CN105603074A

CN105603074A - Qualitative and quantitative microorganism detecting method

Info

Publication number: CN105603074A
Application number: CN201610060852.9A
Authority: CN
Inventors: 彭海
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2016-01-29
Filing date: 2016-01-29
Publication date: 2016-05-25
Anticipated expiration: 2036-01-29
Also published as: CN105603074B

Abstract

The invention discloses a qualitative and quantitative microorganism detecting method and belongs to the technical field of biology. The method comprises the following steps that target microorganism class groups, target microorganisms and non-target organisms in a sample to be detected, and reference microorganisms which do not exist in the sample to be detected are determined; feature regions of the target microorganism class groups and the target microorganisms are designed; multiple amplification primers of the feature regions are designed; after the reference microorganisms and exogenous nucleic acid are added into the sample to be detected, nucleic acid of the microorganisms in the sample to be detected is extracted; the designed multiple primers are utilized for amplifying the nucleic acid of the microorganisms, and amplifying is carried out to obtain a feature sequencing fragment; the feature sequencing fragment is utilized for qualitatively and quantitatively analyzing the microorganisms in the sample to be detected. According to the method, the microorganisms do not need to be precultured or proliferated, the multiple known microorganisms in the sample to be detected can be detected with high flux, high accuracy and high resolution at a time, the detection process is simple and fast, and the flow path is standard.

Description

A kind of microorganism qualitative and quantitative detection method

Technical field

The present invention relates to biological technical field, particularly a kind of detection method of microorganism qualitative and quantitative.

Background technology

Microorganism is ubiquitous, and also very extensive to the detection application of microorganism, comprise agricultural disease qualification and predict,Food safety detection and the detection of medical science pathogen etc.

Existing microorganism qualitative and quantitative detection technique comprises morphology counting, chip detection, 16SrRNA order-checking, grand baseBecause of group order-checking and real-time quantitative PCR (PolymeraseChainReaction, PCR). The inspection of morphology countingSurvey need to be carried out preculture to microorganism, and length consuming time can not be cultivated microorganism and can not be detected, and once only can detect a kind of micro-lifeThing, flux is low, and in the time of counting, amount of sampling is limited, and result is coarse, cannot distinguish planting following taxon. Chip inspectionThe DNA amount of surveying required testing sample is large, need to carry out preculture and enrichment processing to microorganism, and testing result is inaccurate, andCannot do quantitative detection. 16SrRNA order-checking cannot be distinguished planting following taxon. The grand gene order-checking degree of depth hasLimit, very poor for the quantitative accuracy in detection of the microorganism of low content. Real-time quantitative PCR once can only detect a kind of microorganism,Flux is low. In addition, existing method has defect and is, cannot calculate the reliability of microorganism qualitative and quantitative, makes conclusion practicalityProperty is poor.

Summary of the invention

Detect inaccurate problem in order to solve microorganism qualitative and quantitative in prior art, the embodiment of the present invention providesA kind of detection method of microorganism qualitative and quantitative. Described technical scheme is as follows:

The embodiment of the present invention provides a kind of detection method of microorganism qualitative and quantitative, and described method comprises:

Determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and be not present in instituteState the reference microorganism in testing sample;

According to described objective microbe monoid, described objective microbe, described with reference to microorganism and described nontarget organismReference genome sequence, obtain the characteristic area of described objective microbe monoid, described objective microbe characteristic area andThe described characteristic area with reference to microorganism;

The first multiplex amplification primer, the described target that increases of the characteristic area of the described objective microbe monoid of preparation amplification are micro-The 3rd multiplex amplification of the second multiplex amplification primer of biological characteristic area and the described characteristic area with reference to microorganism of amplificationPrimer, is mixed to get described the first multiplex amplification primer, described the second multiplex amplification primer and described the 3rd multiplex amplification primerTo mixing multiplex amplification primer;

Described with reference to microorganism to adding in described testing sample, obtain biased sample;

Extract the nucleic acid of described biased sample;

Utilize the nucleic acid of described mixing multiplex amplification primer and described biased sample to carry out amplified reaction, obtain amplification and produceThing;

Utilize described amplified production to carry out high-flux sequence, obtain high-flux sequence fragment;

According to described high-flux sequence fragment, to described objective microbe monoid and described objective microbe carry out qualitative andQuantitative analysis.

Particularly, number >=1 of described objective microbe monoid, and each described objective microbe monoid comprise >=0Plant described objective microbe;

Described objective microbe be bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw,At least one in algae and protozoan;

Described with reference to microorganism be bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw,At least one in algae and protozoan.

Particularly, the method for the nontarget organism in described definite testing sample comprises: described nontarget organism is determinedFor all biologies except described objective microbe monoid, if can obtain the characteristic area of described objective microbe monoid,Described nontarget organism refers to all biologies except described objective microbe monoid; If can not obtain described objective microbe classGroup's characteristic area, described nontarget organism refers in described biased sample, other except described objective microbe monoidBiological.

Particularly, the characteristic area of described objective microbe monoid is the ginseng of the microorganism in described objective microbe monoidExamine the nucleotide sequence on genome; The sequence of the both sides of the characteristic area of described objective microbe monoid described with reference to genomeIn be unique sequence; The sequence of the both sides of the characteristic area of described objective microbe monoid in described objective microbe monoid notWith conservative between microorganism; Discrimination >=3 of the characteristic area of described objective microbe monoid;

The characteristic area homology of the characteristic area of described objective microbe and described objective microbe monoid; Described target is micro-M2 value >=2 of biological characteristic area, wherein, the characteristic area that m2 value is described objective microbe and described objective microbe classThe minimum of a value of the difference base number described in other in group except described objective microbe between microorganism;

The described characteristic area with reference to microorganism is described with reference to the nucleotide sequence on the reference genome of microorganism; DescribedSequence with reference to the both sides of the characteristic area of microorganism is unique sequence in the described reference genome with reference to microorganism; DescribedWith reference to the sequence of the both sides of the characteristic area of microorganism not thering is homology except described in reference to other biology microorganism.

Further, described discrimination refers to by the arbitrary described target of same described mixing multiplex amplification primer amplification micro-The minimum of a value of the difference base number between the characteristic area of biological group and arbitrary non-characteristic area, wherein, described non-characteristic areaThe amplified production that is described mixing multiplex amplification primer taking the nucleic acid of described biased sample as template, and described non-characteristic area is notFor the characteristic area of described objective microbe monoid, if without described non-characteristic area, described discrimination=3 × L1/4, wherein,L1 is the nucleotide sequence length of the characteristic area of described objective microbe monoid.

Particularly, in the time extracting the nucleic acid of described biased sample, if the content of described testing sample amplifying nucleic acid is too low, existExtract in the process of nucleic acid of described biased sample the exogenous nucleic acid that adds described mixing multiplex amplification primer not increase.

Particularly, the method for qualitative analysis of described objective microbe monoid and described objective microbe is as follows:

The characteristic area of described high-flux sequence fragment and every kind of described objective microbe monoid is compared, work as differenceWhen base number≤n1, compare successfully the characteristic area that corresponding described high-flux sequence fragment is described objective microbe monoidTerritory, wherein, n1 is the fault-tolerant base number of maximum of the feature order-checking fragment of described objective microbe monoid; If described in comparison successfully, judge that described high-flux sequence fragment is described objective microbe monoid when the characteristic area >=1 of objective microbe monoid kindFeature order-checking fragment;

The characteristic area of the described objective microbe monoid of the characteristic area of described objective microbe and every kind of homology is enteredThe standard gene of objective microbe described in difference base composition is extracted in row comparison in the characteristic area of described objective microbeType; In the feature order-checking fragment of described objective microbe monoid, the Reference genotype institute that extracts described objective microbe is correspondingBase, form the test cdna type of described objective microbe; If the test cdna type of described objective microbe and described targetThe difference base number≤n2 of the Reference genotype of microorganism, wherein, feature that n2 is described objective microbe order-checking fragmentLarge fault-tolerant base number, the described high-flux sequence fragment at the test cdna type place of described objective microbe is that described target is micro-Biological feature order-checking fragment;

Using described with reference to microorganism as the described objective microbe monoid that only comprises a described objective microbe, calculateThe feature order-checking fragment of the described objective microbe obtaining, is the described order-checking of the feature with reference to microorganism fragment;

