CN111424102B - Multiple PCR (polymerase chain reaction) rapid detection kit for specific bacteria of cosmetics and detection method thereof - Google Patents
Multiple PCR (polymerase chain reaction) rapid detection kit for specific bacteria of cosmetics and detection method thereof Download PDFInfo
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Abstract
The invention discloses a multiple PCR (polymerase chain reaction) rapid detection kit for characteristic bacteria of cosmetics and a detection method thereof. The method solves the problems of long detection period, complex operation and low specificity and sensitivity of specific pathogenic bacteria in cosmetics in the traditional technology, the designed primer and a multiple PCR system are assembled into a kit for quickly detecting the specific bacteria of the cosmetics, the detection method has good specificity, has no cross reaction with similar reference pathogenic bacteria, has high sensitivity, and can reach the detection limit of 10 after the enrichment of simulated pollution samples of cosmetics of different formulations 0 cfu/mL, meets the requirement of zero detection of specific bacteria of the cosmetics, and is quick and convenient.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiple PCR (polymerase chain reaction) rapid detection kit for specific bacteria of cosmetics and a detection method thereof.
Background
Specific bacteria (special microorganisms) are specific microorganisms that are not detected in cosmetics, including pathogenic bacteria and opportunistic bacteria. The determination of specific bacteria is not uniformly regulated in the world at present, and is different from country to country. At present, China is in accordance with the "cosmetic hygiene code" revised in 2007, and the number of specific bacteria, i.e., undetected pathogenic microorganisms, is 3: pseudomonas aeruginosa, staphylococcus aureus and faecal coliform. The foreign cosmetic quality assurance is mainly performed according to the regulations of the consumer science council (SCCP) of the european union or the Food and Drug Administration (FDA), including salmonella. The faecal coliform group has the same composition as the total coliform group, and the main composition is Escherichia coli, and only Escherichia coli is closely related to human life in the genus.
At present, in addition to the traditional microorganism culture method, some rapid detection means such as a rapid test strip technology, a polymerase chain reaction technology, an adenosine triphosphate bioluminescence detection technology, an electrical impedance technology, a fluorescence photoelectric method, a microorganism volatile organic compound detection technology and the like are developed according to market demands. The traditional microbial detection method needs simple experimental materials and low price, but has complex operation and long time consumption, which is about 4 to 7 days. Other technologies are simple to operate, fast and efficient, but have high cost, are easily interfered by impurities in a sample, only detect the total number of microorganisms and cannot distinguish the species of the microorganisms; PCR technology and LAMP technology are also gradually applied to cosmetic detection, but only a single target pathogen can be detected in one reaction.
Disclosure of Invention
In order to solve the problems of long detection period, complex operation and low specificity and sensitivity of specific pathogenic bacteria in cosmetics in the traditional technology, a primer and a multiple PCR system are designed to assemble a kit, and a kit and a detection method for the multiple PCR rapid detection of specific bacteria in cosmetics are provided.
The invention provides a multiple PCR (polymerase chain reaction) rapid detection kit for specific bacteria of cosmetics, which comprises specific primers of four target bacteria, namely pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and salmonella, a positive quality control product, a negative quality control product and a PCR reaction mixed solution.
Further, the specific primers of the pseudomonas aeruginosa, the staphylococcus aureus, the escherichia coli and the salmonella comprise specific primers ETA-F1 and ETA-R1 for amplifying the pseudomonas aeruginosa, specific primers nuc-F1 and nuc-R1 for amplifying the staphylococcus aureus, specific primers LacZ-F1 and LacZ-R1 for amplifying the escherichia coli, and specific primers invA-F1 and invA-R1 for amplifying the salmonella.
Further, the nucleotide sequences of the specific primers are: 1, 2, 3, 4, 5, 6, 7 and 8.
Further, the PCR reaction mixed solution comprises PCR buffer solution, Taq DNA polymerase and dNTPs.
The invention also discloses a rapid detection method using the kit, which comprises the following steps:
(1) preparing DNA templates of simulated pollution samples of cosmetics in different formulations, which comprises the following steps:
(a) hydrophilic sample: mixing sterile cosmetic water or emulsion sample in sodium salt culture medium, adding mixed bacteria liquid of four target bacteria, diluting in multiple proportion, increasing bacteria overnight, preparing nucleic acid by bacteria increasing liquid chemical cracking method,
(b) hydrophobic sample: adding a lipstick or foundation liquid sample into sterilized mineral oil and sterilized tween80 for homogenization, adding sterilized normal saline, fully and uniformly mixing and adding into a sodium salt culture medium to obtain a mixed solution, adding mixed bacteria liquid of four target bacteria into the mixed solution, sequentially diluting by multiple proportions, enriching overnight, and preparing nucleic acid by a chemical lysis method of enriched bacteria liquid;
(2) configuring a multiple PCR reaction system, wherein the amplification system comprises 5 mu L of PCR reaction mixed solution, 2 mu L of four target bacteria specific primer mixed solution, 1.0 mu L of DNA template extracted in the step (1), and ddH 2 O make up to 25. mu.L.
(3) Performing amplification by adopting a multiplex PCR method;
(4) and (3) carrying out agarose electrophoresis detection on the PCR amplification reaction product, separating 5 mu L of the amplification reaction product by using 1% agarose electrophoresis, and judging the result according to the band absence and the size.
