CN109182567A - A kind of method of real-time fluorescence quantitative PCR that is while detecting 12 kinds of pathogenic bacterias - Google Patents
A kind of method of real-time fluorescence quantitative PCR that is while detecting 12 kinds of pathogenic bacterias Download PDFInfo
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Abstract
The present invention provides the method for real-time fluorescence quantitative PCR that is a kind of while detecting 12 kinds of pathogenic bacterias: collect target pathogenic bacteria specific pathogenetic gene or toxin gene keeps reaction condition consistent as target gene design primer and probe；Extract sample to be tested genomic templates；Template is separately added into the tubule equipped with different special upstream and downstream primers and probe, corresponding quantitative fluorescent PCR reagent is added；Under same wheel quantitative fluorescent PCR circulation, make corresponding primer and probe in respective reaction tube to sample simultaneously, quickly and quantitative detection.Easier, quick, efficient, economical can detect 12 kinds of common causative bacteriums (Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahemolyticus, beta hemolytic streptococcus, small intestine colon Yersinia ruckeri, streptococcus fecalis, Shigella, proteus mirabilis, vibrio fluvialis, campylobacter jejuni, staphylococcus aureus) in drinking-water and food simultaneously.
Description
Technical field
The present invention relates to a kind of pathogenic bacteria detection technique fields, more particularly to a kind of using easy, quick, efficient, warp
The method that the high-throughput Real-Time Fluorescent Quantitative PCR Technique of Ji detects 12 kinds of pathogenic bacterias simultaneously.
Background technique
Production, sanitary wastewater and the human and animal excreta and natural calamity of the mankind all can make some pathogenic bacteria enter water body, draw
The pollution for playing water environment, leads to the propagation of a variety of diseases, such as bacillus dysenteriae and salmonella.At this stage simultaneously, China's food
Safety has been very important problem, this is related to the people's livelihood matter of fundamental importance in China, and food-safety problem also obtains in recent years
The attention of country and people.Also there is an urgent need to realize efficiently and accurately, time saving and energy saving to common pathogen in drinking-water and food in China
Detection method, the detection method for having these advantages is only the guarantee of drinking-water and food safety.
Currently, detecting for common pathogen in drinking-water and food, conventional method needs to carry out to increase repeatedly bacterium, separation training
The operations such as feeding, various physicochemical property identifications, it is also possible to be difficult to cultivate because bacterial concentration is low, not only complex steps, operation
Complexity, and excessive cycle, it is difficult to notify at once as a result, detect speed, in terms of have limitation, together
When be also difficult to realize the requirement of present batch sample detection.And with the rapid development of science and technology and innovation, also gush in recent years
Many new methods are showed, the immunology detection technology including establishing based on the specific reaction of antigen and antibody is for example enzyme-linked to be exempted from
Epidemic disease absorption method (ELISA) etc.；And molecular Biological Detection technology such as polymerase chain reaction (PCR), multiplex PCR, ring mediation
Isothermal amplification technology (LAMP), quantitative fluorescent PCR, genetic chip etc., but there is certain deficiency in them.Regular-PCR can be with
Realize the quick detection to pathogenic bacteria, but sensitivity is low and cannot quantify to pathogenic bacteria；Relative to regular-PCR, multiplex PCR
Multiple object bacterias can be detected in the same system, but be easy to interfere with each other between multiple groups primer, the requirement to primer also compared with
It is high；LAMP technology has many advantages, such as that small reaction system, high sensitivity, specificity are good but complementary may expand between primer in amplification
Non-specific band causes false positive out；Although biochip technology is quickly comprehensive but repeatability is high, stability is poor and operates
It is more complicated with post-processing；Although multiplex PCR, LAMP, genetic chip have ability that is primary while detecting several pathogenic bacteria,
But maximum deficiency is cannot to quantify to pathogenic bacteria.Though quantitative fluorescent PCR can carry out quantitative detection to pathogenic bacteria, by
In primed probe design limitation, so that primary experiment may can only detect a kind of pathogenic bacteria, and the present invention passes through for specificity
Disease-causing gene carries out primer and probe design, and keeps reaction condition consistent, to propose a kind of while detect 12 kinds of pathogenic bacteria
High-throughput real time fluorescence quantifying PCR method.
Summary of the invention
The present invention is to solve the problems, such as existing detection technique and provide a kind of easier, quick, efficient, economical
Can detect 12 kinds of common causative bacterium (Escherichia coli O 157s: H7, listeria monocytogenes, sand in drinking-water and food simultaneously
Door Salmonella, vibrio parahemolyticus, beta hemolytic streptococcus, small intestine colon Yersinia ruckeri, streptococcus fecalis, Shigella, surprise
Different proteus, vibrio fluvialis, campylobacter jejuni, staphylococcus aureus) TaqMan probe high throughput real time fluorescent quantitative
PCR method.