If probability P 5 >=α 5 that the feature of described objective microbe monoid order-checking fragment exists, judges described testing sampleIn there is described objective microbe monoid, wherein, α 5 is probability guarantee; If the feature of described objective microbe monoid order-checking fragmentProbability P 5 < the α 5 existing, judges and in described testing sample, does not have described objective microbe monoid;

If probability P 6 >=α 6 that the feature of described objective microbe order-checking fragment exists, judges in described testing sample and depositsAt described objective microbe, wherein, α 6 is probability guarantee; If the probability P 6 that the feature of described objective microbe order-checking fragment exists< α 6, judges and in described testing sample, does not have described objective microbe;

N1 makes P1≤α 1 and P3≤α 3, and wherein, it is not the feature order-checking sheet of described objective microbe monoid that P1 is oneThe described high-flux sequence fragment of section is mistaken for the feature order-checking fragment of described objective microbe monoid and the false positive that producesProbability; P3 is that the feature order-checking fragment of a described objective microbe monoid is mistaken for and is not described objective microbe monoidFeature order-checking fragment and the false-negative probability that produces; α 1 and α 3 are judgment threshold;

N2 makes P2≤α 2 and P4≤α 4, and wherein, it is not the feature order-checking fragment of described objective microbe that P2 is oneDescribed high-flux sequence fragment is mistaken for the feature order-checking fragment of described objective microbe and the false-positive probability that produces; P4Be the feature order-checking fragment of a described objective microbe be mistaken for be not described objective microbe feature order-checking fragment andThe false-negative probability producing; α 2 and α 4 are judgment threshold;

P5＝1-BINOM.DIST(S1,S1,P1,FALSE)，P6＝1-BINOM.DIST(S3,S3,P2,FALSE)，S1For the quantity of the feature order-checking fragment of the described objective microbe monoid of the characteristic area of all described objective microbe monoidsMedian; S3 is the number of the feature order-checking fragment of the described objective microbe of the characteristic area of all described objective microbeThe median of amount, FALSE is parameter value; BINOM.DIST function returns to the probability of monobasic binomial distribution.

Further, the quantitative analysis method of described objective microbe monoid and described objective microbe is as follows:

Amount M1=Mr × the S1/S2 of described objective microbe monoid, the confidential interval of the amount of described objective microbe monoidFor [M11, M12], wherein, Mr is the described amount with reference to microorganism adding in described testing sample; S2 is all described ginsengsExamine the median of the quantity of the described order-checking of the feature with reference to the microorganism fragment of the characteristic area of microorganism; M11 and M12 are respectivelyThe lower limit of the confidential interval of M1 value and the upper limit;

Amount M2=M1 × the S3/S1 of described objective microbe, the confidential interval of the amount of described objective microbe be [M21,M22], M21 and M22 are respectively lower limit and the upper limit of the confidential interval of M2 value;

M11＝M1×(1-S4/S1)，M12＝M1×(1+S5/S1)，M21＝M2×(1-S6/S3)，M22＝M2×(1+ S7/S3); Wherein, S4 is quantity and the S4=of the feature order-checking fragment of false-positive described objective microbe monoidCRITBINOM (nS, P1, α 9), wherein, nS is the described multiple expansion of calculating the characteristic area of the described objective microbe monoid of S1The quantity of the described high-flux sequence fragment of the described non-characteristic area that increasing primer increases; S5 is that false-negative described target is micro-Quantity and the S5=CRITBINOM (S1, P3, α 9) of the feature order-checking fragment of biological group, wherein, α 9 is probability guarantee; S6 isQuantity and the S6=CRITBINOM (S1, P2, α 10) of the feature order-checking fragment of false-positive described objective microbe, S7 is false cloudyProperty quantity and the S7=CRITBINOM (S3, P4, α 10) of feature order-checking fragment of described objective microbe, wherein, α 10 is generalRate ensures; CRITBINOM function returns to the minimum of a value that makes accumulation binomial distribution be more than or equal to critical value.

Further, P1=BINOM.DIST (n1, m1,1-E, TRUE), P2=BINOM.DIST (n2, m2,1-E,TRUE), P3=1-BINOM.DIST (n1, L1, E, TRUE), P4=1-BINOM.DIST (n2, L2, E, TRUE), wherein, m1 isDescribed discrimination; Described m2 is micro-life described in other of the characteristic area of described objective microbe and described objective microbe monoidThe minimum of a value of difference base between thing; L1 is the length of the characteristic area of described objective microbe monoid; L2 is the micro-life of described targetThe length of the Reference genotype of thing; E is base error rate.

The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: method provided by the invention does not need micro-Biology carries out preculture and propagation, consuming time short, can detect multiple-microorganism simultaneously, and flux is high, and when counting, amount of sampling is large, detects knotFruit is meticulous, can distinguish taxon, without a large amount of DNA and avoided enrichment culture, detection architecture noiselessness andAccurately, high for the quantitative precision of low content microorganism, and, resolution accurate for the testing result of microorganism quantitative and qualitative analysisRate is high, highly sensitive, have probability guarantee, and testing process is simple, fast and standard process.

Detailed description of the invention

For making the object, technical solutions and advantages of the present invention clearer, will do into one embodiment of the present invention belowStep ground is described in detail. In the present invention, the reagent of mark explanation is not conventional commercial reagent, equal in most of biotech companiesCan buy and almost indifference of effect.

The qualification of embodiment mono-, paddy rice bacterial leaf spot bacterium

The embodiment of the present invention one from the Experimental Base of Lingshui, Chinese Academy of Sciences Hainan, the water of bacterial leaf-blight of having got natural infectionRice blade is as testing sample, and the method providing by the embodiment of the present invention is carried out the bacterial leaf spot bacterium in got rice leafQualitative and quantitative analysis.

Step 1, determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and do not depositBe the reference microorganism in testing sample, concrete grammar is as follows:

Number >=1 of objective microbe monoid, and comprise >=0 kind of objective microbe of each objective microbe monoid; OrderMark microorganism can be bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw, algae and primaryAt least one in animal. The object of the embodiment of the present invention is qualification bacterial blight of rice, therefore, is only concerned about in testing sampleBacterial leaf spot bacterium, this bacterial leaf spot Pseudomonas is in bacterium, and its Latin formal name used at school is Xanthomonasoryzaepv.oryzae, and known ginsengExamining genomic paddy rice bacterial leaf spot bacterium biological strain comprises KACC, MAFF, PXO99A, BLS256 and PXO86 totally 5 physiology is littleKind, therefore, these 5 biological strains form the objective microbe monoid of the embodiment of the present invention. In these 5 biological strains,PXO99A biological strain pathogenic the strongest, as objective microbe.

With reference to microorganism can be bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw,At least one in algae and protozoan. Be not present in testing sample with reference to microorganism. With reference to the effect of microorganism be forObjective microbe monoid in testing sample and objective microbe a reference is quantitatively provided. Because Agrobacterium tumefaciems is present inIn root, be not present in blade, so be not present in testing sample, therefore, choose in the present embodiment Agrobacterium tumefaciems and doFor with reference to microorganism, Agrobacterium tumefaciems belongs to bacterium, and its Latin formal name used at school is AgrobacteriumtumefaciensK84.

Particularly, the method for determining the nontarget organism in testing sample comprises: nontarget organism is defined as except targetAll biologies outside microbe groups, if can obtain the characteristic area of objective microbe monoid, nontarget organism refers to except orderAll biologies outside mark microbe groups, wherein, all biologies refer to have with reference to genomic biology, are nontarget organismsThe strictest standard. In the present embodiment, nontarget organism is defined as outside objective microbe monoid all oneself know lifeWhen thing, can find the characteristic area (acquisition process of characteristic area sees below, and the results are shown in Table 1) of bacterial leaf spot bacterium, therefore, this enforcementNontarget organism in example can be the zoic set except bacterial leaf spot bacterium.

