PCR primers, probes, kits and methods for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry

Abstract

The present invention relates to the field of biochemical detection technology, and in particular to PCR primers, probes, methods and kits for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry. The present invention respectively designs 7 pairs of primers and corresponding single-base extension probes, and establishes a nucleic acid mass spectrometry (MALDI-TOF MS) multi-target detection system for common porcine respiratory DNA viruses by optimizing primer concentration and reaction conditions. A multi-target detection system for synchronously identifying seven porcine respiratory DNA viruses, PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV, was established by nucleic acid mass spectrometry (MALDI-TOF MS) technology, which has the characteristics of high throughput, high sensitivity and strong specificity, and provides convenience for monitoring and differential diagnosis of pig-related diseases and quarantine of imported and exported animals.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical detection, and in particular to PCR primers, probes, a kit and a method for detecting seven types of porcine respiratory viruses based on MALDI-TOF mass spectrometry. Background Art

[0002] Porcine viral respiratory diseases and reproductive disorders are important factors restricting the scale and intensive development of modern pig farming. Different porcine respiratory and reproductive disorders often cause mixed infections and secondary infections among pig herds and occur in an endemic manner. More than 80% of pig diseases are caused by two or more pathogens, such as pseudorabies virus (PRV), porcine circovirus 2 (PCV-2), porcine circovirus 3 (PCV-3), porcine bocavirus (PBoV) and African swine fever virus (ASFV). PCV and PBoV can cause respiratory diseases in pigs, PCV and PRV can cause reproductive disorders in pigs; and ASFV can cause pigs to be infected with African swine fever, a highly contagious disease. Its virulent strain has a 100% mortality rate, and its etiology and epidemiology are very complex. Since the pathogenesis of pig diseases caused by these pathogens is similar to clinical symptoms, it poses a great obstacle to disease prevention and control, and makes high-throughput rapid detection of multiple pathogens a key link in disease prevention and control.

[0003] my country has a huge pig farming market. According to statistics, my country's pig farming accounts for more than 40% of the world's total pig farming, and the output value of pig farming is close to 1.3 trillion. With the increase in the scale and intensiveness of the pig farming industry, the probability of mixed infection of pig diseases has also increased. In addition, in recent years, due to the expansion of the prevalence of emerging diseases such as porcine bocavirus and porcine delta coronavirus at home and abroad, as well as the spread of major epidemics such as African swine fever, and mixed infections caused by different genotypes of some pathogens, the difficulty of disease prevention and control has increased further. At this time, if multiple single-target detection methods are used to test samples one by one, the detection cycle and cost will be greatly increased. Developing multi-target high-throughput detection methods and improving detection and diagnosis efficiency will provide stronger guarantees for disease prevention and control.

[0004] At present, the multiple molecular diagnostic technology for animal diseases mainly includes multiple PCR method, multiple TaqMan or SYBR Green fluorescence quantitative RT-PCR method and GeXP multiple PCR technology. For example, the Chinese invention patent applied for by the applicant of the present application (publication number: CN105936944A, publication date: 20160914) discloses a triple fluorescent RT-PCR detection kit and primers and probes for classical swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus H1 subtype. This method realizes rapid and early differential diagnosis of the above three pathogens in pigs. Since the primers and probes have high specificity and sensitivity, the efficiency of early identification detection is improved and the detection method is simplified. The Chinese invention patent applied by the applicant (publication number: CN113322352A, publication date: 20210831) discloses a method, primers, probes and kit for detecting porcine circovirus by triple digital droplet PCR. This method is based on the advantages of ddPCR and establishes a highly sensitive and accurate identification and detection method for different genotypes of porcine circovirus. It can be applied to routine animal quarantine and quarantine monitoring and load titration of animal-derived products with low virus content and complex matrix such as feed, meat products and vaccines. The sensitivity and specificity of the multiplex PCR method are lower than those of the fluorescence method, and the annealing temperature is easily affected by the length of the amplified fragment; although the fluorescence quantitative detection method has high specificity and sensitivity and short detection time, it has the limitation of fluorescence channels and cross-reactivity between multiple primers, which greatly limits the ability of the multiplex fluorescence quantitative PCR method to simultaneously detect four or more targets; GeXP multiplex RT-PCR technology may cause false positive test results due to the high sensitivity of capillary electrophoresis.

[0005] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new biomacromolecule detection technology that combines biomacromolecules with matrix molecules to form co-crystals, and distinguishes and detects samples based on the mass-to-charge ratio after laser bombardment. That is, by combining with multiple PCR, corresponding extension probes (UEPs) are designed for single-base extension of multi-target nucleic acid fragments after amplification, and the extended products are analyzed and detected by MALDI-TOF MS to obtain mass signals of different specific nucleic acid fragments, thereby performing multi-target simultaneous identification and detection on the same sample. MALDI-TOF MS technology can perform multi-target detection on up to 2×386 samples at a time, and each reaction can detect up to 40 targets, greatly improving the efficiency of detection and identification.

