CN106399568A - Multiple PCR detection primers of escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, kit and detection method - Google Patents

Multiple PCR detection primers of escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, kit and detection method Download PDF

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CN106399568A
CN106399568A CN201611068855.3A CN201611068855A CN106399568A CN 106399568 A CN106399568 A CN 106399568A CN 201611068855 A CN201611068855 A CN 201611068855A CN 106399568 A CN106399568 A CN 106399568A
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pcr detection
pseudomonas aeruginosa
escherichia coli
multiple pcr
detection
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文霞
杨秀茳
谢小保
孙廷丽
周少璐
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention discloses multiple PCR detection primers of escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, a kit and a detection method. Specific primers are designed by means of combined utilization of escherichia coli alkaline phosphatase gene, pseudomonas aeruginosa outer membrance proteins gene and staphylococcus aureus heat-resistant nuclease gene sequences, multiple PCR detection is conducted on a sample by means of the specific primers according to the method, and specific detection of the escherichia coli, the pseudomonas aeruginosa and the staphylococcus aureus in the sample can be achieved at a time; the detection time is short, operation is easy and convenient, the detection efficiency can be effectively improved, the requirement of rapid detection is met, the sensitivity is high, the specificity is high, and the multiple PCR detection primers of the escherichia coli, the pseudomonas aeruginosa and the staphylococcus aureus, the kit and the detection method can be applied to microbiological detection work of cosmetics.

Description

The multiplex PCR detection of escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses is drawn Thing, test kit and detection method
Technical field：
The invention belongs to microorganism detection field is and in particular to escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus The multiple PCR detection primer of bacterium, test kit and detection method.
Background technology：
Cosmetics contain nutritious protein, fat, vitamin, inorganic salt etc., pole be beneficial to microorganism growth and Breeding.Microbial growth can cause cosmetics putrid and deteriorated, also can produce toxin or metabolite.These foreign bodies are as change Should former or stimulate former may produce sensitization or stimulation to using position, cause all types of cosmetic dermatosises, for example, connect Tactile property dermatitis, acne, hair damage, photosensitive dermatitiss and skin pigmentation disorder etc., therefore,《Cosmetics health specification》(2007 Year version) in regulation, excrement colibacillus group, Pseudomonas aeruginosa and Staphylococcus aureus must not be detected in every gram or every milliliter product Bacterium, wherein escherichia coli are closely related with human lives a kind of bacterium in excrement colibacillus group, are in people and homoiothermic animal intestinal Normal parasitic bacteria, as the optimal indicator bacteria of fecal pollution, the meaning of escherichia coli detection is maximum.
At present, in detection cosmetics, the method for pathogenetic bacteria is mainly bacteriology's culture, generally needs enriched culture, form The processes such as observation, Physiology and biochemistry identification, complex operation, time and effort consuming, and once can only detect a kind of pathogenetic bacteria it is difficult to expire The demand of sufficient quick detection.And the modern molecular biology method with round pcr as representative, by special primer in thermal cycler In target gene fragment entered with row index multiplication.Signal amplifies, it has also become the choosing of the ideal of the quick inspection of pathogen.Multiplex PCR It is a kind of Novel DNA amplification technique improving on the basis of Standard PCR and growing up, be to add in same reaction system simultaneously Enter a plurality of target DNA fragment of multipair primer amplification, detection, this skill while multiple pathogenic microorganisms be can achieve using this technology Art has been applied in food, drinking water, but more rare in cosmetic field, therefore, in the urgent need to set up quick, efficiently, can Detect the Cosmetics microorganism detection method of multiple pathogenic microorganisms simultaneously.
Content of the invention：
The present invention utilizes the specificity sldh gene of escherichia coli, staphylococcus aureuses and Pseudomonas aeruginosa, respectively Devise specific primer, using multiple PCR technique, 3 kinds of pathogen are used for quickly detecting.By to multiplex PCR detection system Optimization, develop in cosmetics escherichia coli, staphylococcus aureuses and Pseudomonas aeruginosa multiplex PCR detection examination Agent box；Meanwhile, in conjunction with pretreatment technology, establish detection accurately, efficient detection method.
