CN108004335A - A kind of bacterial leaf spot bacterium microspecies isolation and identification method - Google Patents
A kind of bacterial leaf spot bacterium microspecies isolation and identification method Download PDFInfo
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Abstract
The invention discloses a kind of bacterial leaf spot bacterium microspecies isolation and identification method, belong to Race identification technical field.The total nucleic acid of microorganism in extraction sample simultaneously builds high-throughput sequencing library；Find the variant sites on genome；High polymorphic site is screened by sliding translation, and multiplex amplification primer is designed in candidate locus both sides；The single bacterium colony in sample is separated using solid medium and is further purified, to obtain monoclonal bacterium colony；Expanded and be sequenced using the primer of above-mentioned steps design after extracting the genomic DNA of bacterium colony；Parting is carried out based on sequencing result monoclonal bacterium colony, the bacterium colony with different genotype carries out follow-up study as different microspecies.This method need not be known a priori by the information such as microspecies quantity and the abundance in sample, without required Physiology and biochemistry qualification test in traditional screening process, the whole microspecies that need to can be only separately cultured out by high-flux sequence and amplification in sample, process is simple, quick and standard process.
Description
Technical field
The invention belongs to Race identification technical field, and in particular to a kind of bacterial leaf spot bacterium microspecies isolation and identification method.
Background technology
Bacterial leaf spot bacterium every year causes Rice Production huge loss, may cause to have no harvest under serious conditions.Different is white
The withered microspecies of leaf have the extent of injury of rice varieties very big difference.Tackled in cultural control for different bacterial leaf spot microspecies
Strategy is also different, it is thus determined that separating, identifying that what is infected is the first step which microspecies is cultural control.
Microspecies are also known as biological strain, strain etc., refer to do not have notable difference in form, but in physio-biochemical characteristics, culture
The different groups for the same species of microorganism that character, pathogenic etc. have differences.Since these microspecies are in the characteristic such as pathogenic
With obvious difference, therefore generally require to accomplish that the horizontal ability of microspecies is meaningful when doing strain separating and identification.
In the prior art, have at least the following problems：Due to there is no notable difference between each microspecies morphologically, obtaining
It can not still know in sample after taking sample and being separately cultured and include several microspecies, be specifically what microspecies, can only pass through
A series of dilution spreads are tested to obtain single bacterium colony, and carry out a series of Physiology and biochemistry test to differentiate one by one.This leads
Cause three problems, first, there is no any information to required dilution gradient, can only select after the dilution of blindness some gradients into
Row subsequent experimental；Second, some microspecies can be sieved by leakage；Third, picking need to do physiological and biochemical analysis to multiple monoclonal bacterium colonies repeatedly
To screen microspecies.Its process expends a large amount of manpower and materials, and not accurate and comprehensive enough.
The content of the invention
The improving eyesight of this hair is to provide a kind of bacterial leaf spot bacterium microspecies isolation and identification method.
The technical solution adopted in the present invention is：
A kind of bacterial leaf spot bacterium microspecies isolation and identification method, comprises the following steps：
(1) extraction needs the total nucleic acid of separation and the rice leaf identified, then builds high-throughput sequencing library；
(2) sequencing of high overburden depth is carried out to library using the method for high-flux sequence, and sequencing result is compared and is arrived
In the reference gene group of corresponding species；
(3) all variant sites are obtained according to comparison result, the gene of each window is then obtained according to window translation
Type number, calculates the polymorphism of each window, selects polymorphism high and in single copy region as candidate molecular marker site；
(4) conservative region is found in the both sides in each molecular labeling site, amplimer is designed in conservative region；
(5) tablet is applied after the rice leaf for needing to separate and identify being done 10 times of dilutions, picking is single after constant temperature incubation
Bacterium colony and the bacterium colony for obtaining monoclonal after homozygosis on new solid medium tablet；
(6) extract the primer designed with step (4) after the nucleic acid of bacterium colony to be expanded, parting after amplified production is sequenced；
(7) the identical monoclonal of all genotyping results only retains one；If there is part to have found genotype in all clones
In be all not present, then picking colony is purified and repeated the above process again, until obtaining the bacterial leaf spot bacterium microspecies.
Further, needed in the step (1) during the total nucleic acid for the rice leaf that extraction needs are separated and identified abundant
Cell lysis, it is ensured that propose the genome sequence of each microspecies.
