CN102732612A - 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
The present invention relates to the method for a kind of non-marked fluorescent PCR and high resolving power melting curve (HRM) analyzing and testing Enterobacter sakazakii, belong to the microorganism detection field.
Background technology
Enterobacter sakazakii (Enterobacter sakazakii) is a kind of of enterobacteriaceae; Renamed Enterobacter sakazakii as by yellow enterobacter cloacae in 1980; Recently, there is the scholar to advise renaming it as Crow promise Bacillaceae (Cronobacter spp) in the world, 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 to Enterobacter sakazakii biochemical character and rrna intervening sequence etc., and it is divided into two big branches, and its representative strain is respectively the ATCC 51329 and ATCC 29544 of American Type Culture Collecti.
The fluorescent PCR detection technique roughly is divided into two big types: fluorescent probe method (mark fluorescent PCR) and universal optical dye method (non-marked fluorescent PCR).The former then utilizes fluorescently-labeled specific probe or primer to discern masterplate, and advantage is that specificity is higher, is applicable to the single-minded detection of extension increasing sequence, wants high a lot of but detect cost.And the latter utilizes optical dye to combine emitting characteristics to indicate the increase of amplified production with double chain DNA molecule, and its advantage is: need not to design in addition fluorescent probe, simple and easy to do, cost is lower.Wherein, optical dye is divided into unsaturation dyestuff (like Sybr Green) and saturable dye (like LC Green etc.) again.
Unsaturation dyestuffs such as relative Sybr Green; Saturable dyes such as Eva Green, LC Green, SYTO9 have the following advantages: reaction has no restraining effect to 1. saturated dyestuff to PCR; Therefore can before the PCR reaction, add; And other unsaturation optical dye can suppress the PCR reaction as if more joining in the reaction solution, the adding of therefore can only limiting the quantity of, thus restriction is received in the indication that fluorescent PCR reacts.When 2. DNA is high-temperature denatured; The unsaturation optical dye adds in the time of then can causing the dna double chain high-temperature denatured, and the luminescent dye molecule of strand part moves, and the luminescent dye molecule recombine is to the vacant site of double-stranded DNA; Cause fluorescent signal not change; Therefore false negative occurs, specificity descends, and saturable dye then this type of situation can not occur.3. high-temperature stable, for the PCR product of high GC content, the Tm value is higher, and when dna double chain high-temperature digestion, saturated fluorescence dyestuff bind nucleic acid is more stable.
(high-resolution melt, HRM) analytical technology is a kind of up-to-date genetic analysis method that is used for transgenation scanning and gene type of rising abroad in recent years to the high resolution melting curve.It is a kind of efficient sane round pcr; Not limited to by mutating alkali yl site and type; Need not the sequence-specific probe, finish directly operation high resolution melting curve analysis of back, can accomplish sample sudden change, SNP (SNP), methylate, the analysis of gene type etc. at PCR.Because of easy and simple to handle quick, use cost is low, and the result is accurate, has realized real stopped pipe operation, and the HRM technology receives common concern.
HRM has utilized specific dyestuff can insert the characteristic in the dna double chain, writes down melting curve through real-time monitoring temperature-rise period double center chain DNA optical dye with the situation that combines of pcr amplification product, thereby sample is detected.Though the fluorescent PCR appearance and the common fluorescent PCR appearance purchase price of band HRM module are more or less the same, it is high a lot of that its temperature uniformity is wanted.Like common quantitative PCR appearance 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.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 biochemical and morphology characteristics, and common in the world " separation of Enterobacter sakazakii the counting in the powdered infant formulation " issued with U.S. FDA is as classical way.China in 2008 has been also with reference to having made and issued Enterobacter sakazakii check national standard (GBT 4789.40-2008), and carried out revising (GB4789.40-2010) in 2010, standard the Enterobacter sakazakii tradition cultivate detection method and molecular detecting method.But there are shortcomings such as complex operation, length consuming time, easy omission in traditional detection method.In the molecular detecting method, comprised conventional PCR method, but conventional PCR carries out electrophoresis after need increasing, complicated operation and very easily cause pollution.Shortcomings such as probe method PCR then exists the detection cost very high, and the reagent preservation period is short.
