Reagent, kit and detection method for simultaneously detecting four viral animal epidemic disease pathogens

Abstract

The invention provides a reagent, a kit and a detection method for simultaneously detecting four viral animal epidemic disease pathogens, and belongs to the technical field of pathogen detection. According to the invention, a primer probe group used for quadruple fluorescent quantitative PCR is designed according to a PRRSV-N gene, a PRV-gE gene, a CSFV-5 '-UTR gene and an FMDV-3D gene, and the kit is assembled on the basis of the primer probe group. In the embodiment, the reliability of the assembled kit is verified by methods of establishing a standard curve, detecting sensitivity, specificity, inter-batch and intra-batch repeatability, shelf life and the like. The invention also provides a method for simultaneously detecting four porcine viral epidemic diseases by using the kit, the method has the advantages of high sensitivity, good specificity, strong stability, low detection cost, short detection time and the like, and a good technical means is provided for prevention and purification of the four porcine viral epidemic diseases, especially epidemic disease purification of a large-scale pig farm and a pig farm.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen detection, and specifically relates to a reagent, a kit and a detection method for simultaneously detecting four viral animal disease pathogens. Background Art

[0002] With the vigorous development of pig farming in my country, the pig farming model is increasingly intensive and large-scale, but the spread and prevention of diseases have become more complicated, especially the mixed infection of multiple diseases. Porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV) are four viral animal disease pathogens that seriously affect the pig industry. Although the epidemic trends of these four diseases are generally stable and sporadic at this stage, the economic losses caused each year cannot be ignored. Accurate and rapid diagnosis is an effective way to control these four diseases, but with the different degrees of genetic variation in the genomes of the epidemic strains of these four swine disease pathogens and the use of related attenuated vaccines, traditional detection methods have been interfered with to varying degrees.

[0003] In recent years, with the rapid development of fluorescence quantitative PCR technology, the important role of this method in pathogen diagnosis has become increasingly irreplaceable. Multiplex fluorescence quantitative PCR technology is improved and developed on the basis of single fluorescence quantitative PCR technology. Compared with single fluorescence quantitative PCR technology, its reaction is faster, the operation is simpler and the cost is lower. However, many studies at this stage only stay at the stage of establishing fluorescence quantitative PCR methods, and the assembly of the kit has not been completed. There is still a big gap from the application to production practice. The assembly of fluorescence PCR quantitative kit is more suitable for use in large-scale farms when multiple diseases are prevalent. Summary of the invention

[0004] The present invention provides a reagent, a kit and a detection method for simultaneously detecting four viral animal disease pathogens. The reagent and the kit are safe and reliable and can provide more economical and convenient technical support for the disease purification of large-scale pig farms.

[0005] The first invention object of the present invention is to provide a reagent for simultaneously detecting four viral animal disease pathogens, comprising an upstream primer PRRSV-F as shown in SEQ ID No.1 and a downstream primer PRRSV-R as shown in SEQ ID No.2 designed according to the nucleotide sequence of the virus PRRSV;

[0006] It includes an upstream primer PRV-F shown in SEQ ID No.4 and a downstream primer PRV-R shown in SEQ ID No.5, which are designed according to the nucleotide sequence of the virus PRV;

[0007] It includes an upstream primer CSFV-F as shown in SEQ ID No.7 and a downstream primer CSFV-R as shown in SEQ ID No.8 according to the nucleotide sequence designed according to the virus CSFV;

[0008] It includes an upstream primer FMDV-F shown in SEQ ID No.10 and a downstream primer FMDV-R shown in SEQ ID No.11, the nucleotide sequence of which is designed according to the virus FMDV.

[0009] In a preferred embodiment of the present invention, probes designed for viruses PRRSV, PRV, CSFV and FMDV are also included, and the nucleotide sequences of the probes are shown in SEQ ID No.3, SEQ ID No.6, SEQ ID No.9 and SEQ ID No.12 respectively.

[0010] In a preferred embodiment of the present invention, the 5' end of the probe is modified with different fluorescent groups, and the 3' end is modified with a quenching group.

[0011] In a preferred embodiment of the present invention, the fluorescent group is selected from any one of the following: FAM, CY5, ROX and VIC;

[0012] The quenching group is selected from any one of the following: BHQ1 and BHQ2.

[0013] The second inventive object of the present invention is to provide a kit for simultaneously detecting four viral animal disease pathogens, comprising the above-mentioned reagents, and also comprising a positive quality control product, a negative quality control product and a PCR reaction solution.

[0014] In a preferred embodiment of the present invention, the reagent is in the form of a mixed solution, the volume ratio of the primers designed for the viruses PRRSV, PRV, CSFV and FMDV is 0.2:0.6:0.4:0.4, and the final concentrations of the primers in the added system are 0.06 μmol / L, 0.17 μmol / L, 0.11 μmol / L and 0.11 μmol / L, respectively;

[0015] In the mixed solution, the volume ratio of each probe is 0.3:0.3:0.3:0.3, and the final concentration of each probe in the added system is 0.09 μmol / L.

