CN108774644A - It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method - Google Patents
It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method Download PDFInfo
- Publication number
- CN108774644A CN108774644A CN201810681313.6A CN201810681313A CN108774644A CN 108774644 A CN108774644 A CN 108774644A CN 201810681313 A CN201810681313 A CN 201810681313A CN 108774644 A CN108774644 A CN 108774644A
- Authority
- CN
- China
- Prior art keywords
- bacterium colony
- granulated sugar
- yellow twig
- 16srdna
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention provides the bacterium colony detection methods in a kind of granulated sugar tangerine of infection yellow twig, are related to field of bioanalysis.It includes：Using the first primer to the molecular detection primer as Citrus shatangju yellow twig pathogen, PCR amplification is carried out to sample to be tested genome, determines illness plant DNA sample；Build the libraries 16SrDNA of illness plant DNA sample；Illness plant DNA sample is detected using high throughput sequencing technologies and the bacterium colony composition in illness plant DNA sample is analyzed.This detection method can analyze the granulated sugar tangerine for infecting yellow twig under the premise of not being separately cultured plant bacterium colony, and the accuracy of method is high, be easy to implement.
Description
Technical field
The present invention relates to field of bioanalysis, are examined in particular to the bacterium colony in a kind of granulated sugar tangerine of infection yellow twig
Survey method.
Background technology
Citrus shatangju is the important citrus variety in Guangdong, due to the continuous sprawling diffusion of citrus yellow shoot disease in recent years, yellow twig
The entire Citrus shatangju Industrial Security in Guangdong Province has been seriously threatened, and has been counted according to government data, more serious morbidity is Germany and Britain of Qingyuan City
It is regional with Yunfu etc..Morbidity Citrus shatangju belongs to typical Huanglong's disease symptoms, and blade is " mottled type yellow symptom ", non-uniform yellowish green
It is alternate；Fruit is " coppernose fruit ", fruit carpopodium part salmon pink, rest part cyan.
Citrus yellow shoot disease (Citrus Huanglongbing) is to restrict the destructive disease of global citrus industry development.
Research thinks that citrus yellow shoot disease is divided into two kinds, Asia bast bacillus (Candidatus Liberibacter
Asiatricus), African bast bacillus (Ca.L. africanus).Due to bast bacillus can not squamous subculture, hinder
Its pathogenesis and prevention and control research work progress, currently also without the definite high efficiency method of prevention citrus yellow shoot disease.
In plant and plant bacterial community belongs to dynamic generating process, and a kind of morbidity pathogen is usually associated with
Different degrees of microbial state treatment or flora is unbalance.But because there is the problem that can not be separately cultured in most phytobiocoenoses,
It knows little so infecting the composition of the bacterial community after yellow twig about citrus at present, is unfavorable for effectively preventing citrus Huanglong
Disease.
Invention content
The purpose of the present invention is to provide the bacterium colony detection methods in a kind of granulated sugar tangerine of infection yellow twig, pass through this inspection
Survey method, can under the premise of not being separately cultured plant bacterium colony to infect yellow twig granulated sugar tangerine analyze, method it is accurate
Degree is high, is easy to implement.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method comprising：
Using the first primer to the molecular detection primer as Citrus shatangju yellow twig pathogen, sample to be tested genome is carried out
PCR amplification determines illness plant DNA sample；
Build the libraries 16SrDNA of illness plant DNA sample；
Illness plant DNA sample is detected using high throughput sequencing technologies and to the bacterium in illness plant DNA sample
Composition is fallen to be analyzed.
