A method for detecting multiple porcine diarrhea viruses based on tNGS technology and application thereof

Abstract

The application discloses a kind of pig multiple diarrhea virus detection method and application based on tNGS technology.The method realizes the accurate detection of pig epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine delta coronavirus by tNGS technology.The primer set involved is used to amplify the S1 and M genes of PEDV, the N gene of TGEV and the M gene of PDCoV.The application also provides a kit comprising the primer set and a corresponding tNGS detection method.Clinical sample verification shows that the method is completely consistent with the qPCR detection results, has high sensitivity, strong specificity, can simultaneously identify mixed infection and other advantages, and has high detection throughput, controllable cost, is suitable for accurate diagnosis, epidemiological monitoring and prevention and control of diarrhea virus in pig farm, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal disease detection technology, specifically relating to a detection method and application for multiple swine diarrhea viruses based on targeted next-generation sequencing (tNGS) technology, particularly primer sets, kits, and methods for the simultaneous detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Background Technology

[0002] Porcine viral diarrhea is one of the major challenges facing the global pig industry, especially affecting newborn piglets with extremely high morbidity and mortality rates, causing huge economic losses to pig production. The viral pathogens causing diarrhea in piglets are complex and diverse, among which porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are the three most prevalent and widespread pathogens. All three viruses are highly contagious, causing severe diarrhea, vomiting, dehydration, and death in piglets after infection. Clinically, mixed infections are common, making the disease more complex and extremely difficult to control.

[0003] Currently, detection methods for these three porcine diarrhea viruses mainly include traditional virus isolation and culture, serological testing, and molecular biological detection techniques. Virus isolation and culture is the gold standard for diagnosis, but it is cumbersome, time-consuming (usually 3-7 days), and has low sensitivity, making it difficult to meet the needs of rapid clinical diagnosis. Serological testing methods, such as enzyme-linked immunosorbent assay (ELISA), are relatively simple to perform, but they have the risk of cross-reactivity, poor specificity, and cannot distinguish between past and current infections. Molecular biological detection techniques, such as conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), are widely used in clinical testing due to their high sensitivity and specificity. However, these methods typically can only detect one or a few pathogens per reaction, resulting in efficiency bottlenecks such as "one disease, one test" or "one disease, multiple tests," low throughput, high cost, and inability to simultaneously detect mixed infections and potential unknown variants, making them unsuitable for the complex and ever-changing needs of epidemiological monitoring and precise control.

[0004] Targeted next-generation sequencing (tNGS) is an innovative technology that combines ultra-multiplex PCR amplification with high-throughput sequencing, developed in recent years. Its core principle is to design highly specific primers for specific pathogen groups, enrich the target nucleic acid sequence through multiplex PCR, and then perform deep sequencing and analysis using high-throughput sequencing. Compared to metagenomic next-generation sequencing (mNGS), tNGS effectively avoids host nucleic acid interference, significantly improves detection sensitivity and specificity, and significantly reduces sequencing data redundancy and sequencing costs, making it particularly suitable for the accurate detection and identification of low-load pathogens in clinical samples. tNGS technology mainly consists of two routes: multiplex primer amplification (amplicon sequencing) and probe capture. Multiplex primer amplification tNGS (amplicon sequencing) utilizes the specific binding of primers to the target gene, efficiently amplifying the target fragment via PCR before sequencing. The product is singular, fundamentally reducing interference from non-target sequences, and offers advantages such as simple library preparation, short detection cycle, and high sensitivity, making it more widely used.

[0005] Although tNGS technology has shown great potential in the field of pathogen detection, there are currently no reports of specific methods for simultaneously detecting the three major porcine diarrhea viruses: PEDV, TGEV, and PDCoV. Therefore, developing a tNGS detection method that can rapidly, efficiently, and accurately identify PEDV, TGEV, and PDCoV and their mixed infections, and has the potential to monitor variant strains, is of great significance for filling the technological gap in this field and improving the comprehensive prevention and control capabilities of diarrheal diseases in the swine industry in my country and globally. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for detecting multiple porcine diarrhea viruses (PEDV, TGEV, PDCoV) based on tNGS technology.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] In a first aspect, the present invention provides a primer set for detecting multiple porcine diarrhea viruses based on tNGS technology, wherein the multiple porcine diarrhea viruses are porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV); the primer set includes 8 oligonucleotide primers, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.8 in sequence.

