PCR (Polymerase Chain Reaction) primer group for detecting bovine nodule skin disease virus based on multiple amplicon targeted enrichment and nanopore real-time sequencing as well as application and detection product of PCR primer group

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer group for detecting bovine nodule skin disease virus based on multiple amplicon targeted enrichment and nanopore real-time sequencing as well as application and a detection product thereof, and relates to the technical field of biological detection. The PCR primer group comprises primers of which the nucleotide sequences are as shown in SEQ ID NO. 1-90. According to the invention, a multiple PCR primer group is designed based on a bovine nodular skin disease virus genome sequence, and a detection method for combining multiple amplicon targeted enrichment and nanopore real-time sequencing aiming at bovine nodular skin disease pathogens and a corresponding detection product are established according to the multiple PCR primer group. By adopting the detection product provided by the invention, nanopore real-time sequencing can be carried out by aiming at the specific amplified fragment of the primer group of the bovine nodular dermatosis pathogen, so that on-site accurate and rapid detection of the bovine nodular dermatosis pathogen is realized, pathogen detection is carried out from a sequence level, and three viruses, namely LSDV, SPPV and GTPV, are accurately distinguished.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a PCR primer set for detecting bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, as well as its applications and detection products. Background Technology

[0002] Bovine nodular dermatovirus (LSDV), along with sheep poxvirus (SPPV) and goat poxvirus (GTPV), belongs to the genus *Capepoxvirus* of the family Poxviridae and shares up to 96% genomic homology, posing a significant challenge to traditional detection methods in practical applications. While methods such as PCR, qPCR, and ELISA are simple to perform, they not only have limited throughput, but more importantly, the high similarity in gene sequence and biological characteristics among these viruses makes specific identification of LSDV in the field extremely challenging.

[0003] Traditional detection methods limit their application in frontline quarantine scenarios. Frontline quarantine requires accurate screening of target pathogens within a short timeframe, but traditional methods, due to insufficient specificity, may lead to false positives and false negatives, failing to meet the core requirement of reliable results in quarantine work. Furthermore, because they cannot efficiently pinpoint the specific genes of LSDV, traditional methods are also ill-suited to the targeted and rapid technical demands of customs on-site sequencing, making it difficult to achieve accurate LSDV sequence identification in the field. This limitation is even more pronounced in scenarios involving mixed infections with multiple pathogens, potentially delaying control efforts due to misidentification and posing a potential threat to livestock biosecurity. Therefore, there is an urgent need to establish highly specific on-site detection and identification methods for LSDV.

[0004] Multiplex amplicon sequencing (MAS) is a highly efficient and precise gene detection technology. This technology uses specific primers to amplify conserved sequence regions within genes. Multiplex PCR is then used to amplify the target region, and the amplified products are sequenced for analysis, allowing for in-depth study of the genetic information characteristics of specific regions. The overall approach involves targeting and capturing the target region using nanopore sequencing. Detailed analysis of the sequencing results reveals the genetic information of the target region. Applying third-generation targeted sequencing technology, which combines multiplex amplicon targeted enrichment with real-time nanopore sequencing, to the detection of bovine nodular dermatitis (LSDV) holds promise for rapid and accurate detection, and for precise differentiation between LSDV, SPPV, and GTPV. Summary of the Invention

[0005] The purpose of this invention is to provide a PCR primer set for detecting bovine nodular dermatitis virus (BND) based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, along with its applications and detection products, to address the problems existing in the prior art. Using the multiplex PCR primer set provided by this invention, real-time nanopore sequencing can be performed on specific amplified fragments of the primer set targeting the BND pathogen, enabling accurate and rapid on-site detection of the BND pathogen. Furthermore, it allows for pathogen detection at the sequence level and accurate differentiation between LSDV, SPPV, and GTPV viruses.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a PCR primer set for detecting bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, including primers with nucleotide sequences as shown in SEQ ID NO.1-90.

[0008] The present invention also provides the application of the above-mentioned PCR primer set in the preparation of a detection product for bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing.

[0009] Furthermore, the testing product is a reagent kit.

