A multiplex PCR primer set and a multiplex PCR and nanopore sequencing method for detecting a broad spectrum of porcine pathogens.

Abstract

The present invention discloses a super-multiplex PCR primer set and a super-multiplex PCR and nanopore sequencing method for detecting a broad spectrum of porcine pathogens, including 45-plex fluorescence quantitative PCR amplification: using 45 specific primers and probes to perform super-multiplex PCR amplification on the sample to be tested, and detecting 45 porcine pathogen antigens, the sequences of the specific primers and probes are shown in SEQ ID NO: 1 to 135. The present invention can simultaneously detect 96 samples and 45 porcine pathogens in each sample in one reaction well, and the method of the present invention is of great significance for the normal detection of large-scale breeding and the emergency prevention and control when an epidemic occurs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a super-multiplex PCR primer set and a super-multiplex PCR and nanopore sequencing method for detecting a broad spectrum of porcine pathogens. Background Art

[0002] The scale of the breeding industry has reached its peak in recent years. Since 2004, various diseases, such as African swine fever, classical swine fever, porcine reproductive and respiratory syndrome, piglet diarrhea, pseudorabies, foot-and-mouth disease, circovirus type II, etc., have had a huge impact on the pig farming industry, requiring more portable, rapid, accurate and popular detection methods. At this stage, the state requires that large-scale pig farms must be equipped with testing laboratories. This measure can also effectively improve the efficiency of pig farms and do a better job of biosafety prevention and control in a more timely manner. Among the various detection technologies, real-time fluorescence quantitative PCR technology stands out and truly achieves popularization. At the same time, it has the advantages of simple operation, high sensitivity, high specificity, and low cost. It has become the industry's gold standard for detection. However, in recent years, with the continuous mutation or evolution of pathogens, the continuous expansion of the scale of pig farms, and the explosive growth of the number of test samples, traditional PCR, including some other methods based on qPCR such as digital PCR and multiplex fluorescence quantitative PCR, can no longer meet the needs of the current detection environment. Before PCR entered the biological arena as a patented biotechnology in 1985, the first-generation sequencing technology was already introduced in 1977. As time goes by, the development of the sequencing field has not stopped, including the extremely mature second-generation sequencing and the third-generation sequencing that is receiving widespread attention. The third-generation nanopore sequencing is even called "the only answer for the future."

[0003] At present, nanopore sequencing technology based on electrical signal detection is the high-throughput sequencing platform with the simplest experimental operation and the fastest sequencing speed. However, the currently used sequencing methods still have problems such as high error rate and low detection efficiency when trying to quickly detect a large number of samples and multiple pathogens.

[0004] If a broad-spectrum swine pathogen detection method with super-multiplex PCR primer sets and nanopore single-molecule sequencing can be provided, a large number of samples can be quickly detected and most of the swine pathogens in the screening samples can be screened. This is of great significance for routine detection of large-scale breeding and emergency prevention and control when an epidemic occurs. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] As one aspect of the present invention, the present invention provides a method for detecting broad-spectrum porcine pathogens using a super-multiplex PCR primer set and nanopore single-molecule sequencing, which includes 45-plex fluorescent quantitative PCR amplification: using 45 specific primers and probes to perform super-multiplex PCR amplification on the sample to be tested to detect 45 porcine pathogens, and the sequences of the specific primers and probes are shown in SEQ ID NOs: 1 to 135.

[0007] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of pig pathogens described in the present invention: the 45 pig pathogens are: Foot-and-mouth disease virus, African swine fever virus, Porcine epidemic diarrhea virus, Japanese encephalitis virus, Porcine reproductive and respiratory syndrome virus 2, Porcine reproductive and respiratory syndrome virus 1, Classical swine fever virus, Porcine parvovirus, Rotavirus, Porcine deltacoronavirus, Pseudorabies virus, Porcine circovirus 1, Porcine circovirus 2, Streptococcus suis, Porcine parvovirus, Porcine rotavirus, Porcine deltacoronavirus, Porcine pseudorabies virus, Porcine circovirus 1, Porcine circovirus 2, Streptococcus suis, Porcine circovirus 3, Porcine circovirus 4, Porcine circovirus 5, Porcine circovirus 6, Porcine circovirus 7, Porcine circovirus 8, Porcine circovirus 9, Porcine circovirus 10, Porcine circovirus 11, Porcine circovirus 12, Porcine circovirus 13, Porcine circovirus 14, Porcine circovirus 15, Porcine circovirus 16, Porcine circovirus 17, Porcine circovirus 18, Porcine circovirus 19, Porcine circovirus 19, Porcine suis), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, Lawsonia intracellularis, Clostridium perfringens type, Clostridium difficile, Swine vesicular disease, Nipahvirus, Transmissible gastroenteritis virus, Swine influenza virus, Porcine circovirus type IIIcircovirus 3), Bovine viral diarrhea virus, Brucella suis, Mycoplasma hyorhinis, eperythrozoon, salmonella, Seneca Valley Virus, Mycoplasma hyosynoviae, Chlamydia porcine, Swine dysentery, Leptospira species, Trichinella, Cryptosporidium, Toxoplasma gondii, Isospora suis, Ascaris lumbricoides, Sarcoptes scabiei, Cysticerci.

