CN105603076A - Qualitative and quantitative soil microorganism detecting method - Google Patents

Qualitative and quantitative soil microorganism detecting method Download PDF

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CN105603076A
CN105603076A CN201610060991.1A CN201610060991A CN105603076A CN 105603076 A CN105603076 A CN 105603076A CN 201610060991 A CN201610060991 A CN 201610060991A CN 105603076 A CN105603076 A CN 105603076A
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objective microbe
characteristic area
monoid
described objective
fragment
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李论
彭海
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Jianghan University
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Abstract

The invention discloses a qualitative and quantitative soil 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 detection method of edaphon qualitative and quantitative
Technical field
The present invention relates to biological technical field, particularly a kind of detection side of edaphon qualitative and quantitativeMethod.
Background technology
Edaphon is the important indicator of environmental pollution, is also being related to the safety of food, and therefore, soil is micro-Biological accurately qualitative and quantitative detecting & monitoring is very necessary.
Existing edaphon qualitative and quantitative detection technique comprises morphology counting, chip detection, 16SrRNAOrder-checking, grand gene order-checking and real-time quantitative PCR (PolymeraseChainReaction, polymerase chainReaction). Morphology count detection need to be carried out preculture to microorganism, and length consuming time can not be cultivated microorganism notCan detect, once only can detect a kind of microorganism, flux is low, and in the time of counting, amount of sampling is limited, and resultCoarse, cannot distinguish planting following taxon. The DNA amount of the required testing sample of chip detectionGreatly, need to carry out preculture and enrichment processing to microorganism, testing result is inaccurate, and cannot do quantitative inspectionSurvey. 16SrRNA order-checking cannot be distinguished planting following taxon. The grand gene order-checking degree of depth is limited,Quantitative accuracy in detection for the microorganism of low content is very poor. Real-time quantitative PCR once can only detect oneMicroorganism, flux is low. In addition, the total defect of existing method is, cannot calculate microorganism qualitative and quantitativeReliability, makes conclusion poor practicability.
Summary of the invention
Detect inaccurate problem, the embodiment of the present invention in order to solve microorganism qualitative and quantitative in prior artA kind of detection method of edaphon qualitative and quantitative is provided. Described technical scheme is as follows:
The embodiment of the present invention provides a kind of detection method of edaphon qualitative and quantitative, described method bagDraw together:
Determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and do not depositBe the reference microorganism in described testing sample, described testing sample is soil;
According to described objective microbe monoid, described objective microbe, described with reference to microorganism and described non-orderMark biological reference genome sequence, obtain the characteristic area of described objective microbe monoid, described target is micro-Biological characteristic area and the described characteristic area with reference to microorganism;
Described in the first multiplex amplification primer of the characteristic area of the described objective microbe monoid of preparation amplification, amplificationThe second multiplex amplification primer of the characteristic area of objective microbe and the described characteristic area with reference to microorganism of amplificationThe 3rd multiplex amplification primer, by described the first multiplex amplification primer, described the second multiplex amplification primer and instituteState the 3rd multiplex amplification primer and be mixed to get mixing multiplex amplification primer;
Describedly can not increase with reference to microorganism and described mixing multiplex amplification primer to adding in described testing sampleExogenous nucleic acid, 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 and expandVolume increase thing;
Utilize described amplified production to carry out high-flux sequence, obtain high-flux sequence fragment;
According to described high-flux sequence fragment, described objective microbe monoid and described objective microbe are carried outQualitative and quantitative analysis.
Particularly, number >=1 of described objective microbe monoid, and each described objective microbe monoid bagDraw together >=0 kind of described objective microbe;
Described objective microbe is that bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, clothing are formerAt least one in body, conveyor screw, algae and protozoan;
Described is that bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, clothing are former with reference to microorganismAt least one in body, conveyor screw, algae and protozoan.
Particularly, the method for the nontarget organism in described definite testing sample comprises: by raw described non-targetThing is defined as all biologies except described objective microbe monoid, if can obtain described objective microbe classGroup's characteristic area, 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 to described aggregate sampleIn product, other biology except described objective microbe monoid.
Particularly, the characteristic area of described objective microbe monoid is the micro-life in described objective microbe monoidNucleotide sequence on the reference genome of thing; The sequence of the both sides of the characteristic area of described objective microbe monoidDescribed 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 characteristic area of described objective microbe monoidDiscrimination >=3 in territory;
The characteristic area homology of the characteristic area of described objective microbe and described objective microbe monoid; DescribedM2 value >=2 of the characteristic area of objective microbe, wherein, the characteristic area that m2 value is described objective microbeAnd the difference base described in other in described objective microbe monoid except described objective microbe between microorganismThe minimum of a value of number;
The described characteristic area with reference to microorganism is described with reference to the nucleotide sequence on the reference genome of microorganism;The sequence of the both sides of the described characteristic area with reference to microorganism is in the described reference genome with reference to microorganismUnique sequence; The sequence of the both sides of the described characteristic area with reference to microorganism except described with reference to microorganismIn other biology, do not there is homology.
Further, described discrimination refers to by described in same described mixing multiplex amplification primer amplification arbitraryThe minimum of a value of the difference base number between the characteristic area of objective microbe monoid and arbitrary non-characteristic area, wherein,The amplification that described non-characteristic area is described mixing multiplex amplification primer taking the nucleic acid of described biased sample as templateProduct, and described non-characteristic area is not the characteristic area of described objective microbe monoid, if without described non-spyLevy region, described discrimination=3 × L1/4, wherein, L1 is the characteristic area of described objective microbe monoidNucleotide sequence length.