[0009] Preferably, the primer set is used to specifically amplify the characteristic target genes of three viruses; the characteristic target genes are the S1 and M genes of porcine epidemic diarrhea virus, the N gene of porcine transmissible gastroenteritis virus, and the M gene of porcine deltacoronavirus. The nucleotide sequences of the target genes are shown in SEQ ID NO.9 to SEQ ID NO.12, respectively.

[0010] More specifically, the primer set consists of four pairs of specific primers, each pair consisting of an upstream primer and a downstream primer, used to amplify the four target gene fragments mentioned above: The first pair of primers: used to amplify the S1 gene of PEDV, consisting of upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2; The second pair of primers: used to amplify the M gene of PEDV, consisting of upstream primer SEQ ID NO.3 and downstream primer SEQ ID NO.4; The third pair of primers: used to amplify the N gene of TGEV, consisting of upstream primer SEQ ID NO.5 and downstream primer SEQ ID NO.6; The fourth pair of primers: used to amplify the M gene of PDCoV, consisting of upstream primer SEQ ID NO.7 and downstream primer SEQ ID NO.8.

[0011] The primers described in this invention are designed for conserved regions of the three viruses mentioned above, exhibiting high specificity and sensitivity, and capable of simultaneously amplifying four target fragments in a single multiplex PCR reaction. The primers were rigorously selected, taking into account primer structure (avoiding hairpins and dimers), Tm value matching (controlled between 58-62℃), GC content (40%-60%), and amplicon length (150-300 bp) to ensure the high efficiency and uniformity of multiplex PCR amplification.

[0012] Secondly, the present invention provides a kit containing the aforementioned primer set. In addition to the primers shown in SEQ ID NO.1 to SEQ ID NO.8, the kit may also contain other reagents and consumables required for nucleic acid extraction, library construction, high-throughput sequencing, and data analysis, such as, but not limited to: DNA extraction reagents (e.g., lysis buffer, proteinase K, magnetic beads, etc.), RNA reverse transcription reagents (e.g., reverse transcriptase, random primers, dNTPs, etc.), multiplex PCR premix (containing high-fidelity DNA polymerase, dNTPs, Mg²⁺, etc.), sequencing adapter primers, purification magnetic beads (e.g., AMPure XP beads), quantitative reagents (e.g., Qubit dsDNAHS Assay Kit), and negative and positive quality controls (containing plasmids or in vitro transcribed RNA of PEDV, TGEV, and PDCoV target genes). Each component of the kit can be individually packaged and accompanied by detailed instructions for use.

[0013] Thirdly, this invention provides a method for detecting multiple porcine diarrhea viruses based on tNGS technology, the method comprising the following steps: (1) Sample Collection and Nucleic Acid Extraction: Collect anal swabs, feces, intestinal tissue, or environmental samples from the pigs to be tested. For anal swabs, place them in a centrifuge tube containing 1-2 mL of PBS buffer and vortex thoroughly to elute. Take 200 μL of the eluent or fecal suspension and extract total nucleic acids (including DNA and RNA) according to the instructions of a commercially available viral DNA / RNA extraction kit. The extracted RNA fraction needs to be reverse transcribed to obtain cDNA. The reverse transcription reaction system may contain: 5 μL RNA template, 1 μL random primers, 1 μL dNTPs, 4 μL 5× reverse transcription buffer, 1 μL reverse transcriptase, 0.5 μL RNase inhibitor, and nuclease-free water to a final volume of 20 μL. Reaction program: 25℃ for 5 min, 42℃ for 60 min, 70℃ for 15 min. The obtained cDNA is mixed with DNA as a template for subsequent PCR or stored at -20℃ for later use.

