Primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing and use thereof

This application is based upon and claims priority to Chinese Patent Application No. 202211450766.0, filed on Nov. 18, 2022, the entire contents of which are incorporated herein by reference.

The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBRZBC153_Sequence Listing.xml, created on Oct. 26, 2023, and is 379,175 bytes in size.

The present invention relates to the technical field of high throughput of target gene detection, and in particular, to a primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing and use thereof.

Amplicon sequencing is a high-targeting method, and the general idea is to target-capture a target region, then perform next generation sequencing (NGS) and analyze a sequencing result by designing primers near the conserved region of a target gene, performing PCR amplification and further performing sequencing analysis on an amplification product, thereby obtaining corresponding information.

Amplicon sequencing mainly comprises 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing, target region amplicon sequencing, and the like. At present, the amplicon sequencing is mainly applied to analysis of a metagenomics sample, and a sequence of a specific hypervariable region of 16S/18S/ITS determined by a second-generation high-throughput sequencing platform is used to reflect the differences between species of environmental samples in the classification of bacteria, fungi, and archaea, so that the microbial composition in the environments such as ocean, soil, and intestinal excrement is studied. In addition, few studies apply amplicon sequencing to pathogen detection, however, there is no related design for detecting porcine respiratory pathogens at present.

The respiratory diseases of pigs are mainly caused by single or mixed infection of viruses, bacteria and other pathogens, and the diagnosis methods of the pathogens generally comprise nucleic acid detection, antigen detection, and the like. Generally, most of detection means are single aiming at a certain pathogen, and the simultaneous rapid and accurate diagnosis of a plurality of pathogens cannot be implemented. In addition, at present, the most extensive fluorescent quantitative detection means for detecting the epidemic diseases in the pig farms can only detect 3 to 5 pathogens at most, however, the fluorescent quantitative detection means is only a simple diagnosis of pathogenic genes, and cannot further detect pathogenic gene mutations on the basis of disease diagnosis so as to discover a new mutant pathogen.

An objective of the present invention is to provide a primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing, which is used for amplicon sequencing detection analysis method for 42 target genes of 15 porcine respiratory pathogens, has the technical advantages of rapidness, high efficiency, high targeting, strong specificity, and the like, and is suitable for rapid and accurate simultaneous diagnosis of multiple pathogens of diseased pigs in pig farms.

In order to achieve the above objective, the present invention provides the following technical solution.

The present invention provides a primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing, which comprises primers having nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 302, the primer group is used to amplify pathogenic target genes, the pathogenic target genes are as follows:

Preferably, the primer group comprises a primer group 1 and a primer group 2, wherein the primer group 1 comprises primer sequences set forth in singular sequence numbers in SEQ ID NO: 1 to SEQ ID NO: 302, and the primer group 2 comprises primer sequences set forth in double sequence numbers in SEQ ID NO: 1 to SEQ ID NO: 302.

The present invention further provides a multiplex PCR reagent for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing, comprising a PCR reagent 1 and a PCR reagent 2, wherein the PCR reagent 1 comprises the primer group 1, and the PCR reagent 2 comprises the primer group 2.

The present invention mainly aims at a specific primer of the pathogenic gene. However, because the sequencing platform requirements of each sequencing company are inconsistent, other corresponding reagents can be added according to different platforms when the primer sequence is synthesized. The PCR reagent 1 and the PCR reagent 2 further comprise necessary reagents required in the PCR amplification process, and the requirements on an amplification system are different and can be adjusted adaptively.

Preferably, each primer in the primer group 1 has a concentration of 10 nmol/μL in the PCR reagent 1, and each primer in the primer group 2 has a concentration of 10 nmol/μL in the PCR reagent 2.

The present invention further provides a kit for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing, comprising the primer group or the multiplex PCR reagent.

The present invention further provides use of the primer group or the multiplex PCR reagent or the kit in constructing a high-throughput amplicon sequencing library for simultaneous detection of 15 porcine respiratory pathogens.