Nontarget organism is defined as to all biologies except objective microbe monoid, if can not obtain objective microbeThe characteristic area of monoid, nontarget organism refers in biased sample, other biology except objective microbe monoid, to dwindleThe scope of nontarget organism, the possibility of the characteristic area of objective microbe monoid is found in increase. In biased sample, except targetOther biology outside microbe groups can rule of thumb determine, for example, in the present embodiment, biased sample comprises paddy rice and referenceMicroorganism, can not exist animal composition and obligatory parasitism in the microorganism of animal in biased sample, therefore, if by this realityExecute nontarget organism in example be defined as outside objective microbe monoid all oneself know the biochron, cannot obtain objective microbeCharacteristic area, non-objective microbe can be defined as except bacterial leaf spot bacterium, animal, obligatory parasitism are in the microorganism of animalBiological set.

Step 2, according to the reference genome sequence of the reference genome sequence of objective microbe monoid, objective microbe,With reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtain the spy of objective microbe monoidLevy the characteristic area of region, objective microbe and the characteristic area with reference to microorganism, concrete grammar is as follows:

The characteristic area of objective microbe monoid is the core on the reference genome of the microorganism in objective microbe monoidAcid sequence; The sequence of the both sides of the characteristic area of objective microbe monoid is unique sequence in reference to genome; The micro-life of targetThe sequence of the both sides of the characteristic area of thing monoid is conservative between different microorganisms in objective microbe monoid; Objective microbe monoidDiscrimination >=3 of characteristic area. Non-characteristic area is not the characteristic area of objective microbe monoid, and non-characteristic area refers to mixedClose the amplified production of multiplex amplification primer taking the nucleic acid of biased sample as template; Discrimination refers to by same mixing multiplex amplification and drawsThe minimum of a value of the difference base number between characteristic area and arbitrary non-characteristic area of arbitrary objective microbe monoid of thing amplification, ifCharacteristic area nothing but, discrimination=3 × L1/4, wherein, L1 is that the nucleotide sequence of characteristic area of objective microbe monoid is longDegree.

Particularly, the characteristic area of objective microbe monoid is used for representing objective microbe monoid, objective microbe monoidCharacteristic area exist, represent objective microbe monoid exist, the order-checking fragment of the characteristic area of objective microbe monoidQuantity, represents the quantity of objective microbe monoid. The multi-primers of the characteristic area of desirable objective microbe monoid only increasesThe characteristic area of objective microbe monoid, nontarget organism can not increase. This just requires the characteristic area of objective microbe monoidBoth sides sequence, i.e. design of primers region homology not in nontarget organism, so, nontarget organism can not be amplified, not yetCan produce non-characteristic area. Now, between characteristic area and non-characteristic area, can only produce at random identical base, totally 4 kinds of bases, phaseWith being respectively 1/4 and 3/4 from different probability, therefore, discrimination is 3 × L1/4. The characteristic area of objective microbe monoidDiscrimination >=3rd, in order to ensure the false positive rate of feature order-checking fragment judgement of objective microbe monoid and false negative rate allLow, its principle is in table 2. The sequence of the both sides of the characteristic area of objective microbe monoid is different micro-lifes in objective microbe monoidConservative between thing, just can be by the different microorganisms in identical primer amplification objective microbe monoid, to get rid of amplification efficiency pairThe impact of the relative quantification between objective microbe monoid different microorganisms.

The characteristic area homology of the characteristic area of objective microbe and objective microbe monoid; The characteristic area of objective microbeM2 value >=2 in territory, wherein, in the characteristic area that m2 value is objective microbe and objective microbe monoid except objective microbeThe minimum of a value of the difference base number between other microorganism. Other microorganism in the present embodiment refers to except objective microbeOther 4 kinds of biological strain: KACC, the MAFF of the outer objective microbe monoid bacterial leaf spot bacterium of PXO99A biological strain, BLS256 andPXO86, m2 value be objective microbe (PXO99A biological strain) characteristic area respectively with these 4 kinds of biological strain homology region ratios, the minimum of a value in the 4 species diversity base numbers that obtain. In the time that objective microbe qualitative and quantitative is analyzed, emphasis is and orderIn mark microbe groups, other microorganism is distinguished. Objective microbe and objective microbe monoid affiliation are often nearer,Between sequence, similitude is high, therefore, is difficult to distinguish. In the time that objective microbe qualitative and quantitative is analyzed, only paid close attention in amplicon withDiscrepant Reference genotype between other microorganism in objective microbe monoid, the source of having reduced error, thus can be betterGround distinguishes objective microbe in objective microbe monoid. In the time of m2 >=2, judge what order-checking fragment was objective microbeFalse positive rate and the false negative rate of feature order-checking fragment are all lower, therefore, and can be by objective microbe from objective microbe monoidIn distinguish, its principle is in table 2.

Be with reference to the nucleotide sequence on the reference genome of microorganism with reference to the characteristic area of microorganism; With reference to microorganismThe sequence of the both sides of characteristic area is unique sequence in the reference genome with reference to microorganism; With reference to the characteristic area of microorganismThe sequence of both sides not thering is homology in other biology with reference to microorganism.

In the present embodiment, discrimination is the unique selection standard of the characteristic area of objective microbe monoid, according to detecting orderDifference, also can be using the microorganism with specific gene sequence as objective microbe monoid, and by specific gene orderRow are as the characteristic area of objective microbe monoid. For example, can be using micro-as target the microorganism with specific Disease-causing geneBiological group, and characteristic area using this Disease-causing gene as objective microbe, so that according to the type of Disease-causing gene, instruct and useMedicine. Equally, drug resistance gene also can direction of medication usage as specific gene sequence.

The first multiplex amplification primer, the amplification target of the characteristic area of step 3, preparation amplification objective microbe monoid are micro-The second multiplex amplification primer of biological characteristic area and the three multiplex amplification primer of amplification with reference to the characteristic area of microorganism,The first multiplex amplification primer, the second multiplex amplification primer and the 3rd multiplex amplification primer are mixed to get to mixing multiplex amplification to be drawnThing.

Integrating step two and step 3 concrete grammar are as follows:

At ftp: download in //ftp.ncbi.nlm.nih.gov/genomes/ in objective microbe monoid (bacterial leaf spot bacterium)The genome sequences of 5 kinds of biological strains, adopt software Megablast (version 2 .2.26) by their genome and querySequence (reference sequences) compares, in the present embodiment, and the genome sequence that query sequence is PXO99A. MegablastEach parameter of software comparison is set to: parameter-e is set to 1e-5; Parameter-p is set to 0; Can is Shuoed – v and is set to 5000; Parameter-mBe set to 1. After having compared, obtain the homologous sequence between all microorganisms of objective microbe monoid, therefrom only select and existIn query sequence, there is the homologous sequence of 1 time. Taking 110bp as window size, taking 10bp as step-length, the homologous sequence of selectingInside do window translation. The window obtaining for translation each time, relatively obtains at least two kinds of micro-lifes in objective microbe monoidThe base there are differences between thing, intercepts in this window and does to the region that last difference base is stopped from first difference baseFor characteristic area, and add up the quantity of the difference base in this characteristic area. The each development length in both sides to characteristic area isThe region of 160bp-characteristic area length is as the primer field of search, in the primer field of search, search exist length be greater than 20bp andThe region without any base difference between all microorganisms in objective microbe monoid, as the design of primers of characteristic areaDistrict, abandons lacking the characteristic area in design of primers district.

Login multiplex amplification primer designs webpage https online: //ampliseq.com, and at " Applicationtype "Option is selected " DNAHotspotdesigns (single-pool) ". If select in the present embodiment multi-pool, manyHeavy PCR will divide multitube to carry out, and cost can increase to some extent. And the primer of selecting single-pool only needs a multiplex PCRCan, save cost, but the design of primers that shortcoming is some characteristic area may failure, but due to the characteristic area on genomeNumber is more, and design of primers failure in a few features region does not affect result, so the present embodiment is selected single-pool. Will be withThe characteristic area of all objective microbe monoids of upper acquisition and corresponding 100 base N for design of primers district thereof (N represent A,Any one in tetra-kinds of bases of T, C and G) couple together, be generated as the reference genome of a design of primers. At " SelectThegenomeyouwishtouse " select in option, after " Custom ", to upload the reference gene of the design of primers of generationGroup. DNAType option is selected " StandardDNA ", in AddHotspot option, fills in characteristic area in drawing of generatingInitial sum final position in the reference genome of thing design. Finally click " Submittargets " button and submit and obtain order toThe multiplex amplification primer sequence of the characteristic area of mark microbe groups.

