A multi-target detection system and method for porcine respiratory RNA viruses based on MALDI-TOF MS technology

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a multi-target detection system and method for porcine respiratory RNA virus based on MALDI-TOF MS technology. A multi-target detection primer and extension probe for porcine respiratory RNA virus based on MALDI-TOF MS technology, the primer and extension probe comprising: a primer PRRSV and an extension probe UEP.PRRSV for detecting porcine reproductive and respiratory syndrome virus; a primer CSFV and an extension probe UEP.CSFV for detecting classical swine fever virus; a primer FMDV and an extension probe UEP.FMDV for detecting foot-and-mouth disease virus; a primer SIV and an extension probe UEP.SIV for detecting universal swine influenza virus; a primer SIV-H1 and an extension probe UEP.SIV-H1 for detecting swine influenza virus H1 type; a primer SIV-H3 and an extension probe UEP.SIV-H3 for detecting swine influenza virus H3 type. The present invention provides convenience for rapid diagnosis of epidemic diseases and medium- and high-throughput detection of related pathogens, can meet the requirements of import and export animal quarantine and related epidemic disease monitoring, and has certain economic value and public health significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a multi-target detection system and method for porcine respiratory RNA virus based on MALDI-TOF MS technology. Background Art

[0002] The pathogenicity of swine respiratory diseases is complex and is often the result of the interaction of multiple factors such as pathogens, external environment or management conditions. [1] . Due to the large number of pathogens of the syndrome, large-scale farming also leads to rapid virus transmission. In addition, some pigs have similar symptoms after infection, which may cause mixed infection on a large scale. Therefore, the pathogens of porcine respiratory syndrome have always been one of the constraints on the large-scale and intensive development of modern pig farming. Its pathogens include swine influenza virus (Swine Influenza Virus, SIV), porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV), classical swine fever virus (Classical Swine Fever Virus, CSFV) and foot-and-mouth disease virus (Foot-and-Mouth Disease Virus, FMDV). After pigs are infected with PRRSV, they often have symptoms such as fever, dyspnea and reproductive disorders; CSFV, as a Class A infectious disease and a Class I animal epidemic disease, has long been one of the pig epidemic diseases that are monitored by animal prevention and control-related units at all levels of government. It has a huge impact on my country's pig farming industry, and it is difficult to completely eliminate and control it. SIV and FMDV are both zoonotic pathogens. FMDV can cause acute, febrile, highly contagious diseases in even-toed ungulates. Unlike FMDV, SIV does not have the most serious impact on pigs. However, given that pigs play the role of "virus mixers" in the spread of influenza viruses, SIV typing detection is necessary for preventing pig infections and influenza virus monitoring. Rapid diagnosis of pathogens is a prerequisite for controlling the occurrence of the disease, so it is particularly important to establish an efficient method for simultaneous detection of multiple pathogens.

[0003] China is the world's largest producer and consumer of pork. In 2021, domestic pork production accounted for 43.4% of the world's total production. According to the statistics of China's meat consumption structure in 2021, pork accounted for 65.4% of all meat consumption. In recent years, with the continuous development of pig farming, the harm caused by pig diseases has become more and more obvious. Viral diseases caused by different syndrome pathogens such as porcine respiratory and reproductive syndrome and diarrhea syndrome have restricted the further development of the pig farming industry and the pork trading market. Some zoonotic pathogens also pose a threat to public health. For example, influenza virus, because SIV can be divided into subtypes such as H1~H16, N1~N9 according to hemagglutinin (H) and neuraminidase (N), there are multiple virus subtypes after gene rearrangement, and pigs have both sialic acid-2,3-galactoside and sialic acid-2,6-galactoside specific receptors for influenza viruses. Different subtypes of influenza viruses can be recombined and adapted in pigs, causing pigs to be infected with avian influenza (SIV-H5, H7, H9) and human influenza (SIV-H1, H2, H3). In addition, imported pork and its products may also carry pathogens. As a highly lethal infectious disease, ASFV currently has no approved commercial vaccine, and port defense and quarantine are still the primary way to prevent such epidemics from being introduced into the country. It can be seen that it is very necessary to establish and improve a multiple pathogen detection system for key pathogens.

[0004] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new biomacromolecule detection technology that can analyze nucleic acids, peptides, and proteins within a certain mass range. By laser bombarding the processed sample matrix co-crystals, this method can obtain a mass spectrum of macromolecules in the sample based on the molecular weight differences after sample ionization. MALDI-TOF MS is combined with multiple PCR reactions as a nucleic acid mass spectrometry technology. [6] Corresponding extension probes (UEPs) are designed for the fragments after multiple PCR amplification. After the extension probes and the amplification products undergo a single-base extension reaction using ddNTPs digested with shrimp alkaline phosphatase (SAP) as substrates, mass spectrometry can be used to obtain mass signals of multiple groups of specific nucleic acid fragments, thereby completing multi-target detection of samples.