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,In the time of difference base number≤n1, to compare successfully, corresponding described high-flux sequence fragment is that described target is micro-The characteristic area of biological group, wherein, n1 is the maximum of the feature order-checking fragment of described objective microbe monoidFault-tolerant base number; If while comparing characteristic area >=a kind of successful described objective microbe monoid, judge instituteStating high-flux sequence fragment is the feature order-checking fragment of described objective microbe monoid;
By the characteristic area of the described objective microbe monoid of the characteristic area of described objective microbe and every kind of homologyComparing in territory, extracts the micro-life of target described in difference base composition in the characteristic area of described objective microbeThe Reference genotype of thing; In the feature order-checking fragment of described objective microbe monoid, extract described target micro-The biological corresponding base of Reference genotype, forms the test cdna type of described objective microbe; If described inThe difference base number≤n2 of the test cdna type of objective microbe and the Reference genotype of described objective microbe,Wherein, the fault-tolerant base number of maximum of the feature order-checking fragment that n2 is described objective microbe, described target is micro-The feature order-checking sheet that the described high-flux sequence fragment at biological test cdna type place is described objective microbeSection;
Using described with reference to microorganism as the described objective microbe monoid that only comprises a described objective microbe,The feature order-checking fragment of calculating the described objective microbe obtaining, is the described order-checking of the feature with reference to microorganismFragment;
If probability P 5 >=α 5 that the feature of described objective microbe monoid order-checking fragment exists, treats described in judgementIn test sample product, have described objective microbe monoid, wherein, α 5 is probability guarantee; If described objective microbeProbability P 5 < α 5 that the feature order-checking fragment of monoid exists, judges and in described testing sample, does not have described targetMicrobe groups;
If probability P 6 >=α 6 that the feature of described objective microbe order-checking fragment exists, treats test sample described in judgementIn product, have described objective microbe, wherein, α 6 is probability guarantee; If the feature of described objective microbe is surveyedProbability P 6 < α 6 that order fragment exists, 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 spy of described objective microbe monoid that P1 is oneThe described high-flux sequence fragment of levying order-checking fragment is mistaken for the feature order-checking sheet of described objective microbe monoidSection and produce false-positive probability; P3 is that the feature of a described objective microbe monoid checks order fragment by mistakeBe judged to the false-negative probability that is not the feature order-checking fragment of described objective microbe monoid and produce; α 1 and α 3For judgment threshold;
N2 makes P2≤α 2 and P4≤α 4, and wherein, it is not the feature survey of described objective microbe that P2 is oneThe described high-flux sequence fragment of order fragment is mistaken for the feature order-checking fragment of described objective microbe and producesFalse-positive probability; P4 is described in the feature order-checking fragment of a described objective microbe is mistaken for and is notThe feature order-checking fragment of objective microbe and the false-negative probability that produces; 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 the feature order-checking of the described objective microbe monoid of the characteristic area of all described objective microbe monoidThe median of the quantity of fragment; S3 is the micro-life of described target of the characteristic area of all described objective microbeThe median of the quantity of the feature order-checking fragment of thing, FALSE is parameter value; BINOM.DIST function returnsThe 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 amount of described objective microbe monoid put letterInterval is [M11, M12], and wherein, Mr is the described amount with reference to microorganism adding in described testing sample;S2 is the number of the described order-checking of the feature with reference to the microorganism fragment of all described characteristic area with reference to microorganismThe median of amount; M11 and M12 are respectively lower limit and the upper limit of the confidential interval of M1 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 the number of the feature order-checking fragment of false-positive described objective microbe monoidAmount and S4=CRITBINOM (nS, P1, α 9), wherein, nS is the described objective microbe monoid that calculates S1The described high-flux sequence fragment of the described non-characteristic area that the described multiplex amplification primer of characteristic area increasesQuantity; S5 is quantity and the S5=CRIT of the feature order-checking fragment of false-negative described objective microbe monoidBINOM (S1, P3, α 9), wherein, α 9 is probability guarantee; S6 is the spy of false-positive described objective microbeThe quantity and the S6=CRITBINOM (S1, P2, α 10) that levy order-checking fragment, S7 is the micro-life of false-negative described targetQuantity and the S7=CRITBINOM (S3, P4, α 10) of the feature order-checking fragment of thing, wherein, α 10 is probability guarantorBarrier; 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 is described discrimination; Described m2 be described objective microbe characteristic area with described inThe minimum of a value of difference base between microorganism described in other of objective microbe monoid; L1 is described objective microbeThe length of the characteristic area of monoid; L2 is the length of the Reference genotype of described objective microbe; E is baseError 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 needTo carry out preculture and propagation to microorganism, consuming time short, can detect multiple-microorganism simultaneously, flux is high, meterWhen number, amount of sampling is large, and testing result is meticulous, can distinguish taxon, without a large amount of DNA alsoAvoid enrichment culture, detection architecture noiselessness and accurate, high for the quantitative precision of low content microorganism,And testing result for microorganism quantitative and qualitative analysis is accurate, resolution ratio is high, highly sensitive, have a probability guarantee,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, below will be to embodiment of the present inventionBe described in further detail. In the present invention, the reagent of mark explanation is conventional commercial reagent, mostlyNumber biotech company all can buy and almost indifference of effect.
The qualification of embodiment, triangle lake edaphon
Testing sample in the present embodiment is the soil in triangle lake, Wuhan, Hubei, detects the microorganism in soilIt is the situation that is subject to the pollutions such as human and animal excreta for the amount monitoring soil by microorganism.
Step 1, determine objective microbe monoid, objective microbe and nontarget organism in testing sample,And be not present in 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;Objective microbe can be bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, Chlamydia,At least one in conveyor screw, algae and protozoan. The object of the present embodiment is in qualification testing sampleSalmonella enteritidis, the main source of this bacterium is human and animal excreta. Triangle lake next-door neighbour Jianghan University and multiple lifeCommunity, is subject to the possibility of fecal pollution larger. , the Latin formal name used at school of Salmonella enteritidis is Salmonellaenterica,In NCBI (Nationalcenterforbiotechnologyinformation, NCBI)Upper, totally 33 (June 2 2015 deadline of the genomic Salmonella enteritidis biological strain of known referenceDay), 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.
Can be that bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, clothing are former with reference to microorganismAt least one in body, conveyor screw, algae and protozoan. Be not present in testing sample with reference to microorganism.Quantitatively providing for the objective microbe monoid in testing sample and objective microbe with reference to the effect of microorganismA reference. Because Agrobacterium tumefaciems is present in plant roots, thus be not present in testing sample, therefore,Choose in the present embodiment Agrobacterium tumefaciems as with reference to microorganism, 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 asAll biologies except objective microbe monoid, if can obtain the characteristic area of objective microbe monoid,Nontarget organism refers to all biologies except objective microbe monoid, and wherein, all biologies refer to have ginsengExamining genomic biology, is the strictest standard of nontarget organism. In the present embodiment, non-target is rawThing be defined as outside objective microbe monoid all oneself know the biochron, can find objective microbe monoidCharacteristic area (acquisition process of characteristic area sees below, and the results are shown in Table 1), therefore, the non-order in the present embodimentMark biology is the zoic set except objective microbe monoid.