The technical solution adopted by the present invention is that:
A kind of TaqMan probe high throughput real-time fluorescence quantitative PCR detects in drinking-water and food 12 kinds of common causative bacteriums simultaneously
Method mainly includes 12 kinds of special Disease-causing genes of common causative bacterium in the drinking-water and food obtained for literature survey, design
Primer and probe, being optimized according to Tm value and G/C content to the primer and probe of design can be under the same reaction condition
It is tested；Respectively using the gene constructed DNA standard items of the target of doubling dilution as template, corresponding primer and probe is added,
Taqman sonde method quantitative fluorescent PCR is carried out, according to the fluorophor being added in probe, software receives fluorescence signal in real time
Collection, it is accumulative using fluorescence signal, according to the recurring number and initial concentration in each reaction tube when fluorescence signal arrival given threshold
Linear relationship existing for logarithm is implemented to carry out quantitative analysis to starting template.
TaqMan probe high throughput real time fluorescence quantifying PCR method: target pathogenic bacteria specific pathogenetic gene or poison are collected
Plain gene keeps reaction condition consistent as target gene design primer and probe；Extract sample to be tested genomic templates；It will
Template is separately added into the tubule equipped with different special upstream and downstream primers and probe, adds corresponding quantitative fluorescent PCR reagent；
Under same wheel quantitative fluorescent PCR circulation, make corresponding primer and probe in respective reaction tube to sample simultaneously, it is quickly and fixed
Amount detection.
The method specifically includes the following steps:
1) drinking-water and food in common causative bacterial target genes selection and bioinformatic analysis；
2) design of TaqMan high throughput real-time fluorescence quantitative PCR primed probe；
3) 12 kinds of pathogenic bacteria genomes extract, design primer, the specificity verification of probe；
4) DNA standard items are constructed, standard curve are made, according to standard curve determination detection sensitivity；
5) determination of 12 kinds of pathogenic bacteria minimum detection limits.
Target gene selects Escherichia coli O 157: prfA gene, the Salmonella of the rfbE gene of H7, listeria monocytogenes
The hilA gene of bacterium, the toxR gene of vibrio parahemolyticus, beta hemolytic streptococcus scpA gene, small intestine colon yersinia genus
The foxA gene of Salmonella, the ddl gene of streptococcus fecalis, Shigella ipaH gene, the ureR gene of proteus mirabilis, river
Flow the toxR gene of vibrios, the mapA gene of campylobacter jejuni, staphylococcus aureus ebps gene.
The present invention selects the virulence gene being located in bacterial genomes as target gene, although gene between various pathogenic Pseudomonas
Group homology it is very high, and the non-pathogenic type belonged to it on biochemical reaction without any difference, but to pathogenic relevant poison
Power gene specific is high, so selecting virulence base as target gene, high specificity.
The fluorescence quantification PCR primer and probe of 12 kinds of aquatic products pathogenic bacteria can be detected simultaneously, and nucleotide sequence is as follows:
NO:1 ~ 2 rfbE group primer pair SEQ ID, probe SEQ ID NO:3；
NO:4 ~ 5 prfA group primer pair SEQ ID, probe SEQ ID NO:6；
NO:7 ~ 8 hilA group primer pair SEQ ID, probe SEQ ID NO:9；
NO:10 ~ 11 toxR group primer pair SEQ ID, probe SEQ ID NO:12；
NO:13 ~ 14 scpA group primer pair SEQ ID, probe SEQ ID NO:15；
NO:16 ~ 17 foxA group primer pair SEQ ID, probe SEQ ID NO:18；
NO:19 ~ 20 ddl group primer pair SEQ ID, probe SEQ ID NO:21；
NO:22 ~ 23 ipaH group primer pair SEQ ID, probe SEQ ID NO:24；
NO:25 ~ 26 ureR group primer pair SEQ ID, probe SEQ ID NO:27；
NO:28 ~ 29 toxR group primer pair SEQ ID, probe SEQ ID NO:30；
NO:31 ~ 32 mapA group primer pair SEQ ID, probe SEQ ID NO:33；
NO:34 ~ 35 ebps group primer pair SEQ ID, probe SEQ ID NO:36.
