CN115927683A - High-throughput detection primer and detection method for soil-borne pathogenic bacteria - Google Patents
High-throughput detection primer and detection method for soil-borne pathogenic bacteria Download PDFInfo
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- CN115927683A CN115927683A CN202211301159.8A CN202211301159A CN115927683A CN 115927683 A CN115927683 A CN 115927683A CN 202211301159 A CN202211301159 A CN 202211301159A CN 115927683 A CN115927683 A CN 115927683A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention belongs to the technical field of biology, and relates to a high-flux detection primer and a detection method for soil-borne pathogenic bacteria. The invention realizes the high-flux synchronous detection of various soil-borne pathogens; through designing, screening and optimizing the composition and layout of the primers, different pathogenic bacteria genes can be detected under the same amplification condition, and the detection efficiency is improved; the invention can detect the target gene as low as 0.001 percent, and has high detection precision; moreover, the primer sequences of common and seriously harmful soil-borne pathogens are integrated, the advantages of PCR detection are taken into consideration, and various soil-borne pathogens can be rapidly and accurately identified and identified.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a high-flux detection primer and a detection method for soil-borne pathogenic bacteria.
Background
Soil-borne pathogens are a class of microorganisms that compromise crop yield and quality, and are characterized by a wide variety of species that can colonize the soil for long periods of time and cause losses by infecting crops. And the detection of soil-borne pathogenic bacteria at a molecular level can monitor the health level of soil microorganisms in time so as to achieve the aims of rapidly identifying various soil-borne plant diseases and preventing the soil-borne plant diseases.
At present, few gene chip development designs related to soil-borne pathogenic bacteria microorganisms are available, on one hand, the existing gene chip development designs are derived from the existing identification of soil-borne diseases based on crop morphological characterization, and on the other hand, the PCR reaction primers for researching the soil-borne diseases are different in design, different in reaction conditions and difficult to integrate simply, so that the difficulty of chip design is increased. Most of the existing PCR methods for detecting soil-borne pathogenic bacteria by molecular science are characterized in that a database is compared after amplification and sequencing of a single specific primer, and the step of amplification of the single specific primer is long in time consumption in analyzing and treating various soil-borne diseases. The gene chip technology is a molecular level detection by fixing a specific nucleotide sequence on a substrate and using a nucleotide primer combination mode, and has the characteristic of detecting a plurality of gene sequences at one time under a specific program. As more and more soil-borne pathogenic microorganisms need to be studied, a method for simultaneously detecting functional genes of various microorganisms is urgently needed to improve efficiency.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a high-throughput detection molecular marker capable of quickly and efficiently detecting soil-borne pathogenic bacteria in soil.
The purpose of the invention can be realized by the following technical scheme: a high-throughput detection molecular marker for soil-borne pathogenic bacteria comprises upstream and downstream primer sequences shown in SEQ ID NO. 1-86.
In the above-mentioned molecular marker for high-throughput detection of soil-borne pathogenic bacteria, the soil-borne pathogenic bacteria include Verticillium sp, verticillium dahliae, verticillium nigrorum, fusarium oxysporum, cladosporum, sporotrichum clavatum, cladosporum oxysporum, cladosporum rubrum, fusarium supernatum, fusarium equiseti, botrytis cinerea, rhizoctonia solani, ralstonia solani, phyllospora tabaci, alternaria clava, rhizoctonia solani, pseudomona syringae, microsporum pseudomonicola, pseudomona syringae, pseudomona fragrans, acrophyta avenae, alternaria collospora, pseudolycopersicum, pseudobulbus solani, pseudocercosmopsis scabies, podosporum lycopersicum, pseudoceros syringae lacrima, pseudoperonospora solani, pseudosclerotium olea, pseudoperonospora solani, pythium species, pseudoperonospora solani, pseudomyces sphaeroides, at least one species of Pseudomyceliophthora, pseudomyceliophthora fornica solani, pseudomyces verticillioti fornica, pythium fornica, pseudoperonosum fornica, pythrina, pseudoperonospora fornica, pseudoperonosum fornica, and at least one species of Cladosporum.
A high-throughput detection chip for soil-borne pathogenic bacteria comprises the molecular marker.