[0006] MALDI-TOF MS has been widely used in the clinical detection of pathogenic microorganisms, and has shown good prospects in the high-throughput detection and typing diagnosis of pathogens. Liu et al. established a nucleic acid mass spectrometry detection method that can simultaneously detect and distinguish 7 human coronaviruses (HCoV), with a detection limit of 1-5 copies / reaction, providing a technical platform for large-scale screening of HCoV; Liu Hongqian et al. established a multi-target detection technology for human respiratory syndrome-related viruses MALDI-TOF MS, which can simultaneously detect 27 respiratory pathogens with a sensitivity of 10 to 1000 copies / μL. In the diagnosis of animal diseases, Liu et al. established a MALDI-TOF MS synchronous detection method for 10 duck-borne pathogenic viruses, and the minimum detection limit of the system can reach 10 copies / μL. However, there is no public report on the high-throughput rapid detection of multiple pathogens in pigs using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Summary of the invention

[0007] In order to solve the above technical problems, the present invention designs multiple primers and UEP probes for common porcine respiratory DNA viruses such as PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV, and establishes a MALDI-TOF MS high-throughput multi-target technology system that can simultaneously identify 7 common porcine respiratory viruses, providing a new and efficient means for monitoring and differential diagnosis of pig herd-related diseases and quarantine of import and export animals.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0009] A PCR primer and probe for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry, wherein the seven porcine respiratory viruses are pseudorabies virus PRV, porcine circovirus type 2 PCV-2, porcine circovirus type 3 PCV-3, porcine bocavirus type 1 PBoV-G1, porcine bocavirus type 2 PBoV-G2, porcine bocavirus type 3 PBoV-G3 and African swine fever virus ASFV, wherein:

[0010] The sequences of the PCR primers for the porcine pseudorabies virus PRV are shown in SEQ ID No. 1 and SEQ ID No. 2, and the primer sequence of the UEP extension probe is shown in SEQ ID No. 15;

[0011] The sequences of the PCR primers for porcine circovirus type 2 PCV-2 are shown in SEQ ID No. 3 and SEQ ID No. 4, and the primer sequence for the UEP extension probe is shown in SEQ ID No. 16;

[0012] The sequences of the PCR primers for porcine circovirus type 3 PCV-3 are shown in SEQ ID No. 5 and SEQ ID No. 6, and the primer sequence for the UEP extension probe is shown in SEQ ID No. 17;

[0013] The sequences of the PCR primers for porcine bocavirus type 1 PBoV-G1 are shown in SEQ ID No.7 and SEQ ID No.8, and the primer sequence for the UEP extension probe is shown in SEQ ID No.18;

[0014] The sequences of the PCR primers for porcine bocavirus type 2 PBoV-G2 are shown in SEQ ID No.9 and SEQ ID No.10, and the primer sequence for the UEP extension probe is shown in SEQ ID No.19;

[0015] The sequences of the PCR primers for porcine bocavirus type 3 PBoV-G3 are shown in SEQ ID No.11 and SEQ ID No.12, and the primer sequence for the UEP extension probe is shown in SEQ ID No.20;

[0016] The primer PCR sequences of African swine fever virus ASFV are shown in SEQ ID No.13 and SEQ ID No.14, and the primer sequence of the UEP extension probe is shown in SEQ ID No.21.

[0017] Preferably, a 10-base tag is added to the 5′ end of each PCR primer shown in SEQ ID No. 1 to SEQ ID No. 24, and the sequence of the tag is ACGTTGGATG.

[0018] Furthermore, the present invention also discloses a kit for detecting seven types of porcine respiratory viruses based on MALDI-TOF mass spectrometry, and the kit comprises the PCR primers and probes.

[0019] Preferably, the kit also includes reagents required for reverse transcription and PCR reactions.

[0020] Preferably, the concentration of the PCR primer is 0.5-0.7 μM, and the concentration of the UEP extension probe is 3.5-6.5 μM; more preferably, the concentration of the PCR primer of the pseudorabies virus PRV is 0.6 μM, the concentration of the PCR primer of the porcine circovirus type 2 PCV-2 is 0.7 μM, the concentration of the PCR primer of the porcine circovirus type 3 PCV-3 is 0.7 μM, the concentration of the PCR primer of the porcine bocavirus type 1 PBoV-G1 is 0.6 μM, the concentration of the PCR primer of the porcine bocavirus type 2 PBoV-G2 is 0.6 μM, and the concentration of the PCR primer of the porcine bocavirus type 3 PBoV-G3 is The concentration of the PCR primer of African swine fever virus ASFV is 0.5μM; the concentration of the extended probe of pseudorabies virus PRV is 6μM, the concentration of the extended probe of porcine circovirus type 2 PCV-2 is 5μM, the concentration of the extended probe of porcine circovirus type 3 PCV-3 is 5μM, the concentration of the extended probe of porcine bocavirus type 1 PBoV-G1 is 5μM, the concentration of the extended probe of porcine bocavirus type 2 PBoV-G2 is 3.5μM, the concentration of the extended probe of porcine bocavirus type 3 PBoV-G3 is 6.5μM, and the concentration of the extended probe of African swine fever virus ASFV is 5μM.

[0021] Furthermore, the present invention also discloses the use of the PCR primers and probes in preparing a kit for detecting seven types of porcine respiratory viruses based on MALDI-TOF mass spectrometry, wherein the seven types of porcine respiratory viruses are pseudorabies virus PRV, porcine circovirus type 2 PCV-2, porcine circovirus type 3 PCV-3, porcine bocavirus type 1 PBoV-G1, porcine bocavirus type 2 PBoV-G2, porcine bocavirus type 3 PBoV-G3 and African swine fever virus ASFV.

[0022] Furthermore, the present invention also discloses a method for detecting seven types of porcine respiratory viruses based on MALDI-TOF mass spectrometry, wherein the method is a non-disease diagnosis method, and the seven types of porcine respiratory viruses are pseudorabies virus PRV, porcine circovirus type 2 PCV-2, porcine circovirus type 3 PCV-3, porcine bocavirus type 1 PBoV-G1, porcine bocavirus type 2 PBoV-G2, porcine bocavirus type 3 PBoV-G3 and African swine fever virus ASFV, and the method uses the PCR primers and probes or the kit for detection.