First purpose of the present invention is the many of a kind of escherichia coli of offer, Pseudomonas aeruginosa and staphylococcus aureuses Weight PCR detection primer is it is characterised in that described multiple PCR detection primer is as follows：For E. coli phoA gene： phoA-F：5'-GTTTCTACCGCAGAGTTG-3', phoA-R：5'-GACTATGACCAGCGTGTT-3'；False single for Aerugo Born of the same parents' bacterium oprL gene：oprL-F：5'-GGCGTGCTGATGCTCGTA-3', oprL-R：5'-CGCTGACCGCTGCCTTTC-3'； For staphylococcus aureuses nuc gene：nuc-F：5'-ACATAAAGAACCTGCGAC-3', nuc-R：5'- CATTTTTCCATCAGCATA-3'.
Second object of the present invention is the many of a kind of escherichia coli of offer, Pseudomonas aeruginosa and staphylococcus aureuses Weight PCR detection kit is it is characterised in that described multiple PCR detection kit comprises the multiplex PCR described in claim 1 Detection primer.
Described multiple PCR detection kit is also included containing Mg2+PCR reaction buffer, dNTP and Taq DNA polymerization Enzyme.
Third object of the present invention is to provide above-mentioned multiple PCR detection primer or multiple PCR detection kit in inspection Survey the application in escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses.
Fourth object of the present invention is to provide escherichia coli in a kind of cosmetics, Pseudomonas aeruginosa and golden yellow Fructus Vitis viniferae The multi-PCR detection method of coccus is it is characterised in that comprise the following steps：A) cosmetic sample to be measured is carried out increasing bacterium training Support, then extract the genomic DNA of antibacterial as template；B) carried out using the multiple PCR detection primer described in claim 1 many Weight PCR amplification；C) detect multiplexed PCR amplification product, judge to whether there is escherichia coli, Pseudomonas aeruginosa in cosmetic sample And staphylococcus aureuses.
Zengjing Granule that cosmetic sample to be measured is carried out described in described step a) is specially cosmetics to be measured Sample inoculation carries out Zengjing Granule in SCDLP enrichment liquid, and described SCDLP enrichment liquid is：Every liter contains peptone 20g, cattle Meat extract 3g, NaCl 5g, K2HPO42.5g, glucose 3g, histidine 4g, sodium thioglycollate 1g, lecithin 2g and tween 80 10g, balance of distilled water, pH7.2～7.3.
The reaction system of described multiplexed PCR amplification is：DNA profiling 1pg～100ng, contain Mg2+10 × buffer solution 10mM, dNTP 0.2mM, Taq archaeal dna polymerase 1.0U, above-mentioned multiple PCR detection primer phoA-F, phoA-R, oprL-F, Each 0.5 μM of oprL-R, nuc-F and nuc-R, plus ddH2O is 25 μ L to cumulative volume；The reaction condition of described multiplexed PCR amplification For：95 DEG C of denaturations 5min；94 DEG C of degeneration 50s, 54 DEG C of annealing 40s, 72 DEG C of extension 1min20s, carry out 30 circulations；72 DEG C are prolonged Stretch 10min.
The present invention by combine escherichia coli alkaline phosphatase gene (phoA gene), P. aeruginosa bacterial outer membrane protein Gene (oprL gene) and staphylococcus aureuses heat stable nuclease gene (nuc gene) sequence separately design specific primer, According to the method for the present invention, multiplex PCR detection is carried out to sample using this specific primer, can once realize to big in sample The specific detection of enterobacteria, Pseudomonas aeruginosa and staphylococcus aureuses, has detection high specificity, sensitivity height, does not have The advantage such as have primer dimer to be formed, the sensitivity of three couples of primer amplification DNA be respectively 1.59pg/ μ L, 1.69pg/ μ L, 1.34pg/μL.
The present invention provide in cosmetics escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses multiple PCR detection method is also equipped with detecting that simple to operate, sensitivity is high, high specificity, the features such as detection time is short, with traditional detection side Method compares testing cost and the time of can greatling save, and improves efficiency and the accuracy of detection, has extensive market application foreground, It is provided with positive control and blank in the detection method of the present invention it is ensured that the accuracy of result simultaneously.
Brief description：
Fig. 1 is 3 kinds of pathogen substance PCR amplifications, wherein, M representation DNA marker, 1～7 template is large intestine bar The STb gene of bacterium ATCC8039, Pseudomonas aeruginosa ATCC9027 and staphylococcus aureuses ATCC6538,1 primer is phoA- F/phoA-R, 2 primer is oprL-F/oprL-R, and 3 primer is nuc-F/nuc-R, 4 primer be phoA-F/phoA-R and OprL-F/oprL-R, 5 primer is phoA-F/phoA-R and nuc-F/nuc-R, 6 primer be oprL-F/oprL-R and Nuc-F/nuc-R, 7 primer is phoA-F/phoA-R, oprL-F/oprL-R and nuc-F/nuc-R, and 8 is blank.