Further, sequencing number of fragments will reach 200 haplotype datas caused by sequencing in the step (2), it is ensured that institute
There are microspecies can effectively to be detected.
Further, the screening in molecular labeling site carries out in a manner of window translates in the step (3), length of window
It is set to L；Only consideration can completely cover the sequencing fragment of the window during window translates, other sequencing fragments are not
Consider.
Further, the analysis of variant sites is unfolded in units of fragment is sequenced in the step (3), marks every survey
The each base position and its mutation type different from reference gene group in sequence fragment；With identical mutation base position and prominent
The reads of change type is defined as a genotype in the window.
Further, all candidate gene types for obtaining the window, count the sequencing segments per genoid type
Amount, and divided by completely cover the window sequencing fragment total quantity, so as to obtain the frequency of the genotype.
Further, obtained after biased sample completion gene order-checking and accurately obtain all genotype, debug
Genotype.
Preferably, the molecular labeling site chosen in the step (3) has distinctive characteristic fragment in the species, and
Very high polymorphism is shown in the sample of mixing sequencing, the computational methods of polymorphism are：
Further, the molecular labeling chosen in the step (3) is in the genome on single copy region, genome
The genomic fragment that interference is formed to the region is not present in other positions；
Singly the criterion in copy region is：1. judged based on similitude：If reference gene group, then ensure ginseng first
Examining other positions on genome does not have similitude to be more than the section that 90%, matching length is more than 100bp；2. based on sequencing depth
Judge：It is sequenced depth and is no more than close region average sequencing depth ± 3 times standard deviation, and close region refers to upstream and downstream each 1,
The genome area of 000bp.
Further, molecular labeling both sides should have the conservative region for being suitable for design of primers in the step (4)；For length
A sliding window for L is spent, the initial position of window in the genome is set to S, which is E, then window is left
Side conserved region is defined as continuous n that coordinate position does not find variation less than S, in sequencing data and in existing data
Base position, window left side conserved region are defined as coordinate position and are more than E, are not sent out in sequencing data and in existing knowledge
The continuous n base position (n >=50) now to make a variation；
In the conserved region design 3 end of primer do not contain low complex sequence, as polyA T C G.
Further, in the step (5) dilution apply after tablet will according to the bacterium colony of morphological feature picking objective microbe,
And ensure picking is single bacterium colony；Picking monoclonal bacterium colony after further purification, and each position is obtained by bacterium colony PCR
The amplified production of point；The genotype information in each amplification site can be obtained after amplified production is sequenced.Each clone in theory
Each amplification site there was only a genotype, if there is the genotype of two and the above, and the gene and highest of secondary high abundance
The abundance ratio of the genotype of abundance is more than 0.01；It is non-monoclonal bacterium colony then to think the bacterium colony, is needed again homozygous；If abundance ratio
Value is less than 0.01, then it is assumed that the genotype of other high abundance introduces for sequencing mistake, and highest abundance genotype is the amplification position
The real gene type of point；
When making dilution painting tablet, diluted multiple should be using the frequency of minimum genotype as reference value：It can then be made less than the value
Lost into part microspecies in solid medium, each microspecies can not be then ensured higher than the value can form single bacterium colony.
Preferably, compare two monoclonal bacterium colonies it is whether identical when, it is desirable to the genotype all same in each amplification site is
It is considered that both belong to same microspecies, is otherwise difference.
Further, the microspecies quantity in biased sample is disposably determined, to ensure to leak some microspecies of sieve；Each gene
The ratio of type abundance can determine the multiple of dilution spread, improve the efficiency of screening.
The present invention has the following advantages：
The method of the present invention need not be known a priori by the information such as microspecies quantity and abundance in sample, without traditional
Required Physiology and biochemistry qualification test in screening process, need to can be only separately cultured out in sample by high-flux sequence and amplification
Whole microspecies, process is simple, quick and standard process.Examination process to each monoclonal is simple, quick, accurate and logical
Amount is high, is generally applicable to the industry rapid screenings such as clinical, agricultural and food and causes a disease microspecies, and from various complex environment samples
The research of middle separation objective microbe microspecies.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below
It is described in detail on step ground.As not plus specified otherwise, agents useful for same is in the market common agents in the present invention, most of biotechnologys
There is sale in company, and effect is equal.