Wherein rfbE group primer pair and probe are used for Escherichia coli O 157: H7, prfA group primer pair and probe are for detecting
Listeria monocytogenes, hilA group primer pair and probe are used for salmonella, toxR group primer pair and probe for detecting pair
For detecting beta hemolytic streptococcus, foxA group primer pair and probe are used for for hemolytic vibrios, scpA group primer pair and probe
It detects small intestine colon Yersinia ruckeri, ddl group primer pair and probe and is used for detecting streptococcus fecalis, ipaH group primer pair and probe
In detection Shigella, for detecting proteus mirabilis, toxR group primer pair and probe are used for for ureR group primer pair and probe
Vibrio fluvialis, mapA group primer pair and probe are detected for detecting campylobacter jejuni, ebps group primer pair and probe for detecting
Staphylococcus aureus.
Due to carry out the amplification of 12 target gene, primer and probe design comparison simultaneously in the same PCR system
Complexity, and it is higher to the specific requirements of primer and probe amplification region.So primer and probe designs no general primer and visits very much
Needle design is simple, to design excellent primer and probe, needs to carry out expense on the basis of various bioinformatics softwares are assessed
When screening.Since designed primer and probe has the selection specificity of height, when carrying out pathogenetic bacteria detection, it is desirable that
The targeted target sequence site of primer and probe should quite guard, and the method established so just has more broad applicability.
Since designed primer and probe will carry out multiple fluorescence quantitative PCR reaction in next step, draw in design
The corresponding sequence of each target gene is analysed and compared jointly when object and probe.Not only to follow primer and probe design
Rule, probe selection will guard, and primer selection will guard, it is also necessary to avoid mutually dry between each primer of multiplex PCR
It disturbs and is interfered with each other between each probe, also to make Tm value close as far as possible between primer pair inside primer pair, it is ensured that can be
Preferable amplification curve is obtained under identical conditions；Cannot be complementary between primer pair inside each pair of primer, particularly avoid 3 ' ends
Complementation, to reduce the formation of primer dimer.Designed primer and probe sequence is in DNAstar software
Primerselect software, which is analyzed and is submitted to the website NCBI, carries out BLAST analysis, to verify the theory of primer and probe
Specificity.
Primer and probe design principle includes:
A. primer length is generally 15-30bp, and the most commonly used is 18-25bp；
B. G+C content 40%-60% in base content, and upstream and downstream primer sequence G/C content difference is not too big；
C. stable primer dimer and hairpin structure are avoided the formation of；
D. product length is between 100-200 bp；
E. the distance of primer and probe: when design primer and probe, consider design primer and probe on two chains simultaneously,
Taqman probe should close and its upstream primer on same chain；
F. the length of Taqman probe is preferably between 13-30, and Tm value is between 68-70, and the Tm value of probe is than primer
Tm value is higher by 10 DEG C.
Heretofore described primer length is 18-25 bp, and the Tm value of primer is 59 ± 2 DEG C, and the length of probe is 20-25
Bp, the length of amplified production is between 80-200 bp.
The amplification of 12 kinds of pathogenic bacteria specific genes all uses TaKaRa companyPremixEx Taq (Probe qPCR)
(RR390) it is expanded, the system and ingredient of amplification are shown in Table 2, and wherein the dilution of primer and probe calculates according to calculation formula and adds
Enter the amount of TE, be first diluted to 100 μM, then be diluted to using concentration, wherein primer is 10 μM, probe is 5 μM.Quantitative fluorescent PCR
Reaction carried out on 7500 quantitative PCR apparatus of ABI, program is as follows: 95 DEG C initial denaturation 30 seconds, 95 DEG C be denaturalized 5 seconds, 55 DEG C annealing
10 seconds, 72 DEG C extended 30 seconds, 40 circulations.
The present invention also protects multiple fluorescence quantitative PCR reaction system that is a kind of while detecting various pathogens, the reactant
System is 25 μ L, wherein 12.5 μ L of Premix Ex Taq (Probe qPCR), upstream and downstream primer each 1.0 μ L, 1.0 μ of probe
L, 1.0 μ L of sample to be tested DNA profiling, ultrapure water supply 25 μ L；
The pathogenic bacteria are the primer pair and the corresponding pathogenic bacteria that can be detected of probe of selection.
5 ' ends of each probe sequence are modified with reporter group, and 3 ' ends are modified with quenching group, reporter group have VIC,
Cy5, FAM, HEX, NED, JOE, FITC, quenching group have TAMRA, ECLIPSE, DABCYL, BHQ-1, BHQ-2.