Nontarget organism is defined as to all biologies except objective microbe monoid, if can not obtain targetThe characteristic area of microbe groups, nontarget organism refers in biased sample, except objective microbe monoidOther biology, to dwindle the scope of nontarget organism, increase and find the characteristic area of objective microbe monoidPossibility. In biased sample, other biology except objective microbe monoid can rule of thumb determine,For example, in the present embodiment, biased sample comprises the soil in water body, in biased sample, can not have landRaw plant and obligatory parasitism be in the microorganism of terrestrial plant, therefore, if by true nontarget organism in the present embodimentBe decided to be outside objective microbe monoid all oneself know the biochron, cannot obtain the characteristic area of objective microbe,Non-objective microbe can be defined as except objective microbe, terrestrial plant, obligatory parasitism are in terrestrial plantBiological set outside microorganism.
Step 2, according to the reference gene of the reference genome sequence of objective microbe monoid, objective microbeGroup sequence, with reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtainThe characteristic area of objective microbe monoid, the characteristic area of objective microbe and the characteristic area with reference to microorganism,Concrete grammar is as follows:
The characteristic area of objective microbe monoid is on the reference genome of the microorganism in objective microbe monoidNucleotide sequence; The sequence of the both sides of the characteristic area of objective microbe monoid is single in reference to genomeSequence; The sequence of the both sides of the characteristic area of objective microbe monoid is different micro-lifes in objective microbe monoidConservative between thing; Discrimination >=3 of the characteristic area of objective microbe monoid. Non-characteristic area is not the micro-life of targetThe characteristic area of thing monoid, non-characteristic area refers to and mixes multiplex amplification primer taking the nucleic acid of biased sample as mouldThe amplified production of plate; Discrimination refers to the arbitrary objective microbe class by same mixing multiplex amplification primer amplificationThe minimum of a value of difference base number between group's characteristic area and arbitrary non-characteristic area, if characteristic area nothing but,Discrimination=3 × L1/4, wherein, L1 is the nucleotide sequence length of the characteristic area of objective microbe monoid.
Particularly, the characteristic area of objective microbe monoid is used for representing objective microbe monoid, the micro-life of targetThe characteristic area of thing monoid exists, and represents that objective microbe monoid exists, the feature of objective microbe monoidThe quantity of the order-checking fragment in region, represents the quantity of objective microbe monoid. Desirable objective microbe monoidOnly the increase characteristic area of objective microbe monoid of the multi-primers of characteristic area, the non-target that can not increase is rawThing. This just requires the both sides sequence of the characteristic area of objective microbe monoid, and design of primers region is at non-orderHomology not in mark biology, so, nontarget organism can not be amplified, and can not produce non-characteristic area. ThisTime, between characteristic area and non-characteristic area, can only produce at random identical base, totally 4 kinds of bases, identical with notSame probability is respectively 1/4 and 3/4, and therefore, discrimination is 3 × L1/4. The characteristic area of objective microbe monoidDiscrimination >=3rd in territory, for the false positive rate and vacation that ensure that the feature order-checking fragment of objective microbe monoid judgesNegative rate is all lower, and its principle is in table 2. The sequence of the both sides of the characteristic area of objective microbe monoid is in targetConservative between different microorganisms in microbe groups, just can be with in identical primer amplification objective microbe monoidDifferent microorganisms, to get rid of amplification efficiency to the relative quantification between objective microbe monoid different microorganismsImpact.
The characteristic area homology of the characteristic area of objective microbe and objective microbe monoid; Objective microbeM2 value >=2 of characteristic area, wherein, the characteristic area that m2 value is objective microbe and objective microbe monoidThe minimum of a value of the difference base number between interior other microorganism except objective microbe. Other in the present embodimentMicroorganism refers to other biological strain in objective microbe monoid except objective microbe, and m2 value is that target is micro-Biological characteristic area respectively with objective microbe monoid in other biological strain homology region comparison, institute obtainsMinimum of a value in the difference base number obtaining. When objective microbe qualitative and quantitative is analyzed, emphasis is and targetIn microbe groups, other microorganism is distinguished. Objective microbe and objective microbe monoid affiliation are pastToward nearer, between sequence, similitude is high, therefore, is difficult to distinguish. In the time that objective microbe qualitative and quantitative is analyzed,Only pay close attention in amplicon and in objective microbe monoid discrepant Reference genotype between other microorganism, subtractedLack the source of error, thereby can better objective microbe have been distinguished in objective microbe monoid.In the time of m2 >=2, judge false positive rate and the false negative rate of the feature order-checking fragment that order-checking fragment is objective microbeAll lower, therefore, objective microbe can be distinguished from objective microbe monoid, 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 micro-The sequence of the both sides of biological characteristic area is unique sequence in the reference genome with reference to microorganism; ReferenceThe sequence of the both sides of the characteristic area of microorganism not having 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 toThe difference of object detecting, also can be using the microorganism with specific gene sequence as objective microbe monoid,And the characteristic area using specific gene sequence as objective microbe monoid. For example, can be specific by havingThe microorganism of Disease-causing gene is as objective microbe monoid, and spy using this Disease-causing gene as objective microbeLevy region, so that according to the type of Disease-causing gene, direction of medication usage. Equally, drug resistance gene is as specific baseBecause sequence also can direction of medication usage.
The first multiplex amplification primer, the amplification of the characteristic area of step 3, preparation amplification objective microbe monoidThe second multiplex amplification primer of the characteristic area of objective microbe and amplification are with reference to the of the characteristic area of microorganismThree multiplex amplification primers, draw the first multiplex amplification primer, the second multiplex amplification primer and the 3rd multiplex amplificationThing is mixed to get and mixes multiplex amplification primer.
Integrating step two and step 3 concrete grammar are as follows:
At ftp: download different physiology in objective microbe monoid in //ftp.ncbi.nlm.nih.gov/genomes/ littleThe genome sequence of planting, adopts software Megablast (version 2 .2.26) by their genome and querySequence (reference sequences) compares, and in the present embodiment, query sequence is that the upper reception number of NCBI isThe genome sequence of CM001471. Each parameter of Megablast software comparison is set to: parameter-e is set to1e-5; Parameter-p is set to 0; Can is Shuoed – v and is set to 5000; Parameter-m is set to 1. After having compared,Obtain the homologous sequence between all microorganisms of objective microbe monoid, therefrom select only in query sequenceThere is the homologous sequence of 1 time. Taking 110bp as window size, taking 10bp as step-length, the homology order of selectingIn row, do window translation. The window obtaining for translation each time, relatively obtains at least in objective microbe classThe base there are differences between two kinds of microorganisms in group, intercepts in this window from first difference base to finallyThe region that difference base is stopped is as characteristic area, and adds up the quantity of the difference base in this characteristic area.To the each development length in both sides of characteristic area be the region of 160bp-characteristic area length as the primer field of search,In the primer field of search, search exists length to be greater than 20bp and in objective microbe monoid between all microorganismsWithout any the region of base difference, as the design of primers district of characteristic area, abandon lacking design of primers districtCharacteristic area.