[0008] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens of the present invention: the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens are composed of the following steps:

[0009] (1) Sample collection and sample nucleic acid extraction;

[0010] (2) The 45-plex fluorescence quantitative PCR amplification according to claim 1;

[0011] (3) constructing a nanopore sequencing library;

[0012] (4) On-line sequencing and bioinformatics analysis.

[0013] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens described in the present invention: in step (1), the sample includes one or more of porcine nasopharyngeal swabs, oral fluid, blood, blood swabs, various porcine tissue samples, and environmental sampling samples.

[0014] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens described in the present invention: the reaction system for PCR amplification is: 4×CAPITAL qPCR Probe Master Mix 10uL, 20×RTase with RNase lnhibitor 1uL, 500nmol / L primer 5uL, 50nmol / L probe 3uL, deionized water 1uL, and sample nucleic acid 5uL.

[0015] As a preferred solution of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens described in the present invention: the reaction procedure of PCR amplification is: reverse transcription at 50°C for 10 min, pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, extension at 65°C for 3 min, reaction cycle number 40, and all probes are FAM channels.

[0016] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens described in the present invention: the construction of a nanopore sequencing library includes constructing a nanopore sequencing library according to a ligation kit, including PCR product purification, PCR product end repair, label ligation, and adapter ligation.

[0017] As a preferred embodiment of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens described in the present invention: in step (4), the whole genome sequencing includes preparing a sequencing chip, performing quality inspection, and then loading the nanopore sequencing chip into a sequencer for nanopore sequencing.

[0018] Beneficial effects of the invention: The invention provides a broad-spectrum swine pathogen detection method using a super-multiplex PCR primer set and nanopore single-molecule sequencing, which can quickly detect a large number of samples and screen most swine diseases in the samples. The invention can simultaneously detect 96 samples and 45 swine pathogens in each sample in one reaction well. The method of the invention is of great significance for routine detection of large-scale breeding and emergency prevention and control when an epidemic occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0020] Figure 1 This is a flow chart of the super-multiplex PCR primer set and nanopore single-molecule sequencing method for detecting a broad spectrum of porcine pathogens of the present invention. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0022] The present invention provides specific primers and probes for amplifying 45 kinds of swine diseases, and the specific primers for amplifying 45 kinds of swine diseases include 45 pairs in total. The present invention combines the advantages of super-multiplex PCR amplification specific primer enrichment with nanopore sequencing in real time, portability, long read length, and high throughput, and can achieve rapid differential diagnosis of 45 kinds of swine diseases. The sequences are shown in SEQ ID NO: 1-135, and the detection steps are as follows:

[0023] (1) Sample collection and sample nucleic acid extraction;

[0024] (2) 45-plex fluorescence quantitative PCR amplification;

[0025] (3) constructing a nanopore sequencing library;

[0026] (4) On-line sequencing and bioinformatics analysis.

[0027] This technology can now detect 96 samples and 45 porcine pathogens in each sample simultaneously.

[0028] The 45 swine pathogens are: Foot-and-mouth disease virus, African swine fever virus, Porcine epidemic diarrhea virus, Japanese encephalitis virus, Porcine reproductive and respiratory syndrome virus 2, Porcine reproductive and respiratory syndrome virus 1, Classical swine fever virus, Porcine parvovirus, Rotavirus, Porcine deltacoronavirus, Pseudorabies virus, Porcine circovirus 1, Porcine circovirus 2, Streptococcus suis, Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, Lawsonia intracellularis, Clostridium perfringens type, Clostridium difficile, Swine vesicular disease, Nipahvirus, Transmissible gastroenteritis virus, Swine influenza virus, Porcine circovirus 3, Bovine viral diarrheaVirus), Brucella suis, Mycoplasma hyorhinis, eperythrozoon, salmonella, Seneca Valley Virus, Mycoplasma hyosynoviae, Chlamydia diaporcine, Swine dysentery, Leptospira species, Trichinella, Cryptosporidium, Toxoplasma gondii, Isospora suis, Ascaris lumbricoides, Sarcoptes scabiei, Cysticerci.

[0029] The sequences of the 45 pairs of specific primers and probes thereof are shown in Table 1 SEQ ID NO: 1-135.