It is preferred that the reporter group of probe SEQ ID NO:3 is VIC, quenching group TAMRA；
The reporter group of probe SEQ ID NO:6 is FAM, quenching group BHQ-1；
The reporter group of probe SEQ ID NO:9 is FAM, quenching group ECLIPSE；
The reporter group of probe SEQ ID NO:12 is JOE, quenching group DABCYL；
The reporter group of probe SEQ ID NO:15 is Cy5, quenching group TAMRA；
The reporter group of probe SEQ ID NO:18 is HEX, quenching group TAMRA；
The reporter group of probe SEQ ID NO:21 is NED, quenching group ECLIPSE；
The reporter group of probe SEQ ID NO:24 is JOE, quenching group TAMRA；
The reporter group of probe SEQ ID NO:27 is FITC, quenching group DABCYL；
The reporter group of probe SEQ ID NO:30 is VIC, quenching group BHQ-1；
The reporter group of probe SEQ ID NO:33 is FITC, quenching group TAMRA；
The reporter group of probe SEQ ID NO:36 is Cy5, quenching group BHQ-2.
The present invention also provides a kind of kits of real-time fluorescence quantitative PCR for detecting 12 kinds of pathogenic bacterias simultaneously, include
PCR primer as claimed in claim 4 and probe.
The beneficial effects of the present invention are:
1. the primer and probe reactivity worth according to 12 kinds of pathogenic bacteria design synthesis is good, specific outcome is shown
Primer specificity is preferable, and does not have cross reaction between bacterial strain.
2. keeping reaction condition consistent with optimization since the design to primer and probe has carried out screening, detection can be same
One wheel quantitative fluorescent PCR circulation is lower to be carried out, and is realized the detection simultaneously to various pathogens, is improved detection efficiency.
3. being able to achieve and being quantified to detection template, detection sensitivity is higher compared with conventional single PCR or multiplex PCR.
4. being detected using the method that the present invention establishes, has quick, sensitive, accurate, efficient and economic advantage, meet
The requirement of modern detecting.
Detailed description of the invention
Figure 1A -1L is the amplification curve of 12 kinds of pathogenic bacteria.
Fig. 2A -2L is 12 kinds of pathogenic bacteria primed probe specificity verification figures.
Fig. 3 A-3L is 12 kinds of pathogenic bacteria examination criteria curves and sensitivity technique result.
Fig. 4 A-4L is 12 kinds of pathogenic bacteria minimum detection limit testing results.
Specific embodiment
It is specifically described below to further appreciate that the contents of the present invention, feature:
It is of the present invention for using high-throughput Real-Time Fluorescent Quantitative PCR Technique (Taqman sonde method) detect simultaneously 12 kinds of drinking-water and
The method of common causative bacterium in food, comprising the following steps:
Target pathogenic bacteria are determined according to test object or background information, and by automatically retrieving, it is special to obtain more complete pathogenic bacteria
Anisotropic Disease-causing gene or toxin gene, using bioinformatics method and comparative genomics technology, sequence analysis search is tested
Finally conservative target-gene sequence is demonstrate,proved and obtained, primer and probe design is carried out according to the target gene of acquisition, keeps its reaction condition equal
One and consistent；Test sample is handled, genomic templates are extracted；Template is separately added into equipped with different primers and probe
In tubule, corresponding quantitative fluorescent PCR reagent is added；Under same wheel quantitative fluorescent PCR circulation, make primer and spy in every pipe
It is carried out simultaneously, quickly and quantitative detection for corresponding sample.Because fluorescence quantitative PCR instrument has 96 reacting holes, it is possible to
Multiple samples are detected.If containing above-mentioned pathogenic bacteria in sample, will be provided in corresponding primed probe pipe
Amplification curve, by the way that accurate quantitative analysis can be realized compared with standard curve.It so in a relatively short period of time, can be by primary
The quantitative detection of various pathogens is completed in reaction, convenient and efficient.
Pathogenic bacteria detected include 12 kinds of pathogenic bacteria common in drinking-water and food: Escherichia coli O 157: H7, monokaryon increase
Raw Listeria, salmonella, vibrio parahemolyticus, beta hemolytic streptococcus, small intestine colon Yersinia ruckeri, excrement hammer
Bacterium, Shigella, proteus mirabilis, vibrio fluvialis, campylobacter jejuni, staphylococcus aureus.
Special primer and probe design be described in detail: the present invention in various pathogens simultaneously, quickly, quantitative detecting method and
In its system applied, the design of special primer and probe is most important factor in whole system；Primer and probe design
Steps are as follows:
(1) it is carried out using gophers such as MedKIT, LitMiner, BioRAT or according to context searchig method design program
Full-automatic retrieval, obtains more complete pathogenic bacteria specific pathogenetic gene or toxin gene
(2) target-gene sequence is finally guarded using BLAST, ClustalX sequence analysis search validation and acquisition.