Utilize the multiplex amplification primer pair objective microbe monoid of design to utilize BLASTN (BasicLocalAlignmentSearchTool, basic Local Alignment research tool) (version2.2.26) analysis that compares, therefrom chooseSelecting in forward and reverse primer has at least to have a specific primer. By select primer again with the gene of nontarget organismGroup is done BLASTN compare of analysis, checks their whether can increase genomes of nontarget organism. In the present embodiment, non-target is rawThing is all biologies except objective microbe monoid, and the genome of nontarget organism is NCBI (NationalcenterforBiotechnologyinformation, NCBI) NT/NR storehouse(www.ncbi.nlm.nih.gov/). Judge that the standard that primer can increase is: amplification region length is no more than 200bp, primerJoin 5 bases that length is greater than 15bp and primer 3 ' end and there is no base deletion or a mispairing with interior. If primer can not increase any non-Target organism, now, the discrimination m1=3 × L1/4 of the characteristic area of the corresponding objective microbe of primer, if primer can expandIncrease part nontarget organism, just the amplified production of arbitrary nontarget organism of this primer amplification and arbitrary objective microbeThe characteristic area of monoid is compared, and obtains in all comparisons, and the minimum of a value of difference base number is discrimination m1, reservation m1 >=The characteristic area of 3 objective microbe monoid, further remove contain simple repeated sequence or on genome for multicopyCharacteristic area. From the characteristic area of objective microbe monoid retaining, the further characteristic area of selected objective target microbe groupsThe characteristic area of territory select target microorganism.

Further, the method for optimizing of the characteristic area of objective microbe monoid is as follows: by raw to characteristic area and non-targetThe reference genome of thing does BLASTN comparison, removes the characteristic area that has 95% above homology with nontarget organism, will remainRemaining characteristic area in objective microbe and objective microbe monoid, between other microorganism, utilize software muscle (version:V3.6) compare by its default parameters, obtain the minimum of a value of difference base number, i.e. m2 value. Retain the objective microbe of m2 >=2The characteristic area of monoid, from the characteristic area retaining, all larger 2 and 2 are above to select arbitrarily discrimination m1 and m2Characteristic area is simultaneously as the characteristic area of objective microbe monoid and the characteristic area of objective microbe, the multiple expansion of its correspondenceIncrease primer simultaneously as the first multiplex amplification primer and the second multiplex amplification primer.

By with the similar method of the characteristic area of finding objective microbe monoid, obtain with reference to microorganism characteristic area andThe 3rd multiplex amplification primer of its correspondence, below emphasis difference is described, something in common is no longer repeated in this description. Same employing is softPart Megablast (version 2 .2.26) is to comparing with reference to microbial genome and query sequence (reference sequences),Query sequence is the genome sequence of AgrobacteriumtumefaciensK84. After having compared, the reference obtainingIn microbial genome, only in query sequence, there is the unique sequence of 1 time. NT/NR storehouse ratio by unique sequence with NCBIRight, abandon existing the unique sequence of homologous sequence, the nonoverlapping 110bp of random choose from unique sequence in nontarget organismLength as characteristic area, the sequence of its both sides is as design of primers region. Design online webpage in multiplex amplification primerHttps: the multiplex amplification primer in the upper design feature of //ampliseq.com region, further screening has successfully designed multiplex amplificationThe characteristic area of primer, concrete grammar is as follows: remove and contain simple repeated sequence or on genome, be the characteristic area of multicopyTerritory, is BLASTN by the reference genome of remaining characteristic area and nontarget organism and compares, and removes with nontarget organism and existsThe characteristic area of 95% above homology. From the characteristic area remaining, random choose more than 2 and 2 characteristic areaAs the characteristic area with reference to microbe groups, its corresponding multiplex amplification primer is as the 3rd multiplex amplification primer.

By Sangon Biotech's synthetic above the first multiplex amplification primer obtaining, the one by oneEach in two multiplex amplification primers and the 3rd multiplex amplification primer weighs amplimer and is answered by each multiplex amplification primer pairThe template sequence of amplification, template sequence refers to that each multiplex amplification primer is in the amplification region of inserting AddHotspot optionTerritory. According to the operation manual (PartNumber of the StepOne real-time PCR of match Mo Feishier company of the U.S.4376784Rev.E) detect the amplification efficiency of each multiplex amplification primer, only retain amplification efficiency 95%～105% multipleAmplimer, the impact with the difference that reduces amplification efficiency on microorganism qualitative and quantitative. Because amplification efficiency impact is less, because ofThis, objective microbe monoid also can be different from the characteristic area of objective microbe, find respectively separately to facilitate more easilyCharacteristic area. The the first multiplex amplification primer obtaining above, the second multiplex amplification primer and the 3rd multiplex amplification primer are retainedThe multiplex amplification primer getting off designs webpage https online by multiplex amplification primer: the consolidation procedure on //ampliseq.comMerge, obtain and mix multiplex amplification primer, after mixing multiplex amplification primer is synthetic by match Mo Feishier company of the U.S., with liquidBodily form formula provides. The final characteristic area relevant information obtaining of the present embodiment is in table 1. Original position in table 1 and final positionRefer to the initial sum final position on the reference genome of characteristic area in query sequence.

The primer relevant information that table 1 embodiment of the present invention one provides

Step 4, in testing sample, add with reference to microorganism, obtain biased sample, concrete grammar is as follows:

Be not present in testing sample with reference to microorganism, so, can be with reference to microorganism as inner reference, and with treatMicroorganism in test sample product carries out operation repetitive, and objective microbe monoid and objective microbe in testing sample are determinedAmount. Be approximately to extract the nucleic acid of the biased sample of 10ng (DNA) with reference to the addition control of microorganism, high normally to buildFlux sequencing library, meanwhile, is unlikely to again to make with reference to the shared ratio of microorganism excessive with reference to the addition of microorganism, takeToo much high-flux sequence data volume. The reference microorganism of the present embodiment is Agrobacterium tumefaciems, the acquisition methods of biased sample asUnder: the bacterium liquid 0.2mL that is the reference microorganism of 2OD (OD is bacterium liquid maximum absorbance value) by concentration is placed in the centrifuge tube of 1.5mLAfter middle vacuum refrigeration centrifugal drying, adding 100mg testing sample is in rice leaf, grinds to form powdery after adding liquid nitrogen, to obtain final productArrive testing sample and the biased sample with reference to microorganism. Count by blood cell plate, calculate acquisition and add the reference of biased sample micro-Biological amount is 2,000,000.

The nucleic acid of step 5, extraction biased sample, concrete grammar is as follows:

In the time extracting the nucleic acid of described biased sample, if the content of testing sample amplifying nucleic acid too low (lower than 1ug) will affectThe extraction of the nucleic acid of biased sample, can be in the leaching process of the nucleic acid of biased sample, adds and mixes multiplex amplification primerThe exogenous nucleic acid that can not increase. The exogenous nucleic acid adding does not exist and occurring in nature, thereby does not disturb microorganism detection. OutsideRNA contrast association has designed and has verified a set of nucleotide sequence, and they do not exist at occurring in nature, can be used as the invention processExogenous nucleic acid in example, its sequence can be with reference to https: //tools.lifetechnologies.com/content/sfs/Manuals/cms_095047.txt. The addition of exogenous nucleic acid is 1ug left and right, and this addition can ensure the core of biased sampleAcid can normal extraction. In the present embodiment, testing sample is rice leaf, and its nucleic acid content is normal, therefore, does not need to mixedClose and in sample, add exogenous nucleic acid. Utilize plant genome DNA to extract kit (article No.: DP305, production company: day root biochemistryScience and technology (Beijing) Co., Ltd) method that provides by its operation manual extracts the nucleic acid of the biased sample obtaining.

