 IPNV is 502 bp, and the theoretical amplification product size of the specific PCR detection primers SEQ ID No. 6 and SEQ ID No. 7 for type 5 IPNV is 543 bp.

[0089] Example 2: Determination of optimal primer and probe concentrations

[0090] Prepare the reaction system shown in Table 1.

[0091] Table 1. Composition of 20 μL reaction system

[0092]

[0093] Note: The volumes of IPNV-F, IPNV-R, and IPNV-Probe in the table are the same, and the following gradients are set: 0.4 μL, 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, 1.4 μL.

[0094] Among them, 2×Premix Ex Taq comes from the kit Premix Ex Taq TM (Probe qPCR) (Takara, RR390A); forward primer IPNV-F (SEQ ID No.1), reverse primer IPNV-R (SEQ ID No.2), and probe IPNV-Probe (SEQ ID No.3) were synthesized by Jiansu Gene (Harbin, China), with a concentration of 10 μM. The template was RNA of type 1 IPNV ChRtm213 strain was extracted using PrimeScript TM The type 1 IPNV cDNA was obtained by reverse transcription using One Step RT-PCR Kit Ver.2 (Takara, 6210A); DEPC water was a product of Biosharp (BL510B).

[0095] The reaction procedure was carried out according to the standard procedure recommended by the kit, namely: 94°C for 30s; 94°C for 5s, 60°C for 20s (40 cycles). The optimal primer probe concentration in the reaction system was determined based on the minimum number of cycles for the appearance of a positive signal.

[0096] Among them, positive or negative is determined as follows:

[0097] If the Ct value is ≤35 and there is a typical amplification curve, the sample to be tested is determined to be positive, that is, it contains infectious pancreatic necrosis virus;

[0098] If 1) the Ct value is greater than 40, or 2) there is no Ct value and a typical amplification curve, the sample to be tested is determined to be negative, i.e., it does not contain infectious pancreatic necrosis virus;

[0099] If 35<Ct value≤40, repeat the test, if there is still a Ct value (Ct value≤40) and a typical amplification curve, the sample to be tested is determined to be positive, otherwise it is negative.

[0100] The results are as follows Figure 1 As shown, when the concentrations of primers and probes in the reaction system were 0.5, 0.6, and 0.7 μM (corresponding to 1.0, 1.2, and 1.4 μL in Table 1, respectively), the number of cycles was the lowest and there was no significant difference among the three groups. Therefore, 0.5 μM with the least amount was selected as the optimal primer and probe concentration.

[0101] Example 3: Determination of the Optimum Annealing Temperature

[0102] The reaction system was prepared with the optimal primer probe concentration in the reaction system determined in Example 2 (see Table 1), and the annealing extension stage temperature in the standard program was changed to 55, 56.2, 57.1, 58.2, 59.3, 60.4, 61.3, 62.1, 63°C, and the optimal annealing temperature was determined according to the minimum number of cycles for the appearance of a positive signal. The determination of a "positive signal" refers to Example 2.

[0103] The results are as follows Figure 2 As shown, when the annealing temperature is 55°C, the cycle number is significantly lower than that of other groups. Although the cycle number at 56.2°C is not significantly different from that at 55°C, it is also not significantly different from that at 57.1°C. Therefore, 55°C is selected as the optimal annealing temperature.

[0104] Example 4: Specificity Detection

[0105] Using the optimal primers, probe concentrations and annealing temperatures determined in Examples 2 and 3, fluorescent probe PCR was performed on the cDNAs of IPNV type 1 (IPNV 1) ChRtm213, IPNV type 5 (IPNV 5) BJ2020-1, infectious hematopoietic necrosis virus (IHNV) LN15, and viral hemorrhagic septicemia virus (VHSV) (completed with reference to Examples 2 and 3) to verify their specificity.

[0106] The results are as follows Figure 3 As shown, the fluorescence quantitative PCR had specific amplification curves for IPNV 1 and IPNV 5 cDNAs with Ct values ​​≤ 35, while there was no nonspecific amplification reaction for IHNV and VHSV cDNAs.

[0107] Example 5: Sensitivity Detection

[0108] The standard plasmids p-IPNV 1VP2 and p-IPNV 5VP2 containing IPNV 1 or IPNV 5VP2 genes (the original plasmids were the plasmid pIHNV-Sn1203 in the article “Zhao JZ, Xu LM, Zhang ZY, et al. Recovery of recombinant infectious hematopoietic necrosis virus strain Sn1203 using the mammalian cell line BHK-21. J Virol Methods. 2019; 265: 84-90. doi: 10.1016 / j.jviromet.2019.01.002”) were cloned and the VP2 CDS sequence of genomic type I IPNV (SEQ ID No. 8) and type V IPNV (SEQ ID No. 9) were used to replace the VP2 CDS sequence of IPNV I and IPNV V in the pIHNV-Sn1203 plasmid, respectively. CDS of P protein gene) was diluted to 10 1 -10 9 copies / μL, and perform fluorescent probe PCR detection (completed with reference to Examples 2 and 3) using each dilution as a template according to the optimal primer probe concentration and annealing temperature determined in Examples 2 and 3, draw a standard curve and determine the detection limit.