[0030] Table 1

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] Note: A positive fluorescent PCR result indicates that the sample contains one or more of the above 45 pathogens, while a negative result indicates that the sample does not contain any of the above 45 pathogens. If the sample is fluorescently positive, the next step of library construction and sequencing can be continued. If the test is negative, it means that the sample does not contain any important pathogenic microorganisms and no sequencing is required.

[0041] Embodiment 1:

[0042] Optimization of 45-plex fluorescence quantitative PCR and establishment of standard curve:

[0043] 45 pairs of primers and probe dilutions:

[0044] 45 pairs of primer probes were diluted to 100umol / L with TE Buffer. Applicable sample types include nasopharyngeal swabs, environmental samples, serum, blood swabs, and tissue samples.

[0045] Optimization of 45-plex fluorescence quantitative PCR:

[0046] The concentrations of all primers and probes were optimized using the gradient method: primers and probes were diluted to different concentrations according to the gradient, and the total primer probe concentration in the system was proportional to the concentration of a single pair of primers and a single probe. The concentrations of a single pair of primers and a single probe were changed, set into multiple groups, and the same positive plasmid was tested on the machine at the same time. A set of solutions with the same fluorescence intensity and similar Ct value as the single-plex fluorescence quantitative PCR was selected as the optimal solution. The multiplex PCR reaction enzyme was biotechrabbit multiplex fluorescence quantitative PCR enzyme, and the brand of the fluorescence quantitative PCR instrument was Tianlong Technology 96R. The optimized primer probe concentrations were: primer 500nmol / L, probe 50nmol / L. After optimization, the determined reaction system was 20uL: 4×CAPITAL qPCR Probe Master Mix 10uL, 20×RTase with RNase Inhibitor 1uL, total primer volume 5uL, total primer concentration 500nmol / L, total probe volume 3uL, total probe concentration 50nmol / L, deionized water 1uL, sample nucleic acid 5uL; the reaction procedure was as follows: reverse transcription 50℃10min, pre-denaturation 95℃3min, denaturation 95℃10s, extension 65℃3min, reaction cycle number 40, and all probes were FAM channels.

[0047] The brand of the fluorescent quantitative PCR instrument tested is Tianlong 96R. The experimental steps are as follows: turn on the Tianlong 96R fluorescent quantitative PCR instrument and preheat it for 10 minutes, configure the qPCR reaction system according to the above-mentioned system, add the samples to be tested and the positive and negative controls and operate the machine, double-click on the computer to open the Tianlong qPCR amplification software, click the sample setting button on the main interface of the qPCR instrument, select the well where the sample is located with the mouse, click the sample type button in the upper right corner, select "to be tested", select "FAM" in "fluorescence", click run settings, set the program according to the above reaction program, and change the reaction system to 20uL, click "run monitoring", select the instrument model, click "run experiment", and wait for the instrument to end.

[0048] Establishment of 45-plex fluorescence quantitative PCR standard curve:

[0049] Determine the concentration of the synthesized plasmid and calculate the copy number according to the following formula: double-stranded DNA copy number (copies / mL) = 6.02 × 10 23 (copies / mL)×concentration (g / mL) / recombinant plasmid length×660; 45 pathogenic genes were loaded into fish genome plasmids respectively, and the fish genome plasmids were used as the positive control standard of the whole system. The fish genome plasmids were diluted to 3×10 8 copies / mL, and then diluted 10-fold to select 7 concentrations (10 8 copies / mL~10 2 The mixture of 100 copies / mL fish genomic plasmid standards was used as a template, and multiplex fluorescence quantitative PCR amplification was performed according to the optimized reaction system and conditions. A standard curve was established with the logarithm of the standard concentration as the horizontal axis and the Ct value as the vertical axis.

[0050] Forty-five-plex fluorescence quantitative PCR sensitivity experiment:

[0051] All 45 pathogens need to be tested for sensitivity. The dilution method of each positive plasmid is:

[0052] 1. In a clean bench, add 100ul TE Buffer to each positive plasmid powder, vortex mix for 5min, centrifuge for 30s, and mark as the corresponding "plasmid stock solution".

[0053] 2. Take 10ul of each plasmid stock solution into a 1.5ml centrifuge tube, add 990ul TE Buffer, the concentration is 1.134X109 copies / ul, and mark it as "positive plasmid 10 9 ″; Copy number calculation formula: copy number = (6.02×1014)a / 660b (a: stock solution concentration, b: plasmid length).

[0054] 3. Dilution of triple positive plasmid was performed as follows (10 9 ~10 3 Copy / ul):

[0055] ① Take 100ul 10 9 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 8 Copy / ul, labeled as "plasmid 10 8 ″. Vortex for 5 min and centrifuge for 30 s.

[0056] ②Take 100ul 10 8 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 7Copy / ul, labeled as "plasmid 10 7 ″. Vortex for 5 min and centrifuge for 30 s.