[0005] MALDI-TOF technology has the ability to detect single nucleotide polymorphisms (SNPs), and has broad application prospects in genotyping detection. It is currently used for tumor susceptibility gene detection, early screening of genetic diseases, and drug-resistant mutation detection. In addition, MALDI-TOF nucleic acid mass spectrometry technology has been widely studied in the detection of pathogenic microorganisms, such as human respiratory viruses, enteroviruses, sexually related disease viruses, papillomaviruses, herpes and polyomaviruses, and duck syndrome virus detection. The research team previously established a MALDI-TOF MS detection method for seven porcine respiratory DNA viruses (Chinese patent application number: 202211274251X). This method has good stability and specificity, and the detection limit of each pathogen is between 8.65–26.27 copies / μL, which is comparable to the performance of the single-plex fluorescence PCR detection method. Summary of the invention

[0006] In order to solve the above-mentioned technical problems, the present application designs multiple primers and extended probes for porcine respiratory syndrome RNA viruses such as SIV, PRRSV, CSFV and FMDV through target gene fragments, and performs typing detection on the two subtypes of SIV, H1 and H3, which are currently the most prevalent and most harmful. A detection method for 6 common porcine respiratory RNA viruses based on the MALDI-TOF MS method is established, providing a new means for batch quarantine of pigs and virus detection of porcine products.

[0007] In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

[0008] The target gene fragment is used in the preparation of a reagent for multi-target detection of porcine respiratory RNA viruses based on MALDI-TOF MS technology. The porcine respiratory RNA viruses are porcine reproductive and respiratory syndrome virus PRRSV, classical swine fever virus CSFV, foot-and-mouth disease virus FMDV, universal swine influenza virus SIV, swine influenza virus H1 type SIV-H1 and swine influenza virus H3 type SIV-H3. The target gene fragment is as follows:

[0009] 1) The sequence of the target gene fragment of porcine reproductive and respiratory syndrome virus PRRSV is shown in SEQ ID NO.19;

[0010] 2) The sequence of the target gene fragment of classical swine fever virus CSFV is shown in SEQ ID NO.20;

[0011] 3) The sequence of the target gene fragment of foot-and-mouth disease virus FMDV is shown in SEQ ID NO.21;

[0012] 4) The sequence of the target gene fragment of the universal swine influenza virus SIV is shown in SEQ ID NO.22;

[0013] 5) The sequence of the target gene fragment of swine influenza virus H1 type SIV-H1 is shown in SEQ ID NO.23;

[0014] 6) The sequence of the target gene fragment of swine influenza virus H3 type SIV-H3 is shown in SEQ ID NO.24.

[0015] Furthermore, the present invention also discloses a reagent for multi-target detection of porcine respiratory RNA viruses based on MALDI-TOF MS technology. The porcine respiratory RNA viruses are porcine reproductive and respiratory syndrome virus PRRSV, classical swine fever virus CSFV, foot-and-mouth disease virus FMDV, universal swine influenza virus SIV, swine influenza virus H1 type SIV-H1 and swine influenza virus H3 type SIV-H3. The target gene fragments of the reagent are as follows:

[0016] 1) The sequence of the target gene fragment of porcine reproductive and respiratory syndrome virus PRRSV is shown in SEQ ID NO.19;

[0017] 2) The sequence of the target gene fragment of classical swine fever virus CSFV is shown in SEQ ID NO.20;

[0018] 3) The sequence of the target gene fragment of foot-and-mouth disease virus FMDV is shown in SEQ ID NO.21;

[0019] 4) The sequence of the target gene fragment of the universal swine influenza virus SIV is shown in SEQ ID NO.22;

[0020] 5) The sequence of the target gene fragment of swine influenza virus H1 type SIV-H1 is shown in SEQ ID NO.23;

[0021] 6) The sequence of the target gene fragment of swine influenza virus H3 type SIV-H3 is shown in SEQ ID NO.24.

[0022] Preferably, the reagents are primers and extension probes.

[0023] Most preferably, the primers and extension probes include:

[0024] 1) Primers PRRSV and extended probe UEP.PRRSV for detecting porcine reproductive and respiratory syndrome virus PRRSV, wherein the primers PRRSV include: a forward primer PRRSV-F, whose sequence is shown in SEQ ID NO.1, and a reverse primer PRRSV-R, whose sequence is shown in SEQ ID NO.2; the sequence of the extended probe UEP.PRRSV is shown in SEQ ID NO.3;

[0025] 2) Primers CSFV and extension probe UEP.CSFV for detecting classical swine fever virus CSFV, the primers CSFV comprising: a forward primer CSFV-F, whose sequence is shown in SEQ ID NO.4, a reverse primer CSFV-R, whose sequence is shown in SEQ ID NO.5; the sequence of the extension probe UEP.CSFV is shown in SEQ ID NO.6;

[0026] 3) Primers FMDV and extended probe UEP.FMDV for detecting foot-and-mouth disease virus FMDV, the primer FMDV comprising: a forward primer FMDV-F, whose sequence is shown in SEQ ID NO.7, a reverse primer FMDV-R, whose sequence is shown in SEQ ID NO.8; the sequence of the extended probe UEP.FMDV is shown in SEQ ID NO.9;

[0027] 4) Primer SIV and extended probe UEP.SIV for detecting universal swine influenza virus SIV, the primer SIV comprising: a forward primer SIV-F, whose sequence is shown in SEQ ID NO.10, a reverse primer SIV-R, whose sequence is shown in SEQ ID NO.11; the sequence of the extended probe UEP.SIV is shown in SEQ ID NO.12;