[0023] Preferably, the method comprises the following steps:

[0024] 1) Multiplex PCR reaction: Mix the sample with the PCR primer mixture and perform multiplex PCR reaction;

[0025] 2) SAP digestion reaction: The amplified product is digested with shrimp alkaline phosphatase to convert the excess deoxyribonucleoside triphosphates in the PCR step into ddNTPs and finally inactivated;

[0026] 3) Extension reaction: adding the digested product to the extension probe for extension reaction;

[0027] 4) After the reaction, the extension product is transferred to a multi-well plate, placed in a mass spectrometer, and desalted with resin before mass spectrometry detection; data acquisition and analysis are completed by MassARRAY Typer software.

[0028] As a further preferred embodiment, the reaction procedure of the multiplex PCR reaction in step 1) is 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 60 s, and finally 72°C for 5 min; step 2) the amplified product is supplemented to 7 μL with shrimp alkaline phosphatase, the excess deoxyribonucleoside triphosphates in the PCR step are digested into ddNTPs at 37°C for 40 min, and finally inactivated at 85°C for 5 min; step 3) 1 μL of extension probe, 0.2 μL of Inplex buffer and 0.2 μL of Termination mix are added to the digested product, and supplemented to 9 μL with ddH2O; the reaction procedure is 95°C for 30 s, 95°C for 5 s, 52°C for 5 s, and 80°C for 5 s for 40 cycles, and finally extended at 72°C for 3 min; step 4) the extended product after the reaction is completed is transferred to a 384-well plate, supplemented to 25 μL with ddH2O, and centrifuged at 10000 r / min for 2 min.

[0029] Preferably, the method further comprises the following steps: taking 0.5 g of sample respectively, freezing and thawing the sample three times and then grinding the sample sufficiently, placing the sample in PBS buffer at a ratio of 1:10, and centrifuging the sample at 10,000 r / min for 5 minutes to obtain the supernatant; freezing and thawing the whole blood sample three times, and then centrifuging the sample at 10,000 r / min for 5 minutes to obtain the supernatant; purifying and extracting nucleic acid according to the instructions for the reagents provided with the nucleic acid extractor NP968-C, and storing the sample at -20°C for future use.

[0030] According to the conservative sequences of 7 pathogen genes, the present invention designs 7 pairs of primers and corresponding single-base extension probes, and establishes a nucleic acid mass spectrometry (MALDI-TOFMS) multi-target detection system for common porcine respiratory DNA viruses by optimizing primer concentration and reaction conditions. MALDI-TOF MS combined with multiple PCR technology can use mass spectrometry to obtain mass signals of multiple specific nucleic acid fragments, thereby performing multi-target detection on the same sample. By analyzing the mass range of products in the range of 4000 to 10000 Daltons, not only the primer dimers and non-specific amplification products that may exist in the multiple PCR products are avoided, but also the specificity of the detection system is further improved by single-base extension in the extension stage, which makes up for the defects of multiple PCR while maintaining the sensitivity of mass spectrometry detection. In addition, since the primers and extension probes in the multiplex PCR and MALDI-TOF MS detection system have little interference, the system has good expandability. After replacing some primers and extension probes and optimizing primer concentrations, the targets used for detection can be further increased to expand the types of pathogens included in the detection system, which can meet the requirements of rapid and high-throughput quarantine of multiple types of pathogens.

[0031] The MALDI-TOF MS system established by the present invention was methodologically validated, and the results showed that the method had good stability and specificity. Mass spectrometry analysis showed that the 7 target product peaks of the multi-target detection system were only produced when specific virus-positive samples were detected, and there was no cross-reaction with other pathogen detections, and no product peaks appeared, indicating that the method had good specificity for the detection of 7 target viruses. Analysis of the repeatability test results showed that the intra-batch positive compliance rate of each virus in the system at high, medium and low concentrations was ≥98.0%, and the inter-batch compliance rate was ≥98.3%, indicating that the method had good repeatability. Analysis of the detection sensitivity test using standard plasmid as a template showed that the minimum detection limit of each virus of the 7 pathogens in the system was between 8.65 and 26.27 copies / μL, which was equivalent to the fluorescent PCR detection method.

[0032] A total of 108 samples were tested using the MALDI-TOF MS system, of which 34 tissue and whole blood suspected disease samples had a mixed infection rate of 54.76%, indicating that mixed infections caused by multiple viral respiratory disease and reproductive disease pathogens are common in sick pigs and pig herds, among which PCV-2 mixed infection is the most common, up to 67.6%; the total detection rate of the three genotypes of PBoV-G1, G2, and G3 is 47.6% (20 / 46), of which mixed infection with other pathogens accounts for 95% (19 / 20). PCV-2 and PCV-3 were detected in 24 pork samples (16.7%, 35.7%), and the mixed infection rate was 29.17%, indicating that there is a certain degree of porcine circovirus contamination in the current pork market. As a deeply processed product, the pork content of pig-derived feed is not high, and some viruses are removed during the processing stage. The positive detection rate and mixed infection rate of the 42 feed samples were the lowest, at 42.9% and 21.43%, respectively. The high mixed infection rate of pathogens in samples from the breeding and circulation links. In addition, the system has a large throughput for sample detection, with a maximum of 768 samples loaded at the same time, which can be used for large-scale detection of batch samples.

[0033] The MALDI-TOF MS test results of 108 samples were compared and verified with the results of digital PCR and fluorescent PCR. The results showed that the total coincidence rate of positive detection of 7 targets was basically consistent with the results of fluorescent PCR and digital PCR (94.4% to 100%). The difference was mainly reflected in the digital PCR detection of PCV-2 and PCV-3 in feed (94.4%, 96.3%), while the coincidence rate with fluorescent PCR for PRV, PBoV-G1, PBoV-G2, PBoV-G3, and ASFV was higher (100%, 99.1%, 97.2%, 100%, 100%). The reason may be that digital PCR has a higher sensitivity and can theoretically detect single copies of sample targets, even in samples with extremely low viral nucleic acid content, which is more suitable for testing samples containing complex matrices such as feed.