(3) GenBank(http: //ncbi.nlm.nih.gov/entrez is logged in), search obtains more complete pathogenic bacteria
Specific pathogenetic gene or toxin gene.
(2) primer and probe is carried out to target-gene sequence data using biosoftwares such as Primer Express 3.0 to set
Meter, design principle include:
A. primer length is generally 15-30bp, and the most commonly used is 18-25bp
B. G+C content 40%-60% in base content, and upstream and downstream primer sequence G/C content difference is not too big.
C. stable primer dimer and hairpin structure are avoided the formation of
D. product length is between 100-200 bp
E. the distance of primer and probe: when design primer and probe, consider design primer and probe on two chains simultaneously,
Taqman probe should close and its upstream primer on same chain.
F. the length of Taqman probe is preferably between 13-30, and Tm value is between 68-70, and the Tm value of probe is than drawing
The Tm value of object is higher by 10 DEG C
G. the evaluation of primer and probe
(3) according to above design principle, all sequences being suitable for as primer and probe is found out, candidate sequence is delivered to
GenBank carries out BLAST comparison, filters out that specificity is best synthesize.
Heretofore described primer length is 18-25 bp, and the Tm value of primer is 59 ± 2 DEG C, and the length of probe is 20-25
Bp, for the length of amplified production between 80-200 bp, every set primer and probe has similar Tm value, the primer and probe used
Sequence is shown in Table 1.
The primer and probe sequence of 1 12 kinds of pathogenic bacteria specific genes of table
The amplification of 12 kinds of pathogenic bacteria specific genes all uses TaKaRa companyPremixEx Taq (Probe qPCR)
(RR390) it is expanded, the system and ingredient of amplification are shown in Table 2, and wherein the dilution of primer and probe calculates according to calculation formula and adds
Enter the amount of TE, be first diluted to 100 μM, then be diluted to using concentration, wherein primer is 10 μM, probe is 5 μM.Quantitative fluorescent PCR
Reaction carried out on 7500 quantitative PCR apparatus of ABI, program is as follows: 95 DEG C initial denaturation 30 seconds, 95 DEG C be denaturalized 5 seconds, 55 DEG C annealing
10 seconds, 72 DEG C extended 30 seconds, 40 circulations.
2 fluorescent quantitative PCR system component table of table
One, the verifying of 12 kinds of pathogenic bacteria primed probe specificity experiments
1.12 kinds of pathogenic bacteria primed probe amplification capability verifyings
By 12 kinds of pathogenic bacteria by after respective condition of culture culture, take the bacterium solution being incubated overnight according to TaKaRa company
MiniBEST Bacteria Genomic DNA Extraction Kit(CAT#9763, TaKaRa) extract respective gene
Group.Using the genome of extraction as template, respective components are added by 2 amplification system of table, by as follows on 7500 quantitative PCR apparatus of ABI
Program: 95 DEG C initial denaturation 30 seconds, 95 DEG C are denaturalized 5 seconds, and 55 DEG C are annealed 10 seconds, and 72 DEG C extend 30 seconds, and 40 circulations carry out fluorescence and determine
Measure PCR reaction.Amplification curve is shown in Fig. 1, it can be seen from the figure that using 12 kinds of Disease-causing gene Taqman sonde methods of design synthesis
Primer is quantitatively used, amplification curve is good, the result is shown in Figure 1, illustrates that primed probe reactivity worth is good, can be used for fluorescent quantitation
PCR experiment.
2. primed probe is to other 11 kinds of bacterium specificity verifications
In order to verify the specificity of 12 kinds of pathogenic bacteria primed probes, with a kind of primed probe that pathogenic bacteria are special go to examine remaining 11
The genomic templates of kind pathogenic bacteria, amplification system and program are shown in 1, as a result see Fig. 2, do not have between 12 kinds of pathogenic bacteria specific primer probes
There is cross reaction.
Two, the production and detection sensitivity of 12 kinds of DNA of pathogenic standard items, standard curve
1. constructing DNA standard items
Utilize Cloning Transformation (using the pathogenic bacteria genome of extraction as template, the primer for designing synthesis carries out PCR amplification) or synthesis
Gene inserts are connected to building standard items plasmid in pMD-19T carrier by the method for gene, super using NanoDrop-2000
Micro-spectrophotometer measure plasmid concentration and examine purity, according to formula copy number=plasmid concentration * Avogadro constant number/
Molecular weight/molecular length calculates copy number, with the EASY Dilution of TaKaRa company that the DNA standard items gradient of building is dilute
It is interpreted as 106、105、104、103、102、101、100Copies/ μ L, high concentration takes 3 μ L, dilution (EASY when dilution
Dilution) plus 27 μ L, turbula shaker mix centrifugation.