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,Multiplex PCR will divide multitube to carry out, and cost can increase to some extent. And select the primer of single-pool only to needA multiplex PCR, saves cost, but the design of primers that shortcoming is some characteristic area may be failed,But because the number of the characteristic area on genome is more, design of primers failure in a few features region does not affect knotReally, so the present embodiment is selected single-pool. By the feature of all objective microbe monoids that obtain above(N represents in A, T, C and tetra-kinds of bases of G for region and corresponding 100 base N for design of primers district thereofAny one) couple together, be generated as the reference genome of a design of primers. At " SelectthegenomeYouwishtouse " select in option, after " Custom ", to upload the reference genome of the design of primers of generation.DNAType option is selected " StandardDNA ", in AddHotspot option, fills in characteristic area and existsInitial sum final position in the reference genome of the design of primers generating. Finally click " Submittargets "The multiplex amplification primer sequence of the characteristic area of objective microbe monoid is submitted and obtained to button to.
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 selecting in forward and reverse primer has at least to have a specific primer. By select primer again withThe genome of nontarget organism does BLASTN compare of analysis, checks their nontarget organisms that whether can increaseGenome. In the present embodiment, nontarget organism is all biologies except objective microbe monoid, non-orderMark the NT/NR storehouse that biological genome is NCBI. Judge that the standard that primer can increase is: the amplification head of districtDegree is no more than 200bp, and 5 bases that primer matching length is greater than 15bp and primer 3 ' end do not have a base with interiorDisappearance or mispairing. Any nontarget organism if primer can not increase, now, the micro-life of the corresponding target of primerDiscrimination m1=3 × the L1/4 of the characteristic area of thing, the part nontarget organism if primer can increase, just shouldThe characteristic area of the amplified production of arbitrary nontarget organism of primer amplification and arbitrary objective microbe monoid carries outComparison, obtains in all comparisons, and the minimum of a value of difference base number is discrimination m1, retains the order of m1 >=3Mark microbe groups characteristic area, further remove contain simple repeated sequence or on genome for copying moreThe characteristic area of shellfish. From the characteristic area of objective microbe monoid retaining, the further micro-life of selected objective targetThe characteristic area of the characteristic area of thing monoid select target microorganism.
Further, the method for optimizing of the characteristic area of objective microbe monoid is as follows: by characteristic area and non-The reference genome of target organism does BLASTN comparison, removes with nontarget organism and has 95% above homologyThe characteristic area of property, by other the micro-life in objective microbe and objective microbe monoid of remaining characteristic areaBetween thing, utilize software muscle (version: V3.6) to compare by its default parameters, obtain difference base numberMinimum of a value, i.e. m2 value. Retain the characteristic area of the objective microbe monoid of m2 >=2, from the spy who retainsLevy in region, all larger 2 and 2 above characteristic areas are simultaneously to select arbitrarily discrimination m1 and m2As the characteristic area of objective microbe monoid and the characteristic area of objective microbe, the multiplex amplification that it is correspondingPrimer is simultaneously as the first multiplex amplification primer and the second multiplex amplification primer.
Press the method similar with the characteristic area of finding objective microbe monoid, obtain with reference to microorganism featureRegion and the 3rd corresponding multiplex amplification primer thereof, below emphasis difference is described, something in common is no longer heavyMultiple description. The same software Megablast (version 2 .2.26) that adopts is to reference to microbial genome and querySequence (reference sequences) compares, and query sequence is AgrobacteriumtumefaciensK84Genome sequence. After having compared, in the reference microbial genome obtaining, only in query sequenceThere is the unique sequence of 1 time. The NT/NR storehouse of unique sequence and NCBI is compared, abandoned in non-target rawIn thing, there is the unique sequence of homologous sequence, random choose nonoverlapping 110bp length from unique sequenceAs characteristic area, the sequence of its both sides is as design of primers region. In the online design grid of multiplex amplification primerPage https: the multiplex amplification primer in the upper design feature of //ampliseq.com region, further screening has successfully designedThe characteristic area of multiplex amplification primer, concrete grammar is as follows: remove and contain simple repeated sequence or at genomeThe upper characteristic area for multicopy, does the reference genome of remaining characteristic area and nontarget organismBLASTN comparison, removes the characteristic area that has 95% above homology with nontarget organism. From retainingIn the characteristic area coming, more than 2 and 2 characteristic area of random choose is as the spy with reference to microbe groupsLevy region, its corresponding multiplex amplification primer is as the 3rd multiplex amplification primer.
By Sangon Biotech's synthetic above the first multiplex amplification obtaining one by oneEach heavy amplimer in primer, the second multiplex amplification primer and the 3rd multiplex amplification primer and by oftenThe template sequence of the amplification that individual multiplex amplification primer pair is answered, template sequence refers to that each multiplex amplification primer filling outEnter the amplification region of AddHotspot option. According to the StepOne real-time quantitative of match Mo Feishier company of the U.S.The operation manual (PartNumber4376784Rev.E) of PCR instrument detects the expansion of each multiplex amplification primerIncreasing Efficiency, only retains amplification efficiency at 95%～105% multiplex amplification primer, to reduce the poor of amplification efficiencyThe different impact on microorganism qualitative and quantitative. Because amplification efficiency impact is less, therefore, objective microbe classGroup also can be different from the characteristic area of objective microbe, more easily find respectively characteristic area separately to facilitateTerritory. By the first multiplex amplification primer, the second multiplex amplification primer and the 3rd multiplex amplification primer that obtain aboveThe multiplex amplification primer remaining designs webpage https online by multiplex amplification primer: on //ampliseq.comConsolidation procedure merge, obtain and mix multiplex amplification primer, mix multiplex amplification primer silent by U.S.'s matchAfter Fei Shier company is synthetic, provide with liquid form. The final characteristic area relevant information obtaining of the present embodimentIn table 1. Original position in table 1 and final position refer to the reference gene of characteristic area in query sequenceInitial sum final position in group.
The primer relevant information that table 1 the present embodiment provides
Step 4, in testing sample, add with reference to microorganism and mix that multiplex amplification primer can not increase outsideSource nucleic acid, obtains biased sample, and 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 carry out operation repetitive with the microorganism in testing sample, to objective microbe monoid and order in testing sampleMicroorganism carries out quantitatively mark. It is the biased sample that approximately can extract 10ng with reference to the addition control of microorganismNucleic acid (DNA), normally to build high-throughput sequencing library, meanwhile, with reference to the addition of microorganism again notExcessive as for making with reference to the shared ratio of microorganism, take too much high-flux sequence data volume. This enforcementThe acquisition methods of example 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 from threeIn 0.25 gram of the soil sample of getting in lake, angle, mix, obtain testing sample and biased sample with reference to microorganism.Count by blood cell plate, calculate the amount that obtains the reference microorganism that adds biased sample in table 2.
