CN102732612B - Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology - Google Patents
Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology Download PDFInfo
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Abstract
The invention discloses a method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology. The method is characterized by comprising the following steps: A, designing a primer pair according to the OmpA gene of Enterobacter sakazakii; B, after sample DNA is extracted, carrying out non-labeled fluorescence PCR amplification by using the designed primer pair; and C, carrying out HRM typing analysis on PCR amplification products by using a fluorescence ration PCR instrument with an HRM module so as to determine the genotype of Enterobacter sakazakii. The objective of the invention is as follows: to overcome disadvantages in the prior art, the method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology with the advantages of simple and rapid operation, accurate detection results and low usage cost is provided.
Description
Technical field
A kind of method that the present invention relates to non-marked fluorescent PCR and high resolving power melting curve (HRM) analyzing and testing Enterobacter sakazakii, belongs to microorganism detection field.
Background technology
Enterobacter sakazakii (Enterobacter sakazakii) is a kind of of enterobacteriaceae, within 1980, by yellow enterobacter cloacae, rename Enterobacter sakazakii as, recently, there is in the world scholar to advise being renamed as Crow promise Bacillaceae (Cronobacter spp), but do not obtain official's official confirmation.Enterobacter sakazakii can cause serious neonatal meningitis, enterocolitis and mattress mass formed by blood stasis, and mortality ratio is up to more than 50%.There are numerous scholars the world for Enterobacter sakazakii biochemical character and rrna intervening sequence etc., is divided into Liang great branch, and its representative strain is respectively ATCC 51329 and the ATCC 29544 of American Type Culture Collecti.
Fluorescent PCR detection technique is roughly divided into two large classes: fluorescent probe method (mark fluorescent PCR) and universal fluorescence dye method (non-marked fluorescent PCR).The former utilizes fluorescently-labeled specific probe or primer to identify masterplate, and advantage is that specificity is higher, is applicable to the single-minded detection of extension increasing sequence, but testing cost wants high a lot.And the latter utilizes fluorescence dye to be combined luminous characteristic with double chain DNA molecule to indicate the increase of amplified production, its advantage is: without design fluorescent probe in addition, simple and easy to do, cost is lower.Wherein, fluorescence dye is divided into again unsaturation dyestuff (as Sybr Green) and saturable dye (as LC Green etc.).
The unsaturation dyestuffs such as relative Sybr Green, the saturable dyes such as Eva Green, LC Green, SYTO9 have the following advantages: 1. saturated dyestuff reacts without any restraining effect PCR, therefore can before PCR reaction, add, and if more the joining in reaction solution of other unsaturation fluorescence dye can be suppressed PCR reaction, therefore can only limit the quantity of and add, thereby the indication of Fluorescence PCR is received to restriction.When 2. DNA is high-temperature denatured, unsaturation fluorescence dye adds in the time of can causing DNA double chain high-temperature denatured, the luminescent dye molecule of strand part moves, luminescent dye molecule recombine is to the vacant site of double-stranded DNA, cause fluorescent signal not change, therefore occur false negative, specificity declines, and saturable dye there will not be this type of situation.3. high-temperature stable, for the PCR product of high GC content, Tm value is higher, and when DNA double chain high-temperature digestion, saturated fluorescence dyestuff bind nucleic acid is more stable.
High-resolution fusion curve (high-resolution melt, HRM) analytical technology is a kind of up-to-date genetic analysis method for transgenation scanning and gene type of rising abroad in recent years.It is a kind of efficient sane round pcr, not limited to by mutating alkali yl site and type, without sequence-specific probe, directly operation high-resolution fusion curve analysis after PCR finishes, has got final product paired samples sudden change, single nucleotide polymorphism (SNP), has methylated, the analysis of gene type etc.Because of easy and simple to handle quick, use cost is low, and result is accurate, has realized real stopped pipe operation, and HRM technology is subject to common concern.