2. standard curve making
Using the good DNA standard items of gradient dilution as template (generally choosing 5-6 point), according to 2 system of table, added respectively respective
Tubule in, on ABI7500 fluorescence quantitative PCR instrument press 95 DEG C of following reaction condition initial denaturation 30 seconds, 95 DEG C be denaturalized 5 seconds, 55
DEG C annealing 10 seconds, 72 DEG C extend 30 seconds, 40 circulations are reacted.After reaction, it makes Ct value according to amplification curve and copies
The standard curve of the shellfish number truth of a matter.
3. standard curve shows linear relationship preferably (R2>0.98), amplification efficiency is higher (80%<E<120%), is shown in Table 3.It answers
The copy number of gene in actual sample can be quantified with the standard curve of building.
3 12 kinds of pathogenic bacteria examination criteria parameters of curve of table
4. diluting the minimum concentration detection sensitivity of template according to standard curve, Fig. 3 is seen.
Three, the determination of 12 kinds of pathogenic bacteria minimum detection limits
1. the measurement of pathogenic bacteria concentration
Using colony counting method: the bacterium solution being incubated overnight is taken respectively, carries out 10 doubling dilutions with physiological saline (0.9% NaCl),
Each dilution is injected separately into two culture dishes, every ware 1mL.Injection is thoroughly melted, and 45 DEG C of red four nitrogen of addition is then cooled to
The corresponding bacterium culture medium of azoles, is laid flat on the table, is carried out back rotation culture dish, is mixed well it, is cooled to inversion culture extremely
Bacterium colony is grown.Extension rate of the selection clump count between 30-300 is counted, and the bacterium of 2 plates of same concentration is write down
It falls sum and calculates average value, be the CFU number (CFU/mL) in 1mL bacterium solution multiplied by extension rate.
2. preparing DNA profiling
By the bacterium solution of above-mentioned 10 doubling dilution, bacterium solution (such as 10 is taken-1Take 1mL i.e. 100 μ L, 200 μ L), reagent is extracted by genome
Box MiniBEST Bacteria Genomic DNA Extraction Kit(CAT#9763, TaKaRa) carry out genome mention
It takes, using the genomic DNA of extraction as template, with batch the gradient dilution of three points is selected, good DNA standard items are template (aforementioned
Three concentration points are selected in standard curve), respective components are added by 2 amplification system of table, by real on 7500 quantitative PCR apparatus of ABI
It applies program in example 1 and carries out quantitative fluorescent PCR.
3. minimum detection limit result
After fluorescent quantitative PCR experiment, using the method for absolute quantitation, the standard curve constructed using three concentration points, meter
The starting copy number (copies/ μ L) in template is calculated, in conjunction with corresponding bacterial concentration as a result, according to detection sensitivity as a result, determining
Minimum detection limit (CFU/mL).See Fig. 4 A-4L.
Specificity experiments prove, do not have cross reaction between designed primed probe, the detection sensitivity of 12 kinds of bacterium and most
Low detection limit such as table 4.