[0028] 5) Primer SIV-H1 and extended probe UEP.SIV-H1 for detecting swine influenza virus H1 type SIV-H1, wherein the primer SIV-

[0029] H1 includes: a forward primer SIV-H1-F, whose sequence is shown in SEQ ID NO.13, a reverse primer SIV-H1-R, whose sequence is shown in SEQ ID NO.14; the sequence of the extended probe UEP.SIV-H1 is shown in SEQ ID NO.15;

[0030] 6) Primer SIV-H3 and extended probe UEP.SIV-H3 for detecting swine influenza virus H3 type SIV-H3, wherein the primer SIV-

[0031] H3 includes: a forward primer SIV-H3-F, whose sequence is shown in SEQ ID NO.16, and a reverse primer SIV-H3-R, whose sequence is shown in SEQ ID NO.17; the sequence of the extended probe UEP.SIV-H3 is shown in SEQ ID NO.18.

[0032] Furthermore, the present application provides a multi-target detection kit for porcine respiratory RNA virus based on MALDI-TOF MS technology, and the kit includes the reagents described above.

[0033] Furthermore, the present application provides a multi-target detection method for porcine respiratory RNA virus based on MALDI-TOF MS technology, the detection method comprising the following steps:

[0034] 1) After extracting total RNA from the sample and reverse transcribing to obtain cDNA, multiplex PCR reaction was performed using the primers;

[0035] 2) mixing the amplified products of the multiplex PCR reaction with a SAP enzyme mixture to digest the excess dNTPs in the reaction system; mixing the extension probe of claim 1 with the amplified products of the multiplex PCR reaction, and performing a single base extension reaction using the ddNTPs digested with SAP as substrates;

[0036] 3) The extension product obtained in step 2) was detected and analyzed by mass spectrometry; each virus in the system had only a single peak of extension product CSFV: 4 850.2 Da, SIV: 5081.3 Da, PRRSV: 5 701.7 Da, FMDV: 5818.8

[0037] Da, SIV-H3: 6 346.2 Da, SIV-H1: 6 440.2 Da.

[0038] Preferably, the reverse transcription reaction procedure in step 1) is: 30°C for 10 min; 42°C for 15 min; the reaction system is as follows:

[0039]

[0040] Preferably, the multiplex PCR reaction program in step 1) is: 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 72°C for 60 s, for 35 cycles; 72°C for 5 min; the reaction system is as follows:

[0041]

[0042] Preferably, the primer concentrations in the PCR primer mixture are: 0.6 μM SIV, 0.5 μM SIV-H1, 0.5 μM SIV-H3, 0.3 μM PRRSV, 0.5 μM CSFV, 0.6 μM FMDV.

[0043] Preferably, the SAP digestion reaction in step 2) is as follows: adding the amplified product to 2 μL of SAP enzyme mixture, and the reaction procedure is 37° C. for 40 min; 85° C. for 5 min.

[0044] Preferably, the extension reaction in step 2) is as follows: add 1 μL extension probe mixture, 0.2 μL Inplex buffer and 0.2 μL Termination mix to the SAP digestion reaction product, and make up to 9 μL with ddH2O; the reaction procedure is 95°C for 30s; 95°C for 5s, 52°C for 5s, 80°C for 5s, for 40 cycles; 72°C for 3min.

[0045] Preferably, the concentrations of the extension probes in the extension probe mixture are: 6 μM UEP.SIV, 5 μM UEP.SIV-H1, 5 μM UEP.SIV-H3, 5 μM UEP.PRRSV, 3.5 μM UEP.CSFV, 5 μM UEP.FMDV.

[0046] Furthermore, the present application provides the use of the reagent, the kit and the detection method in import and export animal quarantine and related disease monitoring.

[0047] The beneficial technical effects of the present invention are as follows:

[0048] In this application, a multi-target detection method for porcine respiratory RNA viruses based on MALDI-TOF MS technology was established for the first time to detect SIV, SIV-H1, SIV-H3, PRRSV, CSFV and FMDV viruses, further improving the respiratory syndrome pathogen detection system, facilitating the rapid diagnosis of epidemic diseases and the medium- and high-throughput detection of related pathogens, and meeting the requirements of import and export animal quarantine and related epidemic disease monitoring. In addition, this detection method performs typing detection of universal swine influenza viruses and H1 and H3 types, which can further reduce the missed detection rate of SIV and play a vital role in preventing the spread and pandemic of swine influenza. [3] It also has certain economic value and public health significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 :Single reaction test results of 6 viruses (left peak: ddH2O as template; right peak: 10 4copies / μL plasmid as template; A: CSFV; B: FMDV; C: PRRSV; D: SIV; E: SIV-H1; F: SIV-H3).

[0050] Figure 2 : Mass spectrometry results of mixed plasmid and blank control (left peak: ddH2O as template; right peak: 6 targets 10 2 copies / μL mixed plasmid).

[0051] Figure 3 : Specificity test results of 6-plex nucleic acid mass spectrometry system (X: 6-plex detection system target; Y: ddH2O, each virus, and mixed plasmids of 6 targets; Z: signal-to-noise ratio (SNR)).