[0034] The present invention establishes a multi-target detection system for the simultaneous identification of seven porcine respiratory DNA viruses, namely PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV, through nucleic acid mass spectrometry (MALDI-TOF MS) technology. The system has the characteristics of high throughput, high sensitivity and strong specificity, and provides convenience for the monitoring and differential diagnosis of pig herd-related epidemic diseases and the quarantine of import and export animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a diagram of mass spectrometry check after mixing 13 groups of UEP probes. It can be seen that there is interaction when there are 13 groups (miscellaneous peaks at other positions besides the main peak).

[0036] Figure 2-Figure 8 The results of single reaction tests of 7 viruses; red peak: ddH2O is blank control; black peak; Figure 2 :PRV; Figure 3 :PCV-2; Figure 4 :PCV-3; Figure 5 :PBoV-G1; Figure 6 :PBoV-G2; Figure 7 :PBoV-G3; Figure 8 :ASFV.

[0037] Fig. 9 This is a comparison of the mass spectrometry results before and after the initial adjustment of the UEP probe.

[0038] Fig.10 Taking PBoV-G2 as an example, the comparison of primer concentration before and after adjustment (left) and the corresponding extension rate (right, both ≥80%) are shown.

[0039] Fig.11 This is the mass spectrometry result after optimization of primers and UEP probes.

[0040] Fig.12 This is a comparison chart of the mass spectrometry results of the sensitivity test using PCV-2 as an example.

[0041] Fig.13 Comparison of mass spectrometry results with ddH2O, 50 and 100 copies / μL ASFV plasmid as templates; A: ddH2O; B: 50 copies / μL plasmid; C: 100 copies / μL plasmid; the dotted line UEP ASFV and the dotted line T are the molecular masses corresponding to the ASFV extension probe and the extension product, respectively.

[0042] Fig.14 The results of the 7-plex nucleic acid mass spectrometry system specificity test; where: X: 7-plex detection system target; Y: ddH2O, each virus and 100 copies / μL of 7 target mixed plasmid templates; Z: signal-to-noise ratio (SNR).

[0043] Fig.15 This is the combined mass spectrometry results of the mixed plasmid and blank control; red peak: ddH2O as template; black peak: 10 2 copies / μL of mixed plasmid as template.

[0044] Fig.16It is the result of 7-plex nucleic acid mass spectrometry sensitivity test; where: X: 7-plex detection system target; Y: ddH2O and 7 target mixed plasmid templates at different concentrations; Z: the number of positive results of 20 repeated tests. DETAILED DESCRIPTION

[0045] 1. Materials and Methods

[0046] 1.1 Viruses

[0047] Classical swine fever virus inactivated vaccine (CSFV, C strain) and swine influenza virus inactivated vaccine (SIV, TJ strain) were purchased from Wuhan Keqian Biological Co., Ltd., and FMDV inactivated vaccine was purchased from Jinyu Biotechnology Co., Ltd. Porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV, Bartha strain), porcine epidemic diarrhea virus (PEDV, CV777 strain) and PCV 2 (JHSRJ strain) were donated by Zhejiang University, and the positive samples of porcine parvovirus (PPV) were donated by Zhejiang Agricultural and Forestry University. The positive samples of PCV3, PBoV-G1, PBoV-G2, PBoV-G3, etc. were stored in this laboratory. Adenovirus containing the full gene of ASFV-p72 was customized by Bioengineering (Shanghai) Co., Ltd. The samples were purified and extracted according to the instructions of the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit, and the DNA or reverse transcribed cDNA template was stored at -20℃ for later use.

[0048] 1.2 Reagents and instruments

[0049] TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0, PrimeScript TM IV1st strand cDNA Synthesis Mix was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; 10×PCR buffer, SAP Buffer, Iplex Pro Reagent Set were purchased from Agena; fluorescent PCR primers and probes [] Customized by Bioengineering (Shanghai) Co., Ltd.; Viral DNA / RNA Extraction Kit 4.0 and NP968-C Fully Automatic Nucleic Acid Extractor were purchased from Xi'an Tianlong Technology Co., Ltd.; Qubit 4 Fluorometer and ProFlex PCR Instrument were purchased from Thermofisher; DP-TOF Nucleic Acid Mass Spectrometer was from Zhejiang Dipu Diagnostic Technology Co., Ltd.

[0050] 1.3 Design and synthesis of primers and quality probes

[0051] According to the relevant viral gene sequences published in GenBank, multiple sequence comparisons were performed using MEGA-X, and the conserved regions of the gB gene of PRV, the rep gene of PCV-2, the cap gene of PCV-3, the NP1 gene of PBoV, and the P72 gene of ASFV were selected for primer design. ClustalW and MEGA-X were used to compare the gB, gM, and gE genes of 190 PRVs, the ORF1 and ORF2 genes of 98 PCV-2s and 129 PCV-3s, the ORF1 and NP1 genes of 273 PBoV-G1, PBoV-G2, and PBoV-G3, and the B646L encoding P72 protein, the D117L encoding P17 protein, and the CP530 gene of 249 ASFVs.