[0016] In a preferred embodiment of the present invention, the positive quality control product comprises plasmids respectively comprising the sequences shown in SEQ ID No. 13 to SEQ ID No. 16.

[0017] The third invention object of the present invention is to provide a method for detecting four viral animal disease pathogens for non-disease diagnosis purposes, comprising extracting nucleic acid from a sample to be tested, and mixing the extracted nucleic acid of the sample to be tested, a positive quality control product, and a negative quality control product with the reagents and PCR reaction solution in the above-mentioned kit to obtain a quadruple fluorescent quantitative PCR reaction solution;

[0018] The quadruple fluorescence quantitative PCR reaction solution is subjected to fluorescence quantitative PCR amplification, and the detection result is obtained according to the Ct value and amplification curve in the fluorescence quantitative PCR amplification process.

[0019] In a preferred embodiment of the present invention, the procedure of the fluorescent quantitative PCR amplification includes 95°C for 5 min; 95°C for 30 s, 54.8°C for 30 s, and 40 cycles.

[0020] In a preferred embodiment of the present invention, the sample to be tested includes blood, tissue or feed sample.

[0021] Beneficial effects: The present invention designs a primer probe set for quadruple fluorescent quantitative PCR based on the PRRSV-N gene, PRV-gE gene, CSFV-5′-UTR gene and FMDV-3D gene, and assembles a kit based on the primer probe set. The reliability of the assembled kit is verified in the examples by establishing a standard curve, testing sensitivity, specificity, inter-batch and intra-batch repeatability, shelf life and the like.

[0022] The present invention also provides a method for simultaneously detecting four porcine viral diseases using the kit, which has the advantages of high sensitivity, good specificity, strong stability, low detection cost and short detection time, and provides a good technical means for the prevention and purification of these four porcine diseases, especially the purification of diseases in large-scale pig farms and breeding pig farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of temperature screening of PRRSV, PRV, CSFV and FMDV by quadruple fluorescence quantitative PCR method are shown in Figure a: coarse temperature screening by quadruple method; b: fine temperature screening by quadruple method, where Target1: PRRSV, Target2: PRV, Target3: CSFV, Target4: FMDV;

[0024] Figure 2 The results of primer concentration screening for the quadruple fluorescent quantitative PCR method for PRRSV, PRV, CSFV and FMDV are shown in Figure a: PRRSV and FMDV double primer concentration screening, Target1: PRRSV, Target2: FMDV; b: PRRSV, FMDV and PRV triple primer concentration screening, Target1: PRRSV, Target2: PRV, Target3: FMDV; c: PRRSV, FMDV, PRV and CSFV quadruple primer concentration screening, Target1: PRRSV, Target2: PRV, Target3: CSFV, Target4: FMDV;

[0025] Figure 3 The results of the probe concentration screening of the quadruple fluorescent quantitative PCR method for PRRSV, PRV, CSFV and FMDV are shown in Figure a: PRRSV and FMDV double probe concentration screening, Target1: PRRSV, Target2: FMDV; b: PRRSV, FMDV and PRV triple probe concentration screening, Target1: PRRSV, Target2: PRV, Target3: FMDV; c: PRRSV, FMDV, PRV and CSFV quadruple probe concentration screening, Target1: PRRSV, Target2: PRV, Target3: CSFV, Target4: FMDV;

[0026] Figure 4 It is the standard curve of the quadruple fluorescence quantitative PCR method for PRRSV, PRV, CSFV and FMDV, where Target1: PRRSV, Target2: PRV, Target3: CSFV, Target4: FMDV;

[0027] Figure 5 The results of the four-plex fluorescence quantitative PCR method for specific detection of PRRSV, PRV, CSFV and FMDV are shown in Figure a: 45 cycles of non-specific verification of the four-plex fluorescence quantitative PCR method (1-5: PRRSV, PRV, CSFV, FMDV, ddH 2 O); b: Nonspecific validation of 40 cycles of quadruple fluorescence quantitative PCR (1-5: PRRSV, PRV, CSFV, FMDV, ddH 2 O); c: 40 cycles of specific detection by quadruple fluorescence quantitative PCR (1-10: PRRSV, PRV, CSFV, FMDV, PEDV, PoRV, JEV, Mhp, E.coli, ddH 2 O);

[0028] Figure 6 The appearance of the PRRSV, PRV, CSFV and FMDV quadruple fluorescent quantitative PCR kit. In the figure, a: the outside of the kit; b: the inside of the kit, where 1 to 5 represent PCR reaction solution, primer probe mixture, ddH 2 O, negative quality control and positive quality control;

[0029] Figure 7 It is the standard curve of the PRRSV, PRV, CSFV and FMDV quadruple fluorescence quantitative PCR kit, where Target1: PRRSV, Target2: PRV, Target3: CSFV, Target4: FMDV;

[0030] Figure 8 This is a graph showing the specific detection results of the PRRSV, PRV, CSFV and FMDV quadruple fluorescent quantitative PCR kit, in which 1 to 10 represent PRRSV plasmid, PRV plasmid, CSFV plasmid, FMDV plasmid, PEDV nucleic acid, PoRV nucleic acid, JEV nucleic acid, Mhp nucleic acid, E. coli nucleic acid and negative control product, respectively. DETAILED DESCRIPTION