[0052] Figure 4 : Results of SIV-H1, SIV-H3 and SIV-specific tests (A: Using SIV-h1 nucleic acid as a template, the red arrow indicates that SIV (5081.3Da) and SIV-h1 (6440.2Da) are positive. B: Using SIV-h3 nucleic acid as a template, the red arrow indicates that SIV (5081.3Da) and SIV-h3 (6346.2Da) are positive. C: Using SIV-h5 and SIV-h7 nucleic acids as templates, the red arrow indicates that only SIV is positive (5081.3Da)).

[0053] Figure 5 : Results of 6-plex nucleic acid mass spectrometry sensitivity test (X: 6-plex detection system target; Y: mixed plasmids of 6 targets with different concentrations; Z: number of positive results of 10 sets of repeated tests). DETAILED DESCRIPTION

[0054] The following will be combined with the embodiments of the present invention to clarify and fully describe the technical solutions in the embodiments of the present invention, and then further explain the invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Given the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0055] Example 1 Materials and Methods

[0056] Virus:

[0057] FMDV inactivated vaccine (OHM / 02 strain + AKT-111 strain) was purchased from Jinyu Biotechnology Co., Ltd. SIV-H1 inactivated vaccine (TJ strain) was purchased from Wuhan Keqian Biological Co., Ltd. SIV-H5 and H7 inactivated vaccines (Re-12 strain, H7Re3 strain) were purchased from Qingdao Yibang Biotechnology Co., Ltd. CSFV, PRRSV, pseudorabies virus (PRV, Bartha strain), porcine epidemic diarrhea virus (PEDV, CV777 strain) and porcine circovirus type 2 (PCV-2, JH SRJ strain) were kindly donated by Zhejiang University. Porcine parvovirus (PPV) was kindly donated by Zhejiang Agricultural and Forestry University. Porcine bocavirus (PBoV) nucleic acid was preserved in this laboratory. The nucleic acid of the strains and vaccines was purified and extracted according to the instructions of the TaKaRa MiniBEST ViralRNA / DNA Extraction Kit, and the DNA or reverse transcribed cDNA template was stored at -20℃ for later use.

[0058] Reagents and instruments:

[0059] TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0, PrimeScript TM IV1st strand cDNA Synthesis Mix, Premix Ex Taq TM Purchased from Bio-Tech (Beijing) Co., Ltd. dsDNA HS Assay Kit was purchased from Nanjing Novozymes Biotech Co., Ltd.; PCR Reagent Set, SAP Reagent Set, and Iplex Pro Reagent Set were purchased from Agena Bioscience; 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, ProFlex PCR Instrument, and QuantStudio 5 Real-time Fluorescence Quantitative PCR Instrument were purchased from Thermo Fisher; DP-TOF Nucleic Acid Mass Spectrometer was from Zhejiang Dipu Diagnostic Technology Co., Ltd.

[0060] Design and synthesis of primers and quality probes:

[0061] The relevant viral gene sequences included in GenBank were downloaded, and multiple sequence alignment was performed using ClustalW and MEGA-X. Finally, the ORF6 gene of PRRSV, the 5'-UTR gene of CSFV, the 3D gene of FMDV, the M1 gene of SIV, and the conserved regions of the HA genes of SIV-H1 and SIV-H3 were selected as the target fragments (as shown in Table 1).

[0062] Table 1 Selection of target gene fragments

[0063]

[0064]

[0065] Primer 3 and MassARRAY Assay were used based on the selected fragments. Design Suite (Agena) was used to design specific PCR primers and corresponding extension probes (UEP) for virus detection, and Primer-BLAST and MFEprimer 3.1 were used to evaluate the specificity, primer dimer and hairpin structure of the designed multiple primers and probes. The selected viral target genes, primer sequences and quality information are shown in Table 2. Multiple PCR primers and extension probes, as well as qPCR primers and probes used for comparison and verification were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.

[0066] Table 2 Target genes and primer information of CSFV, SIV, PRRSV, FMDV, SIV-H3 and SIV-H1

[0067]

[0068] Standard plasmid establishment:

[0069] The CSFV (NC_002657.1), PRRSV (AF066183.4), FMDV (AY593823.1), SIV (U53169.1), SIV-H1 (JN809161) and SIV-H3 (NC_007366.1) gene sequences published in GenBank were used as templates, and the corresponding positions were selected with reference to the target viral fragments. The PUC-57 standard plasmid was constructed by Sangon Biotech (Shanghai) Co., Ltd. The concentration of each plasmid was measured using Qubit 4, and the concentration was calculated according to (N A × plasmid concentration) / (base number × 660Da / bp) = copies / mL, where N A =6.02×10 23 , and further obtained the copy number of each plasmid: 6.09×10 9 copies / μL (CSFV), 1.24×10 10 copies / μL (PRRSV), 9.56×10 9 copies / μL (FMDV), 1.48×10 10 Copies / μL (SIV), 8.29×10 9 copies / μL (SIV-H1) and 4.12×109 copies / μL (SIV-H3), diluted 10-fold to 10 9 –10 0 There were 10 gradients of copies / μL in total. Six plasmids at the same concentration level were mixed in equal volumes to obtain mixed standard plasmids for each gradient, which were stored at -20°C for future use.