The nucleic acid that step 6, utilization mix multiplex amplification primer and biased sample carries out amplified reaction, obtains amplified production,Concrete grammar is as follows:

Utilize library construction kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345) manyAfter the nucleic acid of heavy pcr amplification biased sample, utilize amplified production to build high-throughput sequencing library. This kit comprises following examinationAgent: 5 × IonAmpliSeqTMHiFiMix, FuPa reagent, conversion reagent, sequence measuring joints solution and DNA ligase. Library structureThe method of building is by the operation manual " IonAmpliSeq of this kit^TMLibraryPreparation " (publication number:MAN0006735, version: A.0) carry out. The amplification system of multiplex PCR is as follows: 5 × IonAmpliSeq^TMHiFiMix4μl、The nucleic acid 10ng of synthetic mixing multiplex amplification primer 4 μ l, the biased sample of extraction and without enzyme water 11 μ l. The amplification of multiplex PCRProgram is as follows: 99 DEG C, and 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) × 25 circulations; 10 DEG C of insulations. Utilize FuPa reagent to disappearMelt after primer unnecessary in multiplex PCR amplified production, then carry out phosphorylation, concrete grammar is: to the amplified production of multiplex PCRIn add 2 μ LFuPa reagent, after mixing, on PCR instrument by the reaction of following program: 50 DEG C, 10 minutes; 55 DEG C, 10 minutes; 60DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is the amplified production solution containing through phosphorylation. By phosphorylationAmplified production connect upper sequence measuring joints, concrete grammar is: in mixture a, add conversion reagent 4 μ L, sequence measuring joints solution 2 μL and DNA ligase 2 μ L after mixing, react by following program on PCR instrument: 22 DEG C, 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of guarantorsDeposit, obtain mixed liquor b. After utilizing the ethanol intermediate processing purifying mixed liquor b of standard, be dissolved in 10 μ L without in enzyme water. Utilize the U.S.Invitrigen company producesDsDNAHSAssayKit (article No. is Q32852) also enters according to its descriptionRow is measured, and obtains after the mass concentration of mixed liquor b, and mixed liquor b after purifying is diluted to 15ng/ml, obtains the about 100pM's of concentrationHigh-throughput sequencing library.

Step 7, utilize amplified production to carry out high-flux sequence, obtain high-flux sequence fragment, concrete grammar is as follows:

Utilize the high-throughput sequencing library and the kit IonPITemplateOT2200Kitv2 (U.S. that obtainInvirtrigen company produces, and article No. is 4485146) ePCR (EmulsionPCR, emulsion polymerization enzyme chain before checking orderReaction) amplification, method of operating is undertaken by the operation manual of this kit. Utilize ePCR product and kit IonPISequencing200Kitv2 (invirtrigen company of the U.S. produces, and article No. is 4485149) is in Proton bis-generations high passOn amount sequenator, carry out high-flux sequence, method of operating is undertaken by the operation manual of this kit. In the present embodiment, high fluxOrder-checking amount is set to 1M order-checking fragment (1M=,100 ten thousand).

According to the primer of order-checking fragment, high-flux sequence fragment is compared to the characteristic area of corresponding objective microbe monoidThe characteristic area of territory, objective microbe and with reference to the characteristic area of microorganism. Remove comparison unsuccessful imperfect with characteristic areaOrder-checking fragment, compare unsuccessful order-checking fragment and mostly be non-specific amplification product, the incomplete order-checking fragment of characteristic area isFinger could not be complete to the Sequence Detection of final position by the original position of the characteristic area in table 1.

Step 8, according to high-flux sequence fragment, objective microbe monoid and objective microbe are carried out to quantitative and qualitative analysisAnalyze, concrete grammar is as follows:

The general principle of microorganism qualitative and quantitative analysis provided by the invention is: characteristic area has represented objective microbe classGroup and objective microbe, if there is the order-checking fragment of characteristic area, show that objective microbe monoid or objective microbe exist, andThe quantity of the order-checking fragment of characteristic area has also represented the quantity of objective microbe monoid and objective microbe. With other microorganismQualitative and quantitative detects different, and the embodiment of the present invention has been calculated the reliability of microorganism qualitative and quantitative, meanwhile, has strengthened conclusionPracticality. The embodiment of the present invention need to first be put relation complicated between parameter in order, could realize the qualitative, quantitative of any microorganismDetect, and obtain reliable conclusion, design parameter of the present invention and reckoning principle thereof are in table 2. Cell, symbol and public affairs in table 2The definition of formula is identical with Excel2010, and wherein, cell " basic parameter " is A1, and other cell is pressed Excel with reference to A12010 rule defines.

Method for qualitative analysis is as follows: high-flux sequence fragment and the characteristic area of every kind of objective microbe monoid are comparedRight, in the time of difference base number≤n1, to compare successfully, corresponding high-flux sequence fragment is the characteristic area of objective microbe monoidTerritory, wherein, n1 is the fault-tolerant base number of maximum of the feature order-checking fragment of objective microbe monoid; If compare the micro-life of successful target, judge that high-flux sequence fragment is the feature order-checking fragment of objective microbe monoid when the characteristic area >=1 of thing monoid kind.

The characteristic area of the objective microbe monoid of the characteristic area of objective microbe and every kind of homology is compared,In the characteristic area of objective microbe, extract the Reference genotype of difference base composition objective microbe, the difference base is hereMicroorganism comparison in characteristic area and any one objective microbe monoid of finger objective microbe, the base there are differencesSummation. In the feature order-checking fragment of objective microbe monoid, extract the corresponding base of Reference genotype of objective microbe,The test cdna type of composition objective microbe; If the Reference genotype of the test cdna type of objective microbe and objective microbeDifference base number≤n2, wherein, the fault-tolerant base number of maximum of the feature order-checking fragment that n2 is objective microbe, objective microbeThe high-flux sequence fragment at the test cdna type place feature order-checking fragment that is objective microbe. Especially, when the micro-life of targetWhile only having comprised an objective microbe in thing monoid, the base number of Reference genotype now and test cdna type is 0,Therefore, the difference base number between them is also 0, no matter n2 is much, all by the test cdna type institute of objective microbeHigh-flux sequence fragment be judged to be the feature order-checking fragment of objective microbe. By above method, obtain respectively target micro-The characteristic fragment number of the characteristic area of biological group and objective microbe, it the results are shown in table 1. In the present embodiment, n1 and n2Value in table 2, its reckoning process sees below.

N1 makes P1≤α 1 and P3≤α 3, and wherein, it is not the feature order-checking fragment of objective microbe monoid that P1 is oneHigh-flux sequence fragment is mistaken for the feature order-checking fragment of objective microbe monoid and the false-positive probability that produces; P3 is oneThe feature order-checking fragment of objective microbe groups is mistaken for and is not that the feature of objective microbe monoid checks order fragment and producesFalse-negative probability; α 1 and α 3 are judgment threshold.

N2 makes P2≤α 2 and P4≤α 4, and wherein, it is not that the feature order-checking fragment of objective microbe is judged by accident that P2 is oneThe false-positive probability producing for the feature order-checking fragment of objective microbe; P4 is the feature order-checking sheet of an objective microorganismSection is mistaken for the false-negative probability that is not the feature order-checking fragment of objective microbe and produce; α 2 and α 4 are judgment threshold;The size of the various threshold values in the embodiment of the present invention need to be determined by reality, and for example, some germ harmfulness is very big, undetected (vacationNegative) will cause serious consequence, so, will control false negative, α 2 and α 4 values are low. If without particular/special requirement, adoptLow false positive and false negative are principle, and the present embodiment belongs to the latter, and α 1 and α 3 values are 0.01%, i.e. about 10,000 featuresThere is 1 false positive or false negative in sequence, its accuracy is very high, why can control so high accuracy, be because ofFor the m1 value in characteristic sequence is larger, be easy to distinguish with other nontarget organism, thereby by false positive rate and false negative rateAll be controlled at a very low level. The value of α 2 and α 4 is 0.5%, about 1,000 characteristic sequences occur 5 false positives orFalse negative, its accuracy is very high as seen. P1=BINOM.DIST (n1, m1,1-E, TRUE), P2=BINOM.DIST (n2, m2,1-E, TRUE), P3=1-BINOM.DIST (n1, L1, E, TRUE), P4=1-BINOM.DIST (n2, L2, E, TRUE), itsIn, m1 is discrimination, specifically refers to the discrimination corresponding to characteristic area of the objective microbe monoid for calculating S1, the present embodimentIn, the value of m1 is in table 1 and table 2; M2 is poor between the characteristic area of objective microbe and other microorganism of objective microbe monoidThe minimum of a value of different base, specifically finger is used for the value of the m2 in the objective microbe characteristic of correspondence region of calculating S3, in the present embodiment,The value of m2 is in table 1 and table 2; L1 is the length of the characteristic area of objective microbe monoid, and in the present embodiment, the value of L1 is in table 2;L2 is the length of the Reference genotype of objective microbe, and in the present embodiment, the value of L2 is in table 2; E is base error rate, its byOrder-checking error rate E1 and natural mutation rate E2 composition, in the present embodiment, the order-checking error rate E1 of PROTON high-flux sequence instrument≤1%, according to our investigation, the aberration rate between the reference genome of microorganism microspecies (as P1-P6 bacterial leaf spot microspecies)Be less than 0.5%, and natural mutation rate is lower than the aberration rate between microspecies, therefore, natural mutation rate E2≤0.5%, for thisThe method applicability of invention is wider, gets E2≤1%, and in the present embodiment, E≤2%, in order to make microorganism in the present embodimentThe probability of the conclusion accuracy of qualitative and quantitative is more reliable, gets the maximum 2% of E value and calculates. By above parameter value substitutionAfter in the formula of P1 and P3, the value of n1 is increased gradually since 0, calculate the value of P1 and P3, in the time of n1=12, calculate P1≤ α 1 and P3≤α 3, therefore, in the present embodiment, n1=12 (in table 2), the P1 that n1=12 is corresponding and the value of P3 are the present embodimentThe value of middle P1 and P3. By similar method, after in the formula of above parameter value substitution P2 and P4, by the value of n2 since 0 byCumulative adding,, calculate the value of P2 and P4, in the time of n2=2, and P2≤α 2, P4≤α 4, therefore, and in the present embodiment, n2=2 (in table 2),The P2 that n2=2 is corresponding and the value of P4 are the value of P2 and P4 in the present embodiment.