Fig. 2 is the sensitivity of primer phoA-F/phoA-R and specific amplification, wherein, M representation DNA Marker, 1～7 template is escherichia coli ATCC8039 genomic DNA, and 1～7 template final concentration is respectively 159.15ng/ μ L, 15.915ng/ μ L, 1.59ng/ μ L, 159.15pg/ μ L, 15.915pg/ μ L, 1.59pg/ μ L and 0.159pg/ μ L, 8 represent copper Green pseudomonass Pseudomonas aeruginosa, 9 represent staphylococcus aureuses Staphylococcus aureus, and 10 Represent pseudomonas putida Pseudomonas putida, 11 represent pseudomonas fluorescens Pseudomonas Fluorescens, 12 represent Burkholderia cepacia Burkholderia cepacia, and 13 represent staphylococcus epidermidiss Staphylococcus Epidermidis, 14 represent staphylococcus saprophyticus Staphylococcus saprophytics, and 15 represent thermophilic Fructus Hordei Germinatus narrow food list Born of the same parents bacterium Stenotrophomonas maltophilia, 16 represent Klebsiella pneumonia Klebsiella pneumoniae, and 17 Represent citrobacter freundii Citrobacter freundii, 18 represent serratia marcescens Serratia marcescens, 19 represent bacillus licheniformis Bacillus lincheniformis, and 20 represent bacillus subtilises Bacillus subtilis.
Fig. 3 is the sensitivity of primer oprL-F/oprL-R and specific amplification, wherein, M representation DNA Marker, 1～7 template is Pseudomonas aeruginosa ATCC9027 genomic DNA, and 1～7 template final concentration is respectively 169.34ng/ μ L, 16.934ng/ μ L, 1.69ng/ μ L, 169.34pg/ μ L, 16.934pg/ μ L, 1.69pg/ μ L and 0.169pg/ μ L, 8 represent escherichia coli Escherichia coli, same Fig. 2 of bacterial strain of 9～20 representatives.
Fig. 4 is the sensitivity of primer nuc-F/nuc-R and specific amplification, wherein, M representation DNA marker, 1 ～7 template is staphylococcus aureuses ATCC6538 genomic DNA, 1～7 template final concentration respectively 134.48ng/ μ L, 13.448ng/ μ L, 1.34ng/ μ L, 134.48pg/ μ L, 13.448pg/ μ L, 1.34pg/ μ L and 0.134pg/ μ L, 8 represent large intestine Bacillus Escherichia coli, 9 represent Pseudomonas aeruginosa Pseudomonas aeruginosa, the bacterial strain of 10～20 representatives Same Fig. 2.
Fig. 5 is primer pair phoA-F/phoA-R, oprL-F/oprL-R and nuc-F/nuc-R to escherichia coli The knot of the sensitive amplification of the STb gene of ATCC8039, Pseudomonas aeruginosa ATCC9027 and staphylococcus aureuses ATCC6538 Really, wherein, M representation DNA marker, 1～7 template final concentration is respectively 151.23ng/ μ L, 15.123ng/ μ L, 1.51ng/ μ L, 151.23pg/ μ L, 15.123pg/ μ L, 1.51pg/ μ L and 0.151pg/ μ L, 8 represent blank.
Fig. 6 is that the multiplex PCR of the escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses in the cosmetics of detection expands Increase result, wherein, M representation DNA marker, 1 represents moisturizer, and 2 represent astringent, and 3 represent facial cream, and 4 represent facial cleaning cream, 5 generations Table cosmetic water, 6 represent foundation emulsion, and 7 represent essence, and 8 represent positive control, and 9 represent negative control.
Specific embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment 1：The design of multiple PCR detection primer and screening
1st, design of primers
Respectively by escherichia coli alkaline phosphatase gene (phoA gene), Pseudomonas aeruginosa outer membrane protein gene (oprL Gene) and staphylococcus aureuses heat stable nuclease gene (nuc gene) as specific detection target sequence, carry out primer and set Meter, filters out 3 pairs of high primers of specificity as follows：
For E. coli phoA gene：phoA-F：5'-GTTTCTACCGCAGAGTTG-3'(such as SEQ ID NO.1 institute Show), phoA-R：5'-GACTATGACCAGCGTGTT-3'(is as shown in SEQ ID NO.2), the fragment length of amplification is 663bp.