[0014] (2) Multiplex PCR Targeted Amplification: Using the nucleic acid (cDNA / DNA mixture) obtained in step (1) as a template, the first round of multiplex PCR amplification was performed using the primer set (SEQ ID NO.1~SEQ ID NO.8) provided in this invention to enrich the target gene fragment. The reaction system was optimized as follows: In a 50 μL system, there was 25 μL of 2× multiplex PCR premix, 2 μL of upstream primer mixture (containing SEQ ID NO.1, 3, 5, 7, with a final concentration of 0.2-0.4 μM for each primer), 2 μL of downstream primer mixture (containing SEQ ID NO.2, 4, 6, 8, with a final concentration of 0.2-0.4 μM for each primer), 5 μL of template nucleic acid, and nuclease-free water to a final volume of 50 μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 10 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 90 seconds, 72℃ extension for 30 seconds, for a total of 30-35 cycles; and finally, 72℃ extension for 7 minutes, followed by storage at 4℃.

[0015] (3) Library construction: Take 25 μL of the first-round PCR product and purify it using 1.0× volume AMPure XP magnetic beads to remove primer dimers and non-specific amplification products. Follow the instructions in the manual for specific procedures. Dissolve the purified product in 25 μL of EB buffer. Take 5 μL of the purified product as a template for the second-round PCR to introduce sequencing adapters and sample tags (Index). The second-round PCR reaction system (50 μL) contains: 25 μL of 2×PCR premix, 2 μL of universal upstream primer (P5) containing the index, 2 μL of universal downstream primer (P7) containing the index, 5 μL of template DNA, and 16 μL of nuclease-free water. The amplification program is as follows: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 20 seconds, for a total of 8-10 cycles; and finally, 72℃ extension for 5 minutes. The second round of PCR products were purified again using 1.0× AMPure XP magnetic beads, and the final library was dissolved in 30 μL EB buffer.

[0016] (4) Library quality control and quantification: Take 1 μL of the purified library and quantify it using a Qubit 4.0 real-time analyzer and a QubitdsDNA HS Assay Kit. Take another 1 μL of the library and use an Agilent 2100 bioanalyzer and a HighSensitivity DNA Kit to detect the fragment size distribution of the library. The main peak should be in the range of 250-400 bp, and there should be no obvious adapter peak (<120 bp).

[0017] (5) High-throughput sequencing: Based on the library quantification results, mix libraries from different samples in equal molar amounts, and adjust the final concentration of the mixed library to 2 nM. Take 10 μL of the mixed library, add 10 μL of 0.1 N NaOH for denaturation, incubate for 5 minutes, then add 980 μL of pre-chilled hybridization buffer and dilute to 20 pM. Load the diluted library into a sequencing kit and perform paired-end 150 bp (PE150) or paired-end 250 bp (PE250) sequencing on an Illumina NovaSeq 6000 or MiSeq sequencer.

[0018] (6) Data Analysis and Result Determination: The raw data from sequencing is analyzed through the following process: Data quality control: The FastP (v0.20.0) software was used to perform quality control on the Raw Data, filtering out low-quality reads (quality value Q<20) and removing connector sequences to obtain Clean Data.

[0019] Alignment and Species Annotation: Clean Data was aligned to a reference database containing the target gene sequences (SEQ ID NO. 9-12) of this invention using BWA (v0.7.17) or Bowtie2 (v2.4.1) software, allowing a maximum of two mismatches. The number of specific reads aligned to each reference sequence was counted, and the relative abundance (the percentage of each viral read to the total number of target reads) was calculated.

[0020] Result Interpretation: For each sample, a positive result is determined when the number of virus-specific reads is ≥ 10. If multiple virus read counts are ≥ 10, a mixed infection is determined. If all virus read counts are < 10, a negative result is determined.

[0021] In some preferred embodiments of the present invention, the annealing temperature of the multiplex PCR in step (2) can be further optimized, for example, by using gradient annealing (55-62°C) to determine the optimal annealing temperature. The primer concentration in step (2) can also be adjusted according to the amplification efficiency to ensure the uniformity of amplification of the four targets.

[0022] In some preferred embodiments of the present invention, in order to improve detection throughput, primers containing different index tags can be used in the second round of PCR to achieve simultaneous sequencing of up to 96 samples.