As the objective microbe monoid that only comprises an objective microbe, calculate the target obtaining micro-with reference to microorganismBiological feature order-checking fragment, is the feature order-checking fragment with reference to microorganism. With reference to the feature sheet of the characteristic area of microorganismHop count is in table 1.

If probability P 5 >=α 5 that the feature of objective microbe monoid order-checking fragment exists, judges and has order in testing sampleMark microbe groups; If probability P 5 < α 5 that the feature of objective microbe monoid order-checking fragment exists, judges in testing sample notHave objective microbe monoid, wherein, α 5 is probability guarantee, and in the present embodiment, α 5 values are 99.99%. P5=1-BINOM.DIST (S1, S1, P1, FALSE), S1 is the objective microbe monoid of the characteristic area of all objective microbe monoidThe median of quantity of feature order-checking fragment, in the present embodiment, the 2nd feature order-checking fragment of objective microbe monoidQuantity is the median of the quantity of the feature order-checking fragment of all objective microbe monoids, so the value of the present embodiment S1 is in table 1With table 2, the computing formula of the value substitution P5 of the value of S1 in the present embodiment and P1 is calculated and obtained P5 >=α 5, therefore, judge this realityExecute in example, have objective microbe monoid in testing sample, FALSE is parameter value, and BINOM.DIST function returns to monobasic binomialThe probability that formula distributes.

If probability P 6 >=α 6 that the feature of objective microbe order-checking fragment exists, judges in testing sample and exists target micro-Biological; If probability P 6 < α 6 that the feature of objective microbe order-checking fragment exists, judges and does not have the micro-life of target in testing sampleThing; α 6 is probability guarantee. In the present embodiment, α 6 values are 99.99%. P6=1-BINOM.DIST (S3, S3, P2, FALSE),BINOM.DIST function returns to the probability of monobasic binomial distribution, and S3 is that the target of characteristic area of all objective microbe is micro-The median of the quantity of biological feature order-checking fragment, in the present embodiment, the 2nd feature order-checking fragment of objective microbeQuantity is the median of the quantity of the feature order-checking fragment of all objective microbes, and the value of its corresponding S3, will in table 1 and table 2In the present embodiment, the computing formula of the value substitution P6 of the value of S3 and P2 is calculated and is obtained P6 >=α 6, therefore, judges in the present embodiment, treatsIn test sample product, there is objective microbe.

In addition, α 5 and α 6 are all that people are fixed according to actual needs, and α 5 and α 6 all can be the same or different, its differenceDepend on actual needs, in the time will strictly controlling certain microorganism, the value of α 5 and α 6 is all larger, otherwise, the value of α 5 and α 6All less. In addition, in the embodiment of the present invention, the value of all a values is all followed this principle.

Quantitative analysis method is as follows: the amount M1=Mr × S1/S2 of objective microbe monoid, wherein, Mr treats test sample for addingThe amount of reference microorganism in product, in the present embodiment, the value of Mr is in table 2; S2 is the characteristic area of all reference microorganismWith reference to the median of the quantity of the feature order-checking fragment of microorganism, in the present embodiment, with reference to the 2nd the feature order-checking of microorganismThe quantity of fragment is the median of the quantity of all features with reference to microorganism order-checking fragments, the value of its corresponding S2 in table 1 andTable 2; By in the computing formula of the value substitution M1 of above parameter and the S1 that obtains by qualitative analysis, calculate and obtain M1 value, treatIn test sample product, the amount of the microorganism in objective microbe monoid is M1=1447461.

The confidential interval of the amount of objective microbe monoid is [M11, M12], and M11 and M12 are respectively the confidential interval of M1 valueLower limit and the upper limit. M11=M1 × (1-S4/S1), M12=M1 × (1+S5/S1), wherein, S4 is the micro-life of false-positive targetQuantity and the S4=CRITBINOM (nS, P1, α 9) of the feature order-checking fragment of thing monoid, S5 is false-negative objective microbe classQuantity and the S5=CRITBINOM (S1, P3, α 9) of group's feature order-checking fragment, wherein, α 9 is probability guarantee, in the present embodiment,α 9 values are that 99.50%, CRITBINOM function returns to the minimum of a value that makes accumulation binomial distribution be more than or equal to critical value, and nS isThe high-flux sequence of the non-characteristic area that the multiplex amplification primer of the characteristic area of the objective microbe monoid of calculating S1 increasesThe quantity of fragment, refers to that other high pass the order-checking of the feature except the objective microbe fragment that multi-primers increases measuresOrder fragment. In the present embodiment, nS is produced by the multiplex amplification primer amplification of the 2nd characteristic area in objective microbe monoidThe quantity of high-flux sequence fragment of non-characteristic area, in the present embodiment, the value of nS is in table 2. By the value generation of the value of nS and P1The formula that enters S4 calculates the value that obtains S4, the formula of the value substitution S5 of the value of the present embodiment S1 and P3 is calculated to the value that obtains S5.Obtain after the value of all parameters in M11 and M12 formula, the value of M11 and M12 in calculating acquisition the present embodiment, and then obtain M1'sConfidential interval, the confidential interval of the amount of objective microbe monoid is [1447461,1447578].

Amount M2=M1 × the S3/S1 of objective microbe, by the above-mentioned formula of value substitution of M1, S3 and S1, obtains the micro-life of targetThe amount M2=1139193 of thing.

The confidential interval of the amount of objective microbe is [M21, M22], M21 and M22 be respectively M2 value confidential interval underLimit and the upper limit; M21=M2 × (1-S6/S3), M22=M2 × (1+S7/S3); Wherein, S6 is false-positive objective microbeQuantity and the S6=CRITBINOM (S1, P2, α 10) of feature order-checking fragment, the feature order-checking that S7 is false-negative objective microbeThe quantity of fragment and S7=CRITBINOM (S3, P4, α 10), wherein, α 10 is probability guarantee; CRITBINOM function returns and makes to tire outLong-pending binomial distribution is more than or equal to the minimum of a value of critical value. In the present embodiment, α 10 values are 99.50%, by the present embodiment S1 andThe value of S3, and the value substitution S6 of P2 and P4 and the computing formula of S7, calculate the value of S6 and S7. Further by S6, S7,The value substitution M21 of M1 and S3 and the computing formula of M22, calculate the value of M21 and M22, and then obtain the amount of objective microbeConfidential interval be [1135436,1139607].