[0109] The standard curve is as follows Figure 4 As shown, in 10 1 -10 9 In the concentration range of 100 copies / μL, the regression equation of the standard curve of IPNV1 detected by fluorescence quantitative PCR is y=-3.510lg copies / μL +39.38, R 2 =0.9993, the regression equation of the standard curve of IPNV 5 is y=-3.584lg copies / μL +38.93, R 2 =0.9993. The horizontal axis is the logarithmic value of the template concentration, and the vertical axis is the Ct value. This indicates that there is a good linear relationship between the logarithmic value of the template concentration and the corresponding cycle number, the reaction system is stable, and the detection efficiency of fluorescent quantitative PCR for IPNV 1 and IPNV 5 is similar.

[0110] Sensitivity test results are as follows Figure 5 As shown, in 10 1 -10 9Within the standard plasmid concentration range of 100 copies / μL, the fluorescence quantitative PCR had typical amplification curves for IPNV 1 and IPNV 5 with Ct values ​​≤ 35, and the minimum detection limit was 10 copies / μL.

[0111] Example 6: Repeatability test

[0112] The optimal primer, probe concentration and annealing temperature determined in Examples 2 and 3 were respectively used for 10 7 , 10 5 , 10 3 The p-IPNV 1VP2 plasmid with 100 copies / μL was subjected to intra-batch and inter-batch reproducibility fluorescent probe PCR detection (as performed in reference to Examples 2 and 3), and the intra-batch and inter-batch coefficients of variation were determined: CV = (standard deviation SD / mean value Mean) × 100%.

[0113] The results are shown in Table 2 and Table 3. Fluorescence quantitative PCR detection 10 7 , 10 5 , 10 3 The intra-batch and inter-batch coefficients of variation of copies / μL were both lower than 1%, indicating that the fluorescence quantitative PCR had good repeatability and stable test results.

[0114] Table 2. Fluorescence quantitative PCR detection of IPNV 1 intra-batch duplication

[0115]

[0116]

[0117] Table 3. Repeatability of IPNV 1 detected by fluorescence quantitative PCR

[0118]

[0119] Example 7: Sample testing and genotyping

[0120] Take 30 identified samples stored in this laboratory, sample numbers 1-30, of which 22 are positive samples and 8 are negative samples. Use the cDNA extracted from the sample as a template, perform fluorescent probe PCR detection according to the optimal primer probe concentration and annealing temperature determined in Examples 2 and 3 (completed with reference to Examples 2 and 3), and determine the detection results after the reaction is completed. Among them, 22 positive samples were 22 known IPNV 1 / 5 isolates (including “19 strains of IPNV 1: GS2017-1, HLJ2019-5, HLJ2019-4, GS2019-2, GS2017-2, XZ2019-2, LN2018-1, HLJ2019-2, JL2019-1, QH2019-1, HLJ2019-1, GS2019-1, HLJ2019-3, LN2019-2, GS2018-1, LN2019-1, ChRtm213, XZ2019-1, GS2019-3” and “3 strains of IPNV 5: GS2020-1, GS2020-2, BJ2020-1", all recorded in "Duan, K.; Zhao, J.; Ren, G.; Shao, Y.; Lu, T.; Xu, L.; Tang, X.; Zhao, W.; Xu, L. Molecular Evolution of Infectious Pancreatic Necrosis Virus in China. Viruses, 2021, 13, 488.").

[0121] The results of fluorescence quantitative PCR test for 30 samples are shown in Table 4. Samples 1 to 22 are IPNV positive samples, and samples 23 to 30 are IPNV negative samples. The virus identification result coincidence rate is 100%. The positive samples were typed and the results are shown in Table 4. Figure 6 As shown, PCR detection was performed on the cDNA of 22 IPNV isolates (19 strains of IPNV type 1 and 3 strains of IPNV type 5 were identified) using IPNV type 1 detection primers and IPNV type 5 detection primers, respectively. The results showed that type 1 detection primers (target band 502bp) and type 5 detection primers (target band 543bp) detected 19 strains of IPNV 1 and 3 strains of IPNV 5, respectively, and the genotyping result consistency rate was 100%.

[0122] Table 4. Test results of 30 samples

[0123]

[0124]

[0125] Note: P: IPNV positive; N: IPNV negative; 1: genotype 1; 5: genotype 5; -: no Ct value or genotype.

