CN117070647A - Compositions for pathogen detection based on high throughput amplicon sequencing - Google Patents
Compositions for pathogen detection based on high throughput amplicon sequencing Download PDFInfo
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- 241001337904 Gordonia <angiosperm> Species 0.000 claims abstract description 5
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Abstract
The invention belongs to the field of biological detection. In particular, to compositions for high throughput amplicon sequencing detection, and more particularly, to compositions for high throughput amplicon sequencing detection of pathogens. The present invention provides a composition for detecting pathogens based on high throughput amplicon sequencing, comprising at least 4 targets as shown below: mycobacterium tuberculosis complex, gordonia, mycoplasma psittaci, mycoplasma pneumoniae, orientia tsutsugamushi, rickettsia typhosa, plasmodium malariae, plasmodium vivax, enamoeba histolytica, and Mycobacterium abscessum. The composition can amplify and enrich a plurality of targets in one tube simultaneously, so that the data volume required by subsequent sequencing is obviously reduced, the analysis work is simplified, and the whole detection is more accurate and efficient.
Description
Technical Field
The invention belongs to the field of biological detection. In particular, to compositions for high throughput amplicon sequencing detection, and more particularly, to compositions for high throughput amplicon sequencing detection of pathogens.
Background
Metagenomics (Metagenomics) is also known as microbial environmental genomics. The method constructs a metagenome library by directly extracting DNA of all microorganisms from an environmental sample, and researches genetic composition and community functions of all microorganisms contained in the environmental sample by utilizing a research strategy of genomics. The metagenome is developed on the basis of microbiology, is independent of the isolated culture of microorganisms, can be used for researching natural products in microorganisms which are difficult to culture or can not be cultured and natural products in a silencing state, and greatly improves the utilization degree of microbial resources in metagenome samples. By performing deep sequencing analysis on metagenomic samples, the true species diversity and genetic diversity in the environment can be revealed or estimated, and the metagenomic samples can be used for discovering novel microbial active substances (or novel genes). Currently, metagenomic studies mainly include two approaches, whole Genome Sequencing (WGS) and targeted resequencing (tNGS).
Unlike whole genome sequencing, targeted resequencing techniques can directly perform targeted enrichment on the genome of interest in the sample to be tested, separating the target genome from the complex background nucleic acid, and resequencing. The method is more economical and efficient, and has higher sensitivity and is more convenient for subsequent data analysis. For example, in clinical sample sequencing data, the background data of human genome may be up to 99%, only 1% of the data is the target sequence, so that a large amount of data is required to ensure the accuracy of the data, if the target sequence is enriched and then sequenced separately, the required data amount can be significantly reduced, and the related work can be greatly simplified in subsequent analysis correspondingly.
Thus, there is a need in the art for a composition that enriches for targets such that the amount of data required for subsequent sequencing is significantly reduced, and that simplifies analysis and allows for accurate and efficient detection of a variety of targets.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for detecting pathogens based on high throughput amplicon sequencing, comprising at least 4 targets in the primer set as follows:
primer groups for amplifying the mycobacterium tuberculosis complex as shown in SEQ ID NO. 1-6;
primer groups for amplifying the gordonia mycobacteria shown as SEQ ID NO. 7-12;
primer groups for amplifying Chlamydia psittaci as shown in SEQ ID NO. 13-20;
primer groups for amplifying mycoplasma pneumoniae shown in SEQ ID NO. 21-28;
primer sets for amplifying Orientia tsutsugamushi as shown in SEQ ID NOS.29 to 32;
primer groups for amplifying the typhoid rickettsia shown as SEQ ID NO. 33-36;
primer groups for amplifying plasmodium malariae as shown in SEQ ID NO. 37-40;
primer groups for amplifying plasmodium vivax as shown in SEQ ID No. 41-44;
primer group for amplifying amoeba histolytica as shown in SEQ ID NO. 45-48; or alternatively
Primer sets for amplifying mycobacterium abscessus shown as SEQ ID NO. 49-62.