[0031] The present invention provides a reagent for simultaneously detecting four viral animal disease pathogens, comprising an upstream primer PRRSV-F as shown in SEQ ID No.1 and a downstream primer PRRSV-R as shown in SEQ ID No.2 designed according to the nucleotide sequence of the virus PRRSV;

[0032] It includes an upstream primer PRV-F shown in SEQ ID No.4 and a downstream primer PRV-R shown in SEQ ID No.5, which are designed according to the nucleotide sequence of the virus PRV;

[0033] It includes an upstream primer CSFV-F as shown in SEQ ID No.7 and a downstream primer CSFV-R as shown in SEQ ID No.8 according to the nucleotide sequence designed according to the virus CSFV;

[0034] It includes an upstream primer FMDV-F shown in SEQ ID No.10 and a downstream primer FMDV-R shown in SEQ ID No.11, the nucleotide sequence of which is designed according to the virus FMDV.

[0035] The primer sets of the present invention are designed based on the conservative genes of the above four viruses. For example, the N gene of PRRSV (EU860248.1, AY150564.1 and KY495780.1), the gE gene of PRV (KJ789182.1, KU962917.1, KX170935.1, MH507059.1, MK622299.1 and MN240565.1), and the CS The 5′UTR gene of FV (OQ150770.1, EF026755.1, KJ197314.1 and MH549009.1), and the 3D gene of FMDV (MG372729.1, MT863268.1, LC036265.1, MF782478.1, OP957418.1 and EF175732.1) were used to find their highly conserved regions, thereby designing and synthesizing the primer pairs shown in Table 1. At the same time, the present invention also designs specific probes for the conserved genes of each virus, and the 5′ end of the probe is modified with different fluorescent groups, and the fluorescent groups are selected from any one of the following: FAM, CY5, ROX and VIC; at the same time, the 3′ end of the probe is modified with a quenching group, and the quenching group is selected from any one of the following: BHQ1 and BHQ2. In one embodiment of the present invention, Shanghai Shenggong Bioengineering Technology Service Co., Ltd. was commissioned to synthesize and modify the primer probe set shown in Table 1.

[0036] Table 1 Primer and probe sequences

[0037]

[0038]

[0039] The present invention also provides a kit for simultaneously detecting four viral animal disease pathogens, comprising the above reagents, a positive quality control product, a negative quality control product and a PCR reaction solution.

[0040] In a preferred embodiment of the present invention, the reagent is in the form of a mixed solution, the volume ratio of the primers designed for the viruses PRRSV, PRV, CSFV and FMDV is 0.2:0.6:0.4:0.4, and the final concentrations of the primers in the added system are 0.06 μmol / L, 0.17 μmol / L, 0.11 μmol / L and 0.11 μmol / L, respectively;

[0041] In the mixed solution, the volume ratio of each probe is 0.3:0.3:0.3:0.3, and the final concentration of each probe in the added system is 0.09 μmol / L.

[0042] In a preferred embodiment of the present invention, the positive quality control product includes plasmids containing sequences shown in SEQ ID No.13 to SEQ ID No.16, respectively, wherein the PRRSV-N gene sequence used is shown in SEQ ID No.13; the PRV-gE gene sequence used is shown in SEQ ID No.14; the CSFV-5′-UTR gene sequence used is shown in SEQ ID No.15; and the FMDV-3D gene sequence used is shown in SEQ ID No.16.

[0043] The PCR amplification reagent used in the kit of the present invention, such as Mix, uses a chemically modified hot-start DNA polymerase and adds a dUTP / UDG anti-contamination system to minimize the impact of amplification product contamination on experimental results. For example, in one embodiment, AceQ Universal U+Probe MasterMix of Novezan Biotechnology is used.

[0044] The kit of the present invention also includes negative quality control products and positive quality control products, which can verify the influence of errors in experimental operation and individual operation differences on experimental results. The positive quality control product uses 10 5 The standard mixed plasmid with 100 copies / μL can not only meet the setting requirements of positive quality control products, but also prevent experimental contamination and ensure the safety of other reagents and laboratories.

[0045] The shelf life of the kit of the present invention is at least 6 months, the freeze-thaw stability is good, and the verification compliance rate is greater than 90%, which meets the kit assembly requirements.

[0046] The present invention also provides a method for detecting four viral animal disease pathogens for non-disease diagnosis purposes, comprising extracting nucleic acid from a sample to be detected, and mixing the extracted nucleic acid of the sample to be detected, a positive quality control product, and a negative quality control product with the reagents and PCR reaction solution in the above-mentioned kit to obtain a quadruple fluorescent quantitative PCR reaction solution;

[0047] The quadruple fluorescence quantitative PCR reaction solution is subjected to fluorescence quantitative PCR amplification, and the detection result is obtained according to the Ct value and amplification curve in the fluorescence quantitative PCR amplification process.

[0048] In a preferred embodiment of the present invention, the program of the fluorescent quantitative PCR amplification includes 95°C for 5 min; 95°C for 30 s, 54.8°C for 30 s, and 45 cycles.