[0023] Fourthly, this invention provides the application of the above-mentioned primer set, kit containing the primer set, or detection method in the preparation of porcine diarrhea virus detection kits, or in the clinical diagnosis, epidemiological monitoring, identification of mixed infections, and screening of variant strains of porcine diarrhea virus. In particular, it is applied in the simultaneous detection and differentiation of single and mixed infections of PEDV, TGEV, and PDCoV, as well as in the monitoring of viral gene mutations and evolutionary analysis.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) High throughput and high efficiency: This invention is the first to apply tNGS technology to the simultaneous detection of three major porcine diarrhea viruses. The differential diagnosis of PEDV, TGEV and PDCoV can be completed in one experiment, which greatly improves the detection throughput and efficiency and overcomes the drawback of the traditional method of "one disease, one test".

[0025] (2) High sensitivity and high specificity: The primer set designed in this invention targets conserved regions of the virus, enabling specific amplification of the target fragment and effectively avoiding non-specific amplification and host nucleic acid interference. Combined with the depth of high-throughput sequencing, the detection sensitivity is significantly higher than that of traditional PCR methods. The results of the examples show that the tNGS detection results are highly consistent with the qPCR results, and it can also stably detect samples with low viral load (high Ct value).

[0026] (3) Accurate identification of mixed infection: tNGS technology can sequence and quantify multiple targets at the same time, and can intuitively reveal whether there is a mixed infection in the sample and the relative abundance of each viral component, providing more comprehensive information for clinical precision medicine and disease prevention and control.

[0027] (4) It has the potential to discover variant strains: Although the primers are designed in conserved regions, the amplicon sequences obtained by sequencing contain all the variation information within the target gene fragment. By comparing with the reference sequence, the variation of viral genes can be monitored, providing basic data for epidemiological tracing and vaccine development.

[0028] (5) Standardized operation process, easy to promote: The method provided by this invention is based on a mature multiplex PCR library preparation and high-throughput sequencing platform. The process is easy to standardize and automate, and is easy to promote and apply in testing institutions and laboratories with sequencing capabilities. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0030] Example 1: Primer Design and Screening This embodiment illustrates the design and screening process of the tNGS primer set for detecting porcine PEDV, TGEV, and PDCoV according to the present invention.

[0031] Target gene selection: Representative whole genome sequences of PEDV, TGEV, and PDCoV were downloaded from the NCBI GenBank database and multiple sequence alignment analysis was performed using MEGA software. The S1 gene (hypervariate region, used for identification) and M gene (conserved region, used for sensitization) of PEDV, the N gene (high abundance expression, conserved) of TGEV, and the M gene (containing virus-specific hypervariate region and species-conserved region) of PDCoV were selected as detection targets.

[0032] Primer design: Multiple candidate primer pairs were designed using Primer3 and Oligo 7 software for the conserved regions of the selected target genes. Primer design principles included: Tm values ​​between 58-62℃, GC content between 40%-60%, and amplicon length between 150-300 bp (facilitating multiplex PCR amplification and library construction). Hairpin structures or dimers should be avoided between primers themselves and between primers.

[0033] Primer name Primer number target genes Target gene sequence number PEDV-S1-F1 SEQ ID NO.1 PEDV-S1 SEQ ID NO.9 PEDV-S1-R1 SEQ ID NO.2 PEDV-M-F2 SEQ ID NO.3 PEDV-M SEQ ID NO.10 PEDV-M-R2 SEQ ID NO.4 TGEV-N-F3 SEQ ID NO.5 TGEV-N SEQ ID NO.11 TGEV-N-R3 SEQ ID NO.6 PDCoV-M-F4 SEQ ID NO.7 PDCoV-M SEQ ID NO.12 PDCoV-M-R4 SEQ ID NO.8 Example 2: Establishment of a method for detecting porcine diarrhea virus based on tNGS technology This embodiment illustrates the specific process of establishing a tNGS detection method using the primer set screened in Example 1.