Compared with prior art, beneficial effects of the present invention for example including：
This detection method provided by the invention, by will there is the first primer compared with high specific to draw to being used as yellow twig
Object filters out the illness plant DNA sample of infection yellow twig Citrus shatangju using the Molecular Detection method of Standard PCR；Pass through high pass again
Measure the bacterium colony composition in sequencing analysis illness plant DNA sample, you can obtain the bacteria flora composition in illness plant, in turn
Theoretical direction is provided for the yellow twig prevention of granulated sugar tangerine.In addition, this detection method, it can be before not being separately cultured plant bacterium colony
It puts and the granulated sugar tangerine for infecting yellow twig is analyzed, the accuracy of method is high, is easy to implement.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is 30 Citrus shatangju sample DNA electrophoresis results in granulated sugar tangerine orchard；
Fig. 2 is PCR product electrophoresis result of 3 pairs of primers in 10 samples；
Fig. 3 is the PCR product electrophoresis results of granulated sugar tangerine orchard normal growth sample and mottled type yellow symptom sample；
Fig. 4 is the 16SrDNA PCR product electrophoresis results of 30 samples.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Present embodiment provide it is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method comprising：
Step S1:Using the first primer to the molecular detection primer as Citrus shatangju yellow twig pathogen, to sample to be tested base
Because of a group progress PCR amplification, illness plant DNA sample is determined；
Further, the base sequence of the first primer pair such as SEQ ID NO:Shown in 1~2, wherein SEQ ID NO:1
Sequence is：GCGCGTATCCAATACGAGCGGCA； SEQ ID NO:2 sequence is：GCCTCGCGACTTCGCAACCCAT.
Step S2：Build the libraries 16SrDNA of illness plant DNA sample；
Further, the step of libraries 16SrDNA of structure illness plant DNA sample include：
Using the second primer pair as primer, 16SrDNA PCR amplifications are carried out to illness plant DNA sample.
Wherein, the gene of the second primer pair such as SEQ ID NO:Shown in 3~4：Sequence SEQ ID NO:3 are
AACMGGATTAGATACCCKG；Sequence SEQ ID NO:4 be AGGGTTGCGCTCGTTG.
The indices of the 16SrDNA after amplification are detected again, meet the selected libraries 16SrDNA of structure standard.
Wherein, the structure standard in the libraries 16SrDNA includes：
A. it is selected in the DNA sample master tape in the libraries 16SrDNA in 10kb or more；
B. OD (260/280) value for being selected in the DNA sample in the libraries 16SrDNA is 1.6~1.8, OD (260/230) value
More than 2.
Preferably, 20 μ L reaction systems of 16SrDNA PCR amplifications include：
0.8 μ L of forward primer (5 μM), 0.8 μ L, FastPfu polymerase of reverse primer (5 μM), 0.4 μ L, template DNA
2 μ L, 5 × FastPfu buffer solutions of 10ng, 2.5mM dNTPs, 4 μ L mend distilled water to 20 μ L.
More preferably, the response procedures of 16SrDNA PCR amplifications include：Annealing temperature is 54~56 DEG C, elongating temperature
It is 70~74 DEG C.
Step S3：Illness plant DNA sample is detected using high throughput sequencing technologies and to illness plant DNA sample
In bacterium colony composition analyzed.
Further, the initial data tested through high throughput sequencing technologies splits through data, the removing of primer sequence,
After splicing and the removing of chimera, the valid data for analyzing bacterium colony composition are obtained.
Preferably, base percentage of the base mass value in valid data more than 20bp and 30bp is all higher than 93%.
Preferably, splicing step includes：Read by base number less than 20bp is filtered processing；By pairs of reads
Splicing one sequence of assembling；And sample differentiation is carried out according to the barcode at sequence head and the tail both ends, it is carried out at the same time sequence direction
Adjustment.
More preferably, will splice parameter when assembled to reads is：The length in minimum overlay region is 10bp；Splicing
The maximum mispairing ratio that the overlapping region of sequence allows is less than 0.2.
The feature and performance of the present invention are described in further detail with reference to embodiments：
Embodiment
The present embodiment provides the bacterium colony detection methods in a kind of granulated sugar tangerine of infection yellow twig, and by illness plant
Bacteria flora forms the comparison between the bacteria flora of healthy plant composition, probes into out various bacteria floras and citrus yellow shoot disease
The correlation of pathogen.