[0049] The samples to be tested in the present invention include blood, tissue or feed samples.

[0050] To further illustrate the present invention, a reagent, a kit and a detection method for simultaneously detecting four viral animal disease pathogens provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The reagents, instruments and reagents used in the examples of the present invention are all conventional materials in the art, and unless otherwise specified, are conventional commercially available materials in the art:

[0052] Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and Escherichia coli (E. coli) were isolated and stored in our laboratory. Live vaccine of porcine mycoplasma pneumonia (Mhp, strain RM48) was purchased from Zhengye Biological Co., Ltd., and live vaccine of porcine epidemic Japanese encephalitis (JEV, strain SA14-14-2) was purchased from China Animal Husbandry Industry Co., Ltd. 130 clinical samples were collected from pig farms around Xinjiang and were specially inactivated;

[0053] QuantStudio5 real-time fluorescence quantifier and NanoDrop2000 micro-nucleic acid analyzer were purchased from Thermo Fisher Scientific (China) Co., Ltd., vortex oscillator was purchased from Shanghai Jingke Laboratory Co., Ltd., AceQ Universal U+ProbeMasterMix was purchased from Nanjing Novezan Life Science Co., Ltd., RNA extraction kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., reverse transcription kit was purchased from Kangwei Century Biotechnology Co., Ltd., 1*TE Buffer was purchased from Shanghai Shenggong Bioengineering Co., Ltd., viral DNA extraction kit, classical swine fever virus real-time fluorescence RT-PCR detection kit, porcine reproductive and respiratory syndrome virus real-time fluorescence RT-PCR detection kit, pseudorabies virus (gE gene) real-time fluorescence PCR detection kit, foot-and-mouth disease virus universal nucleic acid detection kit (fluorescence quantitative method) were all purchased from Hangzhou Bioer Technology Co., Ltd.

[0054] Example 1

[0055] 1. Extraction of viral and clinical sample DNA and RNA and RNA reverse transcription

[0056] The DNA and RNA of viruses and clinical samples were extracted according to the instructions of Biori DNA Extraction Kit and Quanshijin RNA Extraction Kit. The extracted RNA was reverse transcribed according to the instructions of Kangwei Century RNA Reverse Transcription Kit. The cDNA and extracted DNA obtained by reverse transcription were stored in a -20℃ refrigerator.

[0057] 2. Preparation of standard positive plasmid

[0058] Referring to the highly conserved gene sequences of four viruses (SEQ ID No.13 to SEQ ID No.16), Shanghai Shenggong Biotechnology Co., Ltd. was commissioned to synthesize positive plasmids. The original dry powder plasmid was diluted with 40 μL 1×TE Buffer, and its concentration was measured with a micro-nucleic acid detector and converted into copy number using a formula. The four virus positive standard plasmids were diluted to 1×10 10 copies / μL and mixed into a standard mixed positive plasmid at a ratio of 1:1:1:1 and diluted in multiple gradients for later use.

[0059]

[0060] The unit of plasmid concentration is ng / μL.

[0061] 3. Screening of annealing temperature

[0062] The standard mixed positive plasmids of PRRSV, PRV, CSFV and FMDV were used for quadruple fluorescence quantitative PCR reaction. The temperature gradient was designed according to the single fluorescence quantitative PCR method screened in the early stage. A total of 6 gradients were used for coarse screening. Based on the coarse screening results, 6 gradients were designed for fine screening to explore the optimal annealing temperature of the quadruple fluorescence PCR method.

[0063] 35 μL reaction system: 0.4 μL each of upstream primers, 0.4 μL each of downstream primers, 0.4 μL each of probes, 2×AceQUniversal U+Probe MasterMix 20 μL, ddH 2 O 8.2 μL and mixed nucleic acid template 2 μL;

[0064] The reaction procedure and temperature screening of the quadruple fluorescence quantitative PCR method were as follows: UNG enzyme action at 37°C for 2 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 sec, annealing, extension, and fluorescence signal collection at (52, 54, 56, 58, 60, 62)°C for 30 sec, and 45 cycles.

[0065] The reaction procedure and temperature screening of the quadruple fluorescence quantitative PCR method were as follows: UNG enzyme action at 37°C for 2 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 sec, annealing, extension, and fluorescence signal collection at (54, 54.4, 54.8, 55.2, 55.6, 56)°C for 30 sec, and 45 cycles.

[0066] The results are as follows Figure 1As shown in the figure, during the temperature coarse screening process, when the annealing temperature was 56°C, the fluorescence intensity of the PRRSV, PRV, CSFV and FMDV quadruple fluorescence quantitative PCR method was the strongest and the amplification curve was the highest. During the further temperature fine screening process, when the annealing temperature was 54.8°C, the fluorescence intensity of the quadruple method was the strongest and the amplification curve was the highest. Therefore, 54.8°C was selected as the optimal annealing temperature for the quadruple method.

[0067] 4. Screening of primer concentration

[0068] The matrix method was used to screen the optimal primer concentration for the quadruple fluorescence quantitative PCR method. Five primer concentration gradients were set. The double primer concentration was screened first, then the triple primer concentration was screened, and finally the quadruple primer concentration was screened. The original primer concentration was 10 μmol / L (the screening primer concentration was expressed as primer volume). See Tables 2 to 4 for details.