[0034] Nucleic acid extraction and reverse transcription of samples Collected porcine anal swab samples clinically and place them in centrifuge tubes containing 1 mL PBS buffer. Vortex for 30 seconds, let stand for 5 minutes, and collect 200 μL of supernatant. Extract total nucleic acid using the QIAamp Viral RNA Mini Kit (Qiagen, 52906), following the manufacturer's instructions. Elute with 60 μL AVE buffer. Use 11 μL of the eluent for reverse transcription, and store the remainder at -80℃. Reverse transcription was performed using the SuperScript IV First-Strand Synthesis System (Invitrogen, 18091050). The reaction mixture consisted of: 11 μL RNA, 1 μL 50 μM random primers, 1 μL 10 mM dNTPs, heated at 65℃ for 5 minutes, and cooled on ice. Then, add 2 μL 10×RT buffer, 2 μL 100 mM DTT, 1 μL RNaseOUT, 1 μL SuperScript IV reverse transcriptase, and 1 μL nuclease-free water, for a total volume of 20 μL. Reaction program: 23℃ for 10 minutes, 55℃ for 10 minutes, 80℃ for 10 minutes. The obtained cDNA is mixed with the remaining RNA (as total nucleic acid) or used directly for PCR.

[0035] Multiplex PCR Targeted Enrichment Using cDNA obtained from reverse transcription as a template, the first round of multiplex PCR amplification was performed using the four primer pairs listed in Table 1. The reaction system consisted of 25 μL of 2× multiplex PCR premix (containing high-fidelity DNA polymerase, dNTPs, Mg²⁺, etc.); 2 μL of upstream primer mixture (each primer at 10 μM, mixed in equal volumes); 2 μL of downstream primer mixture (each primer at 10 μM, mixed in equal volumes); 5 μL of cDNA template; and 16 μL of nuclease-free water. The PCR amplification program was as follows: 95℃ pre-denaturation for 10 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 90 seconds, 72℃ extension for 30 seconds, for a total of 32 cycles; final extension at 72℃ for 7 minutes; and storage at 4℃.

[0036] Library construction and purification Take 25 μL of the first-round PCR product and purify it using 1.0× AMPure XP magnetic beads (Beckman Coulter, A63881). The specific procedure is as follows: Add 25 μL of magnetic beads, mix well by pipetting, and incubate at room temperature for 5 minutes; place on a magnetic rack for 5 minutes and discard the supernatant; add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, and discard the supernatant; repeat the washing once; air dry at room temperature for 5 minutes; add 25 μL of EB buffer, mix well, and incubate at room temperature for 2 minutes; separate on a magnetic rack and transfer the supernatant to a new tube. Use 5 μL of the purified product as a template for the second-round PCR to introduce the index. The second round of PCR was performed using KAPA HiFi HotStart ReadyMix (Roche, KK2602). The reaction mixture (50 μL) consisted of: 25 μL of 2× ReadyMix, 2 μL of 10 μM P5 primers (containing index), 2 μL of 10 μM P7 primers (containing index), 5 μL of purified DNA, and 16 μL of ddH2O. The amplification program was: 95℃ pre-denaturation for 3 minutes; 98℃ denaturation for 20 seconds, 60℃ annealing for 15 seconds, and 72℃ extension for 30 seconds, for a total of 8 cycles; followed by a 2-minute extension at 72℃. The second round of PCR products were purified again using 1.0× AMPure XP magnetic beads, and the final library was dissolved in 30 μL of EB buffer.

[0037] Library quality control and quantitative analysis The purified library was quantified using a Qubit 4.0 qPCR instrument and a Qubit dsDNA HS Assay Kit. The fragment size distribution of the library was detected using an Agilent 2100 bioanalyzer and a High Sensitivity DNA Kit to ensure the main peak was within the expected range (250-400 bp) and free of adapter contamination.

[0038] High-throughput sequencing Based on the library concentration, each library was diluted to 4 nM, 5 μL of each was mixed, denatured with 0.1 N NaOH, diluted to 1.8 pM with pre-cooled hybridization buffer, and 600 μL was loaded into a MiSeq v2 reagent cartridge for paired-end 150 bp sequencing on the Illumina MiSeq platform.