[0057] ③Take 100ul 10 7 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 6 Copy / ul, labeled as "plasmid 10 6 ″. Vortex for 5 min and centrifuge for 30 s.

[0058] ④Take 100ul 10 6 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 5 Copy / ul, labeled as "plasmid 10 5 ″. Vortex for 5 min and centrifuge for 30 s.

[0059] ⑤Take 100ul 10 5 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 4 Copy / ul, labeled as "plasmid 10 4 ″. Vortex for 5 min and centrifuge for 30 s.

[0060] ⑥Take 100ul 10 4 Copy / ul of each plasmid into a 1.5ml centrifuge tube, add 900ul TE Buffer at a concentration of 10 3 Copy / ul, labeled as "plasmid 10 3 ″. Vortex for 5 min and centrifuge for 30 s.

[0061] After the dilutions were completed in sequence, fluorescent quantitative PCR was performed according to the gradient until the CT value of each positive plasmid could not be detected, and then the minimum detection limit of each positive plasmid was recorded.

[0062] The sensitivity results are shown in the following table:

[0063]

[0064]

[0065]

[0066] Forty-five-plex fluorescence quantitative PCR stability experiment:

[0067] Select 10 5 copies / mL, 10 6 copies / mL and 10 7The plasmid standard with 1000 copies / mL dilution was tested repeatedly (5 μL of three dilutions of the standard was taken as template), and 10 replicates were performed for each, and the coefficient of variation was calculated using the coefficient of variation calculation formula. The results showed that the coefficient of variation of the three gradients was less than 2% (as shown in Table 2), indicating that the method has good repeatability.

[0068] Table 2

[0069] 1 <![CDATA[10 7 ]]> 20.34 <![CDATA[10 6 ]]> 23.71 <![CDATA[10 5 ]]> 27.01 2 <![CDATA[10 7 ]]> 20.33 <![CDATA[10 6 ]]> 23.83 <![CDATA[10 5 ]]> 26.54 3 <![CDATA[10 7 ]]> 20.48 <![CDATA[10 6 ]]> 23.69 <![CDATA[10 5 ]]> 26.77 4 <![CDATA[10 7 ]]> 20.62 <![CDATA[10 6 ]]> 23.59 <![CDATA[10 5 ]]> 26.85 5 <![CDATA[10 7 ]]> 20.61 <![CDATA[10 6 ]]> 23.76 <![CDATA[10 5 ]]> 26.98 6 <![CDATA[10 7 ]]> 21.05 <![CDATA[10 6 ]]> 24 <![CDATA[10 5 ]]> 27.04 7 <![CDATA[10 7 ]]> 20.58 <![CDATA[10 6 ]]> 23.81 <![CDATA[10 5 ]]> 27.05 8 <![CDATA[10 7 ]]> 21.32 <![CDATA[10 6 ]]> 24.02 <![CDATA[10 5 ]]> 26.92 9 <![CDATA[10 7 ]]> 20.64 <![CDATA[10 6 ]]> 23.88 <![CDATA[10 5 ]]> 26.79 10 <![CDATA[10 7 ]]> 20.48 <![CDATA[10 6 ]]> 23.69 <![CDATA[10 5 ]]> 26.78 average value <![CDATA[10 7 ]]> 20.645 <![CDATA[10 6 ]]> 23.798 <![CDATA[10 5 ]]> 26.873 Standard Deviation <![CDATA[10 7 ]]> 0.3114 <![CDATA[10 6 ]]> 0.1389 <![CDATA[10 5 ]]> 0.16 CV% <![CDATA[10 7 ]]> 1.51 <![CDATA[10 6 ]]> 0.58 <![CDATA[10 5 ]]> 0.6

[0070] Clinical sample compliance verification:

[0071] This 45-plex fluorescent quantitative PCR and commercially available detection kits were used to simultaneously detect suspected clinical samples (a total of 103 samples, some of which may be infected with pathogens). Two commercially available detection kits A, B and C were used separately to detect African swine fever virus, porcine epidemic diarrhea virus and foot-and-mouth disease virus.

[0072] After testing, the test results of test kit A on the samples were: 67 out of 103 samples were positive for African swine fever virus, and 36 were negative; the results of box B were: 39 out of 103 samples were positive for porcine epidemic diarrhea virus, and 64 were negative; the results of box C were: 48 out of 103 samples were positive for porcine foot-and-mouth disease virus, and 55 were negative.

[0073] The results of forty-five-plex fluorescence quantitative PCR testing showed that 67 cases were positive for African swine fever virus, 39 cases were positive for porcine epidemic diarrhea, and 48 cases were positive for swine foot-and-mouth disease virus.

[0074] The detection results of this patent are consistent with those of commercial detection kits on the market, indicating that the ultra-multiplex fluorescence quantitative detection method of this patent can be used for the detection of clinical samples with reliable sensitivity and accuracy.