HRM has utilized specific dyestuff can insert the characteristic in DNA double chain, and the combination situation by Real-Time Monitoring temperature-rise period double center chain DNA fluorescence dye and pcr amplification product records melting curve, thereby sample is detected.Although fluorescent PCR instrument and common fluorescent PCR instrument purchase price with HRM module are more or less the same, it is high a lot of that its temperature uniformity is wanted.As common quantitative PCR instrument temperature uniformity ± 0.25 ℃, temperature resolution is 0.1 ℃, and Rotor-Gene 6000 temperature uniformities are ± 0.01 ° of C, and temperature resolution is 0.02 ° of C.The temperature uniformity that it is high and temperature resolution make resolving accuracy can reach the differentiation to single base difference, add the appearance of saturability dyestuff (LC Green, Eva Green etc.), make the universal use of this technology of HRM become possibility.
At present, the detection method of Enterobacter sakazakii mainly contains traditional cultural method and molecular detecting method.Wherein traditional Enterobacter sakazakii method for separating and detecting, depends on biochemistry and Morphological Characteristics, and " separation of Enterobacter sakazakii counting in infant formula powder " that the U.S. FDA of conventionally usining is in the world promulgated is as classical way.China in 2008 is also with reference to having made and issued Enterobacter sakazakii check national standard (GBT 4789.40-2008), and in 2010, carried out revising (GB4789.40-2010), standard Enterobacter sakazakii tradition cultivate detection method and molecular detecting method.But there is complex operation, length consuming time, the easy shortcoming such as undetected in traditional detection method.In molecular detecting method, comprised conventional PCR method, but conventional PCR carries out electrophoresis after need to increasing, complicated operation and very easily cause pollution.There is the shortcomings such as testing cost is very high, and reagent preservation period is short in probe method PCR.
Summary of the invention
The object of the invention is in order to overcome weak point of the prior art, provide a kind of easy and simple to handle, quick, detected result is accurate, and the non-marked fluorescent PCR that use cost is low detects the method for Enterobacter sakazakii in conjunction with HRM analytical technology.
In order to achieve the above object, the present invention adopts following scheme:
Non-marked fluorescent PCR detects a method for Enterobacter sakazakii in conjunction with HRM analytical technology, it is characterized in that comprising the following steps:
A, according to Enterobacter sakazakii OmpA gene design primer pair;
After B, extraction sample DNA, utilize the primer pair of design to carry out the amplification of non-marked fluorescent PCR;
C, application are carried out HRM phenotypic analysis with the quantitative real time PCR Instrument of HRM module to pcr amplification product, determine the genotype of Enterobacter sakazakii.
Further, the present invention is undertaken by following reactions steps:
(1) adopt the softwares such as PRIMER, according to the sequence of GeneBank issue, primer pair for Enterobacter sakazakii OmpA gene design, design non-marked fluorescent PCR amplification Enterobacter sakazakii upstream primer sequence is: 5 ' GGTGAAGGATTTAACCGTGAACTT-3 ' sequence, its downstream primer is: 5 ' GCGCCTCGTTATCATCCAAAT-3 '
(2) preparation of standard substance template
With conventional PCR method amplification Enterobacter sakazakii OmpA gene.PCR product is through 1% gel electrophoresis analysis, adopting PCR product to reclaim test kit reclaims PCR product, PCR product after purifying is connected with carrier pMD18-T, connection product is converted into competent cell, and screening obtains single bacterium colony, select but bacterium colony to containing containing antibiotic liquid nutrient medium, incubated overnight, extract plasmid, the recombinant plasmid dna that extracts of take carries out PCR evaluation as template, and to the recombinant plasmid evaluation of checking order.Extract the positive recombinant plasmid that checking is correct, and measure its concentration, by its 10 times of doubling dilutions.
(3) amplification of non-marked fluorescent PCR and HRM analyze
Extract after Enterobacter sakazakii sample DNA, utilize the primer pair of design to carry out the amplification of non-marked fluorescent PCR; Application is carried out HRM analysis with the quantitative real time PCR Instrument (Gene 6000, the LightCycler of Roche company 480 etc. that comprise Corbett Rotor company) of HRM module to pcr amplification product, determines Tm value and the genotype of amplified production.
In the present invention, prepare the reaction solution 20 μ L that adopt in described quantitative fluorescent PCR reaction, comprise following ingredients:
Described fluorescence dye is selected from DNA saturability dyestuff, and described DNA saturability dyestuff is Eva Green dyestuff, LC
a kind of or both above mixtures in PLUS dyestuff, SYTO 9 dyestuffs; Described dNTP mixed solution is the mixed solution of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mM dGTP; Described PCR damping fluid is TrisCl, KCl, (NH4)
2sO4, MgCl
2in a kind of.
