CN113293238A - Soil virus detection technology based on macroviromics - Google Patents
Soil virus detection technology based on macroviromics Download PDFInfo
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Abstract
The invention provides a soil virus detection technology based on macroviromics, which uses a specific label primer composition for amplification; the label primer comprises a primer part and a label part; the tag moiety is selected from SEQ ID NO. 1-12; the primer part is selected from SEQ ID NO. 13-14. The label primer composition has the advantages of high flux, high speed and long reading length. The pretreatment technology establishes a precise, specific and efficient virus genome extraction method, and has little pollution.
Description
Technical Field
The invention belongs to the field of virus detection methods, and particularly relates to a soil virus detection technology based on macroviromics.
Background
Viruses play an important role in ecosystems as an important component of food networks. Viruses can affect mortality, community structure, evolution, and nutrient cycling of the host. However, due to the limitations of the technical methods, the community composition and functional characteristics of soil viruses are still poorly understood. Currently, most of the soil viruses are detected by adopting a traditional culture medium culture method, and the method is complex in operation, long in time consumption and serious in pollution, so that potential viruses or new viruses are difficult to efficiently and comprehensively find.
The high-throughput sequencing technology can simultaneously carry out sequence determination on DNA molecules, the macroviromics detection method gets rid of the limitation of the prior microorganism separation and pure culture, and the environmental sample is taken as a research object to directly enrich and extract virus genomes from the soil sample and then carry out sequencing and bioinformatics analysis. However, the prior method still has the following problems: (1) because the virus has higher gene mutation, the abundance of the virus in the environment can be underestimated by referring to the primer and probe sequences which are researched; (2) the existing pretreatment technology is insufficient in virus genome extraction, so that a virus genome extraction pretreatment technology is designed; (3) at the present stage, the extraction of soil viruses, the analysis of macro virus groups and the like lack unified technical specifications.
Chinese patent 201210071924.1 discloses a method for detecting H9 subtype avian influenza virus molecules in soil, which comprises the following steps: A. designing a primer pair according to the reported HA gene sequence of the H9 subtype avian influenza virus with the accession number of JN 381629; B. treating a soil sample to be detected: mixing the soil collected at different positions of the same place, and sieving the soil by a 20-mesh analysis sieve to remove stones and weeds in the soil; C. extracting RNA in a soil sample to be detected, and extracting total RNA in soil by using a soil RNA extraction kit; D. reverse transcription of virus RNA, establishing a virus RNA reverse transcription system; E. amplifying a target gene by nested PCR: 1) establishing a nested PCR amplification reaction system; 2) establishing the amplification reaction temperature of the nested PCR outer primer; 3) establishing the amplification reaction temperature of the nested PCR inner side primer: 4) and analyzing the amplification product. The method can quickly and accurately detect the H9 subtype avian influenza virus in the detected soil sample, and is simultaneously suitable for detecting the infection of the H9 subtype avian influenza virus of animals and monitoring the H9 subtype avian influenza virus in the environment. However, this method can only detect a single virus, and the nested PCR amplification conditions are complicated.
Chinese patent 201911112786.5 discloses a method for simultaneously detecting A, B, C three groups of rotaviruses, which designs a pair of specific primers and a specific probe with different fluorescent labels aiming at target genes of A group of rotaviruses, B group of rotaviruses and C group of rotaviruses, simultaneously designs a pair of universal primers, and firstly adopts the specific primers with the universal primer labels to carry out enrichment amplification during PCR amplification; then, the universal primer label is adopted for exponential amplification, and the fluorescence signal of the exponential amplification product is collected for detection. The method can be used for simultaneously detecting the group A rotavirus, the group B rotavirus and the group C rotavirus, has strong specificity, high sensitivity and short detection time, and can be used for the early rapid diagnosis of rotavirus infection and the epidemiological research of rotavirus. However, this method involves a secondary amplification and is complicated in steps.
Disclosure of Invention
In order to solve the problems, the invention provides a soil virus detection technology based on macroviromics, and the prior art is optimized by schemes such as optimizing primer sequences and the like. The macro viromics technology can directly obtain viral genomes from soil environment samples, and finally can perform function annotation on viral genes through high-throughput sequencing, splicing assembly and ORF prediction, so that the understanding on the soil viral functions is greatly enriched.
In one aspect, the invention provides a tagged primer composition.
The tag primer composition is used for a soil virus detection technology based on macroviromics.
The label primer comprises a primer part and a label part.
The tag moiety is selected from SEQ ID NO. 1-12; the primer part is selected from SEQ ID NO. 13-14.
The label comprises a forward primer label and is selected from one or more of SEQ ID NO. 1-6.
The label also comprises a reverse primer label, and is selected from one or more of SEQ ID NO. 7-12.
The primer comprises a forward primer and a reverse primer.
The forward primer is SEQ ID NO. 13.