[0075] Forty-five-plex fluorescence quantitative PCR specificity experiment:

[0076] First, the negative control samples were used for amplification, and there was no complementarity between primers, and the results were all negative. Secondly, 45 different primer probe MIXs were configured, and 44 primer probes were added to each MIX. The same pathogen clinical samples without primer types were used for specificity experiments, and no non-specific characteristics occurred. This shows that the method established in this experiment is highly specific, and there are no mismatches between the 45 primer pairs and their probes. Finally, a pig genome specificity experiment was conducted, and the SPF pig gene was added to the 45-plex fluorescent PCR system. The amplification result was negative, which once again proved its specificity.

[0077] Then proceed to the next step of the experiment. Prepare ten samples, five of which are randomly plasmid positive and five are negative. Experimenter 1 numbers them 1-10, with 1-5 as positive controls and 6-10 as negative controls. Shuffle 1-10 and renumber them as AJ and hand them over to experimenter 2 for testing to see if there will be false positives and false negatives. The test results are as follows (as shown in Table 3):

[0078] Table 3

[0079]

[0080]

[0081] There were no false positives or false negatives in the test results, indicating that the 45-species super-multiplex PCR had high specificity and accuracy.

[0082] Construction of sequencing library:

[0083] Database construction process:

[0084] After the forty-five-plex PCR targeted enrichment, the sequencing library was constructed according to the SQK-NBD114.24 ligation kit (Oxford Nanopore Technologies). The specific operations are as follows:

[0085] PCR product purification:

[0086] Prepare reagents and consumables:

[0087] ① Magnetic beads

[0088] ②Washing solution (Note: add 24mL of anhydrous ethanol before use)

[0089] ③Eluent

[0090] ④1.5mL centrifuge tube

[0091] ⑤Qubit TM Quantitative tube (Invitrogen, catalog number: Q32856)

[0092] ⑥Qubit dsDNA HS Assay Kit (ThermoFisher, Cat. No.: Q32851)

[0093] Place the magnetic beads at room temperature for 30 minutes in advance and vortex to mix thoroughly.

[0094] Transfer the PCR reaction product to a new 1.5 mL centrifuge tube, add 12 μL of magnetic beads to each tube, flick to mix, and place on a rotating mixer for 5 minutes. Centrifuge briefly, place on a magnetic rack for adsorption, and discard the supernatant after the liquid is completely clear.

[0095] Add 200 μL of the prepared washing solution along the tube wall and let it stand for 30 seconds. Centrifuge briefly and place on a magnetic rack for adsorption. Wait until the liquid is completely clear and discard the supernatant. Wash twice in this way;

[0096] Centrifuge briefly and place on a magnetic stand for adsorption. When the liquid is completely clear, remove the remaining washing solution with a pipette, air-dry for 30 seconds (be careful not to dry it too dry to crack), and then add 20 μL of elution solution to rinse the magnetic beads.

[0097] Remove the conical centrifuge tube and gently tap to resuspend the magnetic beads. Place it at room temperature for 2 minutes. After a brief centrifugation, place it back on the magnetic rack. When the solution becomes clear, pipette 20 μL of the clear liquid and transfer it to a new 1.5 mL conical centrifuge tube.

[0098] Take 1 μL of the purified product for Qubit quantification.

[0099] End repair:

[0100] Prepare reagents and consumables:

[0101] ① Magnetic beads

[0102] ②Washing liquid

[0103] ③Eluent

[0104] ④End-repair premix

[0105] ⑤Terminal repair enzyme

[0106] ⑥ Nuclease-free water

[0107] Thaw the magnetic beads at room temperature, vortex thoroughly to mix, and place at room temperature; thaw the components of end repair premix, end repair enzyme, and nuclease-free water, flick to mix, centrifuge briefly, and place on ice;

[0108] In a 0.2mL PCR tube, add 100ng of the purified PCR product. If the volume is less than 12.5μL, add nuclease-free water to make it 12.5μL. Mix the ingredients in the table below, pipette and mix 10 to 20 times, and centrifuge briefly.