Summary of the invention
The objective 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 combines the HRM analytical technology to detect the method for Enterobacter sakazakii.
In order to achieve the above object, the present invention adopts following scheme:
A kind of non-marked fluorescent PCR combines the HRM analytical technology to detect the method for Enterobacter sakazakii, it is characterized in that may further comprise the steps:
A, right according to Enterobacter sakazakii OmpA gene design primer;
Behind B, the extraction sample DNA, utilize designed primer to carrying out the amplification of non-marked fluorescent PCR;
The quantitative real time PCR Instrument of C, application band HRM module carries out the HRM phenotypic analysis to pcr amplification product, confirms the genotype of Enterobacter sakazakii.
Further, the present invention is undertaken by following reactions step:
(1) adopts software such as PRIMER; Sequence according to the GeneBank issue; Primer to Enterobacter sakazakii OmpA gene design is right; 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.The PCR product adopts the PCR product to reclaim test kit the PCR product is reclaimed through 1% gel electrophoresis analysis, and the PCR product behind the purifying is connected with carrier pMD18-T; To connect product and be converted into competent cell, the screening obtain single bacterium colony, select but bacterium colony to containing antibiotic liquid nutrient medium; Incubated overnight; Extracting plasmid, is that template is carried out PCR and identified with the recombinant plasmid dna that extracts, and to the recombinant plasmid evaluation of checking order.Extract the correct positive recombinant plasmid of checking, and measure its concentration, with its 10 times of doubling dilutions.
(3) amplification of non-marked fluorescent PCR and HRM analyze
After extracting the Enterobacter sakazakii sample DNA, utilize designed primer to carrying out the amplification of non-marked fluorescent PCR; The quantitative real time PCR Instrument (comprising the Gene 6000, the LightCycler of Roche company 480 of Corbett Rotor company etc.) of using band HRM module carries out HRM to pcr amplification product to be analyzed, and confirms the Tm value and the genotype of amplified production.
The reaction solution 20 μ L that adopted in the said quantitative fluorescent PCR reaction of preparation among the present invention comprise following ingredients:
Said optical 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; Said dNTP mixed solution is the mixed solution of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mM dGTP; Said PCR damping fluid is TrisCl, KCl, (NH4)
2SO4, MgCl
2In a kind of.
Said non-marked fluorescent PCR combines the HRM technology that Enterobacter sakazakii is detected and confirms that its genotypic response procedures is following:
(4) susceptibility and specificity test
The positive template DNA of 10 times of dilutions is added the previous reaction system, carry out the susceptibility experiment.Adopt other encountered pathogenic bacterias to carry out the specificity checking simultaneously, concrete strain name and numbering are provided with the yin and yang attribute contrast, the specificity of verification method as follows simultaneously.
| Reference strain | Numbering | The result |
| Enterobacter sakazakii (Enterobacter sakazakii) | ATCC51329 | + |
| Enterobacter sakazakii (Enterobacter sakazakii) | ATCC29544 | + |
| Escherichia.coli (ETEC) | ATCC11775 | - |
| Escherichia.coli (ETEC) | ATCC11229 | - |
| Escherchia coli (ETEC) | 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) (monocyte hyperplasia listeria spp) | 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 (Lu Shi yersinia) | ATCC29473 | - |
| Yersinia.Kristensenii (Ke Shi yersinia) | ATCC33639 | - |
| Shigella boydii (Shigella bogdii) | AB200052 | - |
| Campylobacter jejuni (crooked enterobacteria) | ATCC33291 | - |
| Pseudomonas aeruginosa (Pseudomonas aeruginosa bacterium) | ATCC27853 | - |
| Pseudomonas putida (pseudomonas putida) | ATCC49128 | - |
| Clostridium perfringens (clostridium perfringens) | ATCC13124 | - |
Have only Enterobacter sakazakii that smooth amplification curve is arranged in the inventive method, other non-specific pathogenic bacteria does not all have obvious amplification curve, and detection sensitivity reaches 10fg, and has fine repeatability.