Further, the present invention provides a composition based on high throughput amplicon sequencing detection comprising at least 5 targets in the primer set as indicated above.
Further, the present invention provides a composition based on high throughput amplicon sequencing detection comprising at least 6 targets in the primer set as indicated above.
Further, the present invention provides a composition based on high throughput amplicon sequencing detection comprising at least 7 targets in the primer set as indicated above.
Further, the present invention provides a composition based on high throughput amplicon sequencing detection comprising at least 8 targets in the primer set as indicated above.
Further, the present invention provides a composition based on high throughput amplicon sequencing detection comprising at least 9 targets in the primer set as indicated above.
The composition can amplify and enrich a plurality of targets in one tube simultaneously, so that the data volume required by subsequent sequencing is obviously reduced, the analysis work is simplified, and the whole detection is more accurate and efficient.
It is to be noted that if 10 targets can be PCR amplified simultaneously within a tube, it is needless to say that any combination of these 10 targets (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) can be PCR amplified within a tube.
Still further, the present invention provides a composition based on high throughput amplicon sequencing detection, comprising:
primer groups for amplifying the mycobacterium tuberculosis complex as shown in SEQ ID NO. 1-6;
primer groups for amplifying the gordonia mycobacteria shown as SEQ ID NO. 7-12;
primer groups for amplifying Chlamydia psittaci as shown in SEQ ID NO. 13-20;
primer groups for amplifying mycoplasma pneumoniae shown in SEQ ID NO. 21-28;
primer sets for amplifying Orientia tsutsugamushi as shown in SEQ ID NOS.29 to 32;
primer groups for amplifying the typhoid rickettsia shown as SEQ ID NO. 33-36;
primer groups for amplifying plasmodium malariae as shown in SEQ ID NO. 37-40;
primer groups for amplifying plasmodium vivax as shown in SEQ ID No. 41-44;
primer group for amplifying amoeba histolytica as shown in SEQ ID NO. 45-48; and
primer sets for amplifying mycobacterium abscessus shown as SEQ ID NO. 49-62.
The composition provided by the invention can amplify and enrich 10 targets in one tube simultaneously, so that the data volume required by subsequent sequencing is obviously reduced, the analysis work is simplified, and the whole detection is more accurate and efficient.
Further, the primer sets each have a linker sequence to facilitate sequencing.
Further, the upstream primer adapter sequence was ACACGACGCTCTTCCGATCT and the downstream primer adapter sequence was CTTGGCACCCGAGAATTCCA.
In some specific embodiments, the ingredients of the composition are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the present invention provides the use of a composition as described above for the preparation of a kit for high throughput amplicon sequencing detection of a pathogen.
In a third aspect, the invention provides a kit for high throughput amplicon sequencing detection of a pathogen comprising the composition described above.
Further, the kit further comprises at least one of the following: reagents required for nucleic acid extraction, reagents required for nucleic acid amplification, and reagents required for sequencing.
Further, the reagent required for nucleic acid extraction may be a reagent for extracting DNA from blood.
Further, the reagents required for nucleic acid amplification include DNA polymerase, dNTPs, buffer, and Mg 2+ 。
Further, reagents required for sequencing include magnetic beads.
In a fourth aspect, the present invention provides a use of preparing a composition for high throughput amplicon sequencing detection of a pathogen, wherein the detection comprises:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Amplifying using a composition as described above to obtain an amplified product;
3) Processing the amplified products and establishing a library; and
4) Sequencing and analyzing the result.
Further, the conditions of the amplification are:
drawings
FIG. 1 is a flow chart of library construction;
FIG. 2 is a schematic diagram of library construction;
FIG. 3 is a graph of library fragment size results.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1 primers used in the present invention
The primer sets used in the present invention are shown in Table 1.
TABLE 1
Example 2 method of detection of different targets based on high throughput amplicon sequencing
A specific library construction scheme is shown in FIG. 1. The principle is shown in figure 2, in brief, a specific primer is designed based on a target area of pathogenic microorganism, a general sequence is added to the 5' end of all the specific primers, the nucleic acid to be detected is used as a template for first round of amplification, and the obtained amplification product is added with a label primer for second round of amplification to complete library establishment.