[0109] Reagent name Dosage Purified PCR product 12.5μL End Repair Master Mix 1.75μL End repair enzymes 0.75μL Total volume 15μL

[0110] Incubate using a PCR instrument according to the conditions in the table below;

[0111] temperature time 20℃ 5min 65℃ 5min

[0112] After the incubation, transfer the liquid to a 1.5 mL centrifuge tube, add 15 μL of fully vortexed magnetic beads to each tube, cover the centrifuge tube, and flick to mix;

[0113] Place the centrifuge tube on a rotary mixer, incubate at room temperature for 5 minutes, centrifuge briefly, place on a magnetic rack until clarified, and discard the supernatant;

[0114] Keep the magnetic state, add 200μL of the prepared washing solution, be careful not to disturb the magnetic beads during washing, discard the supernatant, and wash twice in this way;

[0115] Centrifuge briefly, place the centrifuge tube back on the magnetic rack, remove the remaining washing solution with a pipette, and air-dry for 30 seconds (be careful not to dry it too much to crack);

[0116] Remove the centrifuge tube, add 10 μL of elution buffer, flick the bottom of the tube to resuspend, centrifuge briefly, incubate at room temperature for 2 minutes, and place on a magnetic stand to absorb until clear;

[0117] Transfer 10 μL of supernatant to a clean 1.5 mL centrifuge tube. This is the unrepaired product. Take 1 μL of the solution and quantify it using Qubit. Record the result.

[0118] Tag connection:

[0119] Prepare reagents and consumables:

[0120] ① Magnetic beads

[0121] ②Washing liquid

[0122] ③Eluent

[0123] ④ Ligase premix 1

[0124] ⑤ Ligation sequencing barcode kit (ONT, catalog number: SQK-NBD114.24)

[0125] ⑥ Centrifuge tube (0.2mL, 1.5mL)

[0126] ⑦Qubit quantitative tube (Invitrogen, catalog number: Q32856)

[0127] ⑧Qubit dsDNA HS Assay Kit (ThermoFisher, Cat. No.: Q32851)

[0128] Thaw the magnetic beads at room temperature, vortex thoroughly to mix, and place at room temperature; thaw the ligase premix 1 and other reagents, flick or pipette to mix, centrifuge briefly, and place on ice;

[0129] Remove the barcode (NB01-24) according to the experimental requirements, mix, and centrifuge. Each sample or quality control requires an independent label number;

[0130] In a 0.2 mL PCR tube, prepare the following reaction;

[0131] Reagent name Dosage End repair products 7.5μl Barcode NB01-24 2.5μl Ligase Master Mix 1 10μl Total volume 20μL

[0132] Mix by pipetting 10-20 times, centrifuge briefly, and incubate at room temperature for 20 minutes;

[0133] After incubation, add 2 μL EDTA (ONT ligation sequencing kit) to each tube, mix by pipetting, and centrifuge briefly;

[0134] Pipette all the ligation products into a 1.5mL centrifuge tube and mix well. Add 0.4 times the total volume of magnetic beads (e.g., add 40μL of magnetic beads to 100μL of the mixed product) for purification; mix thoroughly by pipetting, place on a rotating mixer, and incubate at room temperature for 10 minutes;

[0135] Remove the centrifuge tube, centrifuge it briefly, place it on a magnetic stand for at least 5 minutes until it is clear, and discard the supernatant;

[0136] Keep the magnetic beads in the state of magnetic absorption, add 700μL of the prepared washing solution for washing, be careful not to disturb the magnetic beads during washing, and discard the supernatant. Wash twice in this way;

[0137] Centrifuge briefly, place the centrifuge tube back on the magnetic rack, remove the remaining washing solution with a pipette, and air-dry for 30 seconds (be careful not to dry it too much to crack);

[0138] Remove the centrifuge tube, add 35 μL of elution buffer, flick the bottom of the tube to resuspend, centrifuge briefly, incubate at 37°C for 10 min, take out and flick the tube every 2 min to mix well to ensure elution efficiency;

[0139] Centrifuge briefly and place on a magnetic stand to absorb until clear. Transfer 35 μL of supernatant to a clean 1.5 mL centrifuge tube. This is the tag-linked product.

[0140] Take 1 μL of the ligation product and quantify it with Qubit and record the result. The tag ligation product can be stored at 4°C overnight.

[0141] Notice:

[0142] (1) Each sample corresponds to a labeling reagent, and each nucleic acid sample is processed separately.

[0143] (2) The same chip cannot use the same barcode number. For example, if the barcode 1#-6# is used this time, the chip can use 7#-12 or other barcodes other than 1#-6# next time.

[0144] Connector connection:

[0145] Prepare reagents and consumables:

[0146] ① Magnetic beads

[0147] ② Ligase premix 2

[0148] ③ Ligase 2

[0149] ④ Ligation sequencing kit (ONT, catalog number: SQK-NBD114.24)

[0150] ⑤ Centrifuge tube (0.2mL, 1.5mL)

[0151] ⑥Qubit TM Quantitative tube (Invitrogen, catalog number: Q32856)

[0152] ⑦Qubit dsDNA HS Assay Kit (ThermoFisher, Cat. No.: Q32851)

[0153] Thaw the magnetic beads at room temperature, vortex thoroughly to mix, and place at room temperature; thaw the components of ligase premix 2, ligase 2 and other reagents, flick or pipette to mix, centrifuge briefly, and place on ice;

[0154] In a 1.5 mL centrifuge tube, mix the following reagents in the order shown in the table below. After adding each reagent, pipette and mix 10 to 20 times, mix thoroughly, centrifuge briefly, and incubate at room temperature for 20 minutes.