The reverse primer is SEQ ID NO. 14.
In another aspect, the invention provides a soil virus detection technology based on macroviromics.
The soil virus detection technology comprises the step of amplifying the DNA of a sample by using the label primer composition.
The soil virus detection technology comprises the following steps of pretreating a sample: and (3) sieving the soil sample, uniformly mixing the sieved soil sample with potassium citrate in proportion, oscillating and centrifuging.
The concentration of the potassium citrate is 1% (w/v).
The ratio of the soil sample to the potassium citrate is 1 kg/6.6L.
The soil virus detection technology comprises the following steps:
(1) pretreating a soil sample;
(2) filtering by a tangential flow filtration system;
(3) concentrating by ultrafiltration column, and filtering with sterile filter;
(4) removing free extracellular DNA;
(5) extracting virus DNA;
(6) performing amplification using the aforementioned tag primer composition;
(7) carrying out electrophoresis detection;
(8) and (6) performing detection on the machine.
Preferably, the filtration conditions in step (2) are continuous filtration, sequentially passing through filter columns of 1mm, 0.45 μm and 0.22 μm.
Preferably, the ultrafiltration column in the step (3) is a 30kDa ultrafiltration column.
Preferably, the sterile filter in step (3) has a size of 0.22 μm.
Preferably, the virus concentrated solution DNase I is used for removing free extracellular DNA in the step (4).
Preferably, the step (5) uses a virus DNA extraction kit to extract virus DNA.
In a further aspect, the invention provides the use of the aforementioned tagged primer composition in the preparation of a reagent and/or kit for the detection of a virus in a soil sample.
In a further aspect, the present invention provides the use of the soil virus detection technique described above in the preparation of a reagent and/or kit for the detection of a virus in a soil sample.
In yet another aspect, the present invention provides a kit for detecting a virus in soil.
The kit comprises the label primer composition.
The kit is based on a metagenome method.
The kit also comprises the reagents used in the soil virus detection technology, including but not limited to: 1% (w/v) potassium citrate solution, DNase I, virus DNA extraction reagent and 2 XTaq Plus PCR Master Mix.
The kit also comprises a buffer solution, wherein the buffer solution is a virus DNA extraction buffer solution.
The invention has the beneficial effects that:
1. the primer sequence has the advantages of high flux, high speed and long read length.
2. The pretreatment technology of the patent establishes an accurate, specific and efficient virus genome extraction method, and is less in pollution.
Drawings
FIG. 1 shows the result of PCR gel electrophoresis detection of a sample to be detected.
FIG. 2 shows Solexa sequencing results.
FIG. 3 shows Solexa sequencing results.
It should be noted that fig. 2 and 3 are diagrams illustrating the apparatus, such as making a determination that a modification may affect the result, and thus retaining the gray-scale image of the original image.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 soil virus detection technology based on macroviromics
The method comprises the following steps:
(1) after the fresh soil sample is sieved by a 2mm sieve, 500g of the fresh soil sample is weighed, and is uniformly mixed with 3.3L of 1% (w/v) potassium citrate solution, and the mixture is shaken at 2000rpm for 16 min. The mixture was centrifuged at 5 ℃ and 1600rpm for 8min at low speed.
(2) The supernatant was continuously filtered using a tangential flow filtration system, sequentially through 1mm, 0.45 μm and 0.22 μm filter columns, and the permeate was collected.
(3) The permeate was then concentrated by passage through a 30kDa ultrafiltration column and the retentate was collected. Filtering the concentrated solution with 0.22 μm sterile filter, and repeating for 3 times to remove impurity bacteria contamination during continuous filtration process.
(4) The virus concentrate was then treated with DNase I to remove free extracellular DNA from the soil.
(5) The virus DNA in the concentrated solution was extracted using a virus DNA extraction kit (available from Shanghai towering Biotech Co., Ltd., product number Hhzt-4616).
(6) The sample genome DNA is used as a template, and the primers in the table 1 are used for PCR amplification, wherein the PCR amplification system is shown in table 2, and the PCR amplification conditions are shown in table 3.
TABLE 1 primer and tag sequences
TABLE 2 PCR reaction System
| Composition (I) | Content (μ L) |
| Virus DNA (10 ng/. mu.L) | 2 |
| Forward primer (10. mu.M) | 1 |
| Reverse primer (10. mu.M) | 1 |
| dd water | 8.5 |
| 2×Taq Plus PCR Master Mix | 12.5 |
| Total of | 25 |
TABLE 3 PCR reaction conditions
(7) And detecting the PCR result by 1% agarose gel electrophoresis.
The soil samples in this example were taken from a tobacco bench, and all soil samples were taken from a grape planting field;
viral genome extraction and PCR amplification are carried out on 10 kinds of complete soil, the design combination of primers is shown in the table, and the experimental process is the same as that described above. The detection result is shown in figure 1, wherein a lane 1 is a marker, the DNA size from left to right is about 650bp, and the electrophoresis result shows that the amplification product obtained by adopting the primer combination provided by the invention has clear bands, obvious length difference and no dispersion phenomenon.