1 Experimental reagent and apparatus
The reagents and instrumentation required are shown in tables 2 and 3 below.
TABLE 2
TABLE 3 Table 3
| Name of the name | Branding |
| LabChip GX | caliper |
| Sansureseq1000 | Saint Hunan organism |
| PCR amplification instrument | Hangzhou Bo-day technology |
| Qubit 3.0fluorometer | thermo fisher |
2. Experimental procedure
2.1 Multiplex amplification primer set preparation
The synthesized multiplex amplification primers (100. Mu.M) with the adaptor sequences were mixed in the same volume.
2.2 Sample preparation
Extracting gDNA in clinical samples, standard bacterial liquid and national reference bacterial liquid by using a magnetic bead method blood genome DNA extraction kit, uniformly mixing gDNA in equal quantity, and carrying out multiplex PCR amplification by taking the mixed gDNA or plasmid (fewer drug-resistant gene clinical samples are obtained, so that genes are synthesized on the plasmid) as a template. All clinical samples are from the Save Vial laboratory, and the standard bacterial liquid and the national reference bacterial liquid are purchased outsourced, and the drug-resistant gene plasmid is synthesized by the Bai Lige biotechnology Co.
2.3 first round multiplex amplification
The extracted gDNA (40 ng) or drug-resistant plasmid (diluted to 10≡5 copies/. Mu.l, 5. Mu.l) was used as a template, multiplex PCR amplification was performed with a mixed primer set, the amplification system is shown in Table 4, the reaction system is shown in Table 5, and all reagents were contained in the kit purchased in Ai Jitai kang MultipSeq Library Prep Kit.
TABLE 4 Table 4
| Reagent | Volume(μl) |
| ddH 2 O | 9-x |
| Enhancer buffer NB(1N) | 3.5 |
| Enhancer buffer M | 2.5 |
| Primer pool | 5 |
| Stencil (one tube includes all targets) | x |
| IGT EM808Polymerase Mixture | 10 |
TABLE 5
2.4 purification after first round PCR amplification
Mu.l of PCR product was taken and 27. Mu.l of Nanjinozan DNA clear beads equilibrated to room temperature was added, mixed well with shaking and incubated at room temperature for 5min to bind the DNA to the magnetic beads.
Centrifuging instantly, placing the centrifuge tube on a magnetic rack for 3min until the centrifuge tube is clear, and discarding the supernatant;
adding 50 mu L of YF buffer B into the centrifuge tube, fully oscillating and uniformly mixing, incubating for 5min at room temperature, performing instantaneous centrifugation, placing the centrifuge tube on a magnetic rack for 3min until the centrifuge tube is clear, and discarding the supernatant.
200 mu L of 80% ethanol is added into the centrifuge tube, the magnetic beads are ensured to be completely immersed into the 80% ethanol, the centrifuge tube is kept stand for 1min, and the supernatant is discarded.
200 mu L of 80% ethanol is added into the centrifuge tube, the magnetic beads are ensured to be completely immersed into the 80% ethanol, the centrifuge tube is kept stand for 1min, and the supernatant is discarded.
After sufficient centrifugation, the supernatant was thoroughly removed with a 10. Mu.L pipette and left to stand for 3min to allow the residual ethanol to evaporate thoroughly.
Adding 24 mu l Nuclease free water, shaking, mixing, and standing for 2min.
The centrifuge tube was placed on a magnetic rack for 3min to clarify, 13.5 μl of supernatant was pipetted into a new 200 μl PCR tube.
2.5 round 2 linker sequence PCR reactions
The PCR reaction system of round 2 was prepared as shown in Table 6 using the first round of PCR purified product as a template, and CDIPrimer was included in the purchased MultipSeq CDI Adapter kit, and all other products were included in the purchased Ai Jitai cm MultipSeq Library Prep Kit kit, and the reaction system was as shown in Table 7.