The separation and identification of the bacterial leaf spot bacterium microspecies of rice leaf infection
In the present embodiment, the rice leaf for infecting bacterial leaf spot is material, and the purpose of the present embodiment is that separation identifies blade
Present in bacterial leaf spot bacterium different microspecies.
First, the extraction of the mixed genomic DNA
From leaf spot lesion position clip 1mg blades, liquid nitrogen grinding will be added after its surface clean with deionized water.Then use
PureLink Microbiome purification kit (article No.s：A29790, production unit are limited for the silent winged generation your scientific and technological (China) of match
Company) the total genomic DNA of extraction.
2nd, the structure of high-throughput sequencing library and sequencing
Utilize ultraviolet specrophotometer (NanoDrop oneC, match silent winged scientific and technological (China) Co., Ltd of generation that) measure core
The OD260/280 values of acid are 1.92.Extraction nucleic acid is quantified with Qubit, determines that the DNA concentration of extraction reaches library construction
Amount.Instrument (Covaris M220) is interrupted using Covaris System ultrasonic waves, DNA to be measured is broken into 250bp sizes, so
Afterwards the high-throughput sequencing library of face PCR amplification is built according to the full-length genome library construction Kit of Ion torrent.Using
Ion torrentS5 high-flux sequence instrument is sequenced.
3rd, the screening technique in polymorphic molecular marker site
3.1 sequencing fragments are compared with genome sequence
All sequencing fragments are compared onto the reference gene group of bacterial leaf spot bacterium using bowtie2 (version number 2.1.0), in vain
The version number of the reference gene group of the withered bacterium of leaf is NC_010717.2, and download address is：
https：//www.ncbi.nlm.nih.gov/genome/529Genome_assembly_id=233750,
Alignment parameters are all using default value.
3.2 variant sites are analyzed
Variant sites on genome are counted according to comparison result, method is as follows：Set the size of sliding window as
100bp, window move forward 30bp every time；For each window, the variant sites information of every reads is counted first, such as
Base on genome is A, and it is T that corresponding site in fragment, which is sequenced, then the site is recorded as T；Such as with the alkali on genome
Base information is identical, then is recorded as R.Represent the sequencing fragment on the window using the information of all base positions as an entirety
Genotype.Since the insertion introduced in sequencing procedure, the proportion of missing are higher, especially in simple repeated sequence
Position occur sequencing mistake than regular meeting higher, therefore we neglect all insertions, deletion segment, and appear in simple
All variant sites of duplicate block.
3.3 calculate the polymorphic sex index of each window.
Count the sequencing number of fragments of each genotype, and divided by be completely covered the window sequencing fragment sum,
Then obtain the percentage frequency of the genotype.The polymorphism formula of index of the window is as follows：Its
Middle piFor the frequency of i-th of genotype.If the polymorphic sex index in the window is less than 0.2, which gives up；Assuming that the window
The position that interior first variation occurs is n, and the position that last variation occurs is m, if L=200- (m-n), then in nbp
The conservative region for whether having a segment length to exceed 50bp is looked into the region of (n-L) bp, and the region inspection of mbp to (m+L) bp
In the presence of, it is desirable to any nucleotide variation is not detected by the region, if the region is made there are satisfactory region in both sides
Retain for the polymorphic site of candidate, otherwise give up the window.
The screening in 3.4 molecular labeling sites
The step of window translates forward 30bp, repeat step 3.1-3.3, so as to obtain the candidate point on every chromosome
Sub- marker site.Then 30 sites before being chosen according to the height of polymorphism, then remove site closer to the distance on genome,
Method is as follows：The window for setting length as 10 a, 000bp checks whether there is candidate's polymorphic site in the region, if nothing,
Found again after extending 5,000bp forward；If there is a site, which is retained；If there are multiple sites, select more
The highest site of state property retains.The high polymorphic molecular marker site that the present embodiment filters out is shown in Table 1.
The high polymorphic molecular marker site of 1 bacterial leaf spot bacterium of table
4th, primer design method
Log in LifeTechnology company multiplex amplification primer Photographing On-line webpage https：//ampliseq.com, point
Hit " My References " options, the selection " Add reference " options, in the page jumped out in the page newly jumped out
The reference gene group of oneself is chosen, and clicks on " save ", so as to will be caught in the reference gene group sequence of bacterial leaf spot bacterium used
Go.Then under click " my design " options " start a new design " options, hence into the design of primers page.