Described non-marked fluorescent PCR detects Enterobacter sakazakii in conjunction with HRM technology and determines that its genotypic response procedures is as follows:
(4) susceptibility and specific test
The positive template DNA of 10 times of dilutions is added to previous reaction system, carry out sensitivity experiments.Adopt other encountered pathogenic bacterias to carry out specificity checking, concrete strain name and numbering are as follows simultaneously, yin and yang attribute contrast are set simultaneously, the specificity of verification method.
| Reference strain | Numbering | Result |
| Enterobacter sakazakii(Enterobacter sakazakii) | ATCC51329 | + |
| Enterobacter sakazakii(Enterobacter sakazakii) | ATCC29544 | + |
| Escherichia.coli(colon bacillus) | ATCC11775 | - |
| Escherichia.coli (colon bacillus) | ATCC11229 | - |
| Escherchia coli (colon bacillus) | ATCC25922 | - |
| Enterobacter.aerogenes (enteroaerogen) | ATCC13048 | - |
| Proteus.mirabilis (proteus mirabilis) | ATCC12453 | - |
| Proteus.vulgaris(proteus vulgaris) | ATCC6380 | - |
| Listeria.grayi(Ge Shi Listera) | ATCC25401 | - |
| L (Listeria) .ivanovii Yi Shi Listera) | ATCC19615 | - |
| Listeria.welshimeri(Wei Shi Listera) | ATCC35897 | - |
| Listeria.innocua (listeria innocua) | ATCC33090 | - |
| Listeria.Seeligeri(Si Shi Listera) | ATCC35967 | - |
| Listeria.monocytogenes (4a) (Listeria Monocytogenes) | ATCC19114 | - |
| Enterococcus.faecalis (enterococcus faecalis) | ATCC29212 | - |
| Citrobacter.freundii (citrobacter freundii) | ATCC8090 | - |
| Staphylococcus.aureus(streptococcus aureus) | ATCC25923 | - |
| Staphylococcus.aureus (streptococcus aureus) | ATCC49444 | - |
| Staphylococcus.epidermidis (staphylococcus epidermidis) | ATCC12228 | - |
| Klebsiella.pneumoniae (Klebsiella pneumonia) | ATCC4352 | - |
| Rhodococcus.equi(Rhodococcus equi) | ATCC6939 | - |
| Salmonella.choleraesuis (Salmonella choleraesuls) | ATCC10708 | - |
| Vibrio.parahaemolyticus(Vibrio parahemolyticus) | ATCC17802 | - |
| Salmonella.Enteritidis (D) (Salmonella enteritidis) | ATCC13076 | - |
| Salmonella.Typhimurium(Salmonella typhimurium) | ATCC13311 | - |
| Yersinia.enterocolitica(yersinia entero-colitica) | ATCC27729 | - |
| Yersinia.ruckeri(Y.rucker) | ATCC29473 | - |
| Yersinia.Kristensenii(Yerinia kristensenii) | ATCC33639 | - |
| Shigella boydii(Shigella bogdii) | AB200052 | - |
| The crooked enterobacteria of Campylobacter jejuni() | ATCC33291 | - |
| Pseudomonas aeruginosa(Pseudomonas aeruginosa bacterium) | ATCC27853 | - |
| Pseudomonas putida(pseudomonas putida) | ATCC49128 | - |
| Clostridium perfringens(clostridium perfringens) | ATCC13124 | - |
In the inventive method, only have Enterobacter sakazakii to have smooth amplification curve, other non-specific pathogenic bacteria is all without obvious amplification curve, and detection sensitivity reaches 10fg, and has fine repeatability.
Enterobacter sakazakii can be divided into two gene hypotypes according to HRM analytical results, and two main representative strain ATCC 51329 and ATCC 29544 can be respectively as representative strains.Its TM value is respectively 79.9 ± 0.3 ℃ and 81.2 ± 0.3 ℃.
Between present method doubling dilution template concentrations and Cp value, be good linear relationship, its relation conefficient is 0.987, illustrates that the method has good tolerance range and satisfactory stability.
Detection method of the present invention can complete Enterobacter sakazakii is detected and gene type within 1 day, with respect to the method for other traditional detection, had simplified testing process, had also greatly shortened sense cycle.