[0069] Table 2 Screening of primers for PRRSV and FMDV dual fluorescence quantitative PCR

[0070]

[0071]

[0072] Table 3 Primer screening for triple fluorescence quantitative PCR of PRRSV, FMDV and PRV

[0073] PRV+(PRRSV+FMDV) 1 1 0.2 / 0.2 / 0.6 2 0.4 / 0.2 / 0.6 3 0.6 / 0.2 / 0.6 4 0.8 / 0.2 / 0.6 5 1.0 / 0.2 / 0.6

[0074] Table 4 Primer screening for PRRSV, FMDV, PRV, CSFV quadruple fluorescence quantitative PCR

[0075] CSFV+(PRV+PRRSV+FMDV) 1 1 0.2 / 0.6 / 0.2 / 0.6 2 0.4 / 0.6 / 0.2 / 0.6 3 0.6 / 0.6 / 0.2 / 0.6 4 0.8 / 0.6 / 0.2 / 0.6 5 1.0 / 0.6 / 0.2 / 0.6

[0076] The results are as follows Figure 2As shown in the figure, during the screening of PRRSV and FMDV double primer concentrations, it was found that with the increase of PRRSV primer concentration, the amplification curve of FMDV became more and more oblique, and the typical S-type amplification curve gradually disappeared, proving that the primer concentration of PRRSV would affect the amplification efficiency of FMDV. In order to ensure the amplification efficiency and fluorescence intensity, 0.2μL and 0.4μL were selected as the optimal primer concentrations of PRRSV and FMDV, respectively. During the screening of PRRSV, FMDV and PRV triple primer concentrations, it was found that when the PRV primer concentration was 0.6μL, the fluorescence intensity of the triple method was the strongest. During the screening process of quadruple primer concentrations for PRRSV, FMDV, PRV and CSFV, it was found that when the CSFV primer concentration was 0.4μL, the fluorescence intensity of the quadruple method was the strongest. Therefore, the optimal primer addition amounts of the PRRSV, FMDV, PRV and CSFV quadruple fluorescence quantitative PCR method were 0.2μL, 0.4μL, 0.6μL and 0.4μL, respectively, and the optimal primer concentrations were 0.06μmol / L, 0.11μmol / L, 0.17μmol / L and 0.11μmol / L.

[0077] 5. Screening of probe concentration

[0078] The matrix method was used to screen the optimal probe concentration for the quadruple fluorescence quantitative PCR method. Five concentration gradients were set. The method was the same as the quadruple primer concentration screening. For details, see Tables 5 to 7.

[0079] Table 5 Screening of probe concentrations for PRRSV and FMDV dual fluorescence quantitative PCR method

[0080]

[0081]

[0082] Table 6 Screening of probe concentrations for PRRSV, FMDV, and PRV triple fluorescence quantitative PCR method

[0083] PRV+(PRRSV+FMDV) 1 1 0.1 / 0.3 / 0.3 2 0.2 / 0.3 / 0.3 3 0.3 / 0.3 / 0.3 4 0.4 / 0.3 / 0.3 5 0.5 / 0.3 / 0.3

[0084] Table 7 Screening of probe concentrations for quadruple fluorescence quantitative PCR method

[0085] CSFV+(PRV+PRRSV+FMDV) 1 1 0.1 / 0.3 / 0.3 / 0.3 2 0.2 / 0.3 / 0.3 / 0.3 3 0.3 / 0.3 / 0.3 / 0.3 4 0.4 / 0.3 / 0.3 / 0.3 5 0.5 / 0.3 / 0.3 / 0.3

[0086] The results are as follows Figure 3As shown, with the increase of probe concentration, the fluorescence intensity of the PRRSV and FMDV double, PRRSV, FMDV and PRV triple, and PRRSV, FMDV, PRV and CSFV quadruple methods all increased, and the probe concentration with the strongest fluorescence intensity was 0.5μL. However, considering that the probe cost is relatively high and a lower concentration can meet the experimental requirements, 0.3μL was selected as the optimal probe addition amount for the PRRSV, FMDV, PRV and CSFV quadruple fluorescence quantitative PCR method, and the optimal probe concentration was 0.09μmol / L.

[0087] 6. Construction of standard curve

[0088] The linearity of the quadruple fluorescence quantitative PCR method was better. 3 ~10 7 A total of 5 concentration gradients of standard mixed positive plasmids were used for amplification reaction, and the standard curve was drawn using the software provided with the fluorescence quantification instrument.

[0089] The results are as follows Figure 4 As shown, the regression equation of PRRSV is y = -3.776x + 38.614 (R 2 =0.998), PRV is y=-4.239x+47.172(R 2 =0.995), CSFV is y=-3.935x+43.198(R 2 =0.998), FMDV is y=-3.816x+41.631(R 2 =0.995), PRRSV, PRV, CSFV and FMDV at 10 3 ~10 7 The concentration range of copies / μL showed a good linear relationship, and its R 2 All of them were greater than 0.99, which met the requirements for establishing the fluorescence quantitative PCR method.