For Pseudomonas aeruginosa oprL gene：oprL-F：5'-GGCGTGCTGATGCTCGTA-3'(such as SEQ ID Shown in NO.3), oprL-R：5'-CGCTGACCGCTGCCTTTC-3'(is as shown in SEQ ID NO.4), the fragment length of amplification is 444bp.
For staphylococcus aureuses nuc gene：nuc-F：5'-ACATAAAGAACCTGCGAC-3'(such as SEQ ID Shown in NO.5), nuc-R：5'-CATTTTTCCATCAGCATA-3'(is as shown in SEQ ID NO.6), the fragment length of amplification is 274bp.
2nd, substance PCR sensitivity and specificity experiments
Drawn using a pair in synthetic primer pair phoA-F/phoA-R, oprL-F/oprL-R and nuc-F/nuc-R Thing, two pairs of primers and three pairs of primers are respectively to escherichia coli ATCC8039, Pseudomonas aeruginosa ATCC9027 and golden yellow Fructus Vitis viniferae After tri- plants of bacterial strain mixed in equal amounts of coccus ATCC6538 extract STb gene enter performing PCR amplification, result amplify 663bp, 444bp and The specific fragment of 274bp, amplified fragments size and expection consistent (Fig. 1).
PCR amplification reaction system be：Concentration is the DNA profiling 1 μ L of 10ng/ μ L, contains Mg2+10 × buffer solution 10mM (final concentration), dNTP 0.2mM (final concentration), Taq archaeal dna polymerase 1.0U, upstream and downstream primer each 0.5 μM (final concentration), plus ddH2O is 25 μ L to cumulative volume；PCR amplification reaction condition be：95 DEG C of denaturations 5min；94 DEG C of degeneration 50s, 54 DEG C of annealing 40s, 72 DEG C of extension 1min20s, carry out 30 circulations；72 DEG C of extension 10min.
2.1 substance PCR sensitivity experiments
Using escherichia coli ATCC8039 genomic DNA as DNA profiling, according to final concentration be respectively 159.15ng/ μ L, 15.915ng/ μ L, 1.59ng/ μ L, 159.15pg/ μ L, 15.915pg/ μ L, 1.59pg/ μ L and 0.159pg/ μ L are added, Carry out substance PCR amplification using primer pair phoA-F/phoA-R according to the reaction system of above-mentioned PCR amplification and reaction condition.
Using Pseudomonas aeruginosa ATCC9027 genomic DNA as DNA profiling, it is respectively 169.34ng/ μ according to final concentration L, 16.934ng/ μ L, 1.69ng/ μ L, 169.34pg/ μ L, 16.934pg/ μ L, 1.69pg/ μ L and 0.169pg/ μ L are added Plus, carry out substance PCR expansion using primer pair oprL-F/oprL-R according to the reaction system of above-mentioned PCR amplification and reaction condition Increase.
Using staphylococcus aureuses ATCC6538 genomic DNA as DNA profiling, it is respectively 34.48ng/ according to final concentration μ L, 13.448ng/ μ L, 1.34ng/ μ L, 134.48pg/ μ L, 13.448pg/ μ L, 1.34pg/ μ L and 0.134pg/ μ L are added Plus, carry out substance PCR amplification using primer pair nuc-F/nuc-R according to the reaction system of above-mentioned PCR amplification and reaction condition.
The agarose gel electrophoresiies result of pcr amplification product shows：Primer pair phoA-F/phoA-R, oprL-F/oprL-R Sensitivity with nuc-F/nuc-R is respectively 1.59pg/ μ L, 1.69pg/ μ L, 1.34pg/ μ L (Fig. 2～4).
2.2 substance PCR specificity experiments
It is respectively adopted primer pair phoA-F/phoA-R, oprL-F/oprL-R and nuc-F/nuc-R, according to above-mentioned PCR The reaction system of amplification and reaction condition are respectively to the 11 plants of contaminating strains (Pseudomonas putida isolated from pollution cosmetics Bacterium, pseudomonas fluorescens, Burkholderia cepacia, staphylococcus epidermidiss, staphylococcus saprophyticus, stenotrophomonas maltophilia, Klebsiella pneumonia, citrobacter freundii, serratia marcescens, bacillus licheniformis, bacillus subtilises) enter performing PCR Amplification, result does not all amplify band (Fig. 2～4), above-mentioned three pairs of primer pair escherichia coli ATCC8039, P. aeruginosa is described This 3 kinds of pathogen of bacterium ATCC9027 and staphylococcus aureuses ATCC6538 have good detection specificity and accuracy.