[0039] Data Analysis The raw data from sequencing is analyzed using the following process: (1) Data quality control: Use the fastp software to perform quality control on the raw data, filter low-quality reads and remove connector sequences to obtain clean data.

[0040] (2) Alignment and Species Annotation: Clean Data was aligned to a reference database containing the target gene sequences (SEQ ID NO. 9-12) of this invention using BWA or Bowtie2 software. The number of specific reads aligned to each reference sequence was counted.

[0041] (3) Result determination: For each sample, when the number of specific reads of a certain virus is ≥ 10, it is determined to be positive for that virus.

[0042] Example 3: tNGS detection of clinical samples and comparative validation with qPCR method This embodiment uses the tNGS method established in this invention to detect 22 clinically collected pig anal swab samples, and compares the results with qPCR results to verify the accuracy and reliability of this method.

[0043] Sample Information This study collected 22 clinical anal swab samples, all from multiple regions in Henan and Hubei provinces, China. Four samples came from Guangshui City, Hubei Province (numbered 1958-3, 1633-11, 1808-1, and 1692); three samples came from Macheng City, Hubei Province (numbered 2313, RW16-1, and 030019-1); two samples came from Huanggang City, Hubei Province (numbered 1853-2 and 1974-6); the largest number of samples came from Shangcheng County, Henan Province (numbered SC-7, SC1, SC2, SC3, SC4, SC8, SC10, SC14, SC15, and SC16; note: SC-7, although its number contains a hyphen, belongs to the same regional sequence); and three samples came from Luoshan County, Henan Province (numbered LS7, LS8, and LS9). All samples were anal swabs, and their geographical distribution covered the border area between northern Hubei and southern Henan, exhibiting a clear geographical clustering characteristic. 2. Detection Method tNGS detection: Nucleic acid extraction, library construction, sequencing and data analysis were performed on 22 samples according to the method described in Example 2.

[0044] qPCR detection: For the qPCR detection of PEDV, TGEV, and PDCoV, commercial kits (Sangon Biotech, corresponding to the PEDV (S gene) qPCR kit, TGEV (N gene) qPCR kit, and PDCoV (M gene) qPCR kit respectively) were used and performed on an ABI7500 Fast Real-Time PCR instrument. Reaction system: 2×qPCR Mix 10 μL, primer-probe mixture 1 μL, template 2 μL, ddH2O 7 μL. Reaction program: 95°C for 3 min; 95°C for 5 s, 60°C for 30 s, for 40 cycles. Samples with Ct value ≤ 35 were determined as positive, 35 < Ct < 40 were determined as suspicious (rechecked), and samples without Ct value or Ct ≥ 40 were determined as negative.

[0045] Detection results The detection results of the two methods are compared as shown in Table 2.

[0046] Sample number PED general tNGS detection results (reads) qPCR test results (Ct value) PDcoV universal tNGS detection results (reads) PDcoV qPCR detection results (Ct value) TGE universal tNGS test results (reads) TGEqPCR test results (Ct value) 1958-3 258200 21.824 0 No Ct value 0 No Ct value 1633-11 548793 19.066 0 No Ct value 0 No Ct value 1808-1 502142 20.582 0 No Ct value 0 No Ct value 1692 561106 16.824 0 No Ct value 0 No Ct value SC-7 0 No Ct value 404478 28.480 0 No Ct value 2313 0 No Ct value 1344007 14.746 0 No Ct value RW16-1 11501 34.363 0 No Ct value 0 No Ct value 030019-1 848010 33.402 0 No Ct value 0 No Ct value 1853-2 273763 18.840 0 No Ct value 0 No Ct value 1974-6 1021964 17.113 0 No Ct value 0 No Ct value SC1 0 No Ct value 266183 16.582 0 No Ct value SC2 0 No Ct value 2328293 18.926 0 No Ct value SC3 0 No Ct value 1408999 20.73 0 No Ct value SC4 0 No Ct value 180962 22.066 0 No Ct value SC8 0 No Ct value 26525 25.379 0 No Ct value SC10 0 No Ct value 83222 22.66 0 No Ct value SC14 168726 27.395 0 No Ct value 0 No Ct value SC15 0 No Ct value 145844 26.02 0 No Ct value SC16 0 No Ct value 75585 25.059 0 No Ct value LS7 106689 36.113 0 No Ct value 0 No Ct value LS8 123127 29.871 0 No Ct value 0 No Ct value LS9 0 No Ct value 0 No Ct value 586789 24.03 4. Result analysis: (1) Consistency analysis As can be seen from Table 2, the tNGS detection results are completely consistent with the qPCR detection results. For all samples detected as positive by qPCR, tNGS can detect the corresponding viral reads. For example, samples 1958-3, 1633-11, 1808-1, 1692, RW16-1, 030019-1, 1853-2, 1974-6, SC14, LS7, LS8 are positive for PEDV; samples SC-7, 2313, SC1, SC2, SC3, SC4, SC8, SC10, SC15, SC16 are positive for PDCoV; sample LS9 is positive for TGEV). For all samples detected as negative by qPCR, the tNGS read counts are almost 0. This indicates that the tNGS method established in this invention has extremely high detection accuracy.