[0090] 7. Use nucleic acids extracted from PEDV, PoRV, JEV, Mhp, E.coli, and standard mixed positive plasmids as positive controls, ddH 2 O was used as a negative control for specificity detection to verify the specificity of the quadruple fluorescence quantitative PCR method.

[0091] The experimental results are as follows Figure 5As shown in the figure, it was found that sometimes positive amplification curves with lower fluorescence intensity appeared in addition to the target positive amplification curve during the experiment. In order to verify whether this positive amplification curve with lower fluorescence intensity was caused by nonspecific amplification caused by interference between the quadruple method and other viral, bacterial nucleic acids or self-primer probes, a large number of experimental verifications were carried out. The experimental results showed that the appearance of nonspecific amplification curves was caused by mutual interference between primers and probes in the quadruple method. In order to eliminate this nonspecific amplification, the number of cycles of the method was changed from 45 to 40. Although the reduction in the number of cycles may affect the sensitivity of the method, it can reduce the occurrence of false positives. In the subsequent 40 specific tests, the results showed that the established quadruple method only produced specific amplification curves for PRRSV, PRV, CSFV, and FMDV positive plasmids, while there were no amplification curves for PEDV, PoRV, JEV, Mhp, and E.coli nucleic acids, and the negative control did not show nonspecific amplification caused by primers and probes, indicating that the optimized PRRSV, PRV, CSFV, and FMDV quadruple fluorescence quantitative PCR method has good specificity.

[0092] 8. Repeatability test

[0093] 10 7 , 10 5 , 10 3 The positive plasmids were mixed with 100 copies / μL standard as templates for intra-batch and inter-batch experiments.

[0094] In-batch experiment: three concentrations of standard mixed positive plasmid were tested three times at the same time;

[0095] Repeated experiment between batches: Three concentrations of standard mixed positive plasmids were tested three times at different time periods. SPSS software was used to analyze and sort out the Ct values ​​within and between batches to evaluate the repeatability of the established quadruple fluorescence quantitative PCR method.

[0096] The results are shown in Table 8. The intra-batch coefficient of variation of the method was 0.20% to 1.72%, and the inter-batch coefficient of variation was 0.12% to 1.85%, both less than 5%. This indicates that the constructed quadruple fluorescence quantitative PCR method has good repeatability and meets the requirements for the construction of the fluorescence quantitative PCR method.

[0097] Table 8 Repeatability test of quadruple fluorescence quantitative PCR method

[0098]

[0099] 9. Testing of clinical samples

[0100] 130 clinical samples collected and stored in the laboratory were tested using the quadruple fluorescence quantitative PCR method established by the present invention and compared with the common PCR method.

[0101] The results are shown in Table 9. The quadruple fluorescence quantitative PCR method detected 21 PRRSV positive samples with a positive detection rate of 16.15%; 5 PRV positive samples with a positive detection rate of 3.84%; 12 CSFV positive samples with a positive detection rate of 9.23%; 7 FMDV positive samples with a positive detection rate of 5.38%; 2 PRRSV+CSFV mixed infection positive samples with a positive detection rate of 1.54%, and no other mixed infection was detected. The ordinary PCR method detected 15 PRRSV positive samples with a positive detection rate of 11.53%; 5 PRV positive samples with a positive detection rate of 3.84%; 9 CSFV positive samples with a positive detection rate of 6.92%; 5 FMDV positive samples with a positive detection rate of 3.84%; no mixed infection was detected. In general, the quadruple fluorescence quantitative PCR method established by the present invention is superior to the ordinary PCR method in clinical sample detection.

[0102] Table 9 Detection of clinical samples by quadruple fluorescence quantitative PCR method

[0103]

[0104] Example 2 Development of a quadruple nucleic acid detection kit for PRRSV, PRV, CSFV, and FMDV

[0105] 2.1 Materials, methods and results

[0106] 2.1.1 Experimental samples

[0107] Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and Escherichia coli (E. coli) were isolated and stored in our laboratory. Live vaccine of porcine mycoplasma pneumonia (Mhp, RM48 strain) was purchased from Zhengye Biological Co., Ltd., and live vaccine of porcine epidemic Japanese encephalitis (JEV, SA14-14-2 strain) was purchased from China Animal Husbandry Industry Co., Ltd. 48 clinical samples were collected from pig farms around Xinjiang and were specially inactivated.

[0108] 2.1.2 Preparation of positive and negative quality control products

[0109] Use 10 5 The standard mixed plasmid with a concentration of 1000 copies / μL is used as a positive quality control. The Ct value of the standard plasmid at this concentration meets the requirements for kit assembly and is not likely to cause contamination to the laboratory and kit reagents. It is safe and reliable. An empty plasmid without the target gene is used as a negative quality control.

[0110] 2.1.3 Assembly of the kit and writing of instructions

[0111] The kit includes: PCR reaction solution, primer probe mixture, positive quality control, negative quality control, ddH2 O. Specifically shown in Table 10, the kit is for 50 tests, and the appearance is as Figure 6 shown.