[0052] Select 1 to 2 groups of gene fragments for each virus, and use Primer 3 to design 3 pairs of primers for each, for a total of 21 pairs to be selected. Design Suite (Agena) was used to design specific PCR extension probes (UEPs) corresponding to each set of primers. MFEprimer-3.0 and Primer-Blast were used to evaluate the specificity, dimer, and hairpin structure of single / multiple primers and probes. The relevant primers, probes, and PUC57 vector plasmids of gene fragments were ordered (a total of 13 sets).

[0053] First, 13 primers PRV gB (3 groups), PCV-2rep (2 groups), PCV-3cap (2 groups), PBoV NP1 (3 groups in total), ASFV P72 (3 groups) and their probes were verified:

[0054] 1) After mixing the 13 groups of UEP probes, mass spectrometry check was performed to ensure that there were no problems with the probe quality, such as Figure 1 shown.

[0055] 2) Perform single target extension test with each single primer and its corresponding plasmid, i.e., PCR+agarose gel electrophoresis, to verify the feasibility of amplification and remove those with poor amplification.

[0056] 3) Mix the above primers and the corresponding UEP probes and perform mass spectrometry check to see whether the probe peak is different from that when the above UEP probe is mixed, and remove the UEP and corresponding primers that will bind.

[0057] 4) The screened primers and probes (one set for each of the seven viral targets) were subjected to PCR (single plasmid / mixed plasmid) + agarose gel electrophoresis to test whether there was interaction. Finally, one set of primers and probes corresponding to the PRV gB, PCV-2 rep, PCV-3 cap, PBoV-G1NP1, PBoV-G2 NP1, PBoV-G3 NP1, and ASFV P72 genes was selected.

[0058] Furthermore, a 10-base tag (ACGTTGGATG) was added to the 5′ end of each PCR primer to avoid interference of multiple primers on the mass spectrometry peak position, and Primer-BLAST was used to test the specificity of the designed primers. The selected target genes, primer sequences and quality information are shown in Table 1. All primers and probes were synthesized and purified by Bioengineering (Shanghai) Co., Ltd.

[0059] Table 1 Primers and probes for PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV detected by MALDI-TOF MS

[0060]

[0061] 1.4 Standard plasmid construction

[0062] Based on the gene sequences of PRV (KX880456.1), PCV-2 (AY691169.1), PCV-3 (NC_031753.1), PBoV-G1 (HQ223038.1), PBoV-G2 (NC_024453.2), PBoV-G3 (NC_016032.1) and ASFV (MN715134.1) published in GenBank, the PUC-57 standard plasmids were constructed by Bioengineering (Shanghai) Co., Ltd. The concentration of the plasmids was measured using Qubit 4 (Thermofisher) and the copy number was confirmed, which was 9.30×10 9 copies / μL (PRV), 1.17×10 10 Copies / μL (PCV-2), 1.92×10 10 Copies / μL (PCV-3), 3.63×10 10 Copies / μL (PBoV-G1), 1.18×10 10 Copies / μL (PBoV-G2), 3.89×10 9 copies / μL (PBoV-G3) and 1.74×10 10 copies / μL (ASFV), diluted 10-fold to 10 9 ~100 There were 10 gradients of copies / μL level. In addition, 7 plasmids with the same dilution multiple were mixed in equal volumes to form 10 groups of plasmid mixtures, which were packaged and stored at -20°C for future use.

[0063] 1.5 Single-plex reaction test

[0064] In order to investigate the reaction status of primers and extension probes and the mass spectrometry results, each medium concentration plasmid (10 4 Copies / μL) were used as templates, and single target amplification experiments were performed on single plasmids using single primers and extension probes. At the same time, ddH2O was set as a blank control group.

[0065] 1.6 Optimization of reaction conditions in multiple systems

[0066] Mix the plasmids at medium to high concentrations (10 4 copies / μL) as the template, according to the extension efficiency E=S 产物峰 / (S UPE残留峰 +S 产物峰 ) was used as an indicator to optimize the multiplex PCR amplification system. The initial concentrations of primers and extension probes were 0.5 μM and 5 μM, respectively. The optimal concentrations of multiplex primers and extension probes were adjusted based on E ≥ 0.8, and the detection sensitivity of the system was optimized to obtain a mass signal spectrum with only a single product peak.

[0067] The six-plex PCR reaction system was 5 μl, and PCR amplification was performed with the primers in Table 1. ① Multiplex PCR reaction: 2 μL of plasmid was mixed with 1.2 μL of 10×PCR mix and 1 μl of PCR primer mixture, and the mixture was made up to 5 μL with ddH2O. The reaction procedure was 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 60 s, and finally 72°C for 5 min. ② SAP digestion reaction: The amplified product was made up to 7 μL with shrimp alkaline phosphatase (SAP), and the excess deoxy-ribonucleoside triphosphate (dNTP) in the PCR step was digested into ddNTP at 37°C for 40 min to prevent dNTP from continuing to extend as a reactant in the subsequent steps. Finally, it was inactivated at 85°C for 5 min.

[0068] ③ Extension reaction: Add the digested product to the extension probe in Table 1 for extension reaction. Add 1μL extension probe, 0.2μL Inplex buffer and 0.2μL Termination mix to the digested product, and add ddH2O to 9μL. The reaction program is 95℃30s, 95℃5s, 52℃5s, 80℃5s for 40 cycles, and finally extend at 72℃ for 3min.

[0069] After the reaction, the extension product was transferred to a 384-well plate, filled with ddH2O to 25 μL, and centrifuged at 10,000 r / min for 2 min. The centrifuged 384-well plate was placed in a mass spectrometer, and resin was added for desalting before mass spectrometry detection. Data acquisition and analysis were completed by MassARRAY Typer software.