[0047] (2) Quantitative correlation Correlation analysis was performed on the tNGS read counts and qPCR Ct values of PEDV-positive samples. The results showed a significant negative correlation between the two (Pearson correlation coefficient R = -0.975, p < 0.0001), indicating that the read abundance detected by tNGS can semi-quantitatively reflect the viral load in the samples.

[0048] (3) Ability to detect mixed infections Among the 22 samples in this batch, the tNGS method did not detect mixed infection samples, which is consistent with the qPCR results. However, in theory, this method can accurately identify mixed infection situations by simultaneously detecting multiple targets.

[0049] (4) Sensitivity It is worth noting that even weakly positive samples such as RW16-1 (Ct=34.363) and LS7 (Ct=36.113) were detected by the tNGS method with tens of thousands or even hundreds of thousands of specific reads, proving that the method still has extremely high detection sensitivity even when the viral load is extremely low (high Ct value).

[0050] Example 4: Methodological Performance Evaluation This embodiment is used to evaluate the sensitivity, specificity, and repeatability of the tNGS detection method of the present invention.

[0051] Sensitivity (lower limit of detection) evaluation RNA standards for PEDV, TGEV, and PDCoV (obtained through in vitro transcription, with known copy numbers) were serially diluted 10-fold to a concentration range of 10. 6 copies / μL to 10 0 Copies / μL. 5 μL of each dilution was used as a template for tNGS detection according to the method in Example 2, with each dilution repeated three times. The lowest concentration that could be detected with 100% accuracy was defined as the limit of detection (LOD) for this method. Results showed that the LOD for all three viruses reached 50 copies / reaction.

[0052] Specificity evaluation Using classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine genomic DNA as templates, tNGS detection was performed according to the method in Example 2. The results showed that, except for positive controls for PEDV, TGEV, and PDCoV, no specific reads were detected for other non-target pathogens and porcine genomic DNA, indicating that this method has good specificity and no cross-reactivity with other common porcine pathogens.

[0053] Repeatability evaluation Clinical samples with high, medium, and low viral loads (e.g., sample 1633-11, sample RW16-1, and sample LS9) were selected. In the same experiment, three replicates were set up for tNGS detection (intra-batch replication), and three independent experiments were performed on different 3-day periods (inter-batch replication). The coefficient of variation (CV) of the number of reads obtained for each sample in each test was calculated. The results showed that the intra-batch and inter-batch CVs for all samples were less than 15%, indicating that this method has good stability and reproducibility.

[0054] Example 5: Application in mixed infection simulated samples This embodiment is used to further verify the ability of the method of the present invention to detect mixed infections.

[0055] Simulated sample preparation Four simulated mixed infection samples were prepared by mixing PEDV, TGEV, and PDCoV RNA standards with known copy numbers in different proportions: Sample A (PEDV:PDCoV = 1:1), Sample B (PEDV:TGEV = 10:1), Sample C (PDCoV:TGEV = 1:10), and Sample D (PEDV:TGEV:PDCoV = 1:1:1).