The present invention further provides use of the primer group or the multiplex PCR reagent or the kit in high-throughput amplicon sequencing for simultaneous detection of 15 porcine respiratory pathogens.

The present invention further provides use of the primer group or the multiplex PCR reagent or the kit in detecting 15 porcine respiratory pathogenic genes in a test sample.

The present invention further provides a method for detecting 15 porcine respiratory pathogenic genes in a test sample, which aims at non-disease diagnosis and treatment and comprises the following steps:

1) performing multiplex PCR reagent on a test sample by using the PCR reagent 1 and the PCR reagent 2 in the multiplex PCR reagent separately to obtain a multiplex PCR amplification product 1 and a multiplex PCR amplification product 2; 2) mixing the multiplex PCR amplification product 1 and the multiplex PCR amplification product 2 to prepare an amplicon library; and 3) sequencing the amplicon library to detect 15 porcine respiratory pathogenic genes in the test sample.

The present invention has the technical effects and advantages as follows.

The present invention provides a primer group for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing and use thereof, wherein the pathogens comprise 7 viral pathogens, namely, African swine fever virus, porcine reproductive and respiratory syndrome virus (North American type NA/European type EU), pseudorabies virus, porcine circovirus (type 2 and type 3), swine fever virus, foot and mouth diseases virus, and swine influenza A virus; 4 bacterial pathogens, namely, haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae , and Streptococcus suis ; and 4 other types of pathogens, namely, Eperythrozoon suis, Toxoplasma gondii, Mycoplasma hyorhinis , and Mycoplasma hyopneumoniae . The present invention particularly relates to target genes corresponding to target pathogens, wherein each pathogen comprises 2 to 8 different target genes, and the total number of the target genes is 42, so that the present invention aims to implement the simultaneous and rapid detection of 15 pathogens by sequencing the target genes of the target pathogens, further implements the simultaneous and rapid detection of multiple pathogens of diseased pigs, and closely associate diseased animals, infection pathogens, and the pathogenic target genes.

The present invention comprises a primer combination that is called Primer Pool and comprises 151 pairs of primers in total, a size range of amplicon fragments corresponding to each pair of primers is 100-150 bp, which can efficiently and specifically amplify pathogenic target genes; in addition, the present invention further provides an amplicon sequencing detection analysis method for 42 target genes of 15 porcine respiratory pathogens, which has the technical advantages of rapidness, high efficiency, high targeting, strong specificity, and the like, and is suitable for rapid and accurate simultaneous diagnosis of multiple pathogens of diseased pigs in pig farms.

FIG. 1 is a simplified flow chart of high-throughput amplicon sequencing;

FIG. 2 is a schematic diagram of amplicon sequencing library construction;

FIG. 3 shows details of pathogens and target genes thereof contained in the amplicon sequencing panel;

FIG. 4 shows distribution of target genes detected in Embodiment 1; and

FIG. 5 shows detection results of pathogens in Embodiment 1.

Unless otherwise stated, the methods used in the following examples are conventional methods.

The materials, reagents, and the like used in the following examples can be commercially available unless otherwise stated.

The technical solutions provided by the present invention will be described in detail below with reference to the examples, which, however, should not be construed as limiting the scope of the present invention.

1. Preparation of Primer Group for Simultaneous Detection of 15 Porcine Pathogens Through High-Throughput Targeted Amplicon Sequencing

According to literature reference, common porcine pathogens were selected through statistics and analysis, pathogenic target genes were determined, a complete gene sequence was downloaded on NCBI (https://www.ncbi.nlm.nih.gov/) according to the names of the target genes, a specificity of the target genes was further determined through NCBI Blast, and finally the sequences of the pathogenic target genes were obtained, wherein the nucleotide sequences of the pathogenic target genes were set forth in SEQ ID NO: 303 to SEQ ID NO: 344.