[0010] This invention also provides a detection product for detecting bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, including multiplex amplicon targeted enrichment reagent and nanopore real-time sequencing detection reagent;

[0011] The multiplex amplicon targeting enrichment reagent includes the PCR primer set described above.

[0012] Furthermore, the testing product is a reagent kit.

[0013] Furthermore, the multiplex amplicon targeting enrichment reagent also includes 2× rapid Taq enzyme premix.

[0014] Furthermore, the multiplex amplicon targeting enrichment reagent also includes nuclease-free water.

[0015] Furthermore, the nanopore real-time sequencing detection reagent also includes a barcode and a sequencing library adapter.

[0016] Furthermore, the PCR reaction program of the kit is as follows: 95℃ for 5 min; 95℃ for 15 s, 59℃ for 15 s, 72℃ for 1 min, 72℃ for 30 s, 35 cycles; store at 4℃.

[0017] The present invention discloses the following technical effects:

[0018] This invention designs a multiplex PCR primer set based on the bovine nodular dermatitis virus (BND) genome sequence and establishes a detection method combining multiplex amplicon targeted enrichment and nanopore real-time sequencing for the BND pathogen, along with corresponding detection products. Using the detection products provided by this invention, real-time nanopore sequencing can be performed on specific amplified fragments from the primer set targeting the BND pathogen, enabling accurate and rapid on-site detection of the BND pathogen. Furthermore, bioinformatics analysis methods can be used to analyze third-generation sequencing data, determine infection status based on the relative abundance of pathogen target genes, and perform rapid pathogen identification. The invention also allows for accurate differentiation of three viruses—LSDV, SPPV, and GTPV—at the sequence level. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a statistical graph showing the number of sequencing reads for pathogen detection in each sample in Example 2;

[0021] Figure 2 This is a statistical chart showing the pathogen detection status of each sample in Example 2;

[0022] Figure 3 This is a distribution diagram of the sequence lengths of each sample in Example 2. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The main reagents and instruments used in the following examples are shown in Tables 1 and 2.

[0029] Table 1 Main Reagents

[0030] name factory Item number Rapid Barcode Sequencing Kit - 24 Oxford Nanopore Technologies SQK-RBK114.24 DNA purification magnetic beads Nanjing Novozymes Biotechnology Co., Ltd. N411 MinION sequencing chip Oxford Nanopore Technologies / Double-stranded DNA fluorescence quantitative detection kit Thermo Fisher Scientific Q32851 2× Rapid Taq Enzyme Premix Nanjing Novozymes Biotechnology Co., Ltd. P222 Cleaning kit Oxford Nanopore Technologies /

[0031] Table 2 Main Instruments

[0032]

[0033]

[0034] Example 1: Multiplex PCR Primer Design

[0035] Primers were designed based on the genome sequence of bovine nodular dermatitis virus (version number AF325528.1), and a primer set with an amplicon fragment of 500-600bp was designed to ensure rapid and accurate detection of the pathogen in front-line quarantine scenarios.

[0036] First, primer sets were initially designed based on the LSDV genome sequence. Then, primers located in known important functional regions of the LSDV genome were selected. The bioinformatics software Blastn was used to screen out primers that could detect SPPV and GTPV, but whose homology similarity was less than 99%, as candidate primer sets.

[0037] For each candidate primer pair, LSDV, SPPV, and GTPV DNA templates were added, and singlet conventional PCR amplification was performed. The amplification products were then subjected to DNA agarose gel electrophoresis. The electrophoresis images were observed, and 15 primer pairs that produced clear bands for all three pathogens were retained. These 15 primer pairs were then mixed and amplified without adding DNA template. The amplification products were then subjected to DNA agarose gel electrophoresis. The electrophoresis images showed no bands, indicating that primer dimers did not form between the primers.