[0070] 1.7 Specificity test

[0071] The target viruses or pseudovirus DNAs such as PRV, PCV-2, PCV-3, PBoV and ASFV in 1.1, and the non-target common swine disease virus DNAs or cDNAs such as SIV, PRRSV, CSFV, FMDV and PPV were used as templates to conduct specificity tests of the MALDI-TOF MS multiple detection system. At the same time, the mixed templates of PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV standard plasmids were used as positive controls, and ddH2O was used as negative controls. If there are no extension product peaks for each non-target pathogen, and the extension product peaks corresponding to PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV in the target pathogens and positive controls appear at specific positions, and there is no cross-overlap between the product peaks, then the detection system is considered to have good specificity.

[0072] 1.8 Sensitivity test

[0073] In order to evaluate the sensitivity of this method, 10 4 , 10 3 , 10 2 Seven target-positive plasmids with 1, 20, 30, 40, 50, 25, 10, 5, 2.5, and 1 copy / μL levels were mixed at the same concentration level as templates and then detected and analyzed according to the optimized MALDI-TOF MS multiplex system. Each gradient of mixed plasmids was repeated 20 times, and the lowest concentration at which all 20 repetitions were positive was identified as the minimum detection limit (LOD) of the MALDI-MS multiplex detection system for the virus.

[0074] 1.9 Repeatability test

[0075] The high, medium and low copy numbers (10 6 , 10 4 , 10 2A mixed plasmid standard with 100 copies / μl) was used as a template, and 50 replicates were set for each dilution to evaluate the intra-batch repeatability of the MALDI-TOF MS method for each virus detection result; in addition, the above-mentioned mixed plasmid standard was repeated at 3 different times, and 20 replicates were set for each dilution. The detection results of the plasmids at each dilution of 3 independent tests were recorded to evaluate the inter-batch repeatability of the method.

[0076] 1.10 Sample testing application

[0077] 42 samples of feed containing porcine ingredients, 34 tissue or whole blood samples and 24 fresh pork samples were collected and stored by our laboratory. 8 samples of artificial contamination of ASFV pseudovirus were prepared by 10-fold serial dilution (10 9 ~10 2 The ASFV-p72 adenovirus with 10 copies / μl was added 5μl to 0.5g of negative pork one by one.

[0078] Take 0.5g of feed, tissue, pork or artificial contaminated samples respectively, freeze and thaw three times, grind them thoroughly, put them in PBS buffer at a ratio of 1:10, and centrifuge them at 10000r / min for 5min to obtain the supernatant. After freezing and thawing the whole blood sample three times, centrifuge it at 10000r / min for 5min and obtain the supernatant. Purify and extract nucleic acids according to the instructions of the nucleic acid extractor NP968-C reagents and store them at -20℃ for later use. The MADLI-TOF MS multiple detection system constructed in this study was used to detect PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV in 108 sample nucleic acids, of which 42 feed samples were verified by digital droplet PCR, and the remaining samples were verified by fluorescence quantitative PCR. The positive compliance rate was calculated as the number of true positives / (the number of true positives + the number of false negatives) × 100%; the negative compliance rate was calculated as the number of true negatives / (the number of true negatives + the number of false positives) × 100%; the total compliance rate of the test results was calculated as [(the number of true positives + the number of true negatives) / total number of tests] × 100%, and the results were evaluated.

[0079] 2 Results

[0080] 2.1 Single-plex reaction test

[0081] Take 10 4The single target amplification test was performed using a single primer and an extension probe with 10 copies / μl of the target plasmid as a template. The results showed that each virus had only a single peak of extension product (PRV: 7048.6Da, PCV-2: 6023.9Da, PCV-3: 7181.7Da, PBoV-G1: 7320.6Da, PBoV-G2: 5522.6Da, PBoV-G3: 7376.8Da, ASFV: 6477.3Da), and the corresponding negative The sex control had only a single peak of extended probe peak (PRV: 6801.4Da, PCV-2: 5776.8Da, PCV-3: 6934.5Da, PBoV-G1: 6993.5Da, PBoV-G2: 5235.4Da, PBoV-G3: 7129.6Da, ASFV: 6206.1Da), and no miscellaneous peaks appeared within the range of mass spectrometry detection, indicating that the amplification primers and mass probes of each virus in the 7-plex system can react effectively ( Figure 2-Figure 8 ).

[0082] 2.2 Establishment of multiple reaction system

[0083] The above 7 sets of primers and probes were mixed with plasmids (such as 10 4 , 10 2 copies / μL, which can ensure that the probes can be consumed during normal reactions) as a template, according to the extension efficiency E=S in the mass spectrometry image 产物峰 / (S UPE残留峰 +S 产物峰 ) is used as the indicator to optimize the system:

[0084] 1) Initial adjustment of UEP concentration: First, the UEP probes were mixed at 5 μM and then mass spectrometry was performed to obtain a mass spectrometry image with only probe peaks. The ratio of probes corresponding to each target was adjusted according to the peak height of each group of probes (H min / H max ≥0.6) to obtain the crude concentration of UEP probe, such as Fig. 9 The probe peak heights are similar before and after the initial adjustment of the UEP probe shown.

[0085] 2) Primer concentration adjustment: Mix the primers at an initial concentration of 0.5 μM, and obtain a set of initial mass spectrometry images through PCR-SAP digestion-single base extension. Appropriately increase or decrease the concentration of PCR primers based on whether there is a residual UEP peak in the image (if there is a residual, increase the primer concentration by 20%; if there is no residual peak for some targets, and the amplification efficiency E of several groups of repeats is 100%, reduce the primer concentration to reduce the competition of the primers for the PCR enzyme). After changing the primer concentration, reconfigure new mixed primers and repeat the above process until there is no residual UEP peak and E ≥ 0.8 when the high-concentration plasmid is used as a template, that is, a more suitable primer concentration is obtained. Fig.10As shown, taking PBoV-G2 as an example, the comparison of primer concentration before and after adjustment (left) and the corresponding extension rate (right, all ≥80%).