TABLE 6
| Reagent | Volume(μl) |
| PCR product mixture | 13.5 |
| Enhancer buffer M | 2.5 |
| ddH 2 O | 2 |
| CDIPrimer(5uM each) | 2 |
| IGT EM808Polymerase Mixture | 10 |
TABLE 7
2.6 round 2 PCR reactions followed by purification.
Mu.l of PCR product was taken and 27. Mu.l of Nanjinozan DNA clear beads equilibrated to room temperature was added, mixed well with shaking and incubated at room temperature for 5min to bind the DNA to the magnetic beads.
Centrifuging instantly, placing the centrifuge tube on a magnetic rack for 3min until the centrifuge tube is clear, and discarding the supernatant;
adding 50 mu L of YF buffer B into the centrifuge tube, fully oscillating and uniformly mixing, incubating for 5min at room temperature, performing instantaneous centrifugation, placing the centrifuge tube on a magnetic rack for 3min until the centrifuge tube is clear, and discarding the supernatant.
200 mu L of 80% ethanol is added into the centrifuge tube, the magnetic beads are ensured to be completely immersed into the 80% ethanol, the centrifuge tube is kept stand for 1min, and the supernatant is discarded.
200 mu L of 80% ethanol is added into the centrifuge tube, the magnetic beads are ensured to be completely immersed into the 80% ethanol, the centrifuge tube is kept stand for 1min, and the supernatant is discarded.
After sufficient centrifugation, the supernatant was thoroughly removed with a 10. Mu.L pipette and left to stand for 3min to allow the residual ethanol to evaporate thoroughly.
Adding 24 mu l Nuclease free water, shaking, mixing, and standing for 2min.
The centrifuge tube was placed on a magnetic rack for 3min to clarify, 20. Mu.L of supernatant was pipetted into a new 200. Mu.L PCR tube, where the prepared multiplex PCR library was prepared.
2.7 library quality inspection machine
By usingdsDNAHS Assay Kit library concentrations were determined. The specific operation is as follows:
preparation of a standard: qubit dsDNAHS Master Mix was dispensed into 2 centrifuge tubes of standard at 190. Mu.L, 10. Mu.L of standard Qubit dsDNAHS Standard #1 and Qubit dsDNA HS Standard #2 were added, respectively, and vortexed for further use, taking care that no air bubbles were present.
1 mu L of a sample to be measured is taken, qubit dsDNAHS Master Mix mu L of the sample is uniformly mixed by vortex, and no bubbles are generated.
The prepared standard and sample were left to react at room temperature for 3min, and library concentration was determined using a Qubit 3.0 fluorometer.
The library fragment size was examined using HT DNAHigh Sensitivity Reagent Kit and the fragment size should be centered at 300-500bp as shown in FIG. 3.
2.8 on-machine sequencing
Using GenoLab M sequencing kit V3.0 (FCM-D SE 075-D), on-machine sequencing was performed according to library on-machine instructions.
And (5) placing the library neutralization solution and the hybridization solution in an ice box for pre-cooling for standby.
As a denatured liquid, 0.2M NaOH was prepared.
Library stock was diluted to 4nM with pre-chilled library dilutions, and then library denaturation was performed as described in Table 8 below to prepare a 20pM library.
TABLE 8
The sample was gently vortexed and mixed, and after rapid centrifugation, placed on ice for further use.
A clean 2mL centrifuge tube equipped in the kit is used as a container for loading the library.
The machine is started according to 2.5pM (the machine quality can be adjusted automatically according to the actual sequencing data), 187.5 mu L of 20pM library and 1312.5 mu L of hybridization solution are taken and mixed uniformly, vibrated, mixed uniformly and centrifuged, and placed on ice for standby.
High throughput sequencing was run on a Santhreeq 1000 platform and the library was placed in the library well site of the sequencing kit in the on-machine mode of SE75 +8. The machine was started up at 2M/sample.
2.9 analysis of results
Sequencing results show that the primer set can specifically amplify target sequences and realize pathogen detection under 2M data volume. The data volume required by the experimental method is far smaller than the data volume (20M) required by metagenomic sequencing, so that the accurate and reliable result is ensured, the method is more economical and efficient, and the workload is greatly reduced.