The present invention adopts non-marked fluorescent PCR to have the following advantages in conjunction with HRM analyzing and testing Enterobacter sakazakii method:
1) once can detect 384 samples at most.Be approximately 1/3 of traditional culture assays method its detection time;
2) HRM detection sensitivity is that traditional SYBR Green fluorescent quantitative PCR detection method is more than 10 times;
3) while analyzing by HRM method, sample directly carries out HRM after pcr amplification, and PCR product is without proceeding to other analytical equipment again, and directly in same PCR pipe, analyzes, and realizes stopped pipe operation, avoids crossed contamination.And can complete quantitative analysis and gene type simultaneously.
4) HRM only need design PCR primer, carries out PCR reaction, without sequence-specific probe, without order-checking, is not also subject to the limitation of mutating alkali yl site and type;
5) compare traditional probe method fluorescent PCR, HRM greatly reduces use cost;
6) HRM only detects the variation of fluorescence intensity in PCR sample, does not consume any PCR sample, does not damage pcr amplification product, and PCR product can carry out downstream analysis, as direct purification order-checking.
Accompanying drawing explanation
Fig. 1 is 10 times of dilution Enterobacter sakazakii non-marked fluorescent PCR amplification curve diagrams;
Fig. 2 is the linear relationship chart of 10 times of dilution Enterobacter sakazakii non-marked fluorescent PCR Cp values and concentration;
Fig. 3 is Enterobacter sakazakii non-marked fluorescent PCR specificity lab diagram amplification curve diagram;
Fig. 4 is 11 strain Enterobacter sakazakii Tm calling analysis charts;
Fig. 5 is 11 strain Enterobacter sakazakii Genescan analysis charts.
Embodiment
Below in conjunction with embodiment, the present invention is described further:
The method that non-marked fluorescent PCR of the present invention detects Enterobacter sakazakii in conjunction with HRM analytical technology comprises the following steps:
A, according to Enterobacter sakazakii OmpA gene design primer pair;
After B, extraction sample DNA, utilize the primer pair of design to carry out the amplification of non-marked fluorescent PCR;
C, application are carried out HRM analysis with the quantitative real time PCR Instrument of HRM module to pcr amplification product, determine amplified production Tm value and genotype.
Wherein said Enterobacter sakazakii OmpA gene is Enterobacter sakazakii outer membrane protein gene A, the upstream primer sequence of described primer pair is: 5 ' GGTGAAGGATTTAACCGTGAACTT-3 ', the downstream primer sequence of described primer pair is: 5 ' GCGCCTCGTTATCATCCAAAT-3 '.
Wherein in step B, carry out the reaction solution (the 20 μ L of take are example) composed of the following components in non-marked fluorescent PCR amplification procedure:
Wherein said fluorescence dye is DNA saturability dyestuff, and described DNA saturability dyestuff is Eva Green dyestuff, LC
the mixture of one or more in PLUS dyestuff, SYTO 9 dyestuffs.Described dNTP mixed solution is the mixed solution being comprised of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mM dGTP.Described PCR damping fluid is TrisCl, KCl, (NH4)
2sO4, MgCl
2in a kind of.
Wherein in step B of the present invention, the amplified reaction of non-marked fluorescent PCR amplification carries out according to the following steps:
(1) 92 ℃～96 ℃ denaturation 30s;
(2) 92 ℃～96 ℃ sex change 10～30s, 55-63 ℃ of annealing 10～30s, 72 ℃ of 10～30s, carry out 45～55 circulations altogether;
(3) 72 ℃ are extended 10～30s.
In step C, pcr amplification product is carried out to HRM phenotypic analysis, carries out according to the following steps:
(1) 92 ℃～96 ℃ sex change 1min;
(2) 40 ℃ of renaturation 1min;
(3) 60～65 ℃ of initial melting temperature (Tm)s then, start program heats up and melts to 92 ℃～96 ℃, and detects in real time fluorescent signal, 15～25 times/second in fusion processes.