[0086] 3) UEP concentration optimization: According to the above-mentioned optimal primer concentration and product peak height, make it min / H max Try to be as consistent as possible. Fig.11 As shown, after the primers and UEP probes were optimized, the product peak heights were similar.

[0087] 4) After obtaining the LOD of each target in the sensitivity test, optimize the primer concentration: low concentration (10 2 The minimum detection limit can be further reduced by increasing the concentration of a certain set of primers and probes at the same time. Fig.12 As shown, taking PCV-2 as an example, the original primer probe concentration was 10 2 Copies / μL mixed plasmids could not be detected (top), but PCV-2 could be detected after the comprehensive improvement of PCV-2 primers and UEP probe (bottom).

[0088] In order to obtain clear mass spectrometry signal analysis spectra, the concentrations of each set of primers and extended probes for PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV viruses were optimized. Finally, the optimal concentrations of each primer in the multiplex system were 0.6μM (PRV), 0.7μM (PCV-2), 0.7μM (PCV-3), 0.6μM (PBoV-G1), 0.6μM (PBoV-G2), 0.5μM (PBoV-G3), and 0.5μM (ASFV), and the optimal concentrations of extended probes were 6μM (PRV), 5μM (PCV-2), 5μM (PCV-3), 5μM (PBoV-G1), 3.5μM (PBoV-G2), 6.5μM (PBoV-G3), and 5μM (ASFV).

[0089] In the analysis of the MALDI-TOF MS multiplex reaction system, each target corresponds to two peak positions: the extension probe (UEP) and the single-base extension product. If a single peak appears at the mass position corresponding to the extension product and the SNR is ≥ 6, the test result is positive. If there is no extension product peak or the extension product peak SNR is < 6, the test result is considered negative. Taking ASFV as an example, the extension probe in the negative control reaction system does not undergo an extension reaction, and only the unconsumed probe peak ( Fig.13 -A); at low template concentration (50 copies / μL positive plasmid), the ASFV extension probe is partially consumed, and the ASFV probe peak and product peak exist at the same time ( Fig.13-B); when the plasmid template concentration is 100 copies / μL, the extension reaction is complete, the ASFV extension probe is completely converted into extension products, and only the ASFV product peak exists ( Fig.13 -C), and the difference between it and the ASFV probe peak (6206.1Da) is the molecular weight of the extended single base.

[0090] 2.3 Specificity analysis

[0091] The specificity test of MALDI-TOF MS multiplex system was carried out using target viruses or pseudovirus DNA such as PRV, PCV-2, PCV-3, PBoV and ASFV, as well as other common swine disease virus DNA or cDNA such as SIV, PRRSV, CSFV, FMDV and PPV as templates. The results showed that the seven target viruses in the system produced single peaks at their respective extension products, and the SNR of the signal peaks were ≥6. There was no peak at the extension product of the non-target template and the blank control group (SNR was ≤3), and only the extension probe peak ( Fig.14 ), indicating that this method can accurately identify the target virus and has no cross-reaction with other non-target pathogens, with good specificity.

[0092] 2.4 Sensitivity analysis

[0093] To evaluate the sensitivity of this method, 10 4 , 10 3 , 10 2 The mixed positive plasmids of 7 target viruses at the levels of 50, 25, 10, 5, 2.5, and 1 copies / μL were used for MALDI-TOF MS multiplex system detection and analysis. The lowest concentration at which 20 replicates were positive was the LOD of each target. The results showed that the minimum detection limits of the multiplex system for the 7 target viruses were: PRV2 2.66 copies / μL, PCV-2 26.10 copies / μL, PCV-3 10.66 copies / μL, PBoV-G1 20.15 copies / μL, PBoV-G2 26.27 copies / μL, PBoV-G3 8.65 copies / μL, and ASFV 9.68 copies / μL ( Fig.15 ), the detection concentration of each target in the reaction system can reach 10 1 The copies / μL level is comparable to that of fluorescent PCR.

[0094] 2.5 Repeatability analysis

[0095] Take 10 6 , 10 4 , 10 2 The multiple primer system was repeated 50 times with the mixed plasmid of 10 copies / μl dilution as template. 6 , 10 4The detection results of PRV, PCV-2, PCV-3, PBoV-G1, PBoV-G2, PBoV-G3 and ASFV in the copy / μl level group were all positive (SNR ≥ 6), 10 2 In the copy / μl level group, except for PBoV-G3, which had a positive rate of 98.0%, the other positive rates were all 100%, indicating that the method had good repeatability. In the test results of three different batches of experiments, the positive rates of PRV, PCV-2, PCV-3, PBoV-G2, PBoV-G3, and ASFV were all 100%, and the positive rate of PBoV-G1 was 98.3%, indicating that the method had high inter-batch repeatability (Table 2).

[0096] Table 2 Repeatability test results

[0097]

[0098]

[0099] 2.6 Sample testing application

[0100] The MALDI-TOF MS method was used to detect 108 tissue, feed, pork or artificially contaminated samples. The results are shown in Table 3. Among the 34 tissue samples, 6 viruses other than ASFV were detected in 31 samples, of which 23 tissue samples could detect mixed infection of two or more viruses, accounting for 67.6%; in feed samples, the other 5 targets except ASFV and PRV were detected, and 18 of the 42 samples were positive, of which 9 samples were mixedly infected with two or more viruses (21.4%); PCV-2 and PCV-3 were detected in 15 of the 24 fresh pork samples, of which 7 samples were mixedly infected with the two viruses, and the mixed infection rate was 29.2%; 8 pork samples artificially contaminated with ASFV-p72 pseudovirus at different concentrations were all ASFV positive, which was consistent with the test results of the plasmid standard.