This method specifically includes：
One, the extraction of Citrus shatangju sample gene group total DNA：
1.1 extraction step：
(1) clip 0.1g leaves middle arteries, shred and are placed in the centrifuge tube of 2.0mL, are smashed with homogeneous instrument；
(2) 200 μ L are added in 65 DEG C of heating water bath 25min into the 2.0mL centrifuge tubes equipped with 0.1g bast samples
Buffer PCB, be placed in biological sample homogeneous instrument (Bioprep-24) high speed concussion grinding 6 times, each 30s；
(3) the Buffer PCB and 12 μ L beta -mercaptoethanols of 400 μ L, 65 DEG C of preheatings are added.Mixing is shaken, is placed in 65 DEG C
Water-bath 25min in constant-temperature metal bath, it is primary per 5min mixings；
(4) 20 μ L RNase A (10mg/mL) are added, and room temperature digests 2min, to remove the RNA mixed；
(5) plus the chloroform of 612 μ L and shake mixing, 12,000rpm centrifugation 5min, it is careful draw supernatant water phase to one it is dry
Only (it is sure not to be drawn to the albumin layer of solid liquid interface) in the centrifuge tube of the 1.5mL to sterilize；
(6) isometric Buffer BD are added, overturn mixing 3~5 times, then add isometric absolute ethyl alcohol, after mixing well
Its whole is added in adsorption column with pipettor, is stored at room temperature 2 min, 10,000rpm centrifugation 1min are outwelled in collecting pipe and given up
Liquid；
(7) adsorption column is placed back in collecting pipe, 500 μ L PW Solution, 10,000rpm centrifugation 1min is added,
Outwell waste liquid in collecting pipe；
(8) adsorption column is put back in collecting pipe, 500 μ L Wash Solution, 10,000 rpm is added, centrifuge 1min,
Outwell waste liquid in collecting pipe；
(9) adsorption column is put back in collecting pipe, 12,000rpm blank pipes centrifuge 2min；
(10) adsorption column is taken out, will be put into a new 1.5mL centrifuge tube, 50 are added in adsorbed film center with liquid-transfering gun
μ L TE Buffer stand 3min, 12,000rpm 2 min of centrifugation；
(11) solution at this time in pipe is DNA solution, places it in -20 DEG C of preservations；
(12) take buffer points of 4 μ L DNA solutions and 1 μ 6 × loading of L dark even respectively, the agarose 1% is solidifying
Electrophoresis on glue observes extracted DNA mass in gel imaging system, prepares for follow-up test.
1.2 extraction results：
The extracting of citrus leaf middle arteries genome DNA, electrophoresis result are carried out by plant genome DNA extraction agent box
There is not apparent DNA degradation phenomenon, loading wells as shown in Figure 1, the total DNA of whole samples all obtains apparent band in display
There is no protein contamination totally, illustrates that Citrus shatangju genome DNA extraction is up-to-standard.
Two, the sensitivity analysis of the first primer pair
The selection of 2.1 primers：
3 pairs of primers of selection carry out yellow twig detection, select the highest primer of sensitivity, then carry out yellow twig to collecting sample
Detection, the sequence of this three pairs of primers are as shown in table 1：
1 Citrus shatangju yellow twig detection primer of table
The comparison procedure of 2.2 primer sensitivitys：
To infect the total DNA of citrus yellow shoot disease as positive control, all samples carry out the PCR reactions of 2 different extractings.
After the completion of amplified reaction, take 5 μ L pcr amplification products in 120V electrophoresis 30min on 1% Ago-Gel, finally in gel at
As observing result under system.Such as there is band at 535bp (P1/P2), 400bp (P3/P4), 1167bp (OI1/OI2c), then
Show that the sample is citrus yellow shoot disease positive.
The PCR reaction systems of primer P1/P2, P3/P4 and OI1/OI2c are：The total volume of PCR reactions is 25 μ L, wherein：
Each 1 μ L (10pM/L) of primer, 1 μ L of Citrus shatangju template DNA (~50ng) to be measured, 10 μ L of ddH2O are eventually adding 2 × Taq
PCR Master Mix (3×106U/L)12μL。
The PCR reaction conditions of primer P1/P2 and P3/P4 are：After 94 DEG C of 5min, 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C
1min (30 cycles)；72℃ 10min.The PCR reaction conditions of OI1/OI2c are：After 96 DEG C of 1min, 94 DEG C of 30s, 55 DEG C
30 s, 72 DEG C of 1min (35 cycles), 72 DEG C of 4min.