Enterobacter sakazakii can be divided into two gene hypotypes according to the HRM analytical results, and two main representative strain ATCC 51329 can be respectively as representative strains with ATCC 29544.Its TM value is respectively 79.9 ± 0.3 ℃ and 81.2 ± 0.3 ℃.
Be good linear relationship between present method doubling dilution template concentrations and the Cp value, its relation conefficient is 0.987, explains that this method has good tolerance range and satisfactory stability property.
Detection method of the present invention can be accomplished within 1 day Enterobacter sakazakii is detected and gene type, with respect to the method for other traditional detection, had simplified testing process, had also shortened sense cycle greatly.
The present invention adopts the non-marked fluorescent PCR to combine HRM analyzing and testing Enterobacter sakazakii method to have the following advantages:
1) once can detect 384 samples at most.Approximately be 1/3 of traditional culture assays method its detection time;
2) the HRM detection sensitivity is that traditional SYBR Green fluorescent quantitative PCR detection method is more than 10 times;
When 3) analyzing with the HRM method, sample directly carries out HRM behind pcr amplification, and the PCR product need not to change other analytical equipment again over to, and directly in same PCR pipe, analyzes, and realizes the stopped pipe operation, avoids crossed contamination.And can accomplish quantitative analysis and gene type simultaneously.
4) HRM only need design the PCR primer, carries out the PCR reaction, need not the sequence-specific probe, need not order-checking, does not also receive 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 the PCR sample, does not consume any PCR sample, does not damage pcr amplification product, and the PCR product can carry out downstream analysis, checks order like direct purification.
Description of drawings
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 an Enterobacter sakazakii non-marked fluorescent PCR specificity lab diagram amplification curve diagram;
Fig. 4 is 11 strain Enterobacter sakazakii Tm calling analysiss;
Fig. 5 is 11 strain Enterobacter sakazakii Genescan analysiss.
Embodiment
Below in conjunction with embodiment the present invention is done and to further describe:
The method that non-marked fluorescent PCR of the present invention combines the HRM analytical technology to detect Enterobacter sakazakii may further comprise the steps:
A, right according to Enterobacter sakazakii OmpA gene design primer;
Behind B, the extraction sample DNA, utilize designed primer to carrying out the amplification of non-marked fluorescent PCR;
The quantitative real time PCR Instrument of C, application band HRM module carries out HRM to pcr amplification product to be analyzed, and confirms amplified production Tm value and genotype.
Wherein said Enterobacter sakazakii OmpA gene is Enterobacter sakazakii outer membrane protein gene A; The right upstream primer sequence of said primer is: 5 ' GGTGAAGGATTTAACCGTGAACTT-3 ', the right downstream primer sequence of said primer is: 5 ' GCGCCTCGTTATCATCCAAAT-3 '.
Wherein carry out the reaction solution (is example with 20 μ L) composed of the following components in the non-marked fluorescent PCR amplification procedure among the step B:
Wherein said optical dye is a DNA saturability dyestuff, and described DNA saturability dyestuff is one or more the mixture in Eva Green dyestuff, LC
PLUS dyestuff, SYTO 9 dyestuffs.Said dNTP mixed solution is the mixed solution of being made up of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mM dGTP.Said PCR damping fluid is TrisCl, KCl, (NH4)
2SO4, MgCl
2In a kind of.
The amplified reaction of non-marked fluorescent PCR amplification carries out according to the following steps among the step B wherein according to the invention:
(1) 92 ℃～96 ℃ preparatory sex change 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.
Among the step C pcr amplification product is carried out the HRM phenotypic analysis, carries out according to the following steps:
(1) 92 ℃～96 ℃ sex change 1min;
(2) 40 ℃ of renaturation 1min;
(3) initial then melting temperature (Tm) is 60～65 ℃, and start program heats up fusion to 92 ℃～96 ℃, and in fusion processes, detects fluorescent signal, 15～25 times/second in real time.