In the page jumped out, " " Custom " is being selected in Select genome to use " options, is then being selected in above-mentioned steps
The bacterial leaf spot bacterium reference gene group sequence of biography, then in " Application type " options selection " DNA Hotspot
Designs (single-pool) ".Then " add targets " buttons, it is polymorphic to input each candidate in new interface for selection
Property molecular labeling site start-stop information, then click on " Submit targets " options start design of primers.Design of primers is complete
Whether Cheng Hou, detect 3 ' ends of each primer with the presence of low complex sequence, including continuous multiple A either T or C or G, with
And sequence as similar ATATAT：If any then needing the corresponding site by this primer in the genome in reference gene group
Redesigned after being set to N.The primer sequence that the present embodiment obtains molecular labeling site is shown in Table 2.
2 molecular labeling primer information of table
5th, the single bacterium colony in sample is separated using solid medium
The blade at 1g rice scabs position is taken, surface sterilization is carried out with 75% alcohol；Then ground in mortar
10ml sterile waters are added afterwards, and concussion 1min therefrom takes 1ml to do 10 times of gradient dilutions to 10 after being shaken up-4.According to following formula
Prepare solid medium：Sucrose 10g, sodium glutamate 5g methionine 0.1g, potassium dihydrogen phosphate 1g, ammonium chloride 1g, magnesium chloride 1g, pancreas
Peptone 0.5g, agarose 17g, water 1000ml, pH value are transferred to 6.5.After solid medium heating and melting, in superclean bench
In be poured into glass culture dish and wait its cooled and solidified quietly.Then three solid medium tablets are taken, are added on each tablet
1ml is diluted to the bacterium solution of 10-4, and it was evenly coated on tablet.Tablet after coating is placed on constant temperature at 27 DEG C to stay overnight
Culture.
6th, the acquisition of monoclonal bacterium colony
With collarium is connect from a small amount of microbial cells of single bacterium colony picking, Z-shaped is drawn on new tablet.Tablet weight after line
Newly it is incubated overnight at 27 DEG C.So pass on repeatedly and obtain monoclonal bacterium colony afterwards three times.With sterile toothpick from each monoclonal bacterium
Fall a small amount of thalline of upper picking to be added in following PCR reaction systems：10 × amplification buffer 10ul, dNTP mixture 200ul,
Primer 100pmol, Taq archaeal dna polymerase 2.5ul, Mg2+1.5mmol/L, adds distilled water to 100ul.The amplification program of PCR is such as
Under：95 DEG C, 2 minutes；(95 DEG C, 10 seconds；55 DEG C, 30 seconds) × 25 circulations；4 DEG C of insulations.
7th, the parting in site is respectively expanded
The amplified production in each molecular labeling site is done into Sanger sequencings, and by sequencing result and the position having detected that
All genotype of point compare, and do not have the genotype of base difference then to think that the site belongs to this genotype with it.It is comprehensive
The genotype information for closing each amplification site determines to recognize the monoclonal bacterium colony parting, the identical monoclonal bacterium colony of all partings
To be to belong to same microspecies.The microspecies and its genotype information for the bacterial leaf spot being separated in the present embodiment are shown in Table 3.
Genotype information of the 3 each molecular labeling of table in 5 microspecies identified
The method of the present invention need not be known a priori by the information such as microspecies quantity and abundance in sample, without traditional
Required Physiology and biochemistry qualification test in screening process, need to can be only separately cultured out in sample by high-flux sequence and amplification
Whole microspecies, process is simple, quick and standard process.Examination process to each monoclonal is simple, quick, accurate and logical
Amount is high, is generally applicable to the industry rapid screenings such as clinical, agricultural and food and causes a disease microspecies, and from various complex environment samples
The research of middle separation objective microbe microspecies.
It should be noted last that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although ginseng
The present invention is described in detail according to preferred embodiment, it will be understood by those of ordinary skill in the art that, can be to the present invention
Technical solution technical scheme is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention, it should all cover
Among scope of the presently claimed invention.