The sensitivity analysis result of 2.3 pairs of primers：
Choose in orchard is 10 Citrus shatangju samples that the yellow twig pathogen positive is diagnosed as with Huanglong's disease symptoms, PCR
DNA further compares 3 pairs of primer PCR results (as shown in Fig. 2, wherein a is primer P1/P2 using Standard PCR；B is primer P3/
P4；C is primer OI1/OI2c).Under same template, amplification condition, the PCR results of primer P1/P2 are shown without band；P3/
The amplified band of P4 primers is apparent but individual samples (sample 1 ', sample 5) are without apparent band.The amplification of OI1/OI2c primers
Band is apparent, and all samples are expanded.Therefore, subsequent research is all made of the primer (i.e. OI1/OI2c primers) and is used as sand
The molecular detection primer of sugared tangerine yellow twig pathogen.
Three, illness plant DNA sample and healthy plant DNA sample are screened：
Through the preliminary judging result of field diagnosis, sample 1-15 is normal growth blade in orchard, and sample 16-30 is mottled
Type yellow symptom.PCR amplification detection is carried out to above-mentioned all samples with primer OI1/OI2c.
The results are shown in Figure 3, and compared with field diagnoses, PCR detection accuracies greatly improve, and can not only be tested with trouble
The plant of disease symptoms also can detect that illness but do not show the plant of symptom have high sensitivity.
Four, sample 16SrDNA library constructions：
4.1. experimental method：
In order to ensure the accuracy of follow-up test result and the reliability of data analysis, need to expand using low circulation PCR
Increase and ensure that recurring number is unified, sequencing library is built by 16SrDNA PCR amplifications, whether testing goal stripe size is correct,
Can whether concentration be suitable, carry out subsequent experimental.Select 16SrDNA expand and sequencing region for：799F-1115R, design primer
For 799F/1115R (5'-AACMGGATTAGATACCCKG-3'/5'-AGGGTTGCGCTCGTTG-3').Tool is chosen in this experiment
Sample needed for representative and sequencing is tested, it is ensured that most of sample can amplify in minimum recurring number
The suitable DNA product of concentration.
PCR uses TransStart Fastpfu DNA Polymerase, 20 μ L reaction systems：5× FastPfu
Buffer 4μL,2.5mM dNTPs 2μL,Forward Primer(5μM)0.8 μL,Reverse Primer(5μM)0.8μ
0.4 μ L, Template DNA 10ng of L, FastPfu Polymerase mend ddH2O to 20 μ L.
PCR response procedures are：
a.1×(5min at 95℃)
b.27×(30sec at 95℃；30seconds at 55℃；45sec at 72℃)
C.5min 72 DEG C of at, 10 DEG C of until halted by user
Ensure that subsequent amplicon sequencing diversity analysis is smoothed out, the 16SrDNA indices of sample amplification must
It must be qualified.If master tape reaches 10kb or more, DNA is polluted without impurity such as protein, pigments, and total amount reaches certain requirement, then may be used
Carry out follow-up test；If not up to standard, need to carry out DNA purifying, master tape is made to reach 20kb or more as possible, ensures DNA without albumen
The pollution of the impurity such as matter, pigment, detection method are shown in Table 2.
2 sample detection methods of table
4.2. experimental result：
In order to ensure that the accuracy and reliability of experimental data makes it first all samples to be tested progress DNA purifying are waited for
Reach the requirement for carrying out the sequencing of 16S rDNA amplicons, wherein 16SrDNA library constructions standard is as follows：1) DNA sample master tape exists
10kb or more；2) DNA pollutes (OD (260/280)=1.6~1.8, OD (260/230) without impurity such as albumen, pigments>2 be conjunction
Lattice).