3rd, multiplex PCR detection sensitivity experiment
By escherichia coli ATCC8039, Pseudomonas aeruginosa ATCC9027 and tri- plants of bacterium of staphylococcus aureuses ATCC6538 Extract STb gene after strain mixed in equal amounts as DNA profiling, according to final concentration be respectively 151.23ng/ μ L, 15.123ng/ μ L, 1.51ng/ μ L, 151.23pg/ μ L, 15.123pg/ μ L, 1.51pg/ μ L and 0.15pg/ μ L are added, simultaneously using three to drawing Thing phoA-F/phoA-R, oprL-F/oprL-R and nuc-F/nuc-R are according to the reaction system of above-mentioned PCR amplification and reaction bar Part (step 2) carries out multiplexed PCR amplification.Result is 1.51pg/ μ L (Fig. 5) using the sensitivity of above-mentioned three pairs of primer amplifications.
Embodiment 2：The detection of 3 kinds of pathogenic bacterium in cosmetics
(1) preparation of artificial contamination's sample：Seven different types of cosmetic samples of certain company's censorship, respectively moisturizing Breast, astringent, facial cream, facial cleaning cream, cosmetic water, foundation emulsion and essence, adopt《Cosmetics health specification》Micro- in (2007 editions) Biological detecting method is detected, does not all find microorganism pollution, carries out manually random pathogen contamination, adds big in moisturizer Enterobacteria ATCC8039 and Pseudomonas aeruginosa ATCC9027, adds Pseudomonas aeruginosa ATCC9027 and golden yellow in astringent Staphylococcus A TCC6538, adds escherichia coli ATCC8039 and staphylococcus aureuses ATCC6538 in facial cream, in facial cleaning cream Add escherichia coli ATCC8039, Pseudomonas aeruginosa ATCC9027 and staphylococcus aureuses ATCC6538, add in cosmetic water Enter pseudomonas putida and staphylococcus saprophyticus, in foundation emulsion, add Burkholderia cepacia and staphylococcus epidermidiss, elite Add Klebsiella pneumonia and citrobacter freundii in liquid, thus prepare artificial contamination's sample of each cosmetics.
(2) the antibacterial Zengjing Granule of sample：Take 10g artificial contamination's sample respectively, add (bacterium in 90mL SCDLP enrichment liquid In suspension, the final concentration of each bacterial strain is 102CFU/mL), after fully mixing, it is placed in 150rpm culture in 37 DEG C of shaken cultivation casees 6h, negative control is 100mL SCDLP enrichment liquid, and positive control is to add escherichia coli ATCC8039, Pseudomonas aeruginosa (in bacteria suspension, the final concentration of 3 plants of bacterial strains is equal for the 100mL SCDLP enrichment liquid of ATCC9027 and staphylococcus aureuses ATCC6538 For 102CFU/mL)；The formula of SCDLP enrichment liquid is：Every liter contains peptone 20g, Carnis Bovis seu Bubali cream 3g, NaCl 5g, K2HPO4 2.5g, glucose 2.5g, histidine 5g, sodium thioglycollate 1g, lecithin 2g, tween 80 10g, distilled water 1000mL, adjust PH7.2～7.3,121 DEG C of autoclaving 20min.
(3) extracting genome DNA：Take each 10mL of enrichment liquid after above-mentioned shaken cultivation, thalline is collected by centrifugation, uses physiology salt Water washing 3 times, extracts artificial contamination's sample, the negative control of seven cosmetics respectively using Magen DNA of bacteria extracts kit With bacterial genomes DNA of positive control, concentration is measured using BioSpec-nano ultramicrospectrophotometer, protect in -20 DEG C Deposit standby.
(4) multiplexed PCR amplification：The genomic DNA being extracted with step (3) as template, using primer pair phoA-F/phoA- R, oprL-F/oprL-R and nuc-F/nuc-R carry out multiplexed PCR amplification, and the reaction system of multiplexed PCR amplification is：Cumulative volume is 25 μ L, concentration is the DNA profiling 1 μ L of 10ng/ μ L, and 10 × buffer solution is (containing Mg2+) 10mM, dNTP 0.2mM, Taq DNA polymerization Enzyme 1.0U, each 0.5 μM of primer phoA-F, phoA-R, oprL-F, oprL-R, nuc-F and nuc-R, plus ddH2O complements to 25 μ L； The reaction condition of multiplexed PCR amplification is：95 DEG C of denaturations 5min, 94 DEG C of degeneration 50s, 54 DEG C of annealing 40s, 72 DEG C of extensions 1min20s, carries out 30 circulations, 72 DEG C of extension 10min.