[0155] Reagent name Dosage Tag connection product 30μL Sequencing Adapter(NA) 5μL Ligase Master Mix 2 10μL Ligase 2 5μL Total volume 50μL

[0156] Add 20 μL of fully mixed magnetic beads, pipette to mix, place the centrifuge tube on a rotary mixer, and incubate at room temperature for 10 min;

[0157] Remove the centrifuge tube, centrifuge it momentarily, place it on a magnetic stand and absorb it until it is clear, and discard the supernatant;

[0158] Remove the centrifuge tube from the magnetic rack, add 125 μL SFB, flick to resuspend, and centrifuge briefly. Place on the magnetic rack and absorb until clear, then discard the supernatant. Wash twice in this way;

[0159] Centrifuge briefly, put the centrifuge tube back on the magnetic rack, remove the residual liquid with a pipette, remove the centrifuge tube, add 15 μL EB, flick the bottom of the tube to resuspend, centrifuge briefly, incubate at 37°C for 10 min, take out and flick to mix every 2 min, flick for 10 seconds each time to ensure elution efficiency;

[0160] Centrifuge briefly and place on a magnetic stand for at least 1 minute until clear. Transfer 15 μL of supernatant to a clean 1.5 mL centrifuge tube, which is the prepared nucleic acid library.

[0161] Take 1 μL of the prepared library for Qubit quantification. The ideal total amount of library on the machine is 3.3ng-6.7ng. If the library concentration is too high, it can be diluted with EB; the prepared library needs to be kept on ice;

[0162] Chip loading:

[0163] Prepare reagents and consumables:

[0164] ① Nuclease-free water

[0165] ②Auxiliary buffer

[0166] ③FlOw Cell Flush (FCF) (ONT, Part No.: SQK-NBD114.24)

[0167] ④Flow Cell Tether (FCT) (ONT, Part No.: SQK-NBD114.24)

[0168] ⑤Library Solution (LIS) (ONT, item number: SQK-NBD114.24)

[0169] ⑥Library Beads (LIB) (ONT, item number: SQK-NBD114.24)

[0170] ⑦Sequencing Buffer (SB) (ONT, catalog number: SQK-NBD114.24)

[0171] Sequencing chip SpotON version R10.4.1 (ONT, catalog number: FLO-MIN106D)

[0172] Thaw the auxiliary buffer and the above reagents in the ONT ligation sequencing kit (Cat. No.: FLO-MIN106D) at room temperature in advance, mix and centrifuge, equilibrate the chip at room temperature for about 10 minutes and perform quality inspection (the quality inspection steps are performed according to the ONT manual).

[0173] Prepare Priming Reagent FB in a 1.5 mL centrifuge tube according to the following table:

[0174] Reagent name Dosage Flow Cell Flush (FCF) 1170μL Flow Cell Tether(FCT) 30μL Auxiliary buffer 5μL Total volume 1205μL

[0175] Prepare the sequencing chip: Open the Priming port by rotating 90° clockwise, adjust the 1mL pipette to 200μL, vertically contact the Priming port hole and slowly rotate to 230μL to suck out the bubbles in the pipeline. At this time, there is a small amount of liquid at the tip of the pipette, indicating that the bubbles in the pipeline have been discharged;

[0176] Pipette 800 μL FB and push it into the pipeline at a constant speed from the priming port hole. Note that a small amount of liquid remains at the tip of the gun and equilibrate at room temperature for 5 minutes. While waiting, prepare the library for loading on the machine.

[0177] Mix LIB by pipetting. Note that the microspheres in LIB precipitate very quickly, so they must be quickly aspirated after mixing. Prepare the library for the machine according to the table below. If the prepared nucleic acid library is less than 12 μL, add EB to make up 12 μL.

[0178] Reagent name Dosage Sequencing Buffer (SB) 37.5μl Library Beads (LIB) 25.5μl Nucleic acid library 12μl Total volume 75μl

[0179] After the chip is balanced, open SpotON by gently pushing it outwards to observe whether there are any residual white crystals around the SpotON sample port and SpotONsample cover (which are easy to remain when used for the second time). If there are any, use a 10μL gun to absorb a small amount of FB to dissolve the white crystals and remove the liquid to avoid bubbles caused by evaporation of liquid due to loose sealing;

[0180] Pipette 200 μL FB and push it into the pipeline from the Priming port hole at a constant speed. Note that a small amount of liquid is retained at the tip of the gun so that FB flows out from the SpotON sample port and then falls back, thereby rinsing the area around the SpotON sample port.