[0101] Table 3 MALDI-TOF MS test results of 108 samples

[0102]

[0103]

[0104] Note: *Multiple positive targets were detected in the same sample at the same time; No positive targets were detected in the samples.

[0105] The detection results of the MALDI-TOF MS system were compared and verified with those of digital PCR (feed) and fluorescence PCR (tissue, pork and artificial contamination samples). The positive coincidence rate, negative coincidence rate and total coincidence rate of the seven virus targets were: PRV: 100%, 100%, 100%; PCV-2: 96.2%, 92.9%, 94.4%; PCV-3: 90.9%, 98.5%, 96.3%; PBoV-G1: 100%, 98.9%, 99.1%; PBoV-G2: 81.3%, 100%, 97.2%; PBoV-G3: 100%, 100%, 100% and ASFV: 100%, 100%, 100%. The detection results of the two methods were basically consistent (Table 4).

[0106] Table 4 The coincidence rate between the monitoring results of MALDI-TOF MS multiple detection system and digital PCR and fluorescence PCR

[0107]

Claims

1. A PCR primer and probe for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry, wherein the seven porcine respiratory viruses are pseudorabies virus PRV, porcine circovirus type 2 PCV-2, porcine circovirus type 3 PCV-3, porcine bocavirus type 1 PBoV-G1, porcine bocavirus type 2 PBoV-G2, porcine bocavirus type 3 PBoV-G3 and African swine fever virus ASFV, characterized in that, The sequences of the PCR primers of the porcine pseudorabies virus PRV are shown in SEQ ID No. 1 and SEQ ID No. 2, and the sequence of the UEP extension probe is shown in SEQ ID No. 15; The sequences of the PCR primers for porcine circovirus type 2 PCV-2 are shown in SEQ ID No. 3 and SEQ ID No. 4, and the sequence of the UEP extension probe is shown in SEQ ID No. 16; The sequences of the PCR primers for porcine circovirus type 3 PCV-3 are shown in SEQ ID No. 5 and SEQ ID No. 6, and the sequence of the UEP extension probe is shown in SEQ ID No. 17; The sequences of the PCR primers for porcine bocavirus type 1 PBoV-G1 are shown in SEQ ID No.7 and SEQ ID No.8, and the sequence of the UEP extension probe is shown in SEQ ID No.18; The sequences of the PCR primers of porcine bocavirus type 2 PBoV-G2 are shown in SEQ ID No.9 and SEQ ID No.10, and the sequence of the UEP extension probe is shown in SEQ ID No.19; The sequences of the PCR primers of porcine bocavirus type 3 PBoV-G3 are shown in SEQ ID No.11 and SEQ ID No.12, and the sequence of the UEP extension probe is shown in SEQ ID No.20; The primer PCR sequences of African swine fever virus ASFV are shown in SEQ ID No.13 and SEQ ID No.14, and the sequence of the UEP extension probe is shown in SEQ ID No.

21.

2. The PCR primer and probe according to claim 1, characterized in that: A 10-base tag is added to the 5′ end of each PCR primer shown in SEQ ID No. 1 to SEQ ID No. 14, and the sequence of the tag is ACGTTGGATG.

3. A kit for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry, characterized in that: The kit comprises the PCR primers and probes according to claim 1 or 2.

4. The kit according to claim 3, characterized in that: The kit also includes the reagents needed to perform reverse transcription and PCR reactions.

5. The kit according to claim 3, characterized in that: The concentration of PCR primers for each virus was 0.5-0.7 μM, and the concentration of UEP extension probes for each virus was 3.5-6.5 μM.

6. The kit according to claim 3, characterized in that: The concentration of the PCR primer of the porcine pseudorabies virus PRV is 0.6 μM, the concentration of the PCR primer of the porcine circovirus type 2 PCV-2 is 0.7 μM, the concentration of the PCR primer of the porcine circovirus type 3 PCV-3 is 0.7 μM, the concentration of the PCR primer of the porcine bocavirus type 1 PBoV-G1 is 0.6 μM, the concentration of the PCR primer of the porcine bocavirus type 2 PBoV-G2 is 0.6 μM, the concentration of the PCR primer of the porcine bocavirus type 3 PBoV-G3 is 0.5 μM, and the concentration of the PCR primer of the African swine fever virus ASFV is 0.5 μM; the concentration of the extended probe of the porcine pseudorabies virus PRV is 6 μM, the concentration of the extended probe of the porcine circovirus type 2 PCV-2 is 5 μM, the concentration of the extended probe of the porcine circovirus type 3 PCV-3 is 5 μM, the concentration of the extended probe of the porcine bocavirus type 1 PBoV-G1 is 5 μM, the concentration of the extended probe of porcine bocavirus type 2 PBoV-G2 is 3.5 μM, the concentration of the extended probe of porcine bocavirus type 3 PBoV-G3 is 6.5 μM, and the concentration of the extended probe of African swine fever virus ASFV is 5 μM.

7. Use of the PCR primers and probes described in any one of claims 1-2 in the preparation of a kit for detecting seven porcine respiratory viruses based on MALDI-TOF mass spectrometry, wherein the seven porcine respiratory viruses are pseudorabies virus PRV, porcine circovirus type 2 PCV-2, porcine circovirus type 3 PCV-3, porcine bocavirus type 1 PBoV-G1, porcine bocavirus type 2 PBoV-G2, porcine bocavirus type 3 PBoV-G3 and African swine fever virus ASFV.

Images