Finally, designing, analysis and comparison were performed many times according to the target gene sequences, wherein each gene was designed with 2-4 pairs of specific primers, and the total number of primers was 151 pairs of primers, and the nucleotide sequences of the primers were set forth in SEQ ID NO: 1 to SEQ ID NO: 302. The primers were synthesized by MGI Tech Co., Ltd., according to the company's library construction kit combined with this group of specific primers, the specific adapter sequence “GACATGGCTACGATCCGACTT” (SEQ ID NO: 345) was added to a 5′-end of each forward primer (F primer), the specific adapter sequence “CGCTTGGCCTCCGACTT” (SEQ ID NO: 346) was added to a 5′-end of each reverse primer (R primer), and finally the primer pool sequence for constructing the amplicon sequencing library was obtained.

2. Amplicon Ds DNA Library Construction

1). Sample Processing

The sample types mainly include, but are not limited to, porcine lung tissue, whole blood or serum, nasal swab, or pharyngeal swab, and the sample microbial genome was extracted by using, but not limited to, HiPure MicroBiome DNA Kit (Magen Biotechnology Limited, http://www.magentec.com.cn/), TIANamp Virus DNA/RNA Kit (Tiangen Biochemical Technology Co., Ltd, http://www.tiangen.com.cn/), and GoScript™ Reverse Transcription Mix, Random Primer (Promega Corporation, http://www.promega.com.cn/), and the like. The microbial DNA extracted from a single sample was quantified on ds DNA by using a Qubit™ 4 Fluorometer, and the concentration of each sample was controlled to be within 100 ng/μL.

2). First Cycle of PCR Amplification

The total DNA of the microorganism of a single sample was taken as a template, first cycle of PCR amplification was performed by using a primer pool of an amplicon sequencing library synthesized by MGI Tech Co., Ltd., and the reaction system was as follows:

The first cycle of PCR reaction solution was vortexed 3 times for 3 s each time, instantaneous centrifugation was performed, the reaction solution was collected to a bottom of a tube, then first cycle of PCR amplification was performed, and the reaction procedures were as follows: heated lid at 105° C.; 37° C. for 5 min, 95° C. for 10 min, 1 cycle; 95° C. for 20 s, 64° C. for 1 min, 60° C. for 1 min, 72° C. for 30 s, 13 cycles; and 12° C. Hold. After the reaction was completed, the reaction solution was centrifuged instantaneously and collected to the bottom of the tube.

3). First Cycle of PCR Product Purification

DNA Clean Beads were taken 30 min in advance and placed at room temperature, the DNA Clean Beads were fully shaken and uniformly mixed before used, 30 μL of the DNA Clean Beads were put in 25 clean 1.5 mL centrifuge tubes according to 25 sample volumes, and sample numbers were marked; all 25 μL products corresponding to the numbers of the first cycle of PCR were transferred to centrifuge tubes, the products were gently pipetted by a pipettor at least 10 times until the products were completely mixed, and it was ensured that all liquid and magnetic beads in a suction head were injected into the tubes for the last time, and incubated at room temperature for 5 min.

After instantaneous centrifugation, a 1.5 mL centrifuge tube was placed on a magnetic rack and left to stand for 2-5 min until the liquid was clear, the liquid was carefully sucked by using a pipettor, the supernatant was discarded; a 1.5 mL centrifuge tube was kept on a magnetic rack, 200 μL of freshly prepared 80% ethanol was added to rinse the magnetic beads and the tube wall, the liquid was left to stand for 30 s and carefully sucked, the supernatant was discarded, and this process was repeated again to ensure the liquid in the tube to be thoroughly sucked for the second time; a 1.5 mL centrifuge tube was kept on a magnetic rack, a tube cover was opened and dried at room temperature until the surface of the magnetic beads had no reflection and no crack; a 1.5 mL centrifuge tube was taken and added with 6.5 μL TE Buffer for DNA elution, and the buffer was gently pipetted by a pipettor until the buffer was completely mixed, then incubated at room temperature for 5 min, and subjected to instantaneous centrifugation. In this step, the magnetic beads were not removed.