[0038] Based on the initial 15 primer pairs, the amplification fragment length was increased, resulting in the design of 10 primer pairs with amplification fragments of 800-1100 bp, 10 pairs with amplification fragments of 1500-1700 bp, and 10 pairs with amplification fragments of 2200-2300 bp. This approach ensured pathogen detection while facilitating further analysis of variants. The primer design results were validated using the PrimerBlast online software from the NCBI website to verify the primer specificity. A total of 45 primer pairs were ultimately designed for the target pathogen and divided into 10 groups based on different amplification fragment lengths, as detailed in Table 3.

[0039] Table 345 Primer Pairs

[0040]

[0041]

[0042]

[0043] Example 2: Sample processing, amplicon sequencing library construction, adapter ligation and sequencing, and sequencing data analysis

[0044] 1. Sample processing and PCR amplification

[0045] This embodiment used three types of viral samples: LSDV, SPPV, and GTPV DNA samples. The genomes of the three viruses were extracted and processed using Qubit. TM 4. The concentration of double-stranded DNA (dsDNA) was detected using a fluorescence quantitative PCR instrument, and the concentration of each sample was controlled at around 30 ng / μL.

[0046] Each sample's DNA was used as a template, and subsequent PCR amplification was performed according to different primer groups (see Table 4).

[0047] Table 4. Sample and primer set information used in Example 2

[0048]

[0049]

[0050] The reaction system and procedure for PCR amplification are shown in Tables 5 and 6.

[0051] Table 5 Target Region Amplification Reaction System

[0052]

[0053] Table 6 Target Region Amplification Reaction Procedure

[0054]

[0055] 2. Library preparation

[0056] 2.1 Adding a barcode

[0057] Transfer the sample containing 80 ng of genomic DNA to a 0.2 mL PCR tube. If the sample volume is less than 10 μL, add nuclease-free water to bring the volume to 10 μL. Gently pipette 10 times to avoid unnecessary interruption. Briefly centrifuge. Add 1.5 μL of the rapid ligation barcode, for a total of 11.5 μL. Thoroughly mix the reagents in the tube by pipetting, then briefly centrifuge again. Incubate the PCR tube at 30°C for two minutes, then at 80°C for two minutes, followed by a brief cooling on ice. Briefly centrifuge the PCR tube and collect the liquid at the bottom of the tube.

[0058] 2.2 Sample pooling and purification

[0059] Combine all the barcode-bearing samples into a 2 mL centrifuge tube and record the total volume of liquid in the tube. Vortex to resuspend the AMPure XP magnetic beads (AXP). Add an equal volume of the resuspended AMPure XP magnetic beads to the combined barcode-bearing samples and gently tap the centrifuge tube to mix. Incubate the centrifuge tube at room temperature for 10 minutes on a low-speed rotary mixer. Briefly centrifuge the sample and place it on a magnetic rack to allow the magnetic beads and liquid phase to separate. Keeping the centrifuge tube still on the magnetic rack, remove the supernatant with a pipette. Keeping the tube still on the magnetic rack, wash the magnetic beads with 1 mL of freshly prepared 80% ethanol (prepared with nuclease-free water). Be careful not to disturb the magnetic beads. Remove and discard the ethanol with a pipette. Repeat the above steps once. Briefly centrifuge the centrifuge tube and place it on a magnetic rack. Remove any residual ethanol with a pipette. Allow the magnetic beads to air dry for about 30 seconds, but do not allow them to dry to the point of cracking. Remove the centrifuge tube from the magnetic rack. Resuspend the magnetic beads in 15 μL of elution buffer (EB) for every 24 barcodes used. Incubate at room temperature for 10 minutes. Let the centrifuge tube stand on the magnetic rack for at least one minute, until the magnetic beads and liquid phase separate and the eluent is clear and colorless. Transfer the remaining eluent to a new 1.5 mL centrifuge tube.

[0060] After purification, the amplicon single-stranded DNA (ss DNA) library was constructed and used with Qubit.TM 4. Quantitative Fluorescence Analyzer The ssDNAAssay kit is used to perform quality testing on ssDNA libraries. The concentration of ssDNA libraries is not less than 0.4 ng / μL, which meets the quality control requirements.

[0061] After three steps—sample processing, dsDNA library construction, and ssDNA library construction—the amplicon sequencing library construction was completed.