A method for detecting a high-throughput detection chip for soil-borne pathogenic bacteria, which is characterized by using the molecular marker or the detection chip.
In the detection method of the chip for high-throughput detection of soil-borne pathogenic bacteria, the method comprises the following steps: extracting DNA from soil, distributing the DNA and primers, performing high-throughput quantitative PCR amplification, and analyzing results and data.
In the detection method of the high-throughput detection chip for the soil-borne pathogenic bacteria, the reaction system of the high-throughput quantitative PCR comprises a reaction system 1 and a reaction system 2; wherein the reaction system 1 comprises 600-700 mul LightCycler 480DNA SYBR GreenI Mix and 350-450 mul high-purity water; the reaction system 2 comprises 55-650 mu of LLightCycler 480DNA SYBR Green I Mix, 100-150 mu of bovine serum albumin aqueous solution and 200-300 mu of high-purity water.
In the detection method of the high-throughput detection chip for the soil-borne pathogenic bacteria, the high-throughput quantitative PCR amplification procedure is as follows: pre-denaturation at 90-100 deg.C for 3-8min,40 cycles of denaturation (90-100 deg.C.times.30 s), annealing (55-60 deg.C.times.30 s) and extension (70-75 deg.C. Times.1 min), and finally heating to 95-100 deg.C from 70-75 deg.C.
Compared with the prior art, the invention has the following beneficial effects: the invention realizes the high-flux synchronous detection of various soil-borne pathogens; through designing, screening and optimizing the composition and layout of the primers, different pathogenic bacteria genes can be detected under the same amplification condition, and the detection efficiency is improved; the invention can detect the target gene as low as 0.001 percent, and has high detection precision; moreover, the primer sequences of common and seriously harmful soil-borne pathogens are integrated, the advantages of PCR detection are taken into consideration, and various soil-borne pathogens can be rapidly and accurately identified and identified.
Detailed Description
The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.
Example 1:
s1, use
A soil DNA extraction kit (MP biomedicine, USA) is used for extracting continuous cropping obstacle soil sample DNA, each sample needs 0.5-1.0g of soil, and other types of environment samples are extracted by using corresponding extraction kits according to instructions. DNA quality was determined by measuring the OD260/280 value with a NanoDrop ND-2000 spectrophotometer (Seimer science, USA), with a value between 1.6 and 2.0 indicating DNA availability. DNA concentration use>
A double-stranded DNA kit (Promega, USA) needs at least 10 microliter of 30 ng/microliter DNA sample for one functional gene quantification.
Table 1: DNA quality and concentration detection table
S2, distributing the primers and the DNA samples on a SmartChip nano chip (Waterpen biosystem, USA) by adopting a multi-sample nano distributor (Waterpen biosystem, USA), wherein the chip has 72 x 72 nano holes in total and can support 5184 fluorescent quantitative PCR reactions. The method adopts a scheme of 54 primers multiplied by 96 samples to carry out fluorescent quantitative PCR detection. The materials to be prepared were 20. Mu. Mol/L bovine serum albumin aqueous solution, 2 pieces
384 enzyme-free well plates (Saimer technology, USA),. Sup.>
LightCycler 480DNA SYBR Green I Mix (Roche pharmaceutical, USA), nucleic acid-depleted PCR-grade ultrapure water (Roche pharmaceutical, USA), and->
Chip kit.
Before the primer allocation, PCR reaction solution I (mixed with 699. Mu.L of LightCycler 480DNA SYBR Green I Mix and 419. Mu.L of high purity water) was prepared in advance, and 12.1. Mu.L of PCR reaction solution I and 3.0. Mu.L of primer mixture were added to the corresponding positions of the first 4 rows in the 384-well plate, respectively. Similarly, before the distribution of the DNA samples, PCR reaction solution II was prepared in advance (610. Mu.L of LightCycler 480DNA SYBR Green I Mix, 122. Mu.L of bovine serum albumin aqueous solution and 244. Mu.L of high purity water were mixed), 12.9. Mu.L of PCR reaction solution II and 3.2. Mu.L of DNA samples were added to the corresponding positions in the first 6 columns of the 384-well plate, respectively, and 96 DNA samples were supported in one chip reaction at most.