6.5 μL of the purified product (containing magnetic beads) was stored in a refrigerator at −20° C.

4). Second Cycle of PCR Amplification

The first cycle of PCR purified product was taken as a template, a customized panel provided by MGI Tech Co., Ltd. was subjected to second cycle of PCR amplification, and the reaction system was as follows:

The prepared second cycle of PCR reaction solution was vortexed 3 times for 3 s each time, instantaneous centrifugation was performed, the reaction solution was collected to a bottom of a tube, then second cycle of PCR amplification was performed, and the reaction procedures were as follows: heated lid at 105° C.; 37° C. for 5 min, 95° C. for 10 min, 1 cycle; 95° C. for 20 s, 64° C. for 1 min, 60° C. for 1 min, 72° C. for 30 s, 27 cycles; and 12° C. Hold. After the reaction was completed, the reaction solution was centrifuged instantaneously and collected to the bottom of the tube.

5). Second Cycle of PCR Product Purification

DNA Clean Beads were taken 30 min in advance and placed at room temperature, the DNA Clean Beads were fully shaken and uniformly mixed, 25 μL of the DNA Clean Beads were put in 25 clean 1.5 mL centrifuge tubes according to 25 sample volumes, and sample numbers were marked; all 25 μL products corresponding to the numbers of the second cycle of PCR in the step 4 were transferred to centrifuge tubes, the products were gently pipetted by a pipettor at least 10 times until the products were completely mixed, and it was ensured that all liquid and magnetic beads in a suction head were injected into the tubes for the last time, and incubated at room temperature for 5 min.

After instantaneous centrifugation, a 1.5 mL centrifuge tube was placed on a magnetic rack and left to stand for 2-5 min until the liquid was clear, the liquid was carefully sucked by using a pipettor, the supernatant was discarded; a 1.5 mL centrifuge tube was kept on a magnetic rack, 200 μL of freshly prepared 80% ethanol was added to rinse the magnetic beads and the tube wall, the liquid was left to stand for 30 s and carefully sucked, the supernatant was discarded, and this process was repeated again to ensure the liquid in the tube to be thoroughly sucked for the second time; a 1.5 mL centrifuge tube was kept on a magnetic rack, a tube cover was opened and dried at room temperature until the surface of the magnetic beads had no reflection and no crack; a 1.5 mL centrifuge tube was taken and added with 25 μL TE Buffer for DNA elution, and the buffer was gently pipetted by a pipettor until the buffer was completely mixed, then incubated at room temperature for 5 min, and subjected to instantaneous centrifugation; the 1.5 mL centrifuge tube was placed on the magnetic rack and left to stand for 2-5 min until the liquid was clear, and 23 μL supernatant was transferred to a new PCR tube. The magnetic beads need to be removed clean in this step.

23 μL of the purified product was stored in a refrigerator at −20° C., at this point, amplicon ds DNA library construction was completed.

3. Amplicon Ss DNA Library Construction

1). Quality Inspection of Ds DNA Library and Construction of Ds DNA Mixed Library

The constructed single ds DNA library was quantified by using a Qubit™ 4 Fluorometer according to an operation instruction, wherein the library required that the yield of a final PCR product was greater than or equal to 5 ng/μL; and then according to the quantitative result of the DNA library, the samples to be sequenced were mixed according to the quality of the barcode numbers and the like, wherein the total amount of the mixed final library was required to be 400 ng, the total volume was less than or equal to 48 μL, and the sampling volume of a single library was ensured to be greater than or equal to 1 μL.

After quantification by a Qubit™ 4 Fluorometer, 25 samples all met the quality requirement, and then 25 samples were mixed by calculation. Sampling was performed, wherein the sampling quantity of a single library was enlarged by 20 times, and finally, 25 amplicon ds DNA libraries were constructed into 1 amplicon ds DNA mixed library.