Furthermore, the multiple reaction system in the step (2) is configured to add a positive quality control substance or a negative quality control substance to each batch of test to replace a template for test control.
Further, the amplification conditions for the multiplex PCR method in the step (3) are 94 ℃ and 4 min; 30s at 94 ℃, 30s at 64 ℃ and 45s at 72 ℃ for 32 cycles; finally, extension is carried out for 5min at 72 ℃.
Furthermore, the detection limit of the detection method after the bacteria increase of the simulated pollution samples of the cosmetics in different formulations can reach 10 0 cfu/mL。
Compared with the prior art, the invention has the advantages and the technical effects that: the multiple PCR technology has the advantages that grouped pathogens can be detected simultaneously, the operation is as simple as that of common PCR, so the detection efficiency can be greatly improved, the detection period is shortened, the detection cost is reduced, the economic benefit and the social benefit are remarkable, the kit is particularly suitable for detecting grouped pathogens, and has extremely high application value in the aspect of detection of specific bacteria of cosmetics.
The method for enrichment culture of cosmetics in different formulations and the method for preparing DNA after enrichment culture can effectively shorten the detection time and improve the detection efficiency. Meanwhile, the proportion of the template DNA of dead bacteria in an amplification system can be greatly reduced after enrichment culture, so that the reliability of the quality control of the cosmetics is ensured. In a word, the multiple PCR detection method for the specific bacteria in the cosmetics, provided by the invention, is simple to operate, efficient and sensitive, provides an effective means for quality control of the cosmetics, and can be popularized and applied to rapid detection of the specific bacteria in the cosmetics.
Drawings
FIG. 1 shows the result of the multiplex PCR detection sensitivity test of a simulated lotion sample according to the present invention, wherein M is DL1000 DNA Marker; 1 is a mixed sample of four target bacteria equivalent DNAPositive quality control; 2-8 are respectively bacteria liquid concentration of 10 6 -10 0 cfu/mL of a DNA sample prepared from four bacteria mixed bacterial liquid; 9 is a blank control of the lotion or emulsion sample without adding target bacteria; 10 is ddH 2 And (4) O negative control.
FIG. 2 shows the results of the multiplex PCR detection sensitivity experiment on the simulated emulsion sample of the present invention, wherein M is DL1000 DNA Marker; 1 is positive quality control of four target bacteria equivalent DNA mixed samples; 2-8 are respectively bacteria liquid concentration of 10 6 -10 0 cfu/mL of a DNA sample prepared from four bacteria mixed bacterial liquid; 9 is a blank control of the lotion or emulsion sample without adding target bacteria; 10 is ddH 2 And (4) O negative control.
FIG. 3 shows the result of the multiple PCR detection sensitivity test on the cream-like simulation sample of the present invention, where M is DL1000 DNA Marker; 1 is positive quality control of four target bacteria equivalent DNA mixed samples; 2-8 respectively 100mg of sterilized lipstick added with 10% concentration according to the above enrichment and DNA preparation method 6 -10 0 cfu/mL DNA sample prepared from mixed bacterial liquid of four specific bacteria; 9 is blank control of lipstick without bacteria; 9 is ddH 2 And (4) O negative control.
FIG. 4 shows the single primer specificity verification of the present invention, wherein A is nuc-F1/nuc-R1 single primer specificity verification result; b is ETA-F1/ETA-R1 single primer specificity verification result; c is the single primer specificity verification result of invA-F1/invA-R1; d is the result of single primer specificity verification of LacZ-F1/LacZ-R1. 1-4 are staphylococcus aureus ATCC26003, ATCC26113, ATCC29213 and strain 1 MRSA; 5-6 2 strains of Pseudomonas aeruginosa ATCC27853, ATCC15442, 7-9 strains of Salmonella, Salmonella enterica (CICC 21510), Salmonella typhimurium (S.typhimurium, CMCC50115), Salmonella choleraesuis (S.choleraesuis, ATCC13312), 9-14 strains of Escherichia coli CMCC44102, CVCC1490, ATCC35401, CVCC1562 and CVCC 248. 15-16 are 2 shigella strains: shigella dysenteriae (Shigella dysenteriae, CMCC51252) and Shigella flexneri (s.flexneri, CMCC 51572); 17 Enterobacter sakazakii (Enterobacter sakazakii, CMCC 45401); 18 Enterococcus faecalis (Enterococcus faecalis, ATCC 29212); 19-20 are 2 strains of bacillus: bacillus cereus (CMCC 63301) and Bacillus subtilis (b.subtilis, CMCC 63501); 21 is Listeria monocytogenes (Listeria monocytogenes, GIM 1.347); 22 is Vibrio parahaemolyticus (Vibrio parahaemolyticus, ATCC 17802); 23 Candida albicans (ATCC 10231); and 24 is a clear water control.
FIG. 5 is an electrophoretic analysis of the multiplex PCR-specific amplification products of the present invention, wherein 1-4 are Salmonella, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively; 5-10 are DNA mixed samples with the same quantity of 2 different bacteria respectively; 11-14 are DNA mixed samples with the same quantity of 3 different bacteria; 15 is the DNA mixed sample with the same quantity of the four bacteria; 16-17 are 2 shigella strains: shigella dysenteriae (s.dysenteriae, CMCC51252) and shigella flexneri (s.flexneri, CMCC 51572); 18 enterobacter sakazakii (e