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),By affecting the extraction of nucleic acid of biased sample, can, in the leaching process of the nucleic acid of biased sample, addMix the exogenous nucleic acid that multiplex amplification primer can not increase. The exogenous nucleic acid adding does not exist and occurring in nature,Thereby do not disturb microorganism detection. Outside RNA contrasts association and has designed and verified a set of nucleotide sequence, itDo not exist at occurring in nature, can be used as the exogenous nucleic acid in the embodiment of the present invention, its sequence can be with reference to httPs: //tools.lifetechnologies.com/content/sfs/manuals/cms_09504 7.txt. Adding of exogenous nucleic acidEntering amount is 1ug left and right, and this addition can ensure that the nucleic acid of biased sample can normal extraction. In this enforcementIn example, testing sample is soil, and its nucleic acid content is lower, therefore, in biased sample, adds exogenous nucleic acid,It is the ERCC-00014 gene of the outside RNA contrast of 1ug association design. Utilize soil genomic DNA to carryGet kit (article No.: DP336, production company: TIANGEN Biotech (Beijing) Co., Ltd.) by its behaviourThe method providing as handbook is extracted the nucleic acid of the biased sample obtaining.
The nucleic acid that step 5, utilization mix multiplex amplification primer and biased sample carries out amplified reaction, obtains and expandsVolume increase thing, concrete grammar is as follows:
Utilize library construction kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345)After the nucleic acid of multiplex PCR amplification biased sample, utilize amplified production to build high-throughput sequencing library. This reagentBox comprises following reagent: 5 × IonAmpliSeqTMHiFiMix, FuPa reagent, conversion reagent, order-checking connectSolution and DNA ligase. The method of library construction is by the operation manual " IonAmpliSeq of this kitTMLibraryPreparation " (publication number: MAN0006735, version: A.0) carry out. Multiplex PCRAmplification system is as follows: 5 × IonAmpliSeqTMHiFiMix4 μ l, synthetic mixing multiplex amplification primer 4 μ l,Extract biased sample nucleic acid 10ng and without enzyme water 11 μ l. The amplification program of multiplex PCR is as follows: 99 DEG C,2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) × 25 circulations; 10 DEG C of insulations. Utilize FuPa reagentDigest after primer unnecessary in multiplex PCR amplified production, then carry out phosphorylation, concrete grammar is: Xiang DuoIn the amplified production of heavy PCR, add 2 μ LFuPa reagent, after mixing, anti-by following program on PCR instrumentShould: 50 DEG C, 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a,Mixture a is the amplified production solution containing through phosphorylation. The amplified production of phosphorylation is connected to upper order-checkingJoint, concrete grammar is: to add in mixture a conversion reagent 4 μ L, sequence measuring joints solution 2 μ L andDNA 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 preservations, obtain mixed liquor b. Utilize after the ethanol intermediate processing purifying mixed liquor b of standardBe dissolved in 10 μ L without in enzyme water. Utilize American I nvitrigen company to producedsDNAHSAssayKit (article No. is Q32852) also measures according to its description, obtains after the mass concentration of mixed liquor b,Mixed liquor b after purifying is diluted to 15ng/ml, obtains the high-throughput sequencing library of the about 100pM of concentration.
Step 6, utilize amplified production to carry out high-flux sequence, obtain high-flux sequence fragment, concrete grammarAs follows:
Utilize the high-throughput sequencing library and the kit IonPITemplateOT2200Kitv2 (U.S. that obtainInvirtrigen company produces, and article No. is 4485146) ePCR (EmulsionPCR, breast before checking orderChange polymerase chain reaction) amplification, method of operating is undertaken by the operation manual of this kit. Utilize ePCR productWith kit IonPISequencing200Kitv2 (invirtrigen company of the U.S. produces, and article No. is 4485149)On Proton bis-generations high-flux sequence instrument, carry out high-flux sequence, method of operating is by the operator of this kitVolume carries out. In the present embodiment, high-flux sequence 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 corresponding objective microbe monoidThe characteristic area of characteristic area, objective microbe and with reference to the characteristic area of microorganism. Remove comparison unsuccessfulWith the incomplete order-checking fragment of characteristic area, compare unsuccessful order-checking fragment and mostly be non-specific amplification product,The incomplete order-checking fragment of characteristic area refers to could not be by the original position of the characteristic area in table 1 to stop bitThe Sequence Detection of putting is complete.
Step 7, according to high-flux sequence fragment, carry out qualitative to objective microbe monoid and objective microbeAnd quantitative analysis, concrete grammar is as follows:
The general principle of microorganism qualitative and quantitative analysis provided by the invention is: characteristic area has represented that target is micro-Biological group and objective microbe, if there is the order-checking fragment of characteristic area, show objective microbe monoid orObjective microbe exists, and the quantity of the order-checking fragment of characteristic area has also represented objective microbe monoid and orderThe quantity of mark microorganism. Different from other microorganism qualitative and quantitative detection, the embodiment of the present invention has been calculated micro-The reliability of biological qualitative and quantitative, meanwhile, has strengthened the practicality of conclusion. The embodiment of the present invention needs elder generationPut relation complicated between parameter in order, could realize the qualitative and quantitative analysis of any microorganism, and obtain reliableConclusion, design parameter of the present invention and calculate principle in table 2. Cell, symbol and formula in table 2Define identically with Excel2010, wherein, cell " basic parameter " is A1, and other cell is with reference to A1Define by the rule of Excel2010.
Method for qualitative analysis is as follows: by the characteristic area of high-flux sequence fragment and every kind of objective microbe monoidCompare, in the time of difference base number≤n1, compare successfully, corresponding high-flux sequence fragment is targetThe characteristic area of microbe groups, wherein, n1 is that the maximum of the feature order-checking fragment of objective microbe monoid is heldWrong base number; If while comparing characteristic area >=a kind of successful objective microbe monoid, judge that high pass measuresOrder fragment is the feature order-checking fragment of objective microbe monoid.