16SrDNA PCR amplification results show (Fig. 4), and all samples are preferable on the whole, and each sample has at 400bp
Apparent band.PCR amplification condition carries out the PCR product that amplification obtains each sample after optimized, and the result is shown in tables 3.3, find
Sample 1,4~7,11,17~21,23,25,29PCR product purpose band sizes it is correct, concentration is suitable, can carry out follow-up reality
It tests, although other sample concentrations are relatively low, PCR product purpose band size is correct, or subsequent experimental is prepared.
Five, high-flux sequence and data analysis
5.1. experimentation：
(1) sequencing is completed in Illumina HiSeq microarray datasets；
(2) the lower machine data that Illumina HiSeq microarray datasets obtain are initial data (Raw Data), are then carried out
Raw Data are pre-processed, and specific processing step is as follows：
A. data are split：Barcode sequences and PCR amplification primer sequence are removed by perl script；
B.PE Reads splicings：The lower read of quality is removed (read base numbers are less than 20bp) and is filtered place
Reason；According to region superposition (overlap) between PE reads, pairs of reads is spliced into one sequence of assembling, (assembly parameter：
Minimum overlap length is 10 bp；0.2) the maximum mispairing ratio for splicing the areas the overlap permission of sequence is；According to sequence head
The barcode at tail both ends carries out sample differentiation, is carried out at the same time the adjustment of sequence direction, (parameter
C.Tags goes chimera sequence：The Tags sequences obtained after handling above and database (Gold
database,http://drive5.com/uchime/uchime_download.html) detection chimera sequence is compared
Row, and chimera sequence is removed, obtain final valid data (Effective Tags).
(3) obtained valid data are analyzed using microbiological manipulations taxon (OTU), and to species population
Analysis.
5.2. analysis result：
(1) pretreatment of data：
It is handled by preliminary data using the lower machine data (initial data) that Illumina HiSeq microarray datasets are completed,
Include the fractionation of data, the removing of primer sequence, the splicing of PE Reads, Tags quality evaluations and screening and chimera
Removing, finally obtain valid data (Effective Tags).
This experiment obtains 1,157,151 high quality sequences in total, and the intermediate value reading of each sample is 38,870 (sequence models
It encloses：30,079-44,743) the average length intermediate value reading of the Effective Tags of each sample is 299.52 (sequence contexts：
296.54-300.02).Base mass value is more than 20bp (sequencing error rate is less than 1%) and 30bp in Effective Tags
The base percentage of (sequencing error rate is less than 0.1%) is respectively 96% and 93% or more, as a result shows that sequencing data quality is non-
Chang Gao.
(2) OTU clusters and species colony assay
Species composition and diversity information carry out clustering (acquiescence selection using whole Effective Tags sequences
Identity is 97%), and to form OTU.The representative series for choosing OTU, acquisition object is compared with ribosomal RNA database
Kind annotation information.Based on species annotation information, removal annotation is chloroplaset, mitochondria and cannot annotate the OTU to boundary's rank
And it includes Tags.According to result it can be found that the microorganism ordered sequence using homogeneity more than or equal to 97% carries out
OTU clusters to have obtained quality data, and each sample obtains more than 200 OTUs, almost without can not annotate to boundary's rank or
Annotation is the OTU Tags numbers of chloroplaset and mitochondria, shows the with a high credibility of experimental data, can carry out subsequent analysis.
Absolute abundance based on OTU and annotation information, to each sample in each categorization levels (boundary Kingdom, door
Phylum, guiding principle Class, mesh Order, section Family belong to Genus, plant Species) on sequence number account for the ratio of total sequence number
Example is counted, and the species annotation resolution ratio (annotation to the higher OTU for indicating sample of the ratio belonged to of sample can be effectively assessed
Annotate effect it is better) and sample species complexity (annotation to belong to ratio it is lower indicate sample species complexity it is higher),
As a result show that 30 samples can all annotate category, and ratio is higher, the ratio of annotation to kind is relatively low.