Embodiment 1
Use non-marked fluorescent PCR of the present invention to detect E. sakazakii in Milk Powder in conjunction with HRM analytical technology
DNA extraction
Adopt aseptic technique to take sample 25g, join 225mL nutrient broth and cultivate, cultivate 18h for 36 ± 1 ℃.Get the centrifugal 2min of 1.5mL culture 10000rpm; Throw out adds the TE damping fluid of 500 μ L, and piping and druming makes it Eddy diffusion repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, mixes, in 37 ℃ of incubation 1h; Add 100 μ L5mol/L NaCl, fully mix, then add 80ul CTAB/NaCl solution, 65 ℃ of incubation 10min again after mixing; Add isopyknic phenol/chloroform/primary isoamyl alcohol to mix, centrifugal 4-5min, proceeds to supernatant in a new pipe, adds the Virahol of 0.6-0.8 times of volume, mixes until DNA precipitates gently, and precipitation can be centrifugal a little; Precipitation is with after 70% washing with alcohol of 1mL, and the centrifugal ethanol of abandoning, adds TE solution dissolution precipitation, and-20 ℃ save backup.
Non-marked Fluorescence PCR
Reaction system is as follows
Reaction conditions is with reference to follow procedure:
Pcr amplification product cycle index is analyzed and melting curve analysis
Adopt the quantitative real time PCR Instrument of HRM module to carry out cycle index tracing analysis to pcr amplification product, if sample non-marked fluorescent PCR has obvious amplification curve, and Cp value <38, be judged to the positive, Cp value >38 and <45 repeats once, if still have obvious upcurve, be judged to the positive, Cp value >45 or be judged to feminine gender without upcurve.
Adopt genescan or Tm calling software to carry out HRM analysis to amplified production, determine amplified production Tm value and genotype.
Claims (4)
1. non-marked fluorescent PCR detects a non-diagnostic method for Enterobacter sakazakii in conjunction with HRM analytical technology, it is characterized in that comprising the following steps:
A, according to Enterobacter sakazakii OmpA gene design primer pair;
After B, extraction sample DNA, utilize the primer pair of design to carry out the amplification of non-marked fluorescent PCR;
C, application are carried out HRM analysis with the fluorescent PCR instrument of HRM module to pcr amplification product, determine Tm value and the genotype of amplified production;
Wherein said Enterobacter sakazakii OmpA gene is Enterobacter sakazakii outer membrane protein gene A, the upstream primer sequence of described primer pair is: 5 ' GGTGAAGGATTTAACCGTGAACTT-3 ', and the downstream primer sequence of described primer pair is: 5 ' GCGCCTCGTTATCATCCAAAT-3 ';
In wherein said step B, the amplified reaction of non-marked fluorescent PCR amplification carries out according to the following steps:
(1) 92 ℃～96 ℃ denaturation 30s;
(2) 92 ℃～96 ℃ sex change 10～30s, 55-63 ℃ of annealing 10～30s, 72 ℃ of 10～30s, carry out 40～50 circulations altogether;
(3) 72 ℃ are extended 10～30s;
In wherein said step C, pcr amplification product is carried out to Tm value and gene type assay, carries out according to the following steps:
(1) 92 ℃～96 ℃ sex change 1min;
(2) 40 ℃ of renaturation 1min;
(3) 60～65 ℃ of initial melting temperature (Tm)s then, start program heats up and melts to 95 ℃, and detects in real time fluorescent signal, 15～25 times/second in fusion processes.
2. non-marked fluorescent PCR according to claim 1 detects the non-diagnostic method of Enterobacter sakazakii in conjunction with HRM analytical technology, it is characterized in that carrying out in reaction solution, carrying out in non-marked fluorescent PCR amplification procedure in step B, the reaction solution of wherein preparing 20 μ L is composed of the following components:
3. non-marked fluorescent PCR according to claim 2 detects the non-diagnostic method of Enterobacter sakazakii in conjunction with HRM analytical technology, it is characterized in that described fluorescence dye is DNA saturability dyestuff.
4. non-marked fluorescent PCR according to claim 3 detects the non-diagnostic method of Enterobacter sakazakii in conjunction with HRM analytical technology, it is characterized in that described DNA saturability dyestuff is Eva Green dyestuff, LC
the mixture of one or more in PLUS dyestuff, SYTO9 dyestuff.
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| CN106399559A (en) * | 2016-11-16 | 2017-02-15 | 广东省农业科学院动物卫生研究所 | Primer pair and kit for detecting mycoplasma hyopneumoniae and Syto9 fluorescent quantitative PCR-HRM detecting method |
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