The detection sensitivity and minimum detection limit of 4 12 kinds of bacterium of table
Sequence table
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<222> (1)..(20)
<223>prfA group probe
<400> 6
cgagcaggct accgcatacg 20
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>hilA group primer 1
<400> 7
caacctacga ctcataca 18
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>hilA group primer 2
<400> 8
gcgtaattga tccatgag 18
<210> 9
<211> 25
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(25)
<223>hilA group probe
<400> 9
tcaagaatat ccttaacact gcggc 25
<210> 10
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>toxR group primer 1
<400> 10
cagactcaag ctcaattg 18
<210> 11
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(19)
<223>toxR group primer 2
<400> 11
gctctacgat tgtttctac 19
<210> 12
<211> 25
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(25)
<223>toxR group probe
<400> 12
cttctgataa caatgacgcc tctgc 25
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(21)
<223>scpA group primer 1
<400> 13
cagacattaa agcaaatact g 21
<210> 14
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(19)
<223>scpA group primer 2
<400> 14
tctgctattg tttcttctg 19
<210> 15
<211> 23
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(23)
<223>scpA group probe
<400> 15
agaagacact cctgctaccg aac 23
<210> 16
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>foxA group primer 1
<400> 16
cggtgatgtg aacaatac 18
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>foxA group primer 2
<400> 17
gccatataac gcagaaga 18
<210> 18
<211> 24
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(24)
<223>foxA group probe
<400> 18
catcaatacg ctcaaggaac cacg 24
<210> 19
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(21)
<223>ddl group primer 1
<400> 19
cccatagtaa aggatacata c 21
<210> 20
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>ddl group primer 2
<400> 20
cgctgtgatt tcttctta 18
<210> 21
<211> 25
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(25)
<223>ddl group probe
<400> 21
cctgaatgaa ttgaacacca tgcct 25
<210> 22
<211> 22
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(22)
<223>ipaH group primer 1
<400> 22
tgctcaatgt atcatataat ca 22
<210> 23
<211> 22
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(22)
<223>ipaH group primer 2
<400> 23
gctgatattc atagtcaata ac 22
<210> 24
<211> 24
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(24)
<223>ipaH group probe
<400> 24
aactaaccta cctgaactgc ctgt 24
<210> 25
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(21)
<223>ureR group primer 1
<400> 25
ccatcagatt atgtcattca a 21
<210> 26
<211> 23
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(23)
<223>ureR group primer 2
<400> 26
gaggaaaatg caatttatct tta 23
<210> 27
<211> 25
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(25)
<223>ureR group probe
<400> 27
cacaccctac ccaacattca tttca 25
<210> 28
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(18)
<223>toxR group primer 1
<400> 28
ttcgcagtct aaatttcg 18
<210> 29
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(19)
<223>toxR group primer 2
<400> 29
tccaccatat tttcttacg 19
<210> 30
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(21)
<223>toxR group probe
<400> 30
cgatgtgatt gtcagcacgc c 21
<210> 31
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(21)
<223>mapA group primer 1
<400> 31
tgctcaagtt aatcaaattt c 21
<210> 32
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(19)
<223>mapA group primer 2
<400> 32
ccctttaatc tttgcttca 19
<210> 33
<211> 24
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(24)
<223>mapA group probe
<400> 33
accaccagga ctttcacaag aact 24
<210> 34
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(19)
<223>ebps group primer 1
<400> 34
ccacatgcct ctaataatg 19
<210> 35
<211> 22
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(22)
<223>ebps group primer 2
<400> 35
gcgattttat tttcttttgt ac 22
<210> 36
<211> 22
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<222> (1)..(22)
<223>ebps group probe
<400> 36
atgccatgcc tccaaatatc gc 22
Claims (10)
1. a kind of method for the real-time fluorescence quantitative PCR for detecting 12 kinds of pathogenic bacterias simultaneously, specific steps include: 1) to cause a disease for 12 kinds
The selection and bioinformatic analysis of bacterial target genes；2) design of high throughput fluorescence quantification PCR primer probe；3) 12 kinds
Pathogenic bacteria genome extracts, and the primer of design, probe are to 12 kinds of pathogenic bacteria specificity verifications；4) DNA standard items, production mark are constructed
Directrix curve, according to standard curve determination detection sensitivity；5) measurement of 12 kinds of pathogenic bacteria minimum detection limits.
2. the method according to claim 1, wherein the corresponding target gene of 12 kinds of pathogenic bacteria is respectively Escherichia coli
The rfbE gene of O157:H7, the prfA gene of listeria monocytogenes, the hilA gene of salmonella, vibrio parahemolyticus
ToxR gene, the scpA gene of beta hemolytic streptococcus, the foxA gene of small intestine colon Yersinia ruckeri, streptococcus fecalis
Ddl gene, the ipaH gene of Shigella, the ureR gene of proteus mirabilis, the toxR gene of vibrio fluvialis, jejunum campylobacter
The mapA gene of bacterium, the ebps gene of staphylococcus aureus.
3. according to the method described in claim 2, the Tm value of primer is it is characterized in that, the primer length is 18-25 bp
59 ± 2 DEG C, the length of probe is 20-25 bp, and the length of amplified production is between 80-200 bp.