Example 3 detection results of test samples of the inventive composition
gDNA pooled samples of 10 pathogens were tested as in example 2 using the compositions shown in Table 1. The detection results are shown in Table 9 below. Comparing the off-line data reads with pathogen reference genome by mem algorithm of BWA software, and taking the optimally compared reads with score more than 30 as the reads detected by the method. From the results, it can be seen that the composition of the present invention is capable of amplifying and enriching all targets within a tube, is detected in subsequent sequencing assays, and requires a greatly reduced amount of data.
TABLE 9
Claims (10)
1. A composition for detecting pathogens based on high throughput amplicon sequencing, comprising at least 4 targets in the primer set as follows:
primer groups for amplifying the mycobacterium tuberculosis complex as shown in SEQ ID NO. 1-6;
primer groups for amplifying the gordonia mycobacteria shown as SEQ ID NO. 7-12;
primer groups for amplifying Chlamydia psittaci as shown in SEQ ID NO. 13-20;
primer groups for amplifying mycoplasma pneumoniae shown in SEQ ID NO. 21-28;
primer sets for amplifying Orientia tsutsugamushi as shown in SEQ ID NOS.29 to 32;
primer groups for amplifying the typhoid rickettsia shown as SEQ ID NO. 33-36;
primer groups for amplifying plasmodium malariae as shown in SEQ ID NO. 37-40;
primer groups for amplifying plasmodium vivax as shown in SEQ ID No. 41-44;
primer group for amplifying amoeba histolytica as shown in SEQ ID NO. 45-48; or alternatively
Primer sets for amplifying mycobacterium abscessus shown as SEQ ID NO. 49-62.
2. The composition of claim 1, comprising at least 8 targets in the primer set as set forth above.
3. A composition according to claim 2, comprising:
primer groups for amplifying the mycobacterium tuberculosis complex as shown in SEQ ID NO. 1-6;
primer groups for amplifying the gordonia mycobacteria shown as SEQ ID NO. 7-12;
primer groups for amplifying Chlamydia psittaci as shown in SEQ ID NO. 13-20;
primer groups for amplifying mycoplasma pneumoniae shown in SEQ ID NO. 21-28;
primer sets for amplifying Orientia tsutsugamushi as shown in SEQ ID NOS.29 to 32;
primer groups for amplifying the typhoid rickettsia shown as SEQ ID NO. 33-36;
primer groups for amplifying plasmodium malariae as shown in SEQ ID NO. 37-40;
primer groups for amplifying plasmodium vivax as shown in SEQ ID No. 41-44;
primer group for amplifying amoeba histolytica as shown in SEQ ID NO. 45-48; and
primer sets for amplifying mycobacterium abscessus shown as SEQ ID NO. 49-62.
4. A composition according to any one of claims 1 to 3, wherein the primer sets each bear a linker sequence.
5. The composition of claim 4, wherein the upstream primer adapter sequence is set forth in SEQ ID NO. 63: ACACGACGCTCTTCCGATCT, the sequence of the downstream primer linker is shown in SEQ ID NO. 64: CTTGGCACCCGAGAATTCCA.
6. The composition of claim 5, wherein the components of the composition are present in a mixed form.
7. Use of a composition according to any one of claims 1 to 6 for the preparation of a kit for high throughput amplicon sequencing detection of a pathogen.
8. A kit for high throughput amplicon sequencing detection of a pathogen comprising the composition of any one of claims 1-6.
9. The kit of claim 8, further comprising at least one of: reagents required for nucleic acid extraction, reagents required for nucleic acid amplification, and reagents required for sequencing.
10. Use of a composition for preparing a high throughput amplicon sequencing assay for detecting a pathogen, wherein the assay comprises:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Amplifying using the composition of any one of claims 1 to 6 to obtain an amplified product;
3) Processing the amplified products and establishing a library; and
4) Sequencing and analyzing the result.
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