2). Denaturation of Ds DNA Library to Ss DNA Library

The single ds DNA mixed library obtained in the step 1 was taken as a template, 400 ng of the ds DNA mixed library was placed in a PCR tube, the PCR tube was supplemented with a TE Buffer to a total volume of 48 μL, and a denaturation reaction was performed. The reaction conditions were as follows: heated lid at 105° C., 95° C. for 3 min, and 95° C. Hold. After the reaction was finished, the PCR tube was quickly placed on ice for an ice bath for 2 min, and then ds DNA mixed library in the tube was subjected to instantaneous centrifugation to obtain an ss DNA initial library.

3). Amplicon Ss DNA Library Circularization

The above denatured amplicon ss DNA library was taken as a template, Amplicon library circularization was performed by using MGIEasy circularization kit, and the reaction system was as follows:

The prepared PCR reaction solution was vortexed 3 times for 3 s each time, instantaneous centrifugation was performed, the reaction solution was collected to a bottom of a tube, then PCR amplification was performed, and the reaction procedures were as follows: heated lid at 105° C.; 37° C. for 30 min, and 4° C. Hold. After the reaction was completed, the PCR tube was instantaneously centrifuged and placed on ice to immediately proceed to the reaction in step 4.

4). Ss DNA Library Enzyme Digestion

The circularized amplicon ss DNA library was taken as a template, the reaction in step 3 was followed by the reaction in step 4, and the reaction system was as follows:

The prepared enzyme digestion solution was vortexed 3 times for 3 s each time, instantaneous centrifugation was performed, the reaction solution was collected to a bottom of a tube, then PCR amplification was performed, and the reaction procedures were as follows: heated lid at 105° C.; 37° C. for 30 min, and 4° C. Hold. After the reaction was completed, the reaction solution was centrifuged instantaneously and collected to the bottom of the tube. Then 7.5 μL of Digestion Stop Buffer was immediately added to the PCR tube, vortexed 3 times for 3 s each time, the reaction solution was collected to the bottom of the tube after instantaneous centrifugation, and the whole reaction solution was sucked and transferred to a new 1.5 mL centrifuge tube.

5). Purification of Enzyme Digestion Products

The DNA Clean Beads were taken out in advance during the enzyme digestion reaction and placed at room temperature, and the DNA Clean Beads was fully shaken and mixed uniformly before use; and 170 μL of the mixture was put into the 1.5 mL centrifuge tube filled with the reaction solution and gently pipetted by a pipettor at least 10 times until the mixture was completely mixed, and it was ensured that all liquid and magnetic beads in a suction head were injected into the tubes for the last time, and incubated at room temperature for 10 min.

After instantaneous centrifugation, a 1.5 mL centrifuge tube was placed on a magnetic rack and left to stand for 2-5 min until the liquid was clear, the liquid was carefully sucked by using a pipettor, the supernatant was discarded; a 1.5 mL centrifuge tube was kept on a magnetic rack, 500 μL of freshly prepared 80% ethanol was added to rinse the magnetic beads and the tube wall, the liquid was left to stand for 30 s and carefully sucked, the supernatant was discarded, and this process was repeated again to ensure the liquid in the tube to be thoroughly sucked for the second time; a 1.5 mL centrifuge tube was kept on a magnetic rack, a tube cover was opened and dried at room temperature until the surface of the magnetic beads had no reflection and no crack; a 1.5 mL centrifuge tube was taken and added with 22 μL TE Buffer for DNA elution, and the buffer was gently pipetted by a pipettor until the buffer was completely mixed, then incubated at room temperature for 10 min, and subjected to instantaneous centrifugation; the 1.5 mL centrifuge tube was placed on the magnetic rack and left to stand for 2-5 min until the liquid was clear, and 20 μL supernatant was transferred to a new PCR tube. The magnetic beads need to be removed clean in this step.

20 μL of the purified product was stored in a refrigerator at −20° C., at this point, amplicon ss DNA library was successfully constructed.

4. Amplicon DNB Library Construction

1). Amplicon Ss DNA Equilibrium Library Construction

The conversion formula of the concentration is as follows: C(fmol/μL)=3030*C(ng/μL)/N.