[0062] 3. Adapter ligation and sequencing

[0063] Dilute the rapid sequencing library adapter (RA) in a 1.5 mL centrifuge tube as shown in Table 7 and mix thoroughly by pipetting.

[0064] Table 7. Dilution of adapters for rapid sequencing libraries

[0065]

[0066] Add 1 μL of diluted rapid sequencing library adapter (RA) to 11 μL of barcoded DNA elution buffer. Gently tap the centrifuge tube to mix thoroughly and centrifuge briefly. Incubate at room temperature for 5 minutes, then store on ice for later use in sequencing.

[0067] The prepared library was sequenced using the Rapid Barcode Sequencing Kit-24 (SQK-RBK114.24).

[0068] 4. Sequencing data analysis

[0069] After sequencing is completed, copy the sequencing sample quality test report and the raw sequencing data (fastq format file) of all samples for analysis, statistical processing of test results, and visualization of results.

[0070] First, it should be able to correctly recognize barcodes, accurately distinguish all samples, view sample quality scores, and retain data with Q≥10.

[0071] Sample data were merged by combining multiple sequencing FASTQ files with the same barcode into a single FASTQ file. Bioinformatics analysis of the raw sequencing data was performed using software such as fq2fa, blast, bwa, and samtools, along with customized scripts. This included analyses of alignment success rate, coverage depth, pathogen detection results, and read length distribution. The statistical results of the number of pathogen-detected reads for each sample are shown below. Figure 1 Statistical charts of pathogen detection in each sample (data with fewer than 50 reads filtered out) are shown below. Figure 2 The distribution of sequence lengths for each sample is shown in the figure. Figure 3Testing revealed that all 10 samples accurately identified the corresponding pathogens and matched the corresponding primer sets. Furthermore, the number of detected reads could be used to determine the pathogen infection status.

[0072] In summary, the detection results of this embodiment demonstrate that the sequencing results are accurate and reliable, thus proving that the present invention successfully achieves rapid and accurate diagnosis of bovine nodular dermatitis virus in no more than 3 hours, and is suitable for on-site pathogen detection in quarantine facilities (such as farms, slaughterhouses, and border ports). This indicates that the method provided by the present invention, which combines multiplex amplicon targeted enrichment with nanopore real-time sequencing to achieve rapid on-site detection of bovine nodular dermatitis virus, has been successfully established.

[0073] Example 3

[0074] A detection method for bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing includes:

[0075] Multiplex amplicon targeted enrichment reagents: 45 pairs of primers as shown in Table 1, 2× rapid Taq enzyme premix and nuclease-free water;

[0076] Nanopore real-time sequencing detection reagents: barcodes and sequencing library adapters.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A PCR primer set for detecting bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, characterized in that, Primers, including those with nucleotide sequences as shown in SEQ ID NO.1-90.

2. The application of the PCR primer set as described in claim 1 in the preparation of a detection product for bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing.

3. The application according to claim 2, characterized in that, The testing product is a reagent kit.

4. A detection product for bovine nodular dermatosis virus based on multiplex amplicon targeted enrichment and nanopore real-time sequencing, characterized in that, Including multiplex amplicon targeted enrichment reagents and nanopore real-time sequencing detection reagents; The multiplex amplicon targeting enrichment reagent includes the PCR primer set described in claim 1.

5. The testing product according to claim 4, characterized in that, The testing product is a reagent kit.

6. The testing product according to claim 5, characterized in that, The multiplex amplicon targeted enrichment reagent also includes 2× rapid Taq enzyme premix.

7. The testing product according to claim 6, characterized in that, The multiplex amplicon targeting enrichment reagent also includes nuclease-free water.

8. The testing product according to claim 4, characterized in that, The nanopore real-time sequencing detection reagent also includes a barcode and a sequencing library adapter.

9. The testing product according to claim 4, characterized in that, The PCR reaction program for the kit is as follows: 95℃ for 5 min; 95℃ for 15 s, 59℃ for 15 s, 72℃ for 1 min, 72℃ for 30 s, 35 cycles; store at 4℃.

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