(main presentation door is opposite with the species of rank are belonged to for relative abundance of the species of each categorization levels in each sample
Distribution situation) statistics.In door level, it is found that highest 5 kinds of abundance is not different in health and susceptible sample, is respectively deformed
Bacterium door (Proteobacteria), actinomyces door (A ctinobacteria), Cyanophyta (Cyanobacteria), Bacteroidetes
(Bacteroidetes), acidfast bacilli door (Acidobacteria).Wherein Proteobacteria (Proteobacteria) health and
Abundance in susceptible sample is all highest, followed by actinomyces door (Actinobacteria), and Tenericutes
(Tenericutes) only occur in susceptible sample.On belonging to level, abundance distribution is had any different in healthy and susceptible sample, wherein strong
In health sample before abundance 5 be respectively Methylobacterium (Methylobacterium) (24.48%), Halomonas
(Halomonas) (10.27%), Sphingomonas (Sphingomonas) (6.71%), unwrapping wire spore Pseudomonas
(Actinomycetospora) (4.91%), Curtobacterium (Curtobacterium) (2.36%)；Susceptible sample abundance
Preceding 5 be respectively Methylobacterium (Methylobacterium) (21.24%), Sphingomonas (Sphingomonas)
(9.32%), Halomonas (Halomonas) (5.75%), Candidatus liberibacter category (Ca.L.asiaticus) (5.58%),
Unwrapping wire spore Pseudomonas (Actinomycetospora) (1.50%), and Ca.L.asiaticus, Clostridium,
These three belong in healthy sample abundance close to 0 Turicibacter, and Candidatus liberibacter category in susceptible sample
(Ca.L.asiaticus) abundance ranking the 4th is only second to Methylobacter (Methylobacterium), Sphingomonas
(Sphingomonas), Halomonas (Halomonas).In conclusion healthier and susceptible sample bacterial diversity
It was found that being not different in door level, and find that the type of flora and abundance change in category level.
Finally by OTU phylogenetic analysis, species COMMUNITY STRUCTURE, Alpha Diversity analyses, Beta
Statistical analysis is obtained to draw a conclusion between Diversity analyses, sample sets：
After the 16S rDNA deep sequencings of susceptible and healthy Citrus shatangju, by known to species taxonomy statistical analysis：
(1) in door level, it is found that highest 5 doors of abundance are not different in health and susceptible sample, are respectively deformed
Bacterium door (Proteobacteria), actinomyces door (Actinobacteria), Cyanophyta (Cyanobacteria), Bacteroidetes
(Bacteroidetes), acidfast bacilli door (Acidobacteria).Wherein Proteobacteria (Proteobacteria) health and
Abundance in susceptible sample is all highest, followed by actinomyces door (Actinobacteria), and Tenericutes
(Tenericutes) only occur in susceptible sample.
(2) on belonging to level, abundance distribution is had any different in healthy and susceptible sample, 5 difference before abundance wherein in healthy sample
For Methylobacter (Methylobacterium), Halomonas (Halomonas), Sphingomonas
(Sphingomonas), unwrapping wire spore Pseudomonas (Actinomycetospora), Curtobacterium (Curtobacterium)；
(3) before susceptible sample abundance 5 be respectively Methylobacterium (Methylobacterium), Sphingomonas
(Sphingomonas), Halomonas (Halomonas), Candidatus liberibacter category (Ca.Liberibacter asiaticus),
Unwrapping wire spore Pseudomonas (Actinomycetospora).And Candidatus liberibacter category (Ca.Liberibacter asiaticus), clostridium
Belong to (Clostridium), staphylococcus (Turicibacter) this 3 belongs in healthy sample abundance close to 0.It is worth noting
, yellow twig pathogen is Candidatus liberibacter category (Ca. Liberibacter asiaticus), and susceptible sample belongs to horizontal rich
Degree demonstrates the testing result of Standard PCR.
(4) two categories of fusobacterium Clostridium and staphylococcus Turicibacter come across susceptible sample, this with
Yellow twig pathogen changes micro-ecological environment correlation in plant, such as nutrition composition, and suitable for fusobacterium (Clostridium)
It is grown with staphylococcus (Turicibacter).
