Pig delta coronavirus whole genome sequencing method based on nanopore sequencing

Abstract

The application provides an amplification primer and method for a whole genome of a porcine delta coronavirus. A detection primer set for a novel coronavirus comprises 124 pairs of amplification primers, and specific sequences are shown in SEQ ID NO. 1-248. The detection primer set provided by the application can achieve relatively uniform coverage of the porcine delta coronavirus, and in nanopore sequencing, 100% of the genomic region is covered. The sequencing fragment preparation method for the whole genome of the porcine delta coronavirus provided by the application is simple and easy to operate, has good amplification effect, and nanopore sequencing has the advantage of real-time analysis while sequencing, which can shorten the detection time, can quickly identify and diagnose the infection of the porcine delta coronavirus, obtain the whole genome sequence, and the obtained genome sequence can provide a scientific basis for virus tracing, pathogen variation tracking, new strain identification and early warning.

Description

Technical Field

[0001] This invention relates to the field of animal virology, and provides primers and methods for amplifying the whole genome of porcine deltacoronavirus. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) belongs to the newly identified δ-coronavirus genus within the Coronaviridae family. It was first discovered in 2012 by Hong Kong researchers in pig fecal samples, but in-depth studies on virus isolation and pathogenicity were not conducted at that time. In February 2014, US researchers detected PDCoV in pig farms experiencing diarrhea outbreaks and successfully isolated the virus. Animal experiments confirmed that the isolated strain was highly pathogenic to newborn piglets, with infected piglets exhibiting symptoms such as decreased appetite, vomiting, and severe diarrhea. Subsequently, at least 20 states in the US reported outbreaks of PDCoV. Following the US reports, Canada, South Korea, Thailand, Vietnam, Laos, and other countries detected PDCoV outbreaks in pig diarrhea samples. Reports indicated that the Thai strain was highly pathogenic to piglets, finishing pigs, and sows, leading to a large number of piglet deaths, drawing significant attention from the global pig farming industry. To date, more than 25 provinces and regions in my country have reported diarrhea outbreaks related to PDCoV, demonstrating the widespread presence and rapid spread of the virus in pig farms across the country.

[0003] PDCoV can infect and cause disease in pigs of all ages during production, and it is highly pathogenic to piglets, leading to mass mortality. This disease occurs in all seasons, but is most prevalent in winter and spring. PDCoV continuously evolves and recombines in the field, and the virus can spread across species through intermediate hosts, making it a potential zoonotic infectious disease. Rapid identification and diagnosis of PDCoV infection, and obtaining its genomic information, can provide a basis for early detection, control, and source tracing, playing a crucial role in preventing PDCoV outbreaks. Currently, the main methods for virus detection include virus isolation and culture, immunological detection, and nucleic acid detection; however, none of these methods can obtain the complete genome sequence of the virus or analyze its genomic characteristics. Nanopore sequencing offers advantages such as real-time processing, portability, long read lengths, and high throughput, enabling rapid real-time detection in the field; however, there is currently no sequencing technology specifically designed for PDCoV that leverages the advantages of nanopore sequencing. Therefore, there is an urgent need for a whole-genome sequencing technology for porcine deltacoronavirus based on third-generation nanopore sequencing. Summary of the Invention

[0004] This invention designs specific primers for long PCR amplification of the PDCoV whole genome and further provides a real-time detection method for PDCoV using nanopore sequencing, fully leveraging the advantages of nanopore sequencing in pathogen detection, such as real-time capability, portability, long read length, and high throughput. This detection method can rapidly identify and diagnose PDCoV infection while obtaining its whole genome sequence. The obtained whole genome sequence can provide information for pathogen variation monitoring and molecular epidemiological studies.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A primer set for detecting a novel coronavirus, comprising 124 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1-248.

[0007] The application of the above-mentioned detection primer set in the preparation of products for detecting the novel coronavirus.

[0008] Furthermore, the product includes reagents or kits.

[0009] On the other hand, the present invention provides a detection reagent for the novel coronavirus, comprising the above-mentioned detection primer set.

[0010] Furthermore, the detection reagent includes a first primer set and a second primer set, wherein the first primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1-124; and the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.125-248.

[0011] On the other hand, the present invention provides a novel coronavirus detection kit, comprising the above-mentioned detection primer set.

[0012] Furthermore, the detection kit includes a first primer set and a second primer set, wherein the first primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1-124; and the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.125-248.