[0070] Singleplex reaction assay:

[0071] Only the amplification primers and extension probes of the target were added to the reaction system, and the concentration of each plasmid (10 4 The single target amplification experiments were carried out on the six targets respectively, and the reaction status of each set of target amplification primers and extension probes was verified by mass spectrometry results.

[0072] Example 2 Multiple System Reaction Conditions and Optimization

[0073] The MALDI-TOF MS combined with PCR method mainly involves performing multiple PCR reactions, SAP digestion reactions, and single-base extension reactions on sample DNA or cDNA, and then detecting and analyzing the extension products using a mass spectrometer.

[0074] (1) Reverse transcription reaction: Mix 2 μL template RNA with 4 μL 5× PrimeScript IV cDNA Synthesis Mix and 2 μL Random 6mers, and make up to 20 μL with ddH2O. The reaction procedure is 30°C for 10 min and 42°C for 15 min.

[0075] (2) Multiplex PCR reaction: Mix 2 μL of plasmid or reverse-transcribed cDNA template with 1.2 μL of 10× PCR Buffer Mix and 1 μL of PCR primer mixture, and make up to 20 μL with ddH2O. The reaction procedure is 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, and 72°C for 60 s for 35 cycles; 72°C for 5 min.

[0076] (3) SAP digestion reaction: add the amplified product to 2 μL SAP enzyme mixture, and the reaction procedure is 37°C for 40 min; 85°C for 5 min.

[0077] (4) Extension reaction: Add 1 μL of extension probe mixture, 0.2 μL of Inplex buffer and 0.2 μL of Termination mix to the digestion product, and make up to 9 μL with ddH2O. The reaction procedure is 95°C for 30 s; 95°C for 5 s, 52°C for 5 s, 80°C for 5 s, for 40 cycles; 72°C for 3 min.

[0078] After the reaction, the extension product was transferred to a 384 plate, supplemented with ddH2O to 25 μL, and centrifuged at 8 000 r / min for 2 min. The centrifuged 384 plate was placed in a mass spectrometer and subjected to mass spectrometry detection after resin desalting. Data acquisition and analysis were completed by MassARRAY Typer software.

[0079] According to the extension efficiency E = S 产物峰 / (S UPE残留峰 +S 产物峰 ), taking E≥0.8 as the standard, and considering the relative peak height of the multiple extension probe, the ratio of the multiple primers and extension probes in the system were adjusted respectively. 4 The multiplex system was optimized using 1000 copies / μL mixed plasmid as a template, and primers and extension probes with initial concentrations of 0.5 μM and 5 μM, respectively, to obtain a mass signal spectrum with clear extension probe peaks and product peaks.

[0080] Example 3 Method Evaluation Experiment

[0081] Specificity test:

[0082] Using non-target common swine disease virus genomic nucleic acids such as PCV-2, PEDV, PBoV, PPV and PRV, and target genomic nucleic acids such as SIV-H1, SIV-H3, SIV-H5 / H7, PRRSV, CSFV and FMDV as templates, 10 2 The specificity test of the MALDI-TOF MS multiple detection system was performed with the standard plasmid mixed template of 1000 copies / μL as the positive control and ddH2O as the negative control. If there is no extension product peak for each non-target pathogen, but there is an extension product peak at a specific position for SIV, SIV-H1, SIV-H3, PRRSV, CSFV, FMDV and the mixed plasmid, and there is no overlap between the product peaks, the detection system is considered to have good specificity.

[0083] Sensitivity test:

[0084] To evaluate the sensitivity of this method, the optimized reaction system was used to conduct experiments on mixed plasmids. 4 , 10 3 , 10 2 , 50, 25, 10, 5, 2.5, and 1 copy / μL mixed standard plasmids were used as templates, and each set of gradients was repeated 10 times using the optimized MALDI-TOF MS detection system. The concentration of the plasmid standard that was positive in each repetition was considered to be the minimum detection limit (LOD) of the MALDI-TOFMS detection system for the virus.

[0085] Repeatability test:

[0086] High, medium and low concentrations (10 6 , 10 4 , 10 2 A mixed plasmid standard with 10 copies / μL) was used as a template, and 20 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 detected at 3 different times, with 10 replicates for each concentration, and the detection results of the plasmids at each dilution of 3 independent experiments were recorded to evaluate the inter-batch repeatability of the method.

[0087] Sample testing application:

[0088] 34 tissue samples, 42 samples of feed containing porcine ingredients, and 48 samples of fresh pork were collected and stored by our laboratory. 0.5 g of tissue or fresh pork and feed samples were taken respectively, ground at low temperature, and placed in PBS buffer at a ratio of 1:10. After shaking and mixing, centrifuged at 10,000 r / min for 5 minutes, and the supernatant was taken for nucleic acid extraction. Total RNA was extracted from the samples according to the instructions of the reagents supporting the nucleic acid extractor NP968-C, and PrimeScript was used. TM IV 1st strand cDNA Synthesis Mix was used to reverse transcribe the obtained nucleic acid, and the obtained cDNA was used for detection by MADLI-TOF MS method and fluorescence quantitative PCR method. The fluorescence quantitative PCR method was PRRSV (GB / T 35912-2018. Fluorescence RT-PCR detection method for porcine reproductive and respiratory syndrome virus), CSFV (GB / T 27540-2011. Real-time fluorescence RT-PCR detection method for classical swine fever virus), FMDV (GB / T27528-2011. Real-time fluorescence RT-PCR detection method for foot-and-mouth disease virus), SIV (Zhang Chunming, Qiao Chuanling, Chen Yan, Yang Huanliang, Xin Xiaoguang, Chen Hualan, Ran Duoliang. Establishment of real-time fluorescence quantitative PCR diagnosis method for swine influenza virus M gene [J]. Chinese Journal of Preventive Veterinary Medicine, 2008 (10): 805-810.), SIV-H1 and SIV-H3 (Wang Bo, Wang Huiyu, Zhao Baohua, Luo Jing, Wang Chengmin, He Hongxuan, Han Xueqing. Establishment and application of dual real-time fluorescence quantitative RT-PCR detection method for H1 and H3 subtype swine influenza viruses [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2017, 44(10): 3035-3041.) Six viruses were detected by single target. The results obtained by the two methods were calculated according to (true positive number + true negative number) / total number of detections × 100%, and the total compliance rate was calculated, and the results were evaluated.

[0089] Example 4 Results and Analysis

[0090] Singleplex reaction assay:

[0091] Take 10 4 The single-target amplification test was performed using a single primer and an extension probe, and a mass spectrometry image with mass as the horizontal axis and SNP intensity as the vertical axis was obtained for each target. Figure 1 As shown, each virus in the system has only a single peak of extension product (CSFV: 4 850.2Da, SIV: 5 081.3Da, PRRSV: 5 701.7Da, FMDV: 5 818.8Da, SIV-H3: 6 346.2Da, SIV-H1: 6 440.2Da), and the corresponding negative control has only a single peak of extension probe peak (CSFV: 4 579.0Da, SIV: 4 794.1Da, PRRSV: 5 454.6Da, FMDV: 5 531.6Da, SIV-H3: 6 059.0Da, SIV-H1: 6 169.0Da). The mass spectrometry image is clear and no miscellaneous peaks appear within the mass detection range, indicating that the amplification primers and mass probes of each virus in the system can react effectively.

[0092] Reaction system establishment:

[0093] like Figure 2 As shown in the figure, each virus in the system has two peak positions corresponding to the extension probe (UEP) and the single base extension product in the mass spectrometry image. If the mass spectrometry image in the test result only has a peak at the extension probe, it means that the detection result of this target is negative, and the extension probe does not participate in the reaction; if a peak appears at the extension product (SNR≥6), it means that the detection result of the target is positive. After the concentration of each set of primers and extension probes in the system was optimized, the multiple PCR primers finally used were 0.6μM (SIV), 0.5μM (SIV-H1), 0.5μM (SIV-H3), 0.3μM (PRRSV), 0.5μM (CSFV), and 0.6μM (FMDV), and the optimal concentrations of the extension probes were 6μM (SIV), 5μM (SIV-H1), 5μM (SIV-H3), 5μM (PRRSV-), 3.5μM (CSFV), and 5μM (FMDV).

[0094] Specificity analysis:

[0095] The experimental results after MALDI-TOF MS detection were as follows: using target virus cDNAs such as SIV-H1, SIV-H3, SIV-H5 / H7, PRRSV, CSFV, FMDV, and non-target common swine disease virus DNA or cDNAs as templates. Figure 3The results showed that each target virus in the system produced a single peak at its respective extension product (SNR≥6), the non-target template only had an extension probe peak, and no peak at the extension product (SNR≤3), and the negative control and positive control were established. Among them, SIV-H1 and SIV-H3 had product peaks at their own and SIV extension products, while SIV-H5 / H7 only produced positive at the SIV universal position, and there were no product peaks and interference peaks at the SIV-H1 and SIV-H3 positions ( Figure 4 ), which is consistent with experimental expectations. The results show that this method can accurately identify the target virus and has no cross-reaction with other non-target pathogens, with good specificity.

[0096] Sensitivity analysis and minimum detection limit:

[0097] To evaluate the sensitivity of the detection system, 10 4 ~10 0 The MALDI-TOF MS experiment was performed with a mixed plasmid template of 10 copies / μL. Based on the plasmid concentration that was positive in 10 replicates, the minimum detection limit for each virus was obtained: 6.76 copies / μL for CSFV, 8.22 copies / μL for SIV, 13.73 copies / μL for PRRSV, 21.25 copies / μL for FMDV, 9.15 copies / μL for SIV-H3, and 18.41 copies / μL for SIV-H1 ( Figure 5 ), the detection concentration of each target in the system was less than 25 copies / μL, among which the detection level of CSFV and SIV could reach single copy, indicating that the sensitivity of the system was good.

[0098] Repeatability analysis:

[0099] To verify the repeatability of the system for mixed plasmids at different concentrations, 10 6 , 10 4 , 10 2 The three concentrations of 1000 copies / μL were used as representatives, and the corresponding mixed plasmids were used as templates for 20 replicates each; in addition, the detection was carried out in three time periods, and each concentration was repeated 10 times. The results are shown in Table 2. The high, medium, and low concentration groups (10 6 , 10 4 , 10 2 The detection results of six targets, SIV, SIV-H1, SIV-H3, PRRSV, CSFV and FMDV, in the three different batches of experiments were all positive (SNR≥6), indicating that the method had good intra-batch repeatability. In the detection results of three different batches of experiments, the positive rates of SIV, SIV-H3, PRRSV, FMDV and CSFV were all 100%, and the positive rate of SIV-H1 was 96.7% (29 / 30), indicating that the method had high inter-batch repeatability (Table 3).