Claims (10)
1. a kind of bacterial leaf spot bacterium microspecies isolation and identification method, it is characterised in that comprise the following steps：
(1) extraction needs the total nucleic acid of separation and the rice leaf identified, then builds high-throughput sequencing library；
(2) sequencing of high overburden depth is carried out to library using the method for high-flux sequence, and sequencing result is compared to corresponding
In the reference gene group of species；
(3) all variant sites are obtained according to comparison result, the genotype number of each window is then obtained according to window translation
Mesh, calculates the polymorphism of each window, selects polymorphism high and in single copy region as candidate molecular marker site；
(4) conservative region is found in the both sides in each molecular labeling site, amplimer is designed in conservative region；
(5) rice leaf that will need to separate and identify applies tablet after doing 10 times of dilutions, the single bacterium colony of picking after constant temperature incubation
And the bacterium colony of monoclonal is obtained after homozygosis on new solid medium tablet；
(6) extract the primer designed with step (4) after the nucleic acid of bacterium colony to be expanded, parting after amplified production is sequenced；
(7) the identical monoclonal of all genotyping results only retains one；If there is part to have found genotype in all clones all
It is not present, then picking colony is purified and repeated the above process again, until obtaining the bacterial leaf spot bacterium microspecies.
2. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that carried in the step (1)
Abundant cell lysis is needed during the total nucleic acid for taking the rice leaf for needing to separate and identifying, it is ensured that proposes the genome of each microspecies
Sequence.
3. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that surveyed in the step (2)
Sequencing number of fragments will reach 200 haplotype datas caused by sequence, it is ensured that all microspecies can be detected effectively.
4. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that divide in the step (3)
The screening of sub- marker site carries out in a manner of window translates, and length of window is set to L；Only consider during window translates
The sequencing fragment of the window can completely be covered, other sequencing fragments without considering.
5. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that become in the step (3)
The analysis of ectopic sites is unfolded in units of fragment is sequenced, and marks each alkali different from reference gene group in every sequencing fragment
Base site and its mutation type；One reads with identical mutation base position and mutation type is defined as in the window
A genotype.
6. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 4 or 5, it is characterised in that described acquisition window
Mouthful all candidate gene types, count the sequencing number of fragments per genoid type, and divided by completely cover the sequencing piece of the window
The total quantity of section, so as to obtain the frequency of the genotype.
7. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that choosing in the step (3)
The molecular labeling site taken has a distinctive characteristic fragment in the species, and shows in the sample of mixing sequencing very high
Polymorphism, the computational methods of polymorphism are：
8. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that choosing in the step (3)
Other positions are not present to the region formation interference molecular labeling taken on single copy region, genome in the genome
Genomic fragment；
Singly the criterion in copy region is：1. judged based on similitude：If reference gene group, then ensure to refer to base first
There is no similitude to be more than the section that 90%, matching length is more than 100bp because organizing upper other positions；2. based on sentencing for sequencing depth
It is disconnected：It is sequenced depth and is no more than close region average sequencing depth ± 3 times standard deviation, and close region refers to upstream and downstream each 1,
The genome area of 000bp.
9. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that divide in the step (4)
Son mark both sides should have the conservative region for being suitable for design of primers；For the sliding window that length is L, window is in genome
On initial position be set to S, which is E, then on the left of window conserved region be defined as coordinate position less than S, surveying
Ordinal number does not find the continuous n base position of variation in and in existing data, window left side conserved region is defined as sitting
Cursor position does not find the continuous n base position (n >=50) of variation more than E, in sequencing data and in existing knowledge；
In the conserved region design 3 end of primer do not contain low complex sequence, as polyA T C G.
10. bacterial leaf spot bacterium microspecies isolation and identification method according to claim 1, it is characterised in that dilute in the step (5)
Release that apply will be according to the bacterium colony of morphological feature picking objective microbe after tablet, and ensure picking is single bacterium colony；Into one
Step picking monoclonal bacterium colony, and pass through the amplified production in each site of bacterium colony PCR acquisitions after purification；After amplified production is sequenced i.e.
The genotype information in each amplification site can be obtained.The each amplification site each cloned in theory only has a genotype, if
There is the genotype of two and the above, and the abundance ratio of the genotype of the gene of secondary high abundance and highest abundance is more than 0.1；Then
It is non-monoclonal bacterium colony to think the bacterium colony, is needed again homozygous；If abundance ratio is less than 0.1, then it is assumed that the base of other high abundance
Because type is sequencing mistake introducing, highest abundance genotype is the real gene type in the amplification site；
When making dilution painting tablet, diluted multiple should be using the frequency of minimum genotype as reference value：Less than the value, it will cause portion
Point microspecies are lost in solid medium, and each microspecies can not be then ensured higher than the value can form single bacterium colony.
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