Table 2 is the embodiment of the present invention one microorganism qualitative and quantitative analytical parameters and calculates principle

The qualification of embodiment bis-, human excrement and urine microorganism

Testing sample in the embodiment of the present invention two is human excrement and urine, takes from diagnosis and have the disease of enteron aisle class diseasePeople, the microorganism detecting in its ight soil is to provide foundation for therapeutic scheme. This enforcement is similar with embodiment mono-method, does not mentionMethod, parameter identical with embodiment mono-with result, therefore, no longer repeat.

Step 1, determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and do not depositBe the reference microorganism in testing sample.

The object of the present embodiment is the Salmonella enteritidis in qualification testing sample, and its Latin formal name used at school isSalmonellaenterica, in NCBI (Nationalcenterforbiotechnologyinformation, countryBiotechnology information centre) upper, totally 33 (2015 deadlines of the genomic Salmonella enteritidis biological strain of known referenceJune 2), specifically see http://www.ncbi.nlm.nih.gov/genome/genomegroups/152, these physiology are littlePlant the objective microbe monoid that forms the present embodiment. In these biological strains, SalmonellaentericaSubsp.houtenaestr.ATCCBAA-1581 is pathogenic stronger, as the objective microbe of the present embodiment.

Step 2, according to the reference genome sequence of the reference genome sequence of objective microbe monoid, objective microbe,With reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtain the spy of objective microbe monoidLevy the characteristic area of region, objective microbe and the characteristic area with reference to microorganism. The final characteristic area obtaining of the present embodimentRelevant information is in table 3.

The primer relevant information that table 3 the present embodiment provides

The acquisition methods of the present embodiment biased sample is as follows: be the ginseng of 2OD (OD is bacterium liquid maximum absorbance value) by concentrationThe bacterium liquid 0.2mL that examines microorganism is placed in after the centrifuge tube vacuum refrigeration centrifugal drying of 1.5mL, adds in 100mg testing sample,Mix, obtain testing sample and the biased sample with reference to microorganism. Count by blood cell plate, calculate acquisition and add biased sampleThe amount of reference microorganism in table 4.

In the present embodiment, testing sample is ight soil, and its nucleic acid content is lower, therefore, in biased sample, adds external sourceNucleic acid, i.e. the ERCC-00014 gene of the outside RNA contrast of 1ug association design. Utilize faeces DNA kit (production company: the U.S.MP company, product article No.: 116570200, product English name: FastDNASPINkitforfeces) by its operation manualThe method providing is extracted the nucleic acid of the biased sample obtaining.

The nucleic acid that step 6, utilization mix multiplex amplification primer and biased sample carries out amplified reaction, obtains amplified production,Concrete grammar is with embodiment mono-.

Step 7, utilize amplified production to carry out high-flux sequence, obtain high-flux sequence fragment, the same embodiment of concrete grammarOne.

Step 8, according to high-flux sequence fragment, objective microbe monoid and objective microbe are carried out to quantitative and qualitative analysisAnalyze, concrete grammar is with embodiment mono-.

Wherein, the design parameter of the embodiment of the present invention two and reckoning principle thereof are in table 4. The present embodiment two analysis results are:In testing sample, have objective microbe monoid and objective microbe, wherein, the amount of the microorganism in objective microbe monoid isM1=3942647, confidential interval is [3942647,3943113]; The amount M2=1787805 of objective microbe, confidential intervalFor [1777581,1788849].

Microorganism qualitative and quantitative analytical parameters and reckoning principle thereof that table 4 provides for the embodiment of the present invention two

The qualification of embodiment tri-, triangle Lake Water Body microorganism

Testing sample in the present embodiment is the water body in triangle lake, Wuhan, Hubei, detect microorganism in water body and be forBe subject to the situation of the pollutions such as human and animal excreta by the amount monitoring water body of microorganism. This enforcement is similar with embodiment mono-method, does not carryAnd method, parameter identical with embodiment mono-with result, therefore, no longer repeat.

Step 1, determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and do not depositBe the reference microorganism in testing sample, concrete grammar is identical with embodiment bis-with result.

Step 2, according to the reference genome sequence of the reference genome sequence of objective microbe monoid, objective microbe,With reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtain the spy of objective microbe monoidLevy the characteristic area of region, objective microbe and the characteristic area with reference to microorganism, the final characteristic area obtaining of the present embodimentRelevant information is in table 5.

The primer relevant information that table 5 embodiment of the present invention three provides

10 kilograms of water samplings from triangle lake, select 4000rpm after centrifugal 20 minutes, then use 12000rpm centrifugal 10 minutes,Collection thalline is deep. The bacterium liquid 0.2mL that is the reference microorganism of 2OD (OD is bacterium liquid maximum absorbance value) by concentration is placed inIn the centrifuge tube of 1.5mL after vacuum refrigeration centrifugal drying, add thalline deep in, mix, obtain testing sample with reference to micro-Biological biased sample. Count by blood cell plate, calculate the amount that obtains the reference microorganism that adds biased sample in table 6.

In the present embodiment, testing sample is water body, and its nucleic acid content is lower, therefore, in biased sample, adds external sourceNucleic acid, i.e. the ERCC-00014 gene of the outside RNA contrast of 1ug association design. Utilize bacterial genomes DNA to extract kit (goodsNumber: DP302, production company: TIANGEN Biotech (Beijing) Co., Ltd.) method that provides by its operation manual extracts and obtainsThe nucleic acid of biased sample.

Wherein, the design parameter of the embodiment of the present invention three and reckoning principle thereof are in table 6. The present embodiment analysis result is: treatIn test sample product, have objective microbe monoid and objective microbe, wherein, the amount of the microorganism in objective microbe monoid is M1=126522, confidential interval is [126522,126547]; The amount M2=16192 of objective microbe, confidential interval is[15809，16213]。

Table 6 is embodiment of the present invention three microbe qualitative and quantitative analytical parameters and calculates principle

The detection method that the embodiment of the present invention provides can be applied the many aspects such as agricultural, food, medical science, in different applicationIn, the separate nucleic acid method of microorganism is slightly variant, and for example, plant, food and medical science sample standard deviation have its different genome to carryGet kit, need to be respectively by their operation manual isolating nucleic acid. Except the separation method of microbial nucleic acids, other stepBasic identical, therefore, the detection method versatility that the embodiment of the present invention provides is stronger. The present invention has changed in existing method onceCan only detect a few microorganism, microbiota can only be assigned to kind, quantitatively inaccurate, testing result is without probability guarantee, needsPreculture, sense cycle are long, certain micro-organisms can not be cultivated thereby can not detect, microorganism Culturability is different and determining of causingAmount distortion, the problems such as quantitatively coarse, for microorganism detection provides a kind of comprehensive, accurate, quick, meticulous qualitative and fixedAmount new detecting method.

Claims

1. a detection method for microorganism qualitative and quantitative, is characterized in that, described method comprises:

Determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and be not present in described in treatReference microorganism in test sample product;

According to described objective microbe monoid, described objective microbe, the described ginseng with reference to microorganism and described nontarget organismExamine genome sequence, obtain the characteristic area of described objective microbe monoid, described objective microbe characteristic area and described inWith reference to the characteristic area of microorganism;

The first multiplex amplification primer of the characteristic area of the described objective microbe monoid of preparation amplification, described objective microbe increasesCharacteristic area the second multiplex amplification primer and amplification the described characteristic area with reference to microorganism the 3rd multiplex amplification primer,Described the first multiplex amplification primer, described the second multiplex amplification primer and described the 3rd multiplex amplification primer are mixed to get to mixingMultiplex amplification primer;

Extract the nucleic acid of described biased sample;

Utilize the nucleic acid of described mixing multiplex amplification primer and described biased sample to carry out amplified reaction, obtain amplified production;

According to described high-flux sequence fragment, described objective microbe monoid and described objective microbe are carried out to quantitative and qualitative analysisAnalyze.

2. method according to claim 1, is characterized in that, number >=1 of described objective microbe monoid, and eachComprise >=0 kind of described objective microbe of described objective microbe monoid;

Described objective microbe is bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw, algaeWith at least one in protozoan;

Described is bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia, conveyor screw, algae with reference to microorganismWith at least one in protozoan.

3. method according to claim 1, is characterized in that, the method for the nontarget organism in described definite testing sampleComprise: described nontarget organism is defined as to all biologies except described objective microbe monoid, if can obtain described orderThe characteristic area of mark microbe groups, described nontarget organism refers to all biologies except described objective microbe monoid;If can not obtain the characteristic area of described objective microbe monoid, described nontarget organism refers in described biased sample, except instituteState other biology outside objective microbe monoid.