According to the quantitative result of the Qubit™ 4 Fluorometer, the concentration of the amplicon ss DNA library constructed in this embodiment was greater than or equal to 2 fmol/μL, and the quality control requirement was met. After ss DNA quantification was performed on standard products provided in the Qubit® ssDNA Assay Kit, the standard products were calculated using the following two formulas:

ss DNA input (μL)=44/C(fmol/μL);  (1)

the standard ss DNA input (μL)=16/C(fmol/μL).  (1)

The ss DNA input and the standard ss DNA input were mixed to construct an amplicon ss DNA equilibrium library.

2). Amplicon DNB Library Construction

The amplicon ss DNA equilibrium library in the step 1 was taken as a template, DNB preparation was performed by using an MGISEQ-200RS high-throughput sequencing kit (FCS PE 100), and two cycles of reactions were performed in total, wherein the first cycle of reaction was PCR primer hybridization, and the reaction system was as follows:

The prepared DNB reaction mixed solution 1 was vortexed, uniform mixed, centrifuged for 5 s by using a mini centrifuge, and then placed in a PCR instrument for primer hybridization, wherein the reaction conditions were as follows: heated lid at 105° C., 95° C. for 1 min, 65° C. for 1 min, 40° C. for 1 min, and 4° C. Hold. After the reaction was completed, the solution was subjected to a second cycle of reaction, namely, a DNB rolling circle amplification reaction. Firstly the PCR reaction tube was taken out, centrifuged by a mini-centrifuge for 5 s, and then placed on ice to prepare a second cycle of reaction solution by DNB, wherein the second cycle of reaction system was as follows:

The prepared DNB mixed reaction solution 2 was vortexed by using a vortex oscillator and uniformly mixed, centrifuged for 5 s by using a mini centrifuge, and immediately placed in a PCR instrument for DNB rolling circle amplification, wherein the reaction conditions were as follows: heated lid at 35° C., 30° C. for 25 min, 4° C. Hold. After the reaction was completed, the solution was immediately added with 20 μL of DNB termination buffer solution, and the mixture was slowly pipetted for 5 to 8 times to be well mixed by using a wide-mouthed pipette tip without vortexing or violently pipetting.

3). DNB Quality Inspection

After DNB preparation was completed, 2 μL of DNB mixed reaction solution was subjected to concentration detection by using Qubit® ssDNA Assay Kit and Qubit® Fluorometer instruments, and the concentration of the amplicon DNB=15.5 ng/μL, which met the quality control requirement. DNB was stored at 4° C. for future use and prepared for on-line sequencing.

5. On-Line Sequencing

The prepared DNB was prepared for on-line sequencing according to the instructions of the high-throughput (rapid) sequencing reagent set in MGI Tech, which was the MGISEQ-200RS high-throughput rapid sequencing reagent set (FCS PE100).

The sequencing data were extracted for analysis and comparison, a total summary report was generated after sequencing was completed, the summary report was opened by a webpage and viewed, the summary report shows that the sequencing quality is high, the data result of greater than or equal to Q30 is more than 90%, the Barcode can be normally identified, all samples can be correctly distinguished, and the capture rate of a pathogenic target gene area has certain difference according to the difference of pathogen load in the samples. The sequencing result is accurate and reliable, and the designed amplification primer has high efficiency and good specificity.

According to the sequencing results of the target gene, after data homogenization and calculation of the pathogen load, the detection of the pathogen is judged according to the calculation result of the pathogenic target gene. It has been proved by practice that the pathogenic genes in the panel are detected in the above 25 samples, thus successfully implementing the rapid, accurate, and simultaneous diagnosis of 15 pathogens in the porcine respiratory tract.

Therefore, the method for simultaneous detection of 15 porcine pathogens through high-throughput targeted amplicon sequencing provided by the present invention is successfully established.

The above descriptions are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall fall within the protection scope of the present invention.