Take pcr amplification product 5 μ L 1.0% agarose gel (view of Gold containing stain) electrophoresis under 120V voltage 30min, is subsequently placed in observed result (Fig. 6) in Bio-Red Labworks image acquisition and analysis software.
The electrophoresis result of pcr amplification product shows, pollution has escherichia coli ATCC8039, Pseudomonas aeruginosa ATCC9027 With the cosmetic sample of two or three of this 3 kinds of pathogen of staphylococcus aureuses ATCC6538 (moisturizer, astringent, Facial cream, facial cleaning cream) all can detect each gene respective strap, without pollute this 3 kinds of pathogen cosmetic sample (cosmetic water, Foundation emulsion, essence) band does not occur, illustrate that the multiple PCR detection primer of the present invention can quick, accurately detect that pollution is made up Escherichia coli in product, Pseudomonas aeruginosa and staphylococcus aureuses.
Sequence table
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Claims (7)

1. a kind of escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses multiple PCR detection primer it is characterised in that Described multiple PCR detection primer is as follows：For E. coli phoA gene：phoA-F：5'- GTTTCTACCGCAGAGTTG-3', phoA-R：5'-GACTATGACCAGCGTGTT-3'；For Pseudomonas aeruginosa oprL base Cause：oprL-F：5'-GGCGTGCTGATGCTCGTA-3', oprL-R：5'-CGCTGACCGCTGCCTTTC-3'；For golden yellow Staphylococcuses nuc gene：nuc-F：5'-ACATAAAGAACCTGCGAC-3', nuc-R：5'-CATTTTTCCATCAGCATA- 3'.
2. the multiple PCR detection kit of a kind of escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses, its feature exists In described multiple PCR detection kit comprises the multiple PCR detection primer described in claim 1.
3. multiple PCR detection kit according to claim 2 is it is characterised in that described multiple PCR detection kit Also include containing Mg2+PCR reaction buffer, dNTP and Taq archaeal dna polymerase.
4. the multiple PCR detection primer described in claim 1 or the multiple PCR detection kit described in Claims 2 or 3 are in inspection Survey the application in escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses.
5. in a kind of cosmetics escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses multi-PCR detection method, it is special Levy and be, comprise the following steps：A) Zengjing Granule is carried out to cosmetic sample to be measured, then extract the genomic DNA of antibacterial As template；B) carry out multiplexed PCR amplification using the multiple PCR detection primer described in claim 1；C) detection multiplex PCR expands Volume increase thing, judges to whether there is escherichia coli, Pseudomonas aeruginosa and staphylococcus aureuses in cosmetic sample.
6. multi-PCR detection method according to claim 5 it is characterised in that described in step a) to cosmetic to be measured Product sample carries out Zengjing Granule and is specially cosmetic sample to be measured is inoculated in SCDLP enrichment liquid carrying out Zengjing Granule, institute The SCDLP enrichment liquid stated is：Every liter contains peptone 20g, Carnis Bovis seu Bubali cream 3g, NaCl 5g, K2HPO42.5g, glucose 3g, group ammonia Sour 4g, sodium thioglycollate 1g, lecithin 2g and tween 80 10g, balance of distilled water, pH7.2～7.3.
7. the multi-PCR detection method according to claim 5 or 6 it is characterised in that described multiplexed PCR amplification anti- The system is answered to be：DNA profiling 1pg～100ng, contain Mg2+10 × buffer solution 10mM, dNTP 0.2mM, Taq archaeal dna polymerase Multiple PCR detection primer phoA-F, phoA-R, oprL-F, oprL-R, nuc-F and nuc-R described in 1.0U, claim 1 are each 0.5 μM, plus ddH2O is 25 μ L to cumulative volume；The reaction condition of described multiplexed PCR amplification is：95 DEG C of denaturations 5min；94℃ Degeneration 50s, 54 DEG C of annealing 40s, 72 DEG C of extension 1min20s, carry out 30 circulations；72 DEG C of extension 10min.
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