[0013] On the other hand, the present invention provides a method for preparing a novel coronavirus sequencing fragment, which uses the above-mentioned detection primer set to amplify cDNA obtained by reverse transcription of the sample to be tested, and the amplification product is purified to obtain the sequencing fragment.

[0014] Furthermore, the cDNA obtained by reverse transcription of the sample to be tested was amplified using the first primer set and the second primer set, respectively, and the amplification products were purified to obtain sequencing fragments.

[0015] The first primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.1-124; the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO.125-248.

[0016] Furthermore, the sequencing samples are suitable for second- or third-generation sequencing.

[0017] Furthermore, the samples to be tested include clinical samples or environmental samples;

[0018] Preferably, clinical samples include throat swabs, nasal swabs, blood samples, or lung tissue cells.

[0019] On the other hand, the present invention provides the application of the aforementioned primer set, detection reagent or kit in the non-disease diagnostic detection of porcine deltacoronavirus.

[0020] Furthermore, the above-mentioned testing steps include:

[0021] 1) Extract total DNA from the sample.

[0022] 2) Multiplex PCR amplification was performed on the DNA obtained from the sample to be tested using the first primer set and the second primer set, respectively. The amplification products were purified to obtain sequencing fragments. The first primer set included 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO. 1-124. The second primer set included 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO. 125-248.

[0023] 3) The amplification products of the two sets of primers were combined and purified to obtain the sequencing fragment.

[0024] 4) Sequencing to obtain genome fragment sequences.

[0025] 5) The genome sequence of porcine deltacoronavirus was obtained by comparison with and / or assembly of the reference genome.

[0026] Furthermore, the sequencing described in step 4) is second-generation or third-generation sequencing.

[0027] Furthermore, the non-disease diagnostic test for porcine deltacoronavirus is to detect environmental samples or throat swabs, nasal swabs, blood samples, or lung tissue cells from inanimate biological individuals.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] Based on the publicly available complete genome sequence of PDCoV in the GenBank database of the Center for Biotechnology Information (CBI), 124 pairs of amplification primers were designed according to genomic locations, with specific sequences shown in SEQ ID NO. 1-248. The detection method of this invention can obtain the complete genome sequence information of PDCoV. This invention utilizes the advantages of nanopore sequencing—real-time, portable, long read length, and high throughput—to achieve rapid identification and diagnosis of PDCoV infection; simultaneously, it obtains its complete genome sequence, which can provide scientific basis for virus tracing, pathogen mutation tracking, identification of novel strains, and early warning. Attached Figure Description

[0030] Figure 1 This study shows the sequencing depth coverage of the amplified products from two groups, the 5' end and the 3' end, based on the primer binding position.

[0031] Figure 2 This represents the sequencing depth coverage of two sets of amplified products obtained by randomly combining primer pairs.

[0032] Figure 3 To determine the MinION sequencing depth coverage of PDCoV strain branch 1 samples.

[0033] Figure 4 To determine the MinION sequencing depth coverage of PDCoV strain branch 2 samples.

[0034] The present invention will be further explained below with reference to specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] 1. Primer design for third-generation sequencing and whole-genome amplification of PDCoV

[0038] Based on the publicly available whole genome sequence of PDCoV in the GenBank gene sequence database of the Center for Biotechnology Information (CBI), whole genome multiple sequence alignment analysis was performed using molecular biology tools such as BioEdit to obtain its conserved regions. Primers were designed and validated using Primer-BLAST. Multiple primer pairs were designed for PDCoV genome amplification. Primer information is as follows:

[0039] Table 1. Detection primer sequence involved in this invention.

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] This detection primer set achieves relatively uniform coverage of PDCoV, providing a good technical means for obtaining more uniform and effective PDCoV genome sequencing samples, and has achieved good results in both nanopore sequencing and high-throughput sequencing. This detection primer set is suitable for preparing products for detecting PDCoV and has been widely used. Specifically, it can be a reagent or kit. This invention also protects the reagent or kit, specifically using the above detection primer set as primers to amplify and prepare sequencing fragments of the entire PDCoV genome.

[0047] 2. Establishing a real-time sequencing method for PDCoV using nanopore sequencing

[0048] This embodiment uses two PDCoV strains with different branches and sequences the PDCoV samples using the preparation method provided by this invention. The specific process is as follows:

[0049] 1) One-stranded cDNA synthesis: Take 8 μL of RNA into a new 0.2 mL PCR tube, add 2 μL of RTMix (TaKaRa) on ice, gently tap to mix, and briefly centrifuge to the bottom of the tube, ensuring no air bubbles remain. Place the PCR tube / plate on ice. Run the PCR program on a PCR instrument (25℃ for 2 min; 55℃ for 10 min; 95℃ for 10 min; 4℃ Hold), and store at 4℃.