[0112] Table 10 Components of the Kit

[0113] Component name Loading Quantity(tube) Main ingredients PCR reaction solution 1000μL / tube 1 Taq enzyme, UNG enzyme, dNTPs, PCR buffer Primer probe mixture 220μL / tube 1 Upstream and downstream primers and probes Positive Control 20μL / tube 1 Plasmid containing target gene fragment Negative Control 20μL / tube 1 Empty plasmid without target gene fragment <![CDATA[ddH 2 The]]> 500μL / tube 1 ddH2O without target gene

[0114] Usage method of the kit: Take out and melt the reagents of the kit in the reagent preparation area, and vortex thoroughly for 15 s and centrifuge. Determine the number of reactions N1, N1 = the number of samples to be tested (n1) + the number of quality control products (2) + 1 (loss). Calculate the amount to be added to the reaction mixture, and the calculation method is shown in Table 11. Dispense 33 μL / tube of the mixture into PCR tubes, add 2 μL of positive and negative quality control products and sample templates, and the total reaction system is 35 μL. The collection of fluorescence signals is set as FAM, CY5, ROX, VIC, and the data collection is set at 54.8 °C. The specific reaction procedure is as follows: UNG enzyme acts at 37 °C for 2 min; pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 10 s, annealing, extension, and fluorescence signal collection at 54.8 °C for 30 s, 40 cycles.

[0115] Table 11 Reaction Calculation Method

[0116] Reagent components Amount PCR reaction solution 20μL×N1 Primer probe mixture 4.3μL×N1 <![CDATA[ddH 2 The]]> 8.7μL×N1

[0117] Result determination: Under the condition that the test is valid, samples with Ct value ≤ 35 are positive, indicating positive sample nucleic acid; samples with Ct value showing no or > 40 are negative samples, indicating negative sample nucleic acid; if 35 < Ct value ≤ 40, it is judged as a suspicious sample. For suspicious samples, first look at the amplification curve. If the amplification curve is a logarithmic amplification curve, it is suspiciously positive, otherwise it is judged as negative. For suspiciously positive samples, re-extract nucleic acid and perform the test again. If the repeated amplification curve is a logarithmic amplification curve, it is judged as sample positive, indicating positive sample nucleic acid; otherwise it is judged as sample negative.

[0118] 2.1.4 Construction of the Standard Curve

[0119] Select standard mixed plasmids with 5 concentrations of 10 7 ~10 3 copies / μL for the amplification reaction, and use the software自带 by the fluorescence quantitative instrument to draw the standard curve.

[0120] The results are as Figure 7 shown. The regression equation of PRRSV is y1 = -3.624x + 37.583 (R 2 = 0.999), for PRV it is y2 = -4.322x + 47.568 (R 2 = 0.995), and for CSFV it is y3 = -3.994x + 43.652 (R2 =0.999), FMDV is y4=-3.936x+42.441(R 2 =0.995). The results showed that PRRSV, PRV, CSFV and FMDV were all above 10 7 ~10 3 The range of copies / μL shows a good linear relationship, and its R 2 All >0.99.

[0121] 2.1.5 Specificity detection

[0122] Nucleic acids extracted from PEDV, PoRV, JEV, Mhp, and E. coli, the positive quality control products that come with the quadruple fluorescence quantitative kit were used as positive controls, and the negative quality control products were used as negative controls for specific detection to verify the specificity of the kit.

[0123] The results are as follows Figure 8 As shown, this kit only produces specific amplification curves for PRRSV, PRV, CSFV, and FMDV positive plasmids, but no amplification curves for PEDV, PoRV, JEV, Mhp, and E. coli nucleic acids, indicating that the developed kit has good specificity.

[0124] 2.1.6 Sensitivity test

[0125] Take 10 3 , 10 2 , 10 1 The amplification was performed with 1000 copies / μL standard mixed plasmids, and each concentration was repeated 30 times. The lowest concentration with a detection rate ≥ 95%, that is, the positive plasmid concentration detected 29 times or more, was its minimum detection limit, that is, its sensitivity.

[0126] The results are shown in Table 12. 3 , 10 2 , 10 1 The standard mixed plasmid was repeated 30 times, and the results showed that the lowest concentration for PRRSV detection rate ≥ 95% was 10 1 copies / μL, so its minimum detection limit is 10 1 copies / μL. Similarly, the minimum detection limit of PRV is 10 2 copies / μL, the minimum detection limit of CSFV is 10 1 copies / μL, the minimum detection limit of FMDV is 10 2 Therefore, the developed kit has a higher sensitivity.

[0127] Table 12 Sensitivity test of quadruple fluorescence quantitative PCR kit

[0128]

[0129] 2.1.7 Verification of shelf life

[0130] The kit was stored at -20°C, and the minimum detection limit standard mixed plasmid and positive and negative quality control products were taken out for testing at 0, 1, 2, 3, 4, 5, and 6 months, respectively. Each item was tested once a month to verify the shelf life of the kit. The results are shown in Table 13. The negative quality control products were not detected during this period, and the positive quality control products and the minimum detection limit standard mixed plasmid were all detected, and the Ct value trend was stable, which met the requirements for kit assembly. Therefore, the shelf life of the developed kit is at least 6 months.