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. the bacterium colony detection method in a kind of granulated sugar tangerine of infection yellow twig, which is characterized in that it includes：
Using the first primer to the molecular detection primer as Citrus shatangju yellow twig pathogen, PCR is carried out to sample to be tested genome
Amplification, determines illness plant DNA sample；
Build the libraries 16SrDNA of the illness plant DNA sample；
The illness plant DNA sample is detected using high throughput sequencing technologies and in the illness plant DNA sample
Bacterium colony composition analyzed.
2. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 1, which is characterized in that described the
The base sequence of one primer pair such as SEQ ID NO:Shown in 1~2.
3. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 1, which is characterized in that structure institute
The step of libraries 16SrDNA for stating illness plant DNA sample includes：
Using the second primer pair as primer, 16SrDNA PCR amplifications are carried out to the illness plant DNA sample, wherein described second
The gene of primer pair such as SEQ ID NO:Shown in 3~4；
The indices of the 16SrDNA after amplification are detected again, meet the selected libraries 16SrDNA of structure standard.
4. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 3, which is characterized in that described
The structure standard in the libraries 16SrDNA includes：
A. it is selected in the DNA sample master tape in the libraries 16SrDNA in 10kb or more；
B. OD (260/280) value for being selected in the DNA sample in the libraries 16SrDNA is that 1.6~1.8, OD (260/230) value is big
In 2.
5. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 3, which is characterized in that described
20 μ L reaction systems of 16SrDNA PCR amplifications include：
0.8 μ L of forward primer (5 μM), 0.8 μ L, FastPfu polymerase of reverse primer (5 μM) 0.4 μ L, template DNA 10ng,
2 μ L, 5 × FastPfu buffer solutions of 2.5mM dNTPs, 4 μ L mend distilled water to 20 μ L.
6. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 3, which is characterized in that described
The response procedures of 16SrDNA PCR amplifications include：
Annealing temperature is 54~56 DEG C, and elongating temperature is 70~74 DEG C.
7. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 1, which is characterized in that described in warp
The initial data that high throughput sequencing technologies are tested is removed through data fractionation, the removing of primer sequence, splicing and chimera
After going, the valid data for analyzing the bacterium colony composition are obtained.
8. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 7, which is characterized in that described to have
Base percentage of the base mass value more than 20bp and 30bp in effect data is all higher than 93%.
9. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 8, which is characterized in that the spelling
Connecing step includes：
Read by base number less than 20bp is filtered processing；
Pairs of reads is spliced into one sequence of assembling；
Sample differentiation is carried out according to the barcode at sequence head and the tail both ends, is carried out at the same time the adjustment of sequence direction.
10. the bacterium colony detection method in the granulated sugar tangerine of infection yellow twig according to claim 9, which is characterized in that by institute
It states and is to reads splicings parameter when assembled：The length in minimum overlay region is 10bp；Splice the overlapping region permission of sequence
Maximum mispairing ratio is less than 0.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810681313.6A CN108774644A (en) | 2018-06-27 | 2018-06-27 | It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810681313.6A CN108774644A (en) | 2018-06-27 | 2018-06-27 | It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108774644A true CN108774644A (en) | 2018-11-09 |
Family
ID=64030436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810681313.6A Pending CN108774644A (en) | 2018-06-27 | 2018-06-27 | It is a kind of infection yellow twig granulated sugar tangerine in bacterium colony detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108774644A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112037858A (en) * | 2020-08-20 | 2020-12-04 | 安徽农业大学 | Method for determining main pathogenic bacteria of plants and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589794A (en) * | 2013-11-04 | 2014-02-19 | 中国热带农业科学院环境与植物保护研究所 | Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing |
-
2018
- 2018-06-27 CN CN201810681313.6A patent/CN108774644A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589794A (en) * | 2013-11-04 | 2014-02-19 | 中国热带农业科学院环境与植物保护研究所 | Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing |
Non-Patent Citations (9)
| Title |
|---|
| STEVEN W. KEMBEL等: "Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest", 《PNAS》 * |
| UMA SHANKAR SAGARAM等: "Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus，Using PhyloChip Arrays and 16S rRNA Gene CloneLibrary Sequencing", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 于国萍等: "利用Illumina MiSeq高通量测序技术分析原料乳的菌群分布", 《食品科学》 * |
| 卢占军等: "3种提取单头柑桔木虱体内黄龙病菌DNA方法的比较", 《中国南方果树》 * |
| 李迪强等: "《自然保护区资源调查和标本采集整理共享技术规程》", 31 May 2009 * |
| 王爱华等: "广西柑橘黄龙病植株韧皮部内生细菌多样性分析", 《中国农业科学》 * |
| 胥伟等: "高通量测序研究霉变黑毛茶的真菌多样性", 《茶叶科学》 * |
| 黄宏明等: "不同地理来源砂糖橘黄龙病亚洲种的检测", 《中国园艺学会热带南亚热带果树分会第四届学术研讨会论文集》 * |
| 黄宏明等: "广西砂糖橘黄龙病亚洲种的Nested-PCR检测", 《南方农业学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112037858A (en) * | 2020-08-20 | 2020-12-04 | 安徽农业大学 | Method for determining main pathogenic bacteria of plants and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hornung et al. | Issues and current standards of controls in microbiome research | |
| US11091813B2 (en) | Multitag sequencing ecogenomics analysis | |
| Nakamura et al. | Metagenomic diagnosis of bacterial infections | |
| Guo et al. | Taxonomic precision of different hypervariable regions of 16S rRNA gene and annotation methods for functional bacterial groups in biological wastewater treatment | |
| Toju et al. | Network modules and hubs in plant-root fungal biomes | |
| Caimi et al. | Leptospira spp., a genus in the stage of diversity and genomic data expansion | |
| Chen et al. | Multi-virulence-locus sequence typing identifies single nucleotide polymorphisms which differentiate epidemic clones and outbreak strains of Listeria monocytogenes | |
| Simmon et al. | Genotypic diversity of anaerobic isolates from bloodstream infections | |
| Dahle et al. | Molecular Epidemiology of Mycobacterium tuberculosis in Norwa y | |
| Filliol et al. | Molecular typing of Mycobacterium tuberculosis based on variable number of tandem DNA repeats used alone and in association with spoligotyping | |
| Catara et al. | Trends in molecular diagnosis and diversity studies for phytosanitary regulated Xanthomonas | |
| Carleton et al. | Metagenomic approaches for public health surveillance of foodborne infections: opportunities and challenges | |
| Pruvost et al. | Genetic structure analysis of strains causing citrus canker in I ran reveals the presence of two different lineages of X anthomonas citri pv. citri pathotype A | |
| CN110283903A (en) | Intestinal microflora for Diagnosis of Pancreatic inflammation | |
| Djordjevic et al. | Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing are reliably predicted by restriction fragment length polymorphism analyses of the flaA gene | |
| Schürch et al. | Genomic tracing of epidemics and disease outbreaks | |
| CN106554998A (en) | Depression biomarker and its application | |
| Dominguez et al. | Comparison of whole-genome sequencing and molecular-epidemiological techniques for Clostridium difficile strain typing | |
| Mikkonen et al. | Ecological inference on bacterial succession using curve-based community fingerprint data analysis, demonstrated with rhizoremediation experiment | |
| Kirchberger et al. | Culture‐independent tracking of Vibrio cholerae lineages reveals complex spatiotemporal dynamics in a natural population | |
| Huys et al. | Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa | |
| Sandstedt et al. | Comparison of laboratory-based and phylogenetic methods to distinguish between Haemophilus influenzae and H. haemolyticus | |
| Akhi et al. | Antibiotic sensitivity of Clostridium perfringens isolated from faeces in Tabriz, Iran | |
| Satterfield et al. | A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F | |
| CN110452974A (en) | A kind of detection bacterium 16S rDNA overall length builds library sequencing approach |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181109 |