[0056] Detection and Analysis The above four simulated samples were subjected to tNGS detection according to the method in Example 2, and the distribution ratio of sequencing reads among the three viruses was analyzed.

[0057] result tNGS detection results showed that the corresponding viral reads were detected simultaneously in samples A, B, C, and D, and the proportion of reads was basically consistent with the proportion of viral RNA copies introduced. For example, in sample D, the reads of PEDV, TGEV, and PDCoV accounted for 32.1%, 33.5%, and 34.4% of the total target reads, respectively, very close to the theoretical value of 1:1:1. This result proves that the method of the present invention can not only accurately identify mixed infections, but also semi-quantitatively reflect the relative abundance of each viral component.

[0058] In summary, this invention successfully developed a method for the simultaneous detection of three porcine diarrhea viruses—PEDV, TGEV, and PDCoV—based on tNGS technology. This method offers advantages such as high sensitivity, high specificity, high throughput, quantification, and the ability to identify mixed infections. Compared with existing qPCR methods, it has significant advantages in detection throughput and information content, providing a powerful new technological tool for the accurate diagnosis, epidemiological monitoring, and control of porcine viral diarrhea.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A primer set for detecting multiple porcine diarrhea viruses based on tNGS technology, wherein the multiple porcine diarrhea viruses are porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV); characterized in that, The primer set includes primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

8.

2. The primer set according to claim 1, characterized in that, The primer set was used to specifically amplify the characteristic target genes of three viruses: the S1 and M genes of porcine epidemic diarrhea virus, the N gene of porcine transmissible gastroenteritis virus, and the M gene of porcine deltacoronavirus.

3. The primer set according to claim 2, characterized in that, The nucleotide sequence of the target gene is shown in SEQ ID NO.9~SEQ ID NO.

12.

4. The primer set according to claim 1, characterized in that, The primer set consists of 4 pairs of specific primers, namely: The primer pair used to amplify the PEDV S1 gene consists of SEQ ID NO.1 and SEQ ID NO.2; The primer pair used to amplify the PEDV M gene consists of SEQ ID NO.3 and SEQ ID NO.4; The primer pair used to amplify the TGEV N gene consists of SEQ ID NO.5 and SEQ ID NO.6; The primer pair used to amplify the PDCoV M gene consists of SEQ ID NO.7 and SEQ ID NO.

8.

5. A kit comprising the primer set according to any one of claims 1-4 for detecting multiple porcine diarrhea viruses.

6. The reagent kit according to claim 5, characterized in that, The kit also includes reagents for nucleic acid extraction, library construction, and high-throughput sequencing.

7. A method for detecting multiple porcine diarrhea viruses based on tNGS technology, characterized in that, Includes the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the nucleic acid obtained in step (1) as a template, multiplex PCR amplification is performed using the primer set described in any one of claims 1-4 to obtain an amplicon library of the target gene; (3) Perform high-throughput sequencing on the amplicon library obtained in step (2) to obtain sequencing data; (4) Analyze the sequencing data, count the specific sequence readings that are aligned to each target gene, and determine whether there is a corresponding viral infection in the sample based on the readings.

8. The method according to claim 7, characterized in that, The reaction system for multiplex PCR in step (2) includes: 2× multiplex PCR premix, upstream and downstream primers (SEQ ID NO.1~SEQ ID NO.8) at a concentration of 10 μM, template DNA / cDNA, and the remainder is nuclease-free water.

9. The method according to claim 7, characterized in that, The reaction procedure for multiplex PCR in step (2) is as follows: pre-denaturation at 95℃ for 5-15 minutes; denaturation at 95℃ for 30 seconds, annealing at 55-60℃ for 90 seconds, extension at 72℃ for 30 seconds, for 25-35 cycles; and finally extension at 72℃ for 5-10 minutes.

10. The primer set according to any one of claims 1-4, the kit according to claim 5 or 6, or the method according to any one of claims 7-9, in the preparation of a porcine diarrhea virus detection kit, or in the clinical diagnosis, epidemiological monitoring, identification of mixed infections, and screening of variant strains of porcine diarrhea virus.