[0131] Table 13 Shelf life verification of quadruple fluorescence quantitative PCR kit

[0132]

[0133] Note: ND means not detected

[0134] 2.1.8 Freeze-thaw cycles

[0135] The kit was repeatedly frozen and thawed 0, 5, 10, 15, and 20 times, and the minimum detection limit standard mixed plasmid was detected 3 times, and the number of detections was calculated to verify the stability of the kit. The results are shown in Table 14. After repeated freezing and thawing 0, 5, 10, 15, and 20 times, the kit was able to effectively detect the minimum detection limit standard mixed plasmid for 3 consecutive times, proving that its anti-thaw property meets the kit assembly requirements.

[0136] Table 14 Repeated thawing and freezing experiment of quadruple fluorescence quantitative PCR kit

[0137]

[0138] 2.1.9 Testing of clinical samples

[0139] A total of 48 clinical samples collected and stored in the laboratory were tested using the assembled kit, and compared with four commercially purchased single-plex fluorescence quantitative PCR kits for PRRSV, PRV, CSFV and FMDV, and the compliance rate was calculated.

[0140] The results are shown in Table 15. The positive coincidence rates of the developed kit and the commercial single-plex kit were 95.83% for PRRSV, 100% for PRV, 97.92% for CSFV, and 100% for FMDV. The commercial single-plex kit detected 2 more positive copies of PRRSV and 1 more positive copy of CSFV than the developed kit. The number of positive and negative copies detected by PRV and FMDV was consistent. The coincidence rates were all greater than 90%, meeting the requirements for kit assembly.

[0141] Table 15 Clinical sample testing with quadruple fluorescence quantitative PCR kit

[0142]

[0143] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A reagent for simultaneously detecting four viral animal disease pathogens, characterized in that: It includes an upstream primer PRRSV-F shown in SEQ ID No.1 and a downstream primer PRRSV-R shown in SEQ ID No.2, which are designed according to the nucleotide sequence of the virus PRRSV; It includes an upstream primer PRV-F shown in SEQ ID No.4 and a downstream primer PRV-R shown in SEQ ID No.5, which are designed according to the nucleotide sequence of the virus PRV; It includes an upstream primer CSFV-F as shown in SEQ ID No.7 and a downstream primer CSFV-R as shown in SEQ ID No.8 according to the nucleotide sequence designed according to the virus CSFV; It includes an upstream primer FMDV-F shown in SEQ ID No.10 and a downstream primer FMDV-R shown in SEQ ID No.11, the nucleotide sequence of which is designed according to the virus FMDV.

2. The reagent according to claim 1, characterized in that It also includes probes designed for viruses PRRSV, PRV, CSFV and FMDV, and the nucleotide sequences of each probe are shown in SEQ ID No.3, SEQ ID No.6, SEQ ID No.9 and SEQ ID No.12 respectively.

3. The reagent according to claim 2, characterized in that The 5' end of the probe is modified with different fluorescent groups, and the 3' end is modified with a quenching group.

4. The reagent according to claim 3, characterized in that The fluorescent group is selected from any one of the following: FAM, CY5, ROX and VIC; The quenching group is selected from any one of the following: BHQ1 and BHQ2.

5. A kit for simultaneously detecting four viral animal disease pathogens, characterized in that: The method comprises the reagent according to any one of claims 1 to 4, and further comprises a positive quality control product, a negative quality control product and a PCR reaction solution.

6. The kit according to claim 5, characterized in that The reagent is in the form of a mixed solution, and the volume ratio of the primers designed for the viruses PRRSV, PRV, CSFV and FMDV is 0.2:0.6:0.4:0.4, and the final concentrations of the primers in the added system are 0.06 μmol / L, 0.17 μmol / L, 0.11 μmol / L and 0.11 μmol / L, respectively; In the mixed solution, the volume ratio of each probe is 0.3:0.3:0.3:0.3, and the final concentration of each probe in the added system is 0.09 μmol / L.

7. The kit according to claim 5, characterized in that The positive quality control product includes plasmids containing sequences shown in SEQ ID No.13 to SEQ ID No.16 respectively.

8. A method for detecting four viral animal disease pathogens for non-disease diagnosis purposes, characterized in that: The method comprises extracting nucleic acid from a sample to be tested, and mixing the extracted nucleic acid of the sample to be tested, a positive quality control product, and a negative quality control product with the reagents and PCR reaction solution in the kit according to any one of claims 5 to 7 to obtain a quadruple fluorescent quantitative PCR reaction solution; The quadruple fluorescence quantitative PCR reaction solution is subjected to fluorescence quantitative PCR amplification, and the detection result is obtained according to the Ct value and amplification curve in the fluorescence quantitative PCR amplification process.

9. The method according to claim 8, characterized in that: The program of the fluorescent quantitative PCR amplification includes 95° C. for 5 min; 95° C. for 30 s, 54.8° C. for 30 s, and 40 cycles.

10. The method according to claim 8, characterized in that: The sample to be tested includes blood, tissue or feed sample.

Images