[0127] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A primer probe set for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5, comprising: (A1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2; (A2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end; (A3) A primer pair for identifying infectious pancreatic necrosis virus type 1: consisting of two primers shown in SEQ ID No. 4 and SEQ ID No. 5; (A4) Primer pair for identifying infectious pancreatic necrosis virus type 5: consisting of two primers shown in SEQ ID No. 6 and SEQ ID No.

7.

2. A primer probe set for detecting infectious pancreatic necrosis virus type 1, comprising: (B1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2; (B2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end; (B3) Primer pair for identifying infectious pancreatic necrosis virus type 1: consisting of two primers shown by SEQ ID No.4 and SEQ ID No.

5.

3. A primer probe set for detecting infectious pancreatic necrosis virus type 5, comprising: (C1) A primer pair for detecting infectious pancreatic necrosis virus: consisting of two primers shown in SEQ ID No. 1 and SEQ ID No. 2; (C2) A probe for detecting infectious pancreatic necrosis virus: a fluorescent group is labeled at one end of the single-stranded DNA shown in SEQ ID No. 3, and a quenching group is labeled at the other end; (C3) Primer pair for identifying infectious pancreatic necrosis virus type 5: consisting of two primers shown by SEQ ID No.6 and SEQ ID No.

7.

4. A kit comprising the primer-probe set according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that: The kit containing the primer probe set of claim 1 further contains standard plasmid 1 and standard plasmid 2; The kit containing the primer probe set of claim 2 further contains the standard plasmid 1; The kit containing the primer probe set of claim 3 further contains the standard plasmid 2; The standard plasmid 1 is a plasmid carrying the DNA fragment shown in SEQ ID No.8; The standard plasmid 2 is a plasmid carrying the DNA fragment shown in SEQ ID No.

9.

6. Any of the following applications: (D1) Use of the primer probe set of claim 1 in the preparation of a product for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5; (D2) Use of the primer probe set of claim 2 in the preparation of a product for detecting infectious pancreatic necrosis virus type 1; (D3) Use of the primer probe set described in claim 3 in the preparation of a product for detecting infectious pancreatic necrosis virus type 5.

7. A non-disease diagnostic method for detecting and distinguishing infectious pancreatic necrosis virus type 1 and type 5, comprising the following steps: (E1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA; (E2) using the cDNA as a template, performing fluorescent quantitative PCR amplification using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus based on the Ct value and the amplification curve: If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following tests (E3) and (E4); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further tests are performed; (E3) using the cDNA as a template, performing PCR amplification using the primer pair shown by SEQ ID No.4 and SEQ ID No.5, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 1 as follows: if the amplified product contains a target fragment of 502 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 1; if the amplified product does not contain the target fragment of 502 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 1; (E4) using the cDNA as a template and using the primer pair shown by SEQ ID No.6 and SEQ ID No.7 to perform PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 5 as follows: if the amplified product contains a target fragment of 543 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 5; if the amplified product does not contain the target fragment of 543 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 5.

8. A non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 1, comprising the following steps: (F1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA; (F2) using the cDNA as a template, using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3 to perform fluorescent quantitative PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus according to the Ct value and the amplification curve: If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following test (F3); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further test is performed; (F3) using the cDNA as a template and using the primer pair shown by SEQ ID No.4 and SEQ ID No.5 to perform PCR amplification, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 1 as follows: if the amplified product contains a target fragment of 502 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 1; if the amplified product does not contain the target fragment of 502 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 1.

9. A non-disease diagnostic method for detecting infectious pancreatic necrosis virus type 5, comprising the following steps: (G1) extracting total RNA from the sample to be tested and reversely transcribing it into cDNA; (G2) using the cDNA as a template, performing fluorescent quantitative PCR amplification using the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the probe shown in SEQ ID No. 3, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus according to the Ct value and the amplification curve: If the test result shows that the sample to be tested contains infectious pancreatic necrosis virus, then continue to perform the following test (G3); if the test result shows that the sample to be tested does not contain infectious pancreatic necrosis virus, then no further testing is performed; (G3) Using the cDNA as a template, PCR amplification is performed using the primer pair shown by SEQ ID No.6 and SEQ ID No.7, and then determining whether the sample to be tested contains infectious pancreatic necrosis virus type 5 as follows: if the amplified product contains a target fragment of 543 bp in size, then the sample to be tested contains or is a candidate for containing infectious pancreatic necrosis virus type 5; if the amplified product does not contain the target fragment of 543 bp in size, then the sample to be tested does not contain or is a candidate for not containing infectious pancreatic necrosis virus type 5.

10. The method according to any one of claims 7 to 9, characterized in that: When performing the fluorescent quantitative PCR amplification, the final concentrations of the upstream and downstream primers and the probe in the reaction system are both 5 μM; and / or When performing the fluorescent quantitative PCR amplification, the annealing temperature is 55° C.; and / or The sample to be tested is selected from: infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, infectious pancreatic necrosis virus type 1, and infectious pancreatic necrosis virus type 5.

Images