At the start of the allocation, the mRNA expression analysis mode was selected, 54 primers and 96 sample allocation protocols under MyDesign were selected, and sample allocation was performed. The distribution process is an automated operation process. After the primers and the sample are distributed, the chip is required to be centrifuged, the first centrifugation is 3300 r/min × 5min, and the second centrifugation is 3300 r/min × 15min. And (5) after the centrifugal chip is pasted with a film, waiting for PCR detection.
Table 1: primer sequences for identifying genera
TABLE 2 primer and control partitioning protocol in 384-well plates
Table 3: protocol for distribution of DNA samples in 384-well plates
Table 4: protocol for distribution of DNA samples and controls in 384-well plates
S3, carrying out high-throughput quantitative detection on the chip by using a SmartChip real-time PCR system (WaferGen biological system, USA), wherein the PCR program for detection is pre-denaturation at 95 ℃ for 5min, denaturation at 40 cycles (95 ℃ multiplied by 30S), annealing (57 ℃ multiplied by 30S) and extension (72 ℃ multiplied by 1 min), finally, the temperature is increased from 72 ℃ to 97 ℃, the temperature increase rate is 4 ℃/S, and the dissolution curve of the primer is measured at one measuring point per 0.4 ℃. After the PCR procedure is completed, the software will automatically analyze and export the data.
S4, after the detection is finished, the exported data is screened according to the following conditions: 1. the amplification efficiency is between 90% and 110%; 2. the Cycle Threshold (CT) must be less than 31; 3. three replicates were counted and the amplification was deemed to be efficient. The data after screening calculated the Relative Abundance (RA).
Table 5: relative abundance (%) and average coefficient of variation SV (%) of pathogenic genes
From the results, the chip method can simultaneously detect the gene abundance of 41 pathogenic bacteria functional genes, can detect the relative abundance of the functional genes with the level of 0.001 percent, has high detection precision, improves the detection efficiency and reduces the detection cost. From the average coefficient of variation, the variation range is between 4.01 and 36.26 percent, and the overall accuracy is high.
The technical range of the embodiment of the invention is not exhaustive, and new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiment are also within the scope of the invention; in all the embodiments of the present invention, which are listed or not listed, each parameter in the same embodiment only represents an example (i.e., a feasible embodiment) of the technical solution, and there is no strict matching and limiting relationship between the parameters, wherein the parameters may be replaced with each other without departing from the axiom and the requirements of the present invention, unless otherwise specified.
The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and the technical means also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments, or alternatives may be employed, by those skilled in the art, without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (7)
1. The high-throughput detection molecular marker for the soil-borne pathogenic bacteria is characterized by comprising upstream and downstream primer sequences shown in SEQ ID NO. 1-86.
2. <xnotran> 1 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . </xnotran>
3. A chip for high-throughput detection of soil-borne pathogenic bacteria, comprising the molecular marker of claim 1.
4. A method for detecting a high-throughput detection chip for a soil-borne pathogen, which comprises using the molecular marker of claim 1 or the detection chip of claim 3.
5. The method for detecting the high-throughput detection chip of the soil-borne pathogenic bacteria according to claim 4, wherein the method comprises the following steps: extracting DNA from continuous cropping obstacle soil, then distributing the DNA and primers, carrying out high-throughput quantitative PCR amplification, and finally carrying out result and data analysis.
6. The detection method of the high-throughput detection chip for the soil-borne pathogenic bacteria according to claim 5, wherein the reaction system of the high-throughput quantitative PCR comprises a reaction system 1 and a reaction system 2; wherein the reaction system 1 comprises 600-700 mul LightCycler 480DNA SYBR GreenI Mix and 350-450 mul high-purity water; the reaction system 2 comprises 55-650 mu L of LightCycler 480DNA SYBR Green I Mix, 100-150 mu L of bovine serum albumin aqueous solution and 200-300 mu L of high-purity water.
7. The detection method of the high-throughput detection chip for the soil-borne pathogenic bacteria according to claim 5, wherein the high-throughput quantitative PCR amplification procedure comprises: pre-denaturation at 90-100 deg.C for 3-8min,40 cycles of denaturation (90-100 deg.C.times.30 s), annealing (55-60 deg.C.times.30 s) and extension (70-75 deg.C. Times.1 min), and finally heating to 95-100 deg.C from 70-75 deg.C.
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