4. method according to claim 1, is characterized in that, the characteristic area of described objective microbe monoid is described orderNucleotide sequence on the reference genome of microorganism in mark microbe groups; The characteristic area of described objective microbe monoidThe sequence of both sides described be unique sequence in reference to genome; The order of the both sides of the characteristic area of described objective microbe monoidBe listed in described objective microbe monoid conservative between different microorganisms; The discrimination of the characteristic area of described objective microbe monoid≥3；

The characteristic area homology of the characteristic area of described objective microbe and described objective microbe monoid; Described objective microbeM2 value >=2 of characteristic area, wherein, in the characteristic area that m2 value is described objective microbe and described objective microbe monoidThe minimum of a value of the difference base number described in other except described objective microbe between microorganism;

The described characteristic area with reference to microorganism is described with reference to the nucleotide sequence on the reference genome of microorganism; Described referenceThe sequence of the both sides of the characteristic area of microorganism is unique sequence in the described reference genome with reference to microorganism; Described referenceThe sequence of the both sides of the characteristic area of microorganism is not having homology except described in reference to other biology microorganism.

5. method according to claim 4, is characterized in that, described discrimination refers to by same described mixing multiplex amplificationDifference base number between the characteristic area of the arbitrary described objective microbe monoid of primer amplification and arbitrary non-characteristic areaLittle value, wherein, the expansion that described non-characteristic area is described mixing multiplex amplification primer taking the nucleic acid of described biased sample as templateVolume increase thing, and described non-characteristic area is not the characteristic area of described objective microbe monoid, if without described non-characteristic area,Described discrimination=3 × L1/4, wherein, L1 is the nucleotide sequence length of the characteristic area of described objective microbe monoid.

6. method according to claim 1, is characterized in that, described method also comprises:

In the time extracting the nucleic acid of described biased sample, if the content of described testing sample amplifying nucleic acid is too low, extracting described mixingClose in the process of nucleic acid of sample the exogenous nucleic acid that adds described mixing multiplex amplification primer not increase.

7. method according to claim 1, is characterized in that, described objective microbe monoid and described objective microbeMethod for qualitative analysis is as follows:

The characteristic area of described high-flux sequence fragment and every kind of described objective microbe monoid is compared, when difference baseWhen number≤n1, compare successfully, the characteristic area that corresponding described high-flux sequence fragment is described objective microbe monoid, itsIn, n1 is the fault-tolerant base number of maximum of the feature order-checking fragment of described objective microbe monoid; If compare successful described target, judge that described high-flux sequence fragment is the spy of described objective microbe monoid when the characteristic area >=1 of microbe groups kindLevy order-checking fragment;

The characteristic area of described objective microbe and the characteristic area of the described objective microbe monoid of every kind of homology are comparedRight, in the characteristic area of described objective microbe, extract the Reference genotype of objective microbe described in difference base composition; ?In the feature order-checking fragment of described objective microbe monoid, extract the corresponding alkali of Reference genotype of described objective microbeBase, forms the test cdna type of described objective microbe; If the test cdna type of described objective microbe and the micro-life of described targetThe difference base number≤n2 of the Reference genotype of thing, wherein, the maximum of the feature order-checking fragment that n2 is described objective microbe is heldWrong base number, the described high-flux sequence fragment at the test cdna type place of described objective microbe is described objective microbeFeature order-checking fragment;

Using described with reference to microorganism as the described objective microbe monoid that only comprises a described objective microbe, calculate obtainThe feature order-checking fragment of described objective microbe, be the described order-checking of the feature with reference to microorganism fragment;

If probability P 5 >=α 5 that the feature of described objective microbe monoid order-checking fragment exists, judges in described testing sample and depositsAt described objective microbe monoid, wherein, α 5 is probability guarantee; If the feature of described objective microbe monoid order-checking fragment existsProbability P 5 < α 5, judge and in described testing sample, do not have described objective microbe monoid;

If probability P 6 >=α 6 that the feature of described objective microbe order-checking fragment exists, judges and has institute in described testing sampleState objective microbe, wherein, α 6 is probability guarantee; If probability P 6 < α 6 that the feature of described objective microbe order-checking fragment exists,Judge and in described testing sample, do not have described objective microbe;

N1 makes P1≤α 1 and P3≤α 3, and wherein, it is not the feature order-checking fragment of described objective microbe monoid that P1 is oneDescribed high-flux sequence fragment be mistaken for the feature order-checking fragment of described objective microbe monoid and produce false-positive generalRate; P3 is that the feature order-checking fragment of a described objective microbe monoid is mistaken for the spy who is not described objective microbe monoidThe false-negative probability of levying order-checking fragment and produce; α 1 and α 3 are judgment threshold;

N2 makes P2≤α 2 and P4≤α 4, wherein, P2 be one be not described objective microbe feature order-checking fragment described inHigh-flux sequence fragment is mistaken for the feature order-checking fragment of described objective microbe and the false-positive probability that produces; P4 is oneDescribed in bar, the feature of objective microbe order-checking fragment is mistaken for and is not that the feature of described objective microbe checks order fragment and producesFalse-negative probability; α 2 and α 4 are judgment threshold;

P5=1-BINOM.DIST (S1, S1, P1, FALSE), P6=1-BINOM.DIST (S3, S3, P2, FALSE), S1 is instituteIn the quantity of the feature order-checking fragment of the described objective microbe monoid of the characteristic area of the described objective microbe monoid havingFigure place; S3 is the quantity of the feature order-checking fragment of the described objective microbe of the characteristic area of all described objective microbeMedian, FALSE is parameter value; BINOM.DIST function returns to the probability of monobasic binomial distribution.

8. method according to claim 7, is characterized in that, described objective microbe monoid and described objective microbeQuantitative analysis method is as follows:

Amount M1=Mr × the S1/S2 of described objective microbe monoid, the confidential interval of the amount of described objective microbe monoid is[M11, M12], wherein, Mr is the described amount with reference to microorganism adding in described testing sample; S2 is all described referencesThe median of the quantity of the described order-checking of the feature with reference to the microorganism fragment of the characteristic area of microorganism; M11 and M12 are respectively M1Lower limit and the upper limit of the confidential interval of value;

Amount M2=M1 × the S3/S1 of described objective microbe, the confidential interval of the amount of described objective microbe is [M21, M22],M21 and M22 are respectively lower limit and the upper limit of the confidential interval of M2 value;

M11＝M1×(1-S4/S1)，M12＝M1×(1+S5/S1)，M21＝M2×(1-S6/S3)，M22＝M2×(1+S7/S3); Wherein, S4 is quantity and the S4=CRITBINOM of the feature order-checking fragment of false-positive described objective microbe monoid(nS, P1, α 9), wherein, nS is the described multiplex amplification primer institute of calculating the characteristic area of the described objective microbe monoid of S1The quantity of the described high-flux sequence fragment of the described non-characteristic area of amplification; S5 is false-negative described objective microbe monoidQuantity and the S5=CRITBINOM (S1, P3, α 9) of feature order-checking fragment, wherein, α 9 is probability guarantee; S6 is false-positiveQuantity and the S6=CRITBINOM (S1, P2, α 10) of the feature order-checking fragment of described objective microbe, S7 be false-negative described inQuantity and the S7=CRITBINOM (S3, P4, α 10) of the feature order-checking fragment of objective microbe, wherein, α 10 is probability guarantee; CRITBINOM function returns to the minimum of a value that makes accumulation binomial distribution be more than or equal to critical value.

9. method according to claim 8, is characterized in that, P1=BINOM.DIST (n1, m1,1-E, TRUE), P2=BINOM.DIST(n2,m2,1-E,TRUE)，P3＝1-BINOM.DIST(n1,L1,E,TRUE)，P4＝1-BINOM.DIST(n2, L2, E, TRUE), wherein, m1 is described discrimination; Described m2 is characteristic area and the described target of described objective microbeThe minimum of a value of difference base between microorganism described in other of microbe groups; L1 is the characteristic area of described objective microbe monoidLength; L2 is the length of the Reference genotype of described objective microbe; E is base error rate.