[0100] Table 3 Repeatability test results

[0101]

[0102] Sample testing application:

[0103] The MALDI-TOF MS method was used to detect 34 tissue or whole blood samples, 42 feed samples containing pig-derived ingredients, and 48 pork samples. The results are shown in Table 4. A total of 16 positive samples were detected in 124 samples, all of which were detected in tissue samples. Among the 34 tissue samples, 9 PRRSV samples were detected, and 3 SIV samples were detected, of which 2 and 1 SIV-H1 and SIV-H3 were detected, respectively. None of the six target viruses were detected in 42 feed samples and 48 fresh pork samples. The MALDI-TOF MS method was used to compare the detection results of PRRSV, CSFV, FMDV, SIV, SIV-H1, and SIV-H3 fluorescence quantitative PCR methods. The results showed that the positive detection results of the two methods were basically consistent. Except for the SIV-H1 coincidence rate of 99.2%, the coincidence rates of the other five targets were 100%.

[0104] Table 4 Results and total coincidence rate of MALDI-TOF MS multiple detection system and fluorescence PCR detection

[0105]

[0106]

[0107] Tissues tested by both methods, pork and feed samples.

[0108] When PCR technology represented by multiplex PCR technology is used for the detection of multiple pathogens, it is often limited by cross-reactions such as dimers and hairpin structures generated between multiple sets of primers in the system, or by the limitations of fluorescent dyes and probe fluorescence channels, and it is usually difficult to detect four or more targets simultaneously. When designing multiple primers, the MALDI-TOF MS combined with multiplex PCR technology adopts the homologous tailing (Homo-Tag Assisted Non-Dimer, HAND) strategy to add the same mass tag (ACGTTGGATG) to the 5′ end of each PCR primer, which can effectively reduce the formation of primer dimers and avoid the interference of a small amount of dimers and unreacted primers on the mass probe peak and product peak position in the mass spectrum, thereby better detecting multiple pathogen targets simultaneously. The MALDI-TOF MS combined with multiplex PCR technology can detect nucleic acid fragments in the mass range of nearly 6 000Da, which is suitable for the monitoring of important pathogens in different syndromes of animal diseases in clinical and import and export port situations.

[0109] Through methodological verification, the RNA virus detection method has good stability and specificity, and the detection limit of each target in the system can reach 10 1 The copy / μL level is equivalent to that of single-plex fluorescent PCR. The results of testing tissue and whole blood samples, feed samples and fresh pork samples showed that the MALDI-TOF MS method and the fluorescent quantitative PCR method had a high coincidence rate (99.2%-100%) for sample detection results, which was consistent with the comparison results of the MALDI-TOFMS detection method and qPCR method for porcine respiratory DNA virus established by the previous research group. In this study, both methods detected 9 PRRSV-positive samples and 3 SIV-positive samples (including 1 SIV-H3-positive sample), while CSFV and FMDV were negative. The difference between the two methods is mainly reflected in the detection results of SIV-H1 in tissue samples: 1 tissue sample was detected by MALDI-TOF MS method, showing that both SIV and SIV-H1 were positive; the results of the fluorescent quantitative PCR method showed that the SIV-H1 of the sample was negative (ct value ≥ 38), while SIV was positive, indicating that the SIV-H1 result of the sample was false negative. Later, sequencing confirmed that the sample was indeed a SIV-H1 positive sample. In addition, the two methods did not detect positive results in 42 feed and fresh pork samples. This may be because the pig-derived ingredients in the feed severely damaged the virus structure during deep processing and subsequent storage, resulting in a low content of viral RNA fragments in the sample (≤25 copies / μL); or the pig-derived ingredients used in the feed may not be contaminated by several viruses. The negative results of all 48 fresh pork samples also support this speculation.