The characteristic area of objective microbe and the characteristic area of the objective microbe monoid of every kind of homology are comparedRight, in the characteristic area of objective microbe, extract the Reference genotype of difference base composition objective microbe,The difference base here refers to micro-in the characteristic area of objective microbe and any one objective microbe monoidRelatively biological, the summation of the base there are differences. In the feature order-checking fragment of objective microbe monoid, carryGet the corresponding base of Reference genotype of objective microbe, the test cdna type of composition objective microbe; IfThe difference base number≤n2 of the test cdna type of objective microbe and the Reference genotype of objective microbe, wherein,N2 is the fault-tolerant base number of maximum of the feature order-checking fragment of objective microbe, the test cdna of objective microbeThe feature order-checking fragment that the high-flux sequence fragment at type place is objective microbe. Especially, when the micro-life of targetWhile only having comprised an objective microbe in thing monoid, the base of Reference genotype now and test cdna typeNumber be 0, therefore, the difference base number between them is also 0, no matter n2 is much, all byThe high-flux sequence fragment at the test cdna type place of objective microbe is judged to be the feature order-checking of objective microbeFragment. By above method, obtain respectively the spy of the characteristic area of objective microbe monoid and objective microbeLevy segments, it the results are shown in table 1. In the present embodiment, the value of n1 and n2 is in table 2, its reckoning processSee below.
N1 makes P1≤α 1 and P3≤α 3, and wherein, it is not the feature order-checking of objective microbe monoid that P1 is oneThe high-flux sequence fragment of fragment is mistaken for the feature order-checking fragment of objective microbe monoid and the vacation sun that producesThe probability of property; P3 is that the feature order-checking fragment of an objective microbe groups is mistaken for and is not objective microbeThe feature order-checking fragment of monoid 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 objective microbe that P2 is oneThe false-positive probability that is mistaken for the feature order-checking fragment of objective microbe and produce; P4 is that an objective is micro-Biological feature order-checking fragment is mistaken for the false negative that is not the feature order-checking fragment of objective microbe and produceProbability; α 2 and α 4 are judgment threshold; The size of the various threshold values in the embodiment of the present invention is needed really by realityFixed, for example, some germ harmfulness is very big, and undetected (false negative) will cause serious consequence, so,Will control false negative, α 2 and α 4 values are low. If without particular/special requirement, adopt lower false positive and false negativeFor principle, the present embodiment belongs to the latter, and α 1 and α 3 values are 0.01%, i.e. about 10,000 characteristic sequencesOccur 1 false positive or false negative, its accuracy is very high, why can control so high accuratelyProperty, be because the m1 value in characteristic sequence is larger, be easy to distinguish with other nontarget organism, thus willFalse positive rate and false negative rate are all controlled at a very low level. The value of α 2 and α 4 is 0.5%, largeArticle approximately 1,000, there is 5 false positives or false negative in characteristic sequence, and 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), wherein, m1 is discrimination,Specifically refer to the discrimination corresponding to characteristic area of the objective microbe monoid for calculating S1, in the present embodiment,The value of m1 is in table 1 and table 2; M2 is that other of the characteristic area of objective microbe and objective microbe monoid is micro-The minimum of a value of difference base between biology, specifically refers to the objective microbe characteristic of correspondence region for calculating S3The value of m2, in the present embodiment, the value of m2 is in table 1 and table 2; L1 is the characteristic area of objective microbe monoidThe length in territory, in the present embodiment, the value of L1 is in table 2; L2 is the length of the Reference genotype of objective microbeDegree, in the present embodiment, the value of L2 is in table 2; E is base error rate, its by check order error rate E1 and fromSo mutation rate E2 composition, in the present embodiment, order-checking error rate E1≤1% of PROTON high-flux sequence instrument,According to our investigation, the change between the reference genome of microorganism microspecies (as P1-P6 bacterial leaf spot microspecies)Different rate is generally less than 0.5%, and natural mutation rate is lower than the aberration rate between microspecies, therefore, and natural mutationRate E2≤0.5%, wider for method applicability of the present invention, get E2≤1%, in the present embodiment, E≤2%,In order to make the probability of conclusion accuracy of qualitative and quantitative of microorganism in the present embodiment more reliable, get E valueMaximum 2% calculate. After in the formula of above parameter value substitution P1 and P3, by the value of n1 from0 starts to increase gradually, calculates the value of P1 and P3, in the time of n1=13, calculates P1≤α 1 and P3≤α 3,Therefore, in the present embodiment, n1=13 (in table 2), the P1 that n1=13 is corresponding and the value of P3 are in the present embodimentThe value of P1 and P3. By similar method, after in the formula of above parameter value substitution P2 and P4, by n2Value increase gradually since 0, calculate the value of P2 and P4, in the time of n2=2, P2≤α 2, P4≤α 4,Therefore, in the present embodiment, n2=2 (in table 2), the P2 that n2=2 is corresponding and the value of P4 are P in the present embodiment2 and the value of P4.
With reference to microorganism, as the objective microbe monoid that only comprises an objective microbe, calculating obtainsThe feature order-checking fragment of objective microbe, is the feature order-checking fragment with reference to microorganism. With reference to microorganismThe characteristic fragment number of characteristic area is in table 1 and table 2.
If probability P 5 >=α 5 that the feature of objective microbe monoid order-checking fragment exists, judges in testing sample and depositsAt objective microbe monoid; If probability P 5 < α 5 that the feature of objective microbe monoid order-checking fragment exists, sentencesIn disconnected testing sample, do not have objective microbe monoid, wherein, α 5 is probability guarantee, in the present embodiment, and α 5Value is 99.99%. P5=1-BINOM.DIST (S1, S1, P1, FALSE), S1 is all objective microbesThe median of the quantity of the feature order-checking fragment of the objective microbe monoid of the characteristic area of monoid, in this enforcementIn example, the quantity of the 2nd feature order-checking fragment of objective microbe monoid is all objective microbe monoidsThe median of the quantity of feature order-checking fragment, so the value of the present embodiment S1 is in table 1 and table 2, by this enforcementIn example, the computing formula of the value substitution P5 of the value of S1 and P1 is calculated and is obtained P5 >=α 5, therefore, judges this enforcementIn example, have objective microbe monoid in testing sample, FALSE is parameter value, BINOM.DIST functionReturn to the probability of monobasic binomial distribution.