Use non-marked fluorescent PCR according to the invention to combine the HRM analytical technology to detect Enterobacter sakazakii in the milk powder
DNA extraction
Adopt aseptic technique to take by weighing sample 25g, join the 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 to suspend again repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, and mixing is in 37 ℃ of incubation 1h; Add 100 μ L5mol/L NaCl, fully mixing adds 80ul CTAB/NaCl solution again, 65 ℃ of incubation 10min again behind the mixing; Add isopyknic phenol/chloroform/primary isoamyl alcohol mixing, centrifugal 4-5min changes supernatant in the new pipe over to, adds the Virahol of 0.6-0.8 times of volume, mixes up to DNA precipitating gently, and deposition can be centrifugal a little; After 70% washing with alcohol of deposition with 1mL, the centrifugal ethanol of abandoning adds TE solution dissolution precipitation, and-20 ℃ of preservations are subsequent use.
The reaction of non-marked fluorescent PCR
Reaction system is following
Reaction conditions is with reference to follow procedure:
The pcr amplification product cycle index is analyzed and the melting curve analysis
Adopt the quantitative real time PCR Instrument of HRM module that pcr amplification product is carried out the cycle index tracing analysis, if sample non-marked fluorescent PCR has obvious amplification curve, and Cp value < 38; Be judged to the positive; The Cp value>38 and 45, repeat once, if still have obvious upcurve; Then be judged to the positive, the Cp value>45 or do not have the rising curve and be judged to feminine gender.
Adopt genescan or Tm calling software that amplified production is carried out HRM and analyze, confirm amplified production Tm value and genotype.
Claims (8)
1. a non-marked fluorescent PCR combines the HRM analytical technology to detect the method for Enterobacter sakazakii, it is characterized in that may further comprise the steps:
A, right according to Enterobacter sakazakii OmpA gene design primer;
Behind B, the extraction sample DNA, utilize designed primer to carrying out the amplification of non-marked fluorescent PCR;
The fluorescent PCR appearance of C, application band HRM module carries out HRM to pcr amplification product to be analyzed, and confirms the Tm value and the genotype of amplified production.
2. non-marked fluorescent PCR according to claim 1 combines the HRM analytical technology to detect the method for Enterobacter sakazakii; It is characterized in that described Enterobacter sakazakii OmpA gene is Enterobacter sakazakii outer membrane protein gene A; The right upstream primer sequence of said primer is: 5 ' GGTGAAGGATTTAACCGTGAACTT-3 ', the right downstream primer sequence of said primer is: 5 ' GCGCCTCGTTATCATCCAAAT-3 '.
3. non-marked fluorescent PCR according to claim 1 combines the HRM analytical technology to detect the method for Enterobacter sakazakii; It is characterized in that carrying out among the step B in reaction solution, carrying out in the non-marked fluorescent PCR amplification procedure, the reaction solution that wherein prepares 20 μ L is composed of the following components:
4. non-marked fluorescent PCR according to claim 3 combines the HRM analytical technology to detect the method for Enterobacter sakazakii, it is characterized in that described optical dye is a DNA saturability dyestuff.
5. the HRM analytical technology that combines non-marked fluorescent PCR according to claim 4 detects the method for Enterobacter sakazakii, it is characterized in that described DNA saturability dyestuff is one or more the mixture in Eva Green dyestuff, LC
PLUS dyestuff, SYTO 9 dyestuffs.
6. the HRM analytical technology that combines non-marked fluorescent PCR according to claim 3 detects the method for Enterobacter sakazakii, it is characterized in that the mixed solution of said dNTP mixed solution for being made up of 10mM dATP, 10mM dCTP, 10mM dTTP, 10mM dGTP.
7. combine the HRM analytical technology to detect the method for Enterobacter sakazakii according to claim 1 or 3 described non-marked fluorescent PCRs, it is characterized in that the amplified reaction of non-marked fluorescent PCR amplification among the step B carries out according to the following steps:
(1) 92 ℃～96 ℃ preparatory sex change 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.
8. non-marked fluorescent PCR according to claim 1 combines the HRM analytical technology to detect the method for Enterobacter sakazakii, it is characterized in that among the step C pcr amplification product being carried out 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) initial then melting temperature (Tm) is 60～65 ℃, and start program heats up fusion to 95 ℃, and in fusion processes, detects fluorescent signal, 15～25 times/second in real time.
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