(8) And (5) purifying a PCR product.
Make up the PCR library amplification product to 40. mu.L (original 25. mu.L) with ribozyme-free water, add 30. mu.L (0.9X) magnetic beads for purification, rinse the magnetic beads twice with 80% ethanol solution, finally elute with 15. mu.L of ribozyme-free water, carefully pipette 15. mu.L of supernatant into a new 1.5mL centrifuge tube.
(9) And (5) detecting the concentration of the library.
The concentration of the library is accurately quantified by using the Qubit 3.0, and the sequencing requirement is met when the concentration is more than 5 ng/mu L. The library was individually subjected to fragment distribution assay using the fully automated nucleic acid analysis system Qsep 100.
6. Qualified library samples in quality and concentration were sent to companies for Solexa sequencing and the results were compared. The sequencing results are shown in FIGS. 2 and 3.
The average basic sequencing amount of the sample is more than 3 ten thousand reads, most of the length is 250bp, the quality of the basic group is more than 35, and the sequencing quality of the sample is qualified.
The sequencing alignment results were as follows:
| viral species | Content (wt.) |
| Family of |
40 |
| Circovirus family | |
| 3％ | |
| |
2 |
| Geminivirus family | |
| 2％ | |
| Brachyury phage family | 0.5％ |
| Myoviridae family | 0.7％ |
| Family of DNA viruses | 0.2％ |
Sequence listing
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Claims (10)
1. A soil virus detection technology based on macroviromics is characterized in that specific label primer compositions are used for amplification; the label primer comprises a primer part and a label part; the tag moiety is selected from SEQ ID NO. 1-12; the primer part is selected from SEQ ID NO. 13-14.
2. The soil virus detection technique of claim 1, wherein the soil virus detection technique comprises the steps of:
(1) pretreating a soil sample;
(2) filtering by a tangential flow filtration system;
(3) concentrating by ultrafiltration column, and filtering with sterile filter;
(4) removing free extracellular DNA;
(5) extracting virus DNA;
(6) performing amplification using the tagged primer composition of claim 1;
(7) carrying out electrophoresis detection;
(8) and (6) performing detection on the machine.
3. The soil virus detection technique of claim 1, wherein the primer portion is a forward primer having the sequence of SEQ ID No.13, and the tag portion is selected from the group consisting of SEQ ID nos. 1-6; the primer part is a reverse primer, the sequence is SEQ ID NO.14, and the label part is selected from SEQ ID NO. 7-12.
4. The soil virus detection technique of claim 2, wherein the pretreatment method of the soil sample comprises: and (3) sieving the soil sample, uniformly mixing the sieved soil sample with potassium citrate in proportion, oscillating and centrifuging.
5. The soil virus detection technique of claim 2, wherein the filtration conditions in step (2) are continuous filtration, sequentially passing through a 1mm, 0.45 μm and 0.22 μm filter column; the ultrafiltration column in the step (3) is a 30kDa ultrafiltration column, and the specification of a sterile filter is 0.22 mu m.
6. The soil virus detection technique of claim 4, wherein the concentration of potassium citrate is 1% by weight/volume; the ratio of the soil sample to the potassium citrate is 1 kg/6.6L.
7. A tagged primer composition, wherein said tagged primer comprises a primer portion and a tag portion; the tag moiety is selected from SEQ ID NO. 1-12; the primer part is selected from SEQ ID NO. 1-2.
8. Use of the tagged primer composition of claim 7 in the preparation of a reagent and/or kit for the detection of a virus in soil.
9. Use of the soil virus detection technique of any one of claims 1 to 6 in the manufacture of a reagent and/or kit for the detection of viruses in soil.
10. A soil virus detection kit based on macroviromics, which is characterized in that the soil virus detection kit comprises the labeled primer composition of claim 7.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114300046A (en) * | 2021-12-28 | 2022-04-08 | 武汉百奥维凡生物科技有限公司 | Identification method of new macrovirome viruses |
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| CN106191045A (en) * | 2016-08-08 | 2016-12-07 | 中国科学院北京基因组研究所 | Index and primer for multiple nucleic acid order-checking |
| CN108342451A (en) * | 2018-01-18 | 2018-07-31 | 中国科学院东北地理与农业生态研究所 | To eliminate method of host's DNA pollution for the macro genome parsing of water body environment virus |
| CN112176047A (en) * | 2020-10-30 | 2021-01-05 | 广西壮族自治区农业科学院 | Method for analyzing microbial diversity index in selenium-rich soil |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114300046A (en) * | 2021-12-28 | 2022-04-08 | 武汉百奥维凡生物科技有限公司 | Identification method of new macrovirome viruses |
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