[0181] Mix the prepared library several times by pipetting, take 75 μL and add it dropwise into the SpotON sample port. The loading beads can be seen to be evenly dispersed.

[0182] First close the SpotON sample cover, then turn counterclockwise to close the Priming port, and finally remove the liquid from the pipeline from Wasteport 1 (make sure the Priming port and SpotON sample port are closed). Place it on the sequencer to set up the experiment.

[0183] Sequencing on the machine:

[0184] The sequencing and parameter setting steps were carried out according to the ONT sequencer manual.

[0185] Recommended sequencing data volume: The recommended average single sample data volume is about 100Mb. That is, when the average single sample data volume reaches 100Mb, sequencing can be terminated.

[0186] Chip cleaning: refer to the instructions of ONT sequencing chip (catalog number EXP-WSH004) for operation.

[0187] Data analysis: The data was analyzed according to the normal bioinformatics process (the database contains all swine disease sequences). The analysis process is as follows:

[0188] Use shell language to build a bioinformatics data analysis process for multi-sample parallel processing; this process can interact with the sequencing platform, obtain sequencing data, and perform the following analysis:

[0189] 1. First, Guppy will be used to convert optical information into fastq and perform barcode splitting of sequencing data during the sequencing process, and then remotely transmitted to the analysis platform;

[0190] Then, the fastq files of different samples were merged; the sequencing data of each sample were quality monitored using NanoStat;

[0191] 2. Use minimap2 to align the reads of each sample with the pig whole genome sequence; use samtools to extract non-host sequences based on the alignment information to obtain the sequence after removing the host;

[0192] 3. Convert the fastq sequence after removing the host into fasta sequence using seqtk; use blastn to compare the reads of each sample with the self-built reference database and filter with 80% coverage and 80% similarity; keep the first one of each sequence comparison result; add a header to the comparison result file; add species information according to the subject information on the comparison; use a self-written script to count the number of sequences of each species in the analysis results, and add information such as name, microbial classification, pathogenicity, and pathogenic microbial classification of the Ministry of Agriculture;

[0193] 4. Statistics are collected on the total number of sequences, the number of sequences of each sample, the number of sequences after quality control, the number and proportion of host sequences, and the number and proportion of sequences in the database compared;

[0194] The results were determined by bioinformatics data analysis. If the number of microbial reads in the sample was greater than ten times that of the positive quality control barcode, the sample was determined to be positive for the microorganism.

[0195] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0196] Nanopore single-molecule sequencing accuracy verification:

[0197] Prepare 4 samples, numbered 1-4, sample 1 is the amplified product of African swine fever, sample 2 is the amplified product of porcine epidemic diarrhea virus, sample 3 is the amplified product of Streptococcus suis, and sample 4 is a negative sample. Samples 1-4 are shuffled and randomly renumbered as A, B, C, and D. Experimenter 2 performs library construction and computer operation. Data analysis is performed 1 hour after sequencing, and the analysis results are as follows:

[0198]

Claims

1. A super multiplex PCR primer set, characterized in that: The super multiplex PCR primer set consists of 45 pairs of specific primers and probes, and the sequences of the 45 pairs of specific primers and probes are shown in SEQ ID NOs: 1-135.

2. Use of the super-multiplex PCR primer set according to claim 1 in preparing a super-multiplex PCR and nanopore sequencing kit for detecting a broad spectrum of porcine pathogens, characterized in that: The kit comprises the 45 pairs of specific primers and probes described in claim 1, and the method of using the kit comprises using the 45 pairs of specific primers and probes to perform 45-plex fluorescent quantitative PCR amplification on the sample to be tested to detect 45 types of porcine pathogens.

3. The use according to claim 2, characterized in that: The 45 swine pathogens are: Foot-and-mouth disease virus, African swine fever virus, Porcine epidemic diarrhea virus, Japanese encephalitis virus, Porcine reproductive and respiratory syndrome virus 2, Porcine reproductive and respiratory syndrome virus 1, Classical swine fever virus, Porcine parvovirus, Rotavirus, Porcine deltacoronavirus, Pseudorabies virus, Porcine circovirus 1, Porcine circovirus 2, Streptococcus suis, Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, Lawsonia intracellularis, Clostridium perfringens type, Clostridium difficile, Swine vesicular disease, Nipah virus, Transmissible gastroenteritis virus, Swine influenza virus, Porcine circovirus 3, Bovine viral diarrhea virusvirus), Brucella suis, Mycoplasma hyorhinis, eperythrozoon, salmonella, Seneca Valleyvirus, Mycoplasma hyosynoviae, Chlamydia porcine, swine bacillary dysentery pathogen, Leptospira species, Trichinella, Cryptosporidium, Toxoplasma gondii, Isospora suis, Ascaris lumbricoides, Sarcoptes scabiei, Cysticerci.

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