[0110] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. Primers and extension probes for multi-target detection of porcine respiratory RNA viruses based on MALDI-TOF MS technology, the primers and extension probes comprising: 1) Primers PRRSV and extended probe UEP.PRRSV for detecting porcine reproductive and respiratory syndrome virus PRRSV, wherein the primers PRRSV include: a forward primer PRRSV-F, whose sequence is shown in SEQ ID NO.1, and a reverse primer PRRSV-R, whose sequence is shown in SEQ ID NO.2; the sequence of the extended probe UEP.PRRSV is shown in SEQ ID NO.3; 2) Primer CSFV and extension probe UEP. CSFV for detecting classical swine fever virus CSFV, the primer CSFV comprising: a forward primer CSFV-F, whose sequence is shown in SEQ ID NO.4, a reverse primer CSFV-R, whose sequence is shown in SEQ ID NO.5; the sequence of the extension probe UEP. CSFV is shown in SEQ ID NO.6; 3) Primers FMDV and extended probe UEP. FMDV for detecting foot-and-mouth disease virus FMDV, the primer FMDV comprising: a forward primer FMDV-F, whose sequence is shown in SEQ ID NO.7, a reverse primer FMDV-R, whose sequence is shown in SEQ ID NO.8; the sequence of the extended probe UEP. FMDV is shown in SEQ ID NO.9; 4) Primer SIV and extended probe UEP.SIV for detecting universal swine influenza virus SIV, the primer SIV comprising: forward primer SIV-F, whose sequence is shown in SEQ ID NO.10, reverse primer SIV-R, whose sequence is shown in SEQ ID NO.11; the sequence of the extended probe UEP.SIV is shown in SEQ ID NO.12; 5) Primer SIV-H1 and extension probe UEP.SIV-H1 for detecting swine influenza virus H1 type SIV-H1, the primer SIV-H1 comprises: a forward primer SIV-H1-F, whose sequence is shown in SEQ ID NO.13, and a reverse primer SIV-H1-R, whose sequence is shown in SEQ ID NO.14; the sequence of the extension probe UEP.SIV-H1 is shown in SEQ ID NO.15; 6) Primer SIV-H3 and extended probe UEP.SIV-H3 for detecting swine influenza virus H3 type SIV-H3, the primer SIV-H3 comprises: a forward primer SIV-H3-F, whose sequence is shown in SEQ ID NO.16, and a reverse primer SIV-H3-R, whose sequence is shown in SEQ ID NO.17; the sequence of the extended probe UEP.SIV-H3 is shown in SEQ ID NO.

18.

2. A reagent for multi-target detection of porcine respiratory RNA viruses based on MALDI-TOF MS technology, characterized in that: The porcine respiratory RNA virus is porcine reproductive and respiratory syndrome virus PRRSV, classical swine fever virus CSFV, foot-and-mouth disease virus FMDV, universal swine influenza virus SIV, swine influenza virus H1 type SIV-H1 and swine influenza virus H3 type SIV-H3; the reagent includes a primer and an extension probe, and the primer and extension probe include: 1) Primers PRRSV and extended probe UEP.PRRSV for detecting porcine reproductive and respiratory syndrome virus PRRSV, wherein the primers PRRSV include: a forward primer PRRSV-F, whose sequence is shown in SEQ ID NO.1, and a reverse primer PRRSV-R, whose sequence is shown in SEQ ID NO.2; the sequence of the extended probe UEP.PRRSV is shown in SEQ ID NO.3; 2) Primer CSFV and extension probe UEP. CSFV for detecting classical swine fever virus CSFV, the primer CSFV comprising: a forward primer CSFV-F, whose sequence is shown in SEQ ID NO.4, a reverse primer CSFV-R, whose sequence is shown in SEQ ID NO.5; the sequence of the extension probe UEP. CSFV is shown in SEQ ID NO.6; 3) Primers FMDV and extended probe UEP. FMDV for detecting foot-and-mouth disease virus FMDV, the primer FMDV comprising: a forward primer FMDV-F, whose sequence is shown in SEQ ID NO.7, a reverse primer FMDV-R, whose sequence is shown in SEQ ID NO.8; the sequence of the extended probe UEP. FMDV is shown in SEQ ID NO.9; 4) Primer SIV and extended probe UEP.SIV for detecting universal swine influenza virus SIV, the primer SIV comprising: forward primer SIV-F, whose sequence is shown in SEQ ID NO.10, reverse primer SIV-R, whose sequence is shown in SEQ ID NO.11; the sequence of the extended probe UEP.SIV is shown in SEQ ID NO.12; 5) Primer SIV-H1 and extension probe UEP.SIV-H1 for detecting swine influenza virus H1 type SIV-H1, the primer SIV-H1 comprises: a forward primer SIV-H1-F, whose sequence is shown in SEQ ID NO.13, and a reverse primer SIV-H1-R, whose sequence is shown in SEQ ID NO.14; the sequence of the extension probe UEP.SIV-H1 is shown in SEQ ID NO.15; 6) Primer SIV-H3 and extended probe UEP.SIV-H3 for detecting swine influenza virus H3 type SIV-H3, the primer SIV-H3 comprises: a forward primer SIV-H3-F, whose sequence is shown in SEQ ID NO.16, and a reverse primer SIV-H3-R, whose sequence is shown in SEQ ID NO.17; the sequence of the extended probe UEP.SIV-H3 is shown in SEQ ID NO.

18.

3. A multi-target detection kit for porcine respiratory RNA virus based on MALDI-TOF MS technology, characterized in that: The porcine respiratory RNA viruses are porcine reproductive and respiratory syndrome virus PRRSV, classical swine fever virus CSFV, foot-and-mouth disease virus FMDV, swine influenza virus universal type SIV, swine influenza virus H1 type SIV-H1 and swine influenza virus H3 type SIV-H3, and the kit includes the reagent described in claim 2.

4. The kit according to claim 3, characterized in that The primer concentrations were: 0.6 μM SIV, 0.5 μM SIV-H1, 0.5 μM SIV-H3, 0.3 μM PRRSV, 0.5 μM CSFV, 0.6 μM FMDV; the extension probe concentrations in the extension probe mixture were: 6 μM UEP.SIV, 5 μM UEP.SIV-H1, 5 μM UEP.SIV-H3, 5 μM UEP.PRRSV, 3.5 μM UEP.CSFV, 5 μM UEP.FMDV.

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