4. according to the method described in claim 3, it is characterized in that, real-time fluorescence quantitative PCR primer and probe, nucleotide sequence
It is as follows:
NO:1 ~ 2 rfbE group primer pair SEQ ID, probe SEQ ID NO:3；
NO:4 ~ 5 prfA group primer pair SEQ ID, probe SEQ ID NO:6；
NO:7 ~ 8 hilA group primer pair SEQ ID, probe SEQ ID NO:9；
NO:10 ~ 11 toxR group primer pair SEQ ID, probe SEQ ID NO:12；
NO:13 ~ 14 scpA group primer pair SEQ ID, probe SEQ ID NO:15；
NO:16 ~ 17 foxA group primer pair SEQ ID, probe SEQ ID NO:18；
NO:19 ~ 20 ddl group primer pair SEQ ID, probe SEQ ID NO:21；
NO:22 ~ 23 ipaH group primer pair SEQ ID, probe SEQ ID NO:24；
NO:25 ~ 26 ureR group primer pair SEQ ID, probe SEQ ID NO:27；
NO:28 ~ 29 toxR group primer pair SEQ ID, probe SEQ ID NO:30；
NO:31 ~ 32 mapA group primer pair SEQ ID, probe SEQ ID NO:33；
NO:34 ~ 35 ebps group primer pair SEQ ID, probe SEQ ID NO:36；
Wherein rfbE group primer pair and probe are used for Escherichia coli O 157: H7, prfA group primer pair and probe are for detecting monokaryon
Hyperplasia Listeria, hilA group primer pair and probe are used for salmonella, toxR group primer pair and probe for detecting secondary haemolysis
Property vibrios, scpA group primer pair and probe are used to detect for detecting beta hemolytic streptococcus, foxA group primer pair and probe
Small intestine colon Yersinia ruckeri, ddl group primer pair and probe are for detecting streptococcus fecalis, ipaH group primer pair and probe for examining
Shigella, ureR group primer pair and probe are surveyed for detecting proteus mirabilis, toxR group primer pair and probe for detecting
Vibrio fluvialis, mapA group primer pair and probe are golden yellow for detecting for detecting campylobacter jejuni, ebps group primer pair and probe
Color staphylococcus.
5. according to the method described in claim 3, it is characterized in that, real-time fluorescence quantitative PCR reaction system, the reaction system are
25 μ L, wherein 12.5 μ L of Premix Ex Taq (Probe qPCR), each 1.0 μ L of upstream and downstream primer, 1.0 μ L of probe, to
1.0 μ L of test sample DNA profiling, ultrapure water supply 25 μ L.
6. according to the method described in claim 4, it is characterized in that, real-time fluorescence quantitative PCR response procedures: 95 DEG C of initial denaturations 30
Second, 95 DEG C are denaturalized 5 seconds, and 55 DEG C are annealed 10 seconds, and 72 DEG C extend 30 seconds, 40 circulations.
7. according to the method described in claim 4, it is characterized in that, 5 ' ends of each probe sequence are modified with reporter group, 3 '
End is modified with quenching group.
8. the method according to the description of claim 7 is characterized in that reporter group have VIC, Cy5, FAM, HEX, NED, JOE,
FITC, quenching group have TAMRA, ECLIPSE, DABCYL, BHQ-1, BHQ-2.
9. according to the method described in claim 8, it is characterized in that,
The reporter group of probe SEQ ID NO:3 is VIC, quenching group TAMRA；
The reporter group of probe SEQ ID NO:6 is FAM, quenching group BHQ-1；
The reporter group of probe SEQ ID NO:9 is FAM, quenching group ECLIPSE；
The reporter group of probe SEQ ID NO:12 is JOE, quenching group DABCYL；
The reporter group of probe SEQ ID NO:15 is Cy5, quenching group TAMRA；
The reporter group of probe SEQ ID NO:18 is HEX, quenching group TAMRA；
The reporter group of probe SEQ ID NO:21 is NED, quenching group ECLIPSE；
The reporter group of probe SEQ ID NO:24 is JOE, quenching group TAMRA；
The reporter group of probe SEQ ID NO:27 is FITC, quenching group DABCYL；
The reporter group of probe SEQ ID NO:30 is VIC, quenching group BHQ-1；
The reporter group of probe SEQ ID NO:33 is FITC, quenching group TAMRA；
The reporter group of probe SEQ ID NO:36 is Cy5, quenching group BHQ-2.
10. a kind of kit for the real-time fluorescence quantitative PCR for detecting 12 kinds of pathogenic bacterias simultaneously, comprising just like claim 4 institute
The PCR primer and probe stated.
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| CN113293165A (en) * | 2021-06-21 | 2021-08-24 | 军事科学院军事医学研究院环境医学与作业医学研究所 | HEV specific crRNA based on CRISPR-Cas12a technology, detection kit and application thereof |
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| CN113621720A (en) * | 2021-08-13 | 2021-11-09 | 河北省畜牧兽医研究所 | Multiple fluorescent quantitative PCR method for detecting and identifying 3 food-borne pathogenic bacteria |
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