If probability P 6 >=α 6 that the feature of objective microbe order-checking fragment exists, judges and has order in testing sampleMark microorganism; If probability P 6 < α 6 that the feature of objective microbe order-checking fragment exists, judges in testing sampleThere is not objective microbe; 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 monobasic binomial distributionProbability, S3 is the quantity of the feature order-checking fragment of the objective microbe of the characteristic area of all objective microbeMedian, in the present embodiment, the quantity of the 2nd feature order-checking fragment of objective microbe is all ordersThe median of the quantity of the feature order-checking fragment of mark microorganism, 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, and judgementIn the present embodiment, in testing sample, 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 identical can be not yetWith, actual needs is depended in its difference, in the time will strictly controlling certain microorganism, the value of α 5 and α 6 is allGreatly, on the contrary the value of α 5 and α 6 is all less. In addition, in the embodiment of the present invention, the value of all a values is equalFollow this principle.
Quantitative analysis method is as follows: the amount M1=Mr × S1/S2 of objective microbe monoid, wherein, Mr is for addingThe amount of reference microorganism in testing sample, in the present embodiment, the value of Mr is in table 2; S2 is all referencesThe median of the quantity of the feature order-checking fragment of the reference microorganism of the characteristic area of microorganism, at the present embodimentIn, be all order-checking of the features with reference to microorganism sheets with reference to the quantity of the 2nd feature order-checking fragment of microorganismThe median of the quantity of section, the value of its corresponding S2 is in table 1 and table 2; By above parameter with by qualitative pointAnalyse in the computing formula of value substitution M1 of the S1 of acquisition, calculate and obtain M1 value, in testing sample, orderThe amount of microorganism in mark microbe groups is M1=14354.
The confidential interval of the amount of objective microbe monoid is [M11, M12], and M11 and M12 are respectively M1 valueLower limit and the upper limit of confidential interval. M11=M1 × (1-S4/S1), M12=M1 × (1+S5/S1), wherein, S4 be the quantity of feature order-checking fragment of false-positive objective microbe monoid and S4=CRITBINOM (nS, P1,α 9), S5 is quantity and the S5=CRITBINOM (S of the feature order-checking fragment of false-negative objective microbe monoid1, P3, α 9), wherein, α 9 is probability guarantee, in the present embodiment, α 9 values are 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 is the target of calculating S1The high-flux sequence fragment of the non-characteristic area that the multiplex amplification primer of the characteristic area of microbe groups increasesQuantity, refer to other height the order-checking of the feature except the objective microbe fragment that multi-primers increasesFlux order-checking fragment. In the present embodiment, nS is the multiple of the 2nd characteristic area in objective microbe monoidThe increase quantity of high-flux sequence fragment of the non-characteristic area that produces of amplimer, in the present embodiment, nSValue in table 2. The formula of the value substitution S4 of the value of nS and P1 is calculated to the value that obtains S4, by this enforcementThe formula of the example value of S1 and the value substitution S5 of P3 calculates the value that obtains S5. Obtain M11 and M12 formulaIn after the value of all parameters, calculate the value that obtains M11 and M12 in the present embodiment, and then obtain putting of M1Letter is interval, and the confidential interval of the amount of objective microbe monoid is [14354,14361].
Amount M2=M1 × the S3/S1 of objective microbe, by the above-mentioned formula of value substitution of M1, S3 and S1, obtainsThe amount M2=3136 that obtains objective microbe is individual.
The confidential interval of the amount of objective microbe is [M21, M22], and M21 and M22 are respectively putting of M2 valueBelieve interval lower limit and the upper limit; M21=M2 × (1-S6/S3), M22=M2 × (1+S7/S3); Wherein, S6 isQuantity and the S6=CRITBINOM (S1, P2, α 10) of the feature order-checking fragment of false-positive objective microbe, S7For quantity and the S7=CRITBINOM (S3, P4, α 10) of the feature order-checking fragment of false-negative objective microbe,Wherein, α 10 is probability guarantee; CRITBINOM function returns to that accumulation binomial distribution is more than or equal to is criticalThe minimum of a value of value. In the present embodiment, α 10 values are 99.50%, by the value of the present embodiment S1 and S3, withAnd the value substitution S6 of P2 and P4 and the computing formula of S7, calculate the value of S6 and S7. Further willThe value substitution M21 of S6, S7, M1 and S3 and the computing formula of M22, calculate M21 and M22Value, and then the confidential interval of amount that obtains objective microbe is for [3078,3144].
Table 2 is the present embodiment microorganism qualitative and quantitative analytical parameters and calculates principle
The present invention changed in existing method, once can only detect a few microorganism, can only be by microbiotaAssign to kind, quantitatively inaccurate, testing result without probability guarantee, need that preculture, sense cycle are long, certain slightlyBiological can not cultivate thereby can not detect, the different and quantization distortion that causes, quantitatively thick of microorganism CulturabilityThe problems such as rough, detect for microorganism detection provides a kind of comprehensive, quick, meticulous qualitative and quantitativeNew method.

Claims (8)

1. a detection method for edaphon qualitative and quantitative, is characterized in that, described method comprises:
Determine objective microbe monoid, objective microbe and the nontarget organism in testing sample and do not depositBe the reference microorganism in described testing sample, described testing sample is soil;
According to described objective microbe monoid, described objective microbe, described with reference to microorganism and described non-orderMark biological reference genome sequence, obtain the characteristic area of described objective microbe monoid, described target is micro-Biological characteristic area and the described characteristic area with reference to microorganism;
Described in the first multiplex amplification primer of the characteristic area of the described objective microbe monoid of preparation amplification, amplificationThe second multiplex amplification primer of the characteristic area of objective microbe and the described characteristic area with reference to microorganism of amplificationThe 3rd multiplex amplification primer, by described the first multiplex amplification primer, described the second multiplex amplification primer and instituteState the 3rd multiplex amplification primer and be mixed to get mixing multiplex amplification primer;
Describedly can not increase with reference to microorganism and described mixing multiplex amplification primer to adding in described testing sampleExogenous nucleic acid, 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 and expandVolume increase thing;
Utilize described amplified production to carry out high-flux sequence, obtain high-flux sequence fragment;
According to described high-flux sequence fragment, described objective microbe monoid and described objective microbe are carried outQualitative and quantitative analysis.
2. method according to claim 1, is characterized in that, the number of described objective microbe monoid>=1, and comprise >=0 kind of described objective microbe of each described objective microbe monoid;
Described objective microbe is that bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, clothing are formerAt least one in body, conveyor screw, algae and protozoan;
Described is that bacterium, virus, fungi, actinomyces, Richettsia, mycoplasma, clothing are former with reference to microorganismAt least one in body, conveyor screw, algae and protozoan.
3. method according to claim 1, is characterized in that, the non-order in described definite testing sampleMarking biological method comprises: described nontarget organism is defined as to the institute except described objective microbe monoidHave biology, if can obtain the characteristic area of described objective microbe monoid, described nontarget organism refers to except instituteState all biologies outside objective microbe monoid; If can not obtain the characteristic area of described objective microbe monoidTerritory, described nontarget organism refers in described biased sample, other except described objective microbe monoidBiological.
4. method according to claim 1, is characterized in that, the feature of described objective microbe monoidRegion is the nucleotide sequence on the reference genome of the microorganism in described objective microbe monoid; Described targetThe sequence of the both sides of the characteristic area of microbe groups described be unique sequence in reference to genome; Described orderThe sequence of both sides of the characteristic area of mark microbe groups is in described objective microbe monoid between different microorganismsConservative; 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; DescribedM2 value >=2 of the characteristic area of objective microbe, wherein, the characteristic area that m2 value is described objective microbeAnd the difference base described in other in described objective microbe monoid except described objective microbe between microorganismThe minimum of a value of number;
The described characteristic area with reference to microorganism is described with reference to the nucleotide sequence on the reference genome of microorganism;The sequence of the both sides of the described characteristic area with reference to microorganism is in the described reference genome with reference to microorganismUnique sequence; The sequence of the both sides of the described characteristic area with reference to microorganism except described with reference to microorganismIn other biology, do not there is homology.
5. method according to claim 4, is characterized in that, described discrimination refers to by described in sameMix characteristic area and arbitrary non-characteristic area of the arbitrary described objective microbe monoid of multiplex amplification primer amplificationThe minimum of a value of the difference base number between territory, wherein, described non-characteristic area is described mixing multiplex amplification primerAmplified production taking the nucleic acid of described biased sample as template, and described non-characteristic area is not that described target is micro-The characteristic area of biological group, if without described non-characteristic area, described discrimination=3 × L1/4, wherein, L1For the nucleotide sequence length of the characteristic area of described objective microbe monoid.
6. method according to claim 1, is characterized in that, described objective microbe monoid and described inThe method for qualitative analysis of objective microbe is as follows:
The characteristic area of described high-flux sequence fragment and every kind of described objective microbe monoid is compared,In the time of difference base number≤n1, to compare successfully, corresponding described high-flux sequence fragment is the micro-life of described targetThe characteristic area of thing monoid, wherein, n1 is that the maximum of the feature order-checking fragment of described objective microbe monoid is heldWrong base number; If while comparing characteristic area >=a kind of successful described objective microbe monoid, described in judgementHigh-flux sequence fragment is the feature order-checking fragment of described objective microbe monoid;
By the characteristic area of the described objective microbe monoid of the characteristic area of described objective microbe and every kind of homologyComparing in territory, extracts the micro-life of target described in difference base composition in the characteristic area of described objective microbeThe Reference genotype of thing; In the feature order-checking fragment of described objective microbe monoid, extract described target micro-The biological corresponding base of Reference genotype, forms the test cdna type of described objective microbe; If described inThe difference base number≤n2 of the test cdna type of objective microbe and the Reference genotype of described objective microbe, itsIn, the fault-tolerant base number of maximum of the feature order-checking fragment that n2 is described objective microbe, the micro-life of described targetThe feature order-checking fragment that the described high-flux sequence fragment at the test cdna type place of thing is described objective microbe;
Using described with reference to microorganism as the described objective microbe monoid that only comprises a described objective microbe,The feature order-checking fragment of calculating the described objective microbe obtaining, is the described order-checking of the feature with reference to microorganismFragment;
If probability P 5 >=α 5 that the feature of described objective microbe monoid order-checking fragment exists, judges described to be measuredIn sample, have described objective microbe monoid, wherein, α 5 is probability guarantee; If described objective microbe classProbability P 5 < α 5 that group's feature order-checking fragment exists, judges in described testing sample and does not exist described target micro-Biological group;
If probability P 6 >=α 6 that the feature of described objective microbe order-checking fragment exists, judges described testing sampleIn there is described objective microbe, wherein, α 6 is probability guarantee; If the feature of described objective microbe order-checkingProbability P 6 < α 6 that fragment exists, 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 of described objective microbe monoid that P1 is oneThe described high-flux sequence fragment of order-checking fragment is mistaken for the feature order-checking fragment of described objective microbe monoidAnd the false-positive probability producing; P3 is that the feature order-checking fragment of a described objective microbe monoid is judged by accidentFor not being the false-negative probability that the feature order-checking fragment of described objective microbe monoid produces; α 1 and α 3For judgment threshold;
N2 makes P2≤α 2 and P4≤α 4, and wherein, it is not the feature order-checking of described objective microbe that P2 is oneThe feature order-checking fragment that the described high-flux sequence fragment of fragment is mistaken for described objective microbe producesFalse-positive probability; P4 is that the feature order-checking fragment of a described objective microbe is mistaken for and is not described orderThe false-negative probability of marking the feature order-checking fragment of microorganism and produce; 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 the feature order-checking of the described objective microbe monoid of the characteristic area of all described objective microbe monoidThe median of the quantity of fragment; S3 is the micro-life of described target of the characteristic area of all described objective microbeThe median of the quantity of the feature order-checking fragment of thing, FALSE is parameter value; BINOM.DIST function returnsThe probability of monobasic binomial distribution.
7. method according to claim 6, is characterized in that, described objective microbe monoid and described inThe quantitative analysis method of objective microbe is as follows:
Amount M1=Mr × the S1/S2 of described objective microbe monoid, the amount of described objective microbe monoid put letterInterval is [M11, M12], and wherein, Mr is the described amount with reference to microorganism adding in described testing sample;S2 is the number of the described order-checking of the feature with reference to the microorganism fragment of all described characteristic area with reference to microorganismThe median of amount; M11 and M12 are respectively lower limit and the upper limit of the confidential interval of M1 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 be false-positive described objective microbe monoid feature order-checking fragment quantity andS4=CRITBINOM (nS, P1, α 9), wherein, nS is the feature of calculating the described objective microbe monoid of S1The number of the described high-flux sequence fragment of the described non-characteristic area that the described multiplex amplification primer in region increasesAmount; S5 is quantity and the S5=CRITBIN of the feature order-checking fragment of false-negative described objective microbe monoidOM (S1, P3, α 9), wherein, α 9 is probability guarantee; S6 is that the feature of false-positive described objective microbe is surveyedThe quantity of order fragment and S6=CRITBINOM (S1, P2, α 10), S7 is false-negative described objective microbeQuantity and the S7=CRITBINOM (S3, P4, α 10) of feature order-checking fragment, 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.
8. method according to claim 7, 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 isDifference alkali between microorganism described in other of the characteristic area of described objective microbe and described objective microbe monoidThe minimum of a value of base; 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.
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