[0050] 2) PCR multiplex amplification: The reaction was divided into two parts, tube 1 (first primer set) and tube 2 (second primer set). The same sample was reacted using NEB (NEW ENGLAND BioLabs) High-Fidelity 2X Master Mix under the following conditions: 98℃, 30s; 35 cycles: 98℃, 15s, 65℃, 5min; stored at 4℃.

[0051] 3) PCR product purification: Combine the products from the two amplification pools into a 1.5 ml EP tube. Purify using 0.8× magnetic beads, wash twice with 200 μl of freshly prepared 70% ethanol, and then elute the purified product with nuclease-free water.

[0052] 4) PCR product quality testing: using The dsDNA HS Assay Kit is a double-stranded DNA fluorescence quantitative reagent kit. Follow the instructions of the quantitative kit to quantify the PCR-purified product.

[0053] 5) Third-generation sequencing: Library construction and sequencing of the mixed samples of amplified products were performed using the MinION nanopore sequencer.

[0054] 6) Analysis results: Using bioinformatics methods, the sequencing fragment sequences were aligned to the GenBank reference genome, and the coverage depth of each genomic locus was calculated.

[0055] Experimental Example 1

[0056] The genome was divided into 5' and 3' ends for multiplex PCR amplification. Specifically, primers were divided into a 5' end primer set (PCR tube 1, expected to amplify the 5' end to approximately 12700 bp) and a 3' end primer set (PCR tube 2, expected to amplify the 3' end starting from approximately 12600 bp) based on their binding sites on the genome. Only the PDCoV strain branch 1 sample was sequenced; other conditions remained unchanged from the above experimental steps.

[0057] The results are as follows Figure 1 As shown, the overall read coverage is extremely uneven, with some sites having coverage of less than 100% or even 0%, making it difficult to meet the requirements for whole-genome coverage.

[0058] Experimental Example 2

[0059] The difference from Experiment 1 is that the primer pairs were randomly combined and divided into two tubes, and only the two samples of the PDCoV strain branch were sequenced. Other conditions remained the same as the above experimental steps.

[0060] The results are as follows Figure 2As shown, the overall read coverage is extremely uneven, with some sites having coverage of less than 100% or even 0%, making it difficult to meet the requirements for whole-genome coverage.

[0061] Experimental Example 3

[0062] According to Table 1, the primers are grouped into the first primer group and the second primer group, and the reactions are divided into two groups. Other conditions are carried out according to the above experimental steps.

[0063] The results showed that this amplification system achieved 100% coverage of the PDCoV genome, with all amplified regions achieving a coverage depth of >100-fold. MinION sequencing results are as follows: Figures 3-4 As shown ( Figure 3 This is a sample from PDCoV strain branch 1. Figure 4 (This refers to a sample from PDCoV strain branch 2). The results demonstrate that the standard evaluation criteria of this invention meet the requirements, providing good quality control for obtaining more uniform and effective PDCoV genome sequencing samples.

[0064] The above results indicate that multiplex PCR combined with nanopore sequencing still requires the design of primers of appropriate length and at appropriate sites, and the primer pair combination needs to be verified to achieve the best results.

[0065] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for detecting porcine deltacoronavirus, characterized in that, The detection primer set includes a first primer set and a second primer set; wherein, the first primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO .1-124; and the second primer set includes 62 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NO .125-248.

2. The use of the detection primer set according to claim 1 in the preparation of products for detecting porcine deltacoronavirus.

3. The application according to claim 2, characterized in that, The products include reagents or kits.

4. A detection reagent for porcine deltacoronavirus, characterized in that, Includes the detection primer set as described in claim 1.

5. A detection kit for porcine deltacoronavirus, characterized in that, Includes the detection primer set as described in claim 1.

6. A method for preparing a sequencing fragment of porcine deltacoronavirus, characterized in that, The cDNA obtained by reverse transcription of the sample to be tested was amplified using the first primer set and the second primer set as described in claim 1, and the amplification products were purified to obtain sequencing fragments.

7. The use of the primer set of claim 1, the detection reagent of claim 4, or the kit of claim 5 in the non-disease diagnostic detection of porcine deltacoronavirus.

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