CN105567831A - Qualitative and quantitative detection method for food microorganisms - Google Patents

Qualitative and quantitative detection method for food microorganisms Download PDF

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CN105567831A
CN105567831A CN201610061063.7A CN201610061063A CN105567831A CN 105567831 A CN105567831 A CN 105567831A CN 201610061063 A CN201610061063 A CN 201610061063A CN 105567831 A CN105567831 A CN 105567831A
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target microorganism
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microorganism
character zone
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CN105567831B (en
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陈利红
方治伟
彭海
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Jianghan University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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Abstract

The invention a qualitative and quantitative detection method for food microorganisms, and belongs to the field of biotechnology. The method includes the following steps that target microorganism groups, target microorganisms and non-target microorganisms in a sample to be detected and reference microorganisms not existing in the sample to be detected are determined; characteristic regions of the target microorganism groups and the target microorganisms are designed; multiplex amplification primers of the characteristic regions are designed; nucleic acid of the microorganisms in the sample to be detected is extracted after the reference microorganisms and exogenous nucleic acid are added into the sample to be detected; nucleic acid of the microorganisms is amplified with the designed multiplex amplification primers to obtain characteristic sequence fragments; the microorganisms in the sample to be detected are qualitatively and quantitatively analyzed with the characteristic sequence fragments. By means of the method, multiple known microorganisms in the sample to be detected can be detected at a time with high throughput, high accuracy and a high identification rate without pre-culturing or multiplying the microorganisms, the detection process is simple and fast, and the procedures are standard.

Description

A kind of detection method of food microorganisms qualitative and quantitative
Technical field
The present invention relates to biological technical field, particularly a kind of detection method of meat-based food microorganism qualitative and quantitative.
Background technology
Food easily pollutes by multiple harmful microorganism, works the mischief to the life and health of people, and therefore, food microorganisms accurately qualitative and quantitative detection are very necessary.
Existing food microorganisms qualitative and quantitative detection technique comprises morphology counting, chip detection, 16SrRNA order-checking, grand gene order-checking and real-time quantitative PCR (PolymeraseChainReaction, polymerase chain reaction).Morphology count detection needs to carry out preculture to microorganism, length consuming time, and uncultured microorganisms can not detect, once only can detect a kind of microorganism, flux is low, and when counting, amount of sampling is limited, and result is coarse, cannot distinguish the following taxon of kind.The DNA amount of the testing sample needed for chip detection is large, and need to carry out preculture and enrichment process to microorganism, detected result is inaccurate, and cannot do detection by quantitative.16SrRNA order-checking cannot be distinguished the following taxon of kind.The grand gene order-checking degree of depth is limited, and the detection by quantitative accuracy for the microorganism of low levels is very poor.Real-time quantitative PCR once can only detect a kind of microorganism, and flux is low.In addition, existing method has defect and is, cannot calculate the reliability of microorganism qualitative and quantitative, makes conclusion poor practicability.
Summary of the invention
Detecting inaccurate problem to solve microorganism qualitative and quantitative in prior art, embodiments providing a kind of detection method of meat-based food microorganism qualitative and quantitative.Described technical scheme is as follows:
Embodiments provide a kind of detection method of meat-based food microorganism qualitative and quantitative, described method comprises:
The reference microorganism determined target microorganism monoid, target microorganism and nontarget organism in testing sample and be not present in described testing sample, described testing sample is food;
According to described target microorganism monoid, described target microorganism, described reference the genome sequence with reference to microorganism and described nontarget organism, obtain the character zone of the character zone of described target microorganism monoid, the character zone of described target microorganism and described reference microorganism;
3rd multiplex amplification primer of the preparation amplification first multiplex amplification primer of character zone of described target microorganism monoid, the second multiplex amplification primer of the character zone of the described target microorganism that increases and the described character zone with reference to microorganism that increases, is mixed to get mixing multiplex amplification primer by described first multiplex amplification primer, described second multiplex amplification primer and described 3rd multiplex amplification primer;
Add described with reference to microorganism 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 amplified production;
Utilize described amplified production to carry out high-flux sequence, obtain high-flux sequence fragment;
According to described high-flux sequence fragment, qualitative and quantitative analysis is carried out to described target microorganism monoid and described target microorganism.
Particularly, number >=1 of described target microorganism monoid, and each described target microorganism monoid comprises >=0 kind of described target microorganism;
Described target microorganism is at least one in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon;
Described reference microorganism is at least one in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon.
Particularly, the method of the described nontarget organism determined in testing sample comprises: described nontarget organism is defined as all biologies except described target microorganism monoid, if can obtain the character zone of described target microorganism monoid, then described nontarget organism refers to all biologies except described target microorganism monoid; If can not obtain the character zone of described target microorganism monoid, then described nontarget organism refers in described biased sample, and other except described target microorganism monoid is biological.
Particularly, the character zone of described target microorganism monoid is the nucleotide sequence on the reference genome of microorganism in described target microorganism monoid; The sequence of the both sides of the character zone of described target microorganism monoid is unique sequence in described reference genome; The sequence of the both sides of the character zone of described target microorganism monoid is conservative between different microorganisms in described target microorganism monoid; Discrimination >=3 of the character zone of described target microorganism monoid;
The character zone of described target microorganism and the character zone homology of described target microorganism monoid; M2 value >=2 of the character zone of described target microorganism, wherein, the minimum value of the distinguishing base number described in other in the character zone that m2 value is described target microorganism and described target microorganism monoid except described target microorganism between microorganism;
The described character zone 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 character zone with reference to microorganism is unique sequence in the reference genome of described reference microorganism; The sequence of the both sides of the described character zone with reference to microorganism does not have homology in other biology except described reference microorganism.
Further, described discrimination refers to the minimum value by the distinguishing base number between the character zone of the arbitrary described target microorganism monoid of same described mixing multiplex amplification primer amplification and arbitrary non-character zone, wherein, described non-character zone is the amplified production that described mixing multiplex amplification primer is template with the nucleic acid of described biased sample, and described non-character zone is not the character zone of described target microorganism monoid, if without described non-character zone, then described discrimination=3 × L1/4, wherein, L1 is the nucleotide sequence length of the character zone of described target microorganism monoid.
Particularly, when extracting the nucleic acid of described biased sample, if the content of described testing sample amplifying nucleic acid is too low, then, in the process of nucleic acid extracting described biased sample, add the exogenous nucleic acid that described mixing multiplex amplification primer can not increase.
Particularly, the method for qualitative analysis of described target microorganism monoid and described target microorganism is as follows:
The character zone of described high-flux sequence fragment with often kind of described target microorganism monoid is compared, as distinguishing base number≤n1, then comparison success, corresponding described high-flux sequence fragment is the character zone of described target microorganism monoid, wherein, n1 is the maximum fault-tolerant base number of the feature sequenced fragments of described target microorganism monoid; If during character zone >=a kind of the successful described target microorganism monoid of comparison, then judge that described high-flux sequence fragment is the feature sequenced fragments of described target microorganism monoid;
The character zone of the character zone of described target microorganism with the described target microorganism monoid of often kind of homology is compared, in the character zone of described target microorganism, extracts the Reference genotype that distinguishing base forms described target microorganism; On the feature sequenced fragments of described target microorganism monoid, extract the base corresponding to Reference genotype of described target microorganism, form the test cdna type of described target microorganism; If the distinguishing base number≤n2 of the Reference genotype of the test cdna type of described target microorganism and described target microorganism, wherein, n2 is the maximum fault-tolerant base number of the feature sequenced fragments of described target microorganism, then the described high-flux sequence fragment at the test cdna type place of described target microorganism is the feature sequenced fragments of described target microorganism;
Using described reference microorganism as the described target microorganism monoid only comprising a described target microorganism, calculate the feature sequenced fragments of the described target microorganism obtained, be the described feature sequenced fragments with reference to microorganism;
If probability P 5 >=α 5 that the feature sequenced fragments of described target microorganism monoid exists, then judge to there is described target microorganism monoid in described testing sample, wherein, α 5 is probability guarantee; If the probability P 5< α 5 that the feature sequenced fragments of described target microorganism monoid exists, then judge in described testing sample, to there is not described target microorganism monoid;
If probability P 6 >=α 6 that the feature sequenced fragments of described target microorganism exists, then judge to there is described target microorganism in described testing sample, wherein, α 6 is probability guarantee; If the probability P 6< α 6 that the feature sequenced fragments of described target microorganism exists, then judge there is not described target microorganism in described testing sample;
N1 makes P1≤α 1 and P3≤α 3, wherein, and the false-positive probability that the feature sequenced fragments that the described high-flux sequence fragment of P1 to be one be not the feature sequenced fragments of described target microorganism monoid is mistaken for described target microorganism monoid produces; P3 is that the feature sequenced fragments of a described target microorganism monoid is mistaken for is not the false-negative probability that the feature sequenced fragments of described target microorganism monoid produces; α 1 and α 3 is judgment threshold;
N2 makes P2≤α 2 and P4≤α 4, wherein, and the false-positive probability that the feature sequenced fragments that the described high-flux sequence fragment of P2 to be one be not the feature sequenced fragments of described target microorganism is mistaken for described target microorganism produces; P4 is that the feature sequenced fragments of a described target microorganism is mistaken for is not the false-negative probability that the feature sequenced fragments of described target microorganism produces; α 2 and α 4 is judgment threshold;
P5=1-BINOM.DIST (S1, S1, P1, FALSE), P6=1-BINOM.DIST (S3, S3, P2, FALSE), S1 is the median of the quantity of the feature sequenced fragments of the described target microorganism monoid of the character zone of all described target microorganism monoids; S3 is the median of the quantity of the feature sequenced fragments of the described target microorganism of the character zone of all described target microorganisms, and FALSE is parameter value; BINOM.DIST function returns the probability of unitary binomial distribution.
Further, the quantitative analysis method of described target microorganism monoid and described target microorganism is as follows:
Amount M1=Mr × the S1/S2 of described target microorganism monoid, the fiducial interval of the amount of described target microorganism monoid is [M11, M12], and wherein, Mr is the described amount with reference to microorganism added in described testing sample; S2 is the median of the quantity of the described feature sequenced fragments with reference to microorganism of all described character zones with reference to microorganism; M11 and M12 is respectively lower limit and the upper limit of the fiducial interval of M1 value;
Amount M2=M1 × the S3/S1 of described target microorganism, the fiducial interval of the amount of described target microorganism is [M21, M22], M21 and M22 is respectively lower limit and the upper limit of the fiducial 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 quantity of the feature sequenced fragments of false-positive described target microorganism monoid and S4=CRITBINOM (nS, P1, α 9), wherein, the quantity of the described high-flux sequence fragment of the described non-character zone that the described multiplex amplification primer that nS is the character zone calculating the described target microorganism monoid of S1 increases; S5 is the quantity of the feature sequenced fragments of false-negative described target microorganism monoid and S5=CRITBINOM (S1, P3, α 9), and wherein, α 9 is probability guarantee; S6 is the quantity of the feature sequenced fragments of false-positive described target microorganism and S6=CRITBINOM (S1, P2, α 10), S7 is the quantity of the feature sequenced fragments of false-negative described target microorganism and S7=CRITBINOM (S3, P4, α 10), wherein, α 10 is probability guarantee; CRITBINOM function returns the minimum value making accumulation binomial distribution be more than or equal to threshold 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; The minimum value of distinguishing base between microorganism described in other of the character zone that described m2 is described target microorganism and described target microorganism monoid; L1 is the length of the character zone of described target microorganism monoid; L2 is the length of the Reference genotype of described target microorganism; 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 to carry out preculture and propagation to microorganism, consuming time short, multiple-microorganism can be detected simultaneously, flux is high, during counting, amount of sampling is large, detected result is meticulous, can distinguish taxon, avoid enrichment culture without the need to a large amount of DNA, detection architecture noiselessness and accurately, quantitative precision for low levels microorganism is high, and it is accurate for the detected result of microorganism quantitative and qualitative analysis, resolving power is high, highly sensitive, there is probability guarantee, testing process is simple, fast and standard process.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail.The reagent not marking explanation in the present invention is conventional commercial reagent, all can buy and effect almost indifference in most of biotech company.
Embodiment one
The qualification of pork contaminating microorganisms
Testing sample is food, and the testing sample in the present embodiment is the raw pork that Zhuankou market, Wuhan is bought, and detects in testing sample the microorganism that may pollute and can determine this raw pork edible whether safety.
Step one, the reference microorganism determined target microorganism monoid, target microorganism and nontarget organism in testing sample and be not present in testing sample, concrete grammar is as follows:
Number >=1 of target microorganism monoid, and each target microorganism monoid comprises >=0 kind of target microorganism; Target microorganism can be at least one in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon.The object of the present embodiment is the Salmonella enteritidis in qualification testing sample, is the main germ of food poisoning.The Classification system of Salmonella enteritidis is Salmonellaenterica, at NCBI (Nationalcenterforbiotechnologyinformation, NCBI) on, the genomic Salmonella enteritidis physiological strain of known reference totally 33 (June 2 2015 dead line), specifically see http://www.ncbi.nlm.nih.gov/genome/genomegroups/152, these physiological strains form the target microorganism monoid of the present embodiment.In these physiological strains, Salmonellaentericasubsp.houtenaestr.ATCCBAA-1581 is pathogenic comparatively strong, as the target microorganism of the present embodiment.
With reference at least one that microorganism can be in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon.Be not present in testing sample with reference to microorganism.Quantitatively provide a reference for the target microorganism monoid in testing sample and target microorganism with reference to the effect of microorganism.Because agrobacterium tumefaciens is present in roots of plants, so be not present in testing sample, therefore, choose agrobacterium tumefaciens in the present embodiment as reference microorganism, its Classification system is AgrobacteriumtumefaciensK84.
Particularly, determine that the method for the nontarget organism in testing sample comprises: nontarget organism is defined as all biologies except target microorganism monoid, if the character zone of target microorganism monoid can be obtained, then nontarget organism refers to all biologies except target microorganism monoid, wherein, all biologies refer to have with reference to genomic biology, are the strictest standards of nontarget organism.In the present embodiment, nontarget organism is defined as outside target microorganism monoid all oneself know the biochron, (acquisition process of character zone sees below can to find the character zone of target microorganism monoid, the results are shown in Table 1), therefore, the nontarget organism in the present embodiment is the zoic set except target microorganism monoid.
Nontarget organism is defined as all biologies except target microorganism monoid, if the character zone of target microorganism monoid can not be obtained, then nontarget organism refers in biased sample, other except target microorganism monoid is biological, to reduce the scope of nontarget organism, increase the possibility finding the character zone of target microorganism monoid.In biased sample, other biology except target microorganism monoid can rule of thumb be determined, such as, in the present embodiment, biased sample comprises meat and reference microorganism, then can not there is plant and obligatory parasitism in biased sample in the microorganism of plant, therefore, if nontarget organism in the present embodiment is defined as outside target microorganism monoid all oneself know the biochron, cannot obtain the character zone of target microorganism, then non-targeted microorganism can be defined as the set except target microorganism, plant, the obligatory parasitism biology except the microorganism of plant.
Step 2, according to the reference genome sequence of the reference genome sequence of target microorganism monoid, target microorganism, with reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtain the character zone of target microorganism monoid, the character zone of target microorganism and the character zone with reference to microorganism, concrete grammar is as follows:
The character zone of target microorganism monoid is the nucleotide sequence on the reference genome of the microorganism in target microorganism monoid; The sequence of the both sides of the character zone of target microorganism monoid is unique sequence in reference genome; The sequence of the both sides of the character zone of target microorganism monoid is conservative between different microorganisms in target microorganism monoid; Discrimination >=3 of the character zone of target microorganism monoid.Non-character zone is not the character zone of target microorganism monoid, and non-character zone refers to the amplified production that mixing multiplex amplification primer is template with the nucleic acid of biased sample; Discrimination refers to the minimum value by the distinguishing base number between the character zone of arbitrary target microorganism monoid of same mixing multiplex amplification primer amplification and arbitrary non-character zone, if character zone nothing but, then discrimination=3 × L1/4, wherein, L1 is the nucleotide sequence length of the character zone of target microorganism monoid.
Particularly, the character zone of target microorganism monoid is used for representing target microorganism monoid, and the character zone of target microorganism monoid exists, then represent target microorganism monoid and exist, the quantity of the sequenced fragments of the character zone of target microorganism monoid, represents the quantity of target microorganism monoid.The multi-primers of the character zone of desirable target microorganism monoid only increases the character zone of target microorganism monoid, and can not increase nontarget organism.This just requires the both sides sequence of the character zone of target microorganism monoid, i.e. design of primers region not homology in nontarget organism, and so, nontarget organism can not be amplified, and can not produce non-character zone.Now, can only produce identical base at random between character zone with non-character zone, base totally 4 kinds, identical from different probability is respectively 1/4 and 3/4, and therefore, discrimination is 3 × L1/4.Discrimination >=3 of the character zone of target microorganism monoid are that the false positive rate that judges of the feature sequenced fragments in order to ensure target microorganism monoid and false negative rate are all lower, and its principle is in table 2.The sequence of the both sides of the character zone of target microorganism monoid is conservative between different microorganisms in target microorganism monoid, just can by the different microorganisms in identical primer amplification target microorganism monoid, to get rid of the impact of amplification efficiency on the relative quantification between target microorganism monoid different microorganisms.
The character zone of target microorganism and the character zone homology of target microorganism monoid; M2 value >=2 of the character zone of target microorganism, wherein, m2 value is the minimum value of the distinguishing base number between the character zone of target microorganism and interior other microorganism except target microorganism of target microorganism monoid.Other microorganism in the present embodiment refers to other physiological strain except target microorganism in target microorganism monoid, m2 value is that the character zone of target microorganism compares with other physiological strain homology region in target microorganism monoid respectively, the minimum value in the distinguishing base number obtained.During target microorganism quantification and qualification, emphasis distinguishes with other microorganism in target microorganism monoid.Target microorganism and target microorganism monoid sibship are often comparatively near, and between sequence, similarity is high, therefore, are difficult to distinguish.When target microorganism quantification and qualification, only to pay close attention in amplicon and discrepant Reference genotype between other microorganism in target microorganism monoid, decrease the source of error, thus can better target microorganism be distinguished in target microorganism monoid.When m2 >=2, judge that sequenced fragments is that the false positive rate of the feature sequenced fragments of target microorganism and false negative rate are all lower, therefore, target microorganism can be distinguished from target microorganism monoid, its principle is in table 2.
Character zone with reference to microorganism is with reference to the nucleotide sequence on the reference genome of microorganism; Sequence with reference to the both sides of the character zone of microorganism is unique sequence in reference to the reference genome of microorganism; Sequence with reference to the both sides of the character zone of microorganism does not have homology in reference to other biology except microorganism.
In the present embodiment, discrimination is the sole selection criteria of the character zone of target microorganism monoid, according to the difference of testing goal, also the microorganism of specific gene sequence can will be had as target microorganism monoid, and using the character zone of specific gene sequence as target microorganism monoid.Such as, can will have the microorganism of specific Disease-causing gene as target microorganism monoid, and using the character zone of this Disease-causing gene as target microorganism, so that according to the type of Disease-causing gene, direction of medication usage.Equally, drug resistance gene also can direction of medication usage as specific gene sequence.
First multiplex amplification primer, the second multiplex amplification primer and the 3rd multiplex amplification primer, with reference to the 3rd multiplex amplification primer of the character zone of microorganism, are mixed to get mixing multiplex amplification primer by step 3, the first multiplex amplification primer of character zone of preparation amplification target microorganism monoid, the second multiplex amplification primer of the character zone of amplification target microorganism and amplification.
Integrating step two and step 3 concrete grammar as follows:
At ftp: the genome sequence downloading the different physiological strains in target microorganism monoid in //ftp.ncbi.nlm.nih.gov/genomes/, software Megablast (version 2 .2.26) is adopted their genome and query sequence (reference sequences) to be compared, in the present embodiment, query sequence is NCBI receives number genome sequence being CM001471.Each optimum configurations of Megablast software comparison is: parameter-e is set to 1e-5; Parameter-p is set to 0; Can Shuo – v is set to 5000; Parameter-m is set to 1.After comparison completes, obtain the homologous sequence between all microorganisms of target microorganism monoid, therefrom select the homologous sequence only occurring 1 time in query sequence.Be window size with 110bp, take 10bp as step-length, in the homologous sequence selected, do window translation.For the window that translation each time obtains, relatively obtain the base that at least there are differences between two kinds of microorganisms in target microorganism monoid, to intercept in this window from first distinguishing base to the region that last distinguishing base stops as character zone, and add up the quantity of the distinguishing base in this character zone.The each development length in both sides to character zone is that the region of 160bp-character zone length is as the primer field of search, in the primer field of search, there is length and be greater than 20bp and region without any base difference between all microorganisms in target microorganism monoid in search, as the design of primers district of character zone, abandon the character zone lacking design of primers district.
Log in multiplex amplification primer Photographing On-line webpage https: //ampliseq.com, select " DNAHotspotdesigns (single-pool) " at " Applicationtype " option.If select multi-pool in the present embodiment, then multiplex PCR will divide multitube to carry out, and cost can increase to some extent.And select the primer of single-pool only to need a multiplex PCR, save cost, but the design of primers that shortcoming is some character zone may be failed, but because the number of the character zone on genome is more, the design of primers failure of a few features region does not affect result, so the present embodiment selects single-pool.The character zone of all target microorganism monoids obtained above and the design of primers district of correspondence thereof 100 base N (N represent in A, T, C and G tetra-kinds of bases any one) are coupled together, is generated as the reference genome of a design of primers.Select " Custom " in " Selectthegenomeyouwishtouse " option after, upload the reference genome of the design of primers of generation.DNAType option selects " StandardDNA ", in AddHotspot option, fills in the initial sum final position of character zone in the reference genome of the design of primers generated.Finally click " Submittargets " button submit to and obtain the multiplex amplification primer sequence of the character zone of target microorganism monoid.
The multiplex amplification primer pair target microorganism monoid of design is utilized to utilize BLASTN (BasicLocalAlignmentSearchTool, basic Local Alignment Search Tool) (version2.2.26) to compare analysis, therefrom select in forward and reverse primer and have at least to have a specific primer.Select primer is done BLASTN compare of analysis with the genome of nontarget organism again, checks whether they can increase the genome of nontarget organism.In the present embodiment, nontarget organism is all biologies except target microorganism monoid, and the genome of nontarget organism is the NT/NR storehouse of NCBI.Judge that the standard that primer can increase is: amplification region length is no more than 200bp, primer matching length is greater than 15bp and does not have base deletion or mispairing within primer 3 ' 5 bases of holding.Any nontarget organism if primer can not increase, now, discrimination m1=3 × the L1/4 of the character zone of the target microorganism corresponding to primer, the part nontarget organism if primer can increase, then just the amplified production of arbitrary nontarget organism of this primer amplification and the character zone of arbitrary target microorganism monoid are compared, obtain in all comparisons, the minimum value of distinguishing base number is discrimination m1, retain the character zone of target microorganism monoid of m1 >=3, remove containing simple repeated sequence further or be the character zone of multiple copied on genome.From the character zone of the target microorganism monoid retained, the character zone of further selected objective target microbe groups the character zone of select target microorganism.
Further, the preferred method of the character zone of target microorganism monoid is as follows: the reference genome of character zone and nontarget organism is done BLASTN comparison, remove the character zone that there is more than 95% homology with nontarget organism, in target microorganism and target microorganism monoid, between other microorganism, utilized by remaining character zone software muscle (version: V3.6) to compare by its default parameters, obtain the minimum value of distinguishing base number, i.e. m2 value.Retain the character zone of the target microorganism monoid of m2 >=2, from the character zone retained, select arbitrarily all larger character zone of more than 2 and 2 of discrimination m1 and m2 simultaneously as the character zone of target microorganism monoid and the character zone of target microorganism, the multiplex amplification primer of its correspondence is simultaneously as the first multiplex amplification primer and the second multiplex amplification primer.
By the method similar with the character zone finding target microorganism monoid, obtain the 3rd multiplex amplification primer with reference to microorganism character zone and correspondence thereof, below emphasis difference is described, something in common is repeated description no longer.Same employing software Megablast (version 2 .2.26) compares to reference to microbial genome and query sequence (reference sequences), and query sequence is the genome sequence of AgrobacteriumtumefaciensK84.After comparison completes, obtain with reference in microbial genome, in query sequence, only occur the unique sequence of 1 time.By the NT/NR storehouse comparison of unique sequence and NCBI, abandon the unique sequence that there is homologous sequence in nontarget organism, from unique sequence the nonoverlapping 110bp length of random choose as character zone, the sequence of its both sides is as design of primers region.In the multiplex amplification primer in the upper DESIGNED FEATURE region of multiplex amplification primer Photographing On-line webpage https: //ampliseq.com, further screening successfully devises the character zone of multiplex amplification primer, concrete grammar is as follows: remove containing simple repeated sequence or be the character zone of multiple copied on genome, the reference genome of remaining character zone and nontarget organism is done BLASTN comparison, removes the character zone that there is more than 95% homology with nontarget organism.From the character zone remained, random choose more than 2 and 2 character zones are as the character zone with reference to microbe groups, and the multiplex amplification primer of its correspondence is as the 3rd multiplex amplification primer.
The template sequence of amplification being synthesized each the heavy amplimer in the first multiplex amplification primer of above acquisition, the second multiplex amplification primer and the 3rd multiplex amplification primer one by one by Sangon Biotech (Shanghai) Co., Ltd. and answered by each multiplex amplification primer pair, template sequence refers to that each multiplex amplification primer is in the amplification region inserting AddHotspot option.The amplification efficiency of each multiplex amplification primer is detected according to the operational manual (PartNumber4376784Rev.E) of the StepOne real-time PCR of match Mo Feishier company of the U.S., only retain amplification efficiency 95% ~ 105% multiplex amplification primer, to reduce the difference of amplification efficiency to the impact of microorganism qualitative and quantitative.Because amplification efficiency impact is less, therefore, target microorganism monoid also can be different from the character zone of target microorganism, more easily find respective character zone respectively to facilitate.The multiplex amplification primer the first multiplex amplification primer obtained above, the second multiplex amplification primer and the 3rd multiplex amplification primer remained is by multiplex amplification primer Photographing On-line webpage https: the consolidation procedure on //ampliseq.com merges, obtain mixing multiplex amplification primer, mixing multiplex amplification primer provides after matching the synthesis of Mo Feishier company by the U.S. in liquid form.The character zone relevant information that the present embodiment finally obtains is in table 1.Zero position in table 1 and final position refer to the initial sum final position on the reference genome of character zone in query sequence.
The primer relevant information that table 1 embodiment of the present invention one provides
Step 4, add with reference to microorganism in testing sample, obtain biased sample, concrete grammar is as follows:
Be not present in testing sample with reference to microorganism, so, with reference to microorganism as internal reference, and parallel running can be carried out with the microorganism in testing sample, the target microorganism monoid in testing sample and target microorganism are carried out quantitatively.Add-on with reference to microorganism controls the nucleic acid (DNA) of the biased sample for approximately extracting 10ng, normally to build high-throughput sequencing library, simultaneously, add-on with reference to microorganism is unlikely to again to make the ratio with reference to shared by microorganism excessive, takies too much high-flux sequence data volume.The acquisition methods of the present embodiment biased sample is as follows: be after the bacterium liquid 0.2mL of the reference microorganism of 2OD (OD is bacterium liquid maximum absorbance value) is placed in the centrifuge tube vacuum freezing centrifugal drying of 1.5mL by concentration, add in 100mg testing sample and pork, grind to form powdery after adding liquid nitrogen, namely obtain testing sample and the biased sample with reference to microorganism.Counted by blood cell plate, calculate acquisition and add the amount of the reference microorganism of biased sample in table 2.
The nucleic acid of step 5, extraction biased sample, concrete grammar is as follows:
When extracting the nucleic acid of described biased sample, if the content of testing sample amplifying nucleic acid too low (lower than 1ug), to the extraction of the nucleic acid of biased sample be affected, then in the leaching process of the nucleic acid of biased sample, the exogenous nucleic acid that mixing multiplex amplification primer can not increase can be added.The exogenous nucleic acid added does not exist and occurring in nature, does not thus disturb microorganism detection.Outside RNA contrasts association and devises and demonstrate a set of nucleotide sequence, they do not exist at occurring in nature, can as the exogenous nucleic acid in the embodiment of the present invention, its sequence can with reference to https: //tools.lifetechnologies.com/content/sfs/manuals/cms_09504 7.txt.The add-on of exogenous nucleic acid is about 1ug, and this add-on can ensure that the nucleic acid of biased sample can normal extraction.In the present embodiment, testing sample is pork, and its nucleic acid content is normal, therefore, does not need to add exogenous nucleic acid in biased sample.Fast DNA extraction detection kit (article No.: KG203, production company: TIANGEN Biotech (Beijing) Co., Ltd.) is utilized to extract the nucleic acid of the biased sample obtained by the method that its operational manual provides.
The nucleic acid of step 6, utilization mixing multiplex amplification primer and biased sample carries out amplified reaction, and obtain amplified production, concrete grammar is as follows:
After utilizing the nucleic acid of library construction Kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345) multiplexed PCR amplification biased sample, amplified production is utilized to build high-throughput sequencing library.This test kit comprises following reagent: 5 × IonAmpliSeq tMhiFiMix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase.The method of library construction presses the operational manual " IonAmpliSeq of this test kit tMlibraryPreparation " (publication number: MAN0006735, version: A.0) carry out.The amplification system of multiplex PCR is as follows: 5 × IonAmpliSeq tMthe nucleic acid 10ng of the mixing multiplex amplification primer 4 μ l of HiFiMix4 μ l, synthesis, the biased sample of extraction 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.After utilizing FuPa reagent to digest primer unnecessary in multiplexed PCR amplification product, then carry out phosphorylation, concrete grammar is: in the amplified production of multiplex PCR, add 2 μ LFuPa reagent, after mixing, by following program reaction in PCR instrument: 50 DEG C, and 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is containing the amplified production solution through phosphorylation.The amplified production of phosphorylation is connected upper sequence measuring joints, and concrete grammar is: in mixture a, add transferring reagent 4 μ L, sequence measuring joints solution 2 μ L and DNA ligase 2 μ L, after mixing, by following program reaction in PCR instrument: 22 DEG C, and 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed solution b.10 μ L are dissolved in without in enzyme water after utilizing the ethanol precipitation methods purifying mixed solution b of standard.American I nvitrigen company is utilized to produce dsDNAHSAssayKit (article No. is Q32852) also measures according to its specification sheets, after obtaining the mass concentration of mixed solution b, mixed solution b after purifying is diluted to 15ng/ml, obtains the high-throughput sequencing library that concentration is about 100pM.
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 test kit IonPITemplateOT2200Kitv2 (invirtrigen company of U.S. production that obtain, article No. is 4485146) check order before ePCR (EmulsionPCR, emulsion polymerization enzyme chain reaction) amplification, working method is undertaken by the operational manual of this test kit.(invirtrigen company of the U.S. produces to utilize ePCR product and test kit IonPISequencing200Kitv2, article No. is 4485149) on Proton bis-generation high-flux sequence instrument, carry out high-flux sequence, working method is undertaken by the operational manual of this test kit.In the present embodiment, high-flux sequence amount is set to 1M sequenced fragments (1M=,100 ten thousand).
According to the primer of sequenced fragments, by the comparison of high-flux sequence fragment to the corresponding character zone of target microorganism monoid, the character zone of target microorganism and the character zone with reference to microorganism.Remove the unsuccessful and incomplete sequenced fragments of character zone of comparison, the unsuccessful sequenced fragments of comparison mostly is non-specific amplification product, and the incomplete sequenced fragments of character zone refers to could not be complete to the sequential detection of final position by the zero position of the character zone in table 1.
Step 8, according to high-flux sequence fragment, carry out qualitative and quantitative analysis to target microorganism monoid and target microorganism, concrete grammar is as follows:
The ultimate principle of microorganism qualitative and quantitative analysis provided by the invention is: character zone represents target microorganism monoid and target microorganism, if the sequenced fragments in existing characteristics region, show that target microorganism monoid or target microorganism exist, and the quantity of the sequenced fragments of character zone also represent the quantity of target microorganism monoid and target microorganism.Detect different from other microorganism qualitative and quantitative, the embodiment of the present invention calculates the reliability of microorganism qualitative and quantitative, meanwhile, enhances the practicality of conclusion.The embodiment of the present invention needs first to put in order relation complicated between parameter, could realize the qualitative and quantitative analysis of any microorganism, and obtain reliable conclusion, and design parameter of the present invention and reckoning principle thereof are in table 2.Cell in table 2, symbol are identical with Excel2010 with the definition of formula, and wherein, cell " basic parameter " is A1, and other cell defines by the rule of Excel2010 with reference to A1.
Method for qualitative analysis is as follows: compared by the character zone of high-flux sequence fragment with often kind of target microorganism monoid, as distinguishing base number≤n1, then comparison success, corresponding high-flux sequence fragment is the character zone of target microorganism monoid, wherein, n1 is the maximum fault-tolerant base number of the feature sequenced fragments of target microorganism monoid; If during character zone >=a kind of the successful target microorganism monoid of comparison, then judge that high-flux sequence fragment is the feature sequenced fragments of target microorganism monoid.
The character zone of the character zone of target microorganism with the target microorganism monoid of often kind of homology is compared, the Reference genotype of distinguishing base composition target microorganism is extracted in the character zone of target microorganism, here distinguishing base refers to that the character zone of target microorganism compares with the microorganism in any one target microorganism monoid, the summation of the base that there are differences.On the feature sequenced fragments of target microorganism monoid, extract the base corresponding to Reference genotype of target microorganism, the test cdna type of composition target microorganism; If the distinguishing base number≤n2 of the Reference genotype of the test cdna type of target microorganism and target microorganism, wherein, n2 is the maximum fault-tolerant base number of the feature sequenced fragments of target microorganism, then the high-flux sequence fragment at the test cdna type place of target microorganism is the feature sequenced fragments of target microorganism.Especially, when only containing a target microorganism in target microorganism monoid, the base number of Reference genotype now and test cdna type is 0, therefore, distinguishing base number between them is also 0, no matter then n2 is much, all the high-flux sequence fragment at the test cdna type place of target microorganism is judged to be the feature sequenced fragments of target microorganism.By above method, obtain the characteristic fragment number of the character zone of target microorganism monoid and target microorganism respectively, it the results are shown in table 1.In the present embodiment, the value of n1 and n2 is in table 2, and its reckoning process sees below.
N1 makes P1≤α 1 and P3≤α 3, wherein, and the false-positive probability that the feature sequenced fragments that the high-flux sequence fragment of P1 to be one be not the feature sequenced fragments of target microorganism monoid is mistaken for target microorganism monoid produces; P3 is that the feature sequenced fragments of an objective microbe groups is mistaken for is not the false-negative probability that the feature sequenced fragments of target microorganism monoid produces; α 1 and α 3 is judgment threshold.
N2 makes P2≤α 2 and P4≤α 4, wherein, and the false-positive probability that the feature sequenced fragments that the feature sequenced fragments of P2 to be one be not target microorganism is mistaken for target microorganism produces; P4 is that the feature sequenced fragments of an objective microorganism is mistaken for is not the false-negative probability that the feature sequenced fragments of target microorganism produces; α 2 and α 4 is judgment threshold; The size of the various threshold values in the embodiment of the present invention is determined by reality need, and such as, some germ hazardness is very big, and undetected (false negative) will cause serious consequence, so, will control false negative, and α 2 and α 4 is worth low.If without particular requirement, lower false positive and false negative is then adopted to be principle, the present embodiment belongs to the latter, α 1 and α 3 value are 0.01%, and namely 1 false positive or false negative appear in about 10,000 characteristic sequences, and its accuracy is very high, why can control so high accuracy, be because the m1 value in characteristic sequence is comparatively large, be easy to distinguish with other nontarget organism, thus false positive rate and false negative rate are all controlled a very low level.The value of α 2 and α 4 is 0.5%, and namely 5 false positives or false negative appear in about 1,000 characteristic sequences, 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, concrete finger is for discrimination corresponding to the character zone of the target microorganism monoid calculating S1, and in the present embodiment, the value of m1 is in table 1 and table 2, m2 is the minimum value of distinguishing base between the character zone of target microorganism and other microorganism of target microorganism monoid, and specifically refer to the value of the m2 in the target microorganism characteristic of correspondence region for calculating S3, in the present embodiment, the value of m2 is in table 1 and table 2, L1 is the length of the character zone of target microorganism monoid, and in the present embodiment, the value of L1 is in table 2, L2 is the length of the Reference genotype of target microorganism, and in the present embodiment, the value of L2 is in table 2, E is base error rate, it is made up of the error rate E1 and natural mutation rate E2 that checks order, in the present embodiment, order-checking error rate E1≤1% of PROTON high-flux sequence instrument, according to our investigation, aberration rate between the reference genome of microorganism microspecies (as P1-P6 bacterial leaf spot microspecies) is generally less than 0.5%, and natural mutation rate be lower than microspecies between aberration rate, therefore, natural mutation rate E2≤0.5%, in order to method applicability of the present invention is wider, get E2≤1%, then in the present embodiment, E≤2%, in order to the probability of the conclusion accuracy making the qualitative and quantitative of microorganism in the present embodiment is more reliable, the maximum value 2% of getting E value calculates.Above parameter value is substituted into after in the formula of P1 and P3, the value of n1 is increased gradually from 0, calculate the value of P1 and P3, as n1=13, calculate P1≤α 1 and P3≤α 3, therefore, in the present embodiment, n1=13 (see table 2), the value of P1 and P3 that n1=13 is corresponding is the value of P1 and P3 in the present embodiment.By similar method, above parameter value is substituted into after in the formula of P2 and P4, the value of n2 is increased gradually from 0, calculates the value of P2 and P4, as n2=2, P2≤α 2, P4≤α 4, therefore, in the present embodiment, n2=2 (see table 2), the value of P2 and P4 that n2=2 is corresponding is the value of P2 and P4 in the present embodiment.
With reference to microorganism as the target microorganism monoid only comprising a target microorganism, calculate the feature sequenced fragments of the target microorganism obtained, be the feature sequenced fragments with reference to microorganism.The characteristic fragment number of the character zone of reference microorganism is in table 1 and table 2.
If probability P 5 >=α 5 that the feature sequenced fragments of target microorganism monoid exists, then judge to there is target microorganism monoid in testing sample; If the probability P 5< α 5 that the feature sequenced fragments of target microorganism monoid exists, then judge there is not target microorganism monoid in testing sample, wherein, α 5 is probability guarantee, and in the present embodiment, α 5 value is 99.99%.P5=1-BINOM.DIST (S1, S1, P1, FALSE), S1 is the median of the quantity of the feature sequenced fragments of the target microorganism monoid of the character zone of all target microorganism monoids, in the present embodiment, the quantity of the 2nd feature sequenced fragments of target microorganism monoid is the median of the quantity of the feature sequenced fragments of all target microorganism monoids, so the value of the present embodiment S1 is in table 1 and table 2, the calculation formula value of the value of S1 in the present embodiment and P1 being substituted into P5 calculates and obtains P5 >=α 5, therefore, judge in the present embodiment, target microorganism monoid is there is in testing sample, FALSE is parameter value, BINOM.DIST function returns the probability of unitary binomial distribution.
If probability P 6 >=α 6 that the feature sequenced fragments of target microorganism exists, then judge to there is target microorganism in testing sample; If the probability P 6< α 6 that the feature sequenced fragments of target microorganism exists, then judge to there is not target microorganism in testing sample; α 6 is probability guarantee.In the present embodiment, α 6 value is 99.99%.P6=1-BINOM.DIST (S3, S3, P2, FALSE), BINOM.DIST function returns the probability of unitary binomial distribution, S3 is the median of the quantity of the feature sequenced fragments of the target microorganism of the character zone of all target microorganisms, in the present embodiment, the quantity of the 2nd feature sequenced fragments of target microorganism is the median of the quantity of the feature sequenced fragments of all target microorganisms, the value of the S3 of its correspondence is in table 1 and table 2, the calculation formula value of the value of S3 in the present embodiment and P2 being substituted into P6 calculates and obtains P6 >=α 6, therefore, judge in the present embodiment, target microorganism is there is in testing sample.
In addition, α 5 and α 6 is all that people are fixed according to actual needs, and α 5 and α 6 all can be the same or different, actual needs is depended in its difference, and when strictly controlling certain microorganism, the value of α 5 and α 6 is all larger, otherwise the value of α 5 and α 6 is all less.In addition, the value of all in embodiment of the present invention a values all follows this principle.
Quantitative analysis method is as follows: the amount M1=Mr × S1/S2 of target microorganism monoid, and wherein, Mr is the amount of the reference microorganism added in testing sample, and in the present embodiment, the value of Mr is in table 2; S2 is the median of the quantity of the feature sequenced fragments of the reference microorganism of the character zone of all reference microorganisms, in the present embodiment, quantity with reference to the 2nd the feature sequenced fragments of microorganism is the median of the quantity of all feature sequenced fragments with reference to microorganism, and the value of the S2 of its correspondence is in table 1 and table 2; Substitute in the calculation formula of M1 by above parameter and the value of S1 that obtained by qualitative analysis, calculate and obtain M1 value, namely in testing sample, the amount of the microorganism in target microorganism monoid is that M1=85 is individual.
The fiducial interval of the amount of target microorganism monoid is [M11, M12], M11 and M12 is respectively lower limit and the upper limit of the fiducial interval of M1 value.M11=M1 × (1-S4/S1), M12=M1 × (1+S5/S1), wherein, S4 is the quantity of the feature sequenced fragments of false-positive target microorganism monoid and S4=CRITBINOM (nS, P1, α 9), S5 is the quantity of the feature sequenced fragments of false-negative target microorganism monoid and S5=CRITBINOM (S1, P3, α 9), wherein, α 9 is probability guarantee, in the present embodiment, α 9 value is 99.50%, CRITBINOM function returns the minimum value making accumulation binomial distribution be more than or equal to threshold value, the quantity of the high-flux sequence fragment of the non-character zone that the multiplex amplification primer that nS is the character zone calculating the target microorganism monoid of S1 increases, namely other high-flux sequence fragment except the feature sequenced fragments of target microorganism that multi-primers increases is referred to.In the present embodiment, the quantity of the high-flux sequence fragment of the non-character zone that nS produces for the multiplex amplification primer amplification of the 2nd character zone in target microorganism monoid, in the present embodiment, the value of nS is in table 2.The formulae discovery value of the value of nS and P1 being substituted into S4 obtains the value of S4, and the formulae discovery value of the value of the present embodiment S1 and P3 being substituted into S5 obtains the value of S5.After obtaining the value of all parameters in M11 and M12 formula, calculate the value obtaining M11 and M12 in the present embodiment, and then obtain the fiducial interval of M1, namely the fiducial interval of the amount of target microorganism monoid is [85,85].
Amount M2=M1 × the S3/S1 of target microorganism, substitutes into above-mentioned formula by the value of M1, S3 and S1, obtains amount M2=24 of target microorganism.
The fiducial interval of the amount of target microorganism is [M21, M22], M21 and M22 is respectively lower limit and the upper limit of the fiducial interval of M2 value; M21=M2 × (1-S6/S3), M22=M2 × (1+S7/S3); Wherein, S6 is the quantity of the feature sequenced fragments of false-positive target microorganism and S6=CRITBINOM (S1, P2, α 10), S7 is the quantity of the feature sequenced fragments of false-negative target microorganism and S7=CRITBINOM (S3, P4, α 10), wherein, α 10 is probability guarantee; CRITBINOM function returns the minimum value making accumulation binomial distribution be more than or equal to threshold value.In the present embodiment, α 10 value is 99.50%, and by the value of the present embodiment S1 and S3, and the value of P2 and P4 substitutes into the calculation formula of S6 and S7, calculates the value of S6 and S7.Further the value of S6, S7, M1 and S3 is substituted into the calculation formula of M21 and M22, calculate the value of M21 and M22, and then the fiducial interval of the amount obtaining target microorganism is [22,24].
The microorganism quantification and qualification parameter that table 2 provides for the present embodiment one and calculate principle
The qualification of embodiment two, romaine lettuce contaminating microorganisms
Testing sample in the present embodiment is the romaine lettuce that Zhuankou market, Wuhan is bought, and detects in testing sample the microorganism that may pollute and can determine that this romaine lettuce edible does not particularly boil edible whether safety.The embodiment of the present invention two is similar with the method for embodiment one, and the method do not mentioned, parameter are identical with embodiment one with result.
Step one, the reference microorganism determined target microorganism monoid, target microorganism and nontarget organism in testing sample and be not present in testing sample, concrete grammar and result are with embodiment one.
Step 2, according to the reference genome sequence of the reference genome sequence of target microorganism monoid, target microorganism, with reference to the reference genome sequence of microorganism and the reference genome sequence of nontarget organism, obtain the character zone of target microorganism monoid, the character zone of target microorganism and the character zone with reference to microorganism, concrete grammar and result are with embodiment one.
Wherein, distinguished by target microorganism from target microorganism monoid, its principle is in table 4.
Step 3, the first multiplex amplification primer of character zone of preparation amplification target microorganism monoid, the second multiplex amplification primer of the character zone of amplification target microorganism and amplification are with reference to the 3rd multiplex amplification primer of the character zone of microorganism, first multiplex amplification primer, the second multiplex amplification primer and the 3rd multiplex amplification primer are mixed to get mixing multiplex amplification primer, concrete grammar, with embodiment one, the results are shown in Table 3.
The primer relevant information that table 3 the present embodiment two provides
Step 4, add with reference to microorganism in testing sample, obtain biased sample, concrete grammar is as follows:
Be after the bacterium liquid 0.2mL of the reference microorganism of 2OD (OD is bacterium liquid maximum absorbance value) is placed in the centrifuge tube vacuum freezing centrifugal drying of 1.5mL by concentration, add in 100mg testing sample and romaine lettuce, grind to form powdery after adding liquid nitrogen, namely obtain testing sample and the biased sample with reference to microorganism.Counted by blood cell plate, calculate acquisition and add the amount of the reference microorganism of biased sample in table 4.
The nucleic acid of step 5, extraction biased sample, concrete grammar is as follows:
Testing sample is romaine lettuce, and its nucleic acid content is normal, therefore, does not need to add exogenous nucleic acid in biased sample.Plant genome DNA is utilized to extract the nucleic acid of the biased sample that test kit (article No.: DP305, production company: TIANGEN Biotech (Beijing) Co., Ltd.) obtains by the method extraction that its operational manual provides.
The nucleic acid of step 6, utilization mixing multiplex amplification primer and biased sample carries out amplified reaction, and obtain amplified production, concrete grammar is with embodiment one.
Step 7, utilize amplified production to carry out high-flux sequence, obtain high-flux sequence fragment, concrete grammar is with embodiment one.
Step 8, according to high-flux sequence fragment, qualitative and quantitative analysis is carried out to target microorganism monoid and target microorganism.
Adopt identical with embodiment one, the different parameter adopted from examples of implementation one in method with the results are shown in table 3 and table 4, the result of determination in the present embodiment is as follows: there is target microorganism in testing sample; The amount of the microorganism in target microorganism monoid is M1=147, and fiducial interval is [147,147]; The amount M2=20 of target microorganism is individual, and fiducial interval is [15,20].
Table 4 is the present embodiment two microorganism quantification and qualification parameter and calculates principle
The present invention changes in existing method once can only detect a few microorganism, microbiota can only be assigned to kind, quantitatively inaccurate, detected result without probability guarantee, need that preculture, sense cycle are long, certain micro-organisms can not cultivate thus can not detect, microbial drug tolerance is different and the quantization distortion caused, the problems such as quantitatively coarse, for microorganism detection provides a kind of comprehensive, quick, meticulous qualitative and quantitative new detecting method.

Claims (9)

1. a detection method for food microorganisms qualitative and quantitative, is characterized in that, described method comprises:
The reference microorganism determined target microorganism monoid, target microorganism and nontarget organism in testing sample and be not present in described testing sample, described testing sample is food;
According to described target microorganism monoid, described target microorganism, described reference the genome sequence with reference to microorganism and described nontarget organism, obtain the character zone of the character zone of described target microorganism monoid, the character zone of described target microorganism and described reference microorganism;
3rd multiplex amplification primer of the preparation amplification first multiplex amplification primer of character zone of described target microorganism monoid, the second multiplex amplification primer of the character zone of the described target microorganism that increases and the described character zone with reference to microorganism that increases, is mixed to get mixing multiplex amplification primer by described first multiplex amplification primer, described second multiplex amplification primer and described 3rd multiplex amplification primer;
Add described with reference to microorganism 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 amplified production;
Utilize described amplified production to carry out high-flux sequence, obtain high-flux sequence fragment;
According to described high-flux sequence fragment, qualitative and quantitative analysis is carried out to described target microorganism monoid and described target microorganism.
2. method according to claim 1, is characterized in that, number >=1 of described target microorganism monoid, and each described target microorganism monoid comprises >=0 kind of described target microorganism;
Described target microorganism is at least one in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon;
Described reference microorganism is at least one in bacterium, virus, fungi, actinomycetes, rickettsia, mycoplasma, chlamydozoan, spirochete, algae and protozoon.
3. method according to claim 1, it is characterized in that, the method of the described nontarget organism determined in testing sample comprises: described nontarget organism is defined as all biologies except described target microorganism monoid, if can obtain the character zone of described target microorganism monoid, then described nontarget organism refers to all biologies except described target microorganism monoid; If can not obtain the character zone of described target microorganism monoid, then described nontarget organism refers in described biased sample, and other except described target microorganism monoid is biological.
4. method according to claim 1, is characterized in that, the character zone of described target microorganism monoid is the nucleotide sequence on the reference genome of the microorganism in described target microorganism monoid; The sequence of the both sides of the character zone of described target microorganism monoid is unique sequence in described reference genome; The sequence of the both sides of the character zone of described target microorganism monoid is conservative between different microorganisms in described target microorganism monoid; Discrimination >=3 of the character zone of described target microorganism monoid;
The character zone of described target microorganism and the character zone homology of described target microorganism monoid; M2 value >=2 of the character zone of described target microorganism, wherein, the minimum value of the distinguishing base number described in other in the character zone that m2 value is described target microorganism and described target microorganism monoid except described target microorganism between microorganism;
The described character zone 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 character zone with reference to microorganism is unique sequence in the reference genome of described reference microorganism; The sequence of the both sides of the described character zone with reference to microorganism does not have homology in other biology except described reference microorganism.
5. method according to claim 4, it is characterized in that, described discrimination refers to the minimum value by the distinguishing base number between the character zone of the arbitrary described target microorganism monoid of same described mixing multiplex amplification primer amplification and arbitrary non-character zone, wherein, described non-character zone is the amplified production that described mixing multiplex amplification primer is template with the nucleic acid of described biased sample, and described non-character zone is not the character zone of described target microorganism monoid, if without described non-character zone, then described discrimination=3 × L1/4, wherein, L1 is the nucleotide sequence length of the character zone of described target microorganism monoid.
6. method according to claim 1, is characterized in that, described method also comprises:
When extracting the nucleic acid of described biased sample, if the content of described testing sample amplifying nucleic acid is too low, then, in the process of nucleic acid extracting described biased sample, add the exogenous nucleic acid that described mixing multiplex amplification primer can not increase.
7. method according to claim 1, is characterized in that, the method for qualitative analysis of described target microorganism monoid and described target microorganism is as follows:
The character zone of described high-flux sequence fragment with often kind of described target microorganism monoid is compared, as distinguishing base number≤n1, then comparison success, corresponding described high-flux sequence fragment is the character zone of described target microorganism monoid, wherein, n1 is the maximum fault-tolerant base number of the feature sequenced fragments of described target microorganism monoid; If during character zone >=a kind of the successful described target microorganism monoid of comparison, then judge that described high-flux sequence fragment is the feature sequenced fragments of described target microorganism monoid;
The character zone of the character zone of described target microorganism with the described target microorganism monoid of often kind of homology is compared, in the character zone of described target microorganism, extracts the Reference genotype that distinguishing base forms described target microorganism; On the feature sequenced fragments of described target microorganism monoid, extract the base corresponding to Reference genotype of described target microorganism, form the test cdna type of described target microorganism; If the distinguishing base number≤n2 of the Reference genotype of the test cdna type of described target microorganism and described target microorganism, wherein, n2 is the maximum fault-tolerant base number of the feature sequenced fragments of described target microorganism, then the described high-flux sequence fragment at the test cdna type place of described target microorganism is the feature sequenced fragments of described target microorganism;
Using described reference microorganism as the described target microorganism monoid only comprising a described target microorganism, calculate the feature sequenced fragments of the described target microorganism obtained, be the described feature sequenced fragments with reference to microorganism;
If probability P 5 >=α 5 that the feature sequenced fragments of described target microorganism monoid exists, then judge to there is described target microorganism monoid in described testing sample, wherein, α 5 is probability guarantee; If the probability P 5< α 5 that the feature sequenced fragments of described target microorganism monoid exists, then judge in described testing sample, to there is not described target microorganism monoid;
If probability P 6 >=α 6 that the feature sequenced fragments of described target microorganism exists, then judge to there is described target microorganism in described testing sample, wherein, α 6 is probability guarantee; If the probability P 6< α 6 that the feature sequenced fragments of described target microorganism exists, then judge there is not described target microorganism in described testing sample;
N1 makes P1≤α 1 and P3≤α 3, wherein, and the false-positive probability that the feature sequenced fragments that the described high-flux sequence fragment of P1 to be one be not the feature sequenced fragments of described target microorganism monoid is mistaken for described target microorganism monoid produces; P3 is that the feature sequenced fragments of a described target microorganism monoid is mistaken for is not the false-negative probability that the feature sequenced fragments of described target microorganism monoid produces; α 1 and α 3 is judgment threshold;
N2 makes P2≤α 2 and P4≤α 4, wherein, and the false-positive probability that the feature sequenced fragments that the described high-flux sequence fragment of P2 to be one be not the feature sequenced fragments of described target microorganism is mistaken for described target microorganism produces; P4 is that the feature sequenced fragments of a described target microorganism is mistaken for is not the false-negative probability that the feature sequenced fragments of described target microorganism produces; α 2 and α 4 is judgment threshold;
P5=1-BINOM.DIST (S1, S1, P1, FALSE), P6=1-BINOM.DIST (S3, S3, P2, FALSE), S1 is the median of the quantity of the feature sequenced fragments of the described target microorganism monoid of the character zone of all described target microorganism monoids; S3 is the median of the quantity of the feature sequenced fragments of the described target microorganism of the character zone of all described target microorganisms, and FALSE is parameter value; BINOM.DIST function returns the probability of unitary binomial distribution.
8. method according to claim 7, is characterized in that, the quantitative analysis method of described target microorganism monoid and described target microorganism is as follows:
Amount M1=Mr × the S1/S2 of described target microorganism monoid, the fiducial interval of the amount of described target microorganism monoid is [M11, M12], and wherein, Mr is the described amount with reference to microorganism added in described testing sample; S2 is the median of the quantity of the described feature sequenced fragments with reference to microorganism of all described character zones with reference to microorganism; M11 and M12 is respectively lower limit and the upper limit of the fiducial interval of M1 value;
Amount M2=M1 × the S3/S1 of described target microorganism, the fiducial interval of the amount of described target microorganism is [M21, M22], M21 and M22 is respectively lower limit and the upper limit of the fiducial 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 quantity of the feature sequenced fragments of false-positive described target microorganism monoid and S4=CRITBINOM (nS, P1, α 9), wherein, the quantity of the described high-flux sequence fragment of the described non-character zone that the described multiplex amplification primer that nS is the character zone calculating the described target microorganism monoid of S1 increases; S5 is the quantity of the feature sequenced fragments of false-negative described target microorganism monoid and S5=CRITBINOM (S1, P3, α 9), and wherein, α 9 is probability guarantee; S6 is the quantity of the feature sequenced fragments of false-positive described target microorganism and S6=CRITBINOM (S1, P2, α 10), S7 is the quantity of the feature sequenced fragments of false-negative described target microorganism and S7=CRITBINOM (S3, P4, α 10), wherein, α 10 is probability guarantee; CRITBINOM function returns the minimum value making accumulation binomial distribution be more than or equal to threshold 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; The minimum value of distinguishing base between microorganism described in other of the character zone that described m2 is described target microorganism and described target microorganism monoid; L1 is the length of the character zone of described target microorganism monoid; L2 is the length of the Reference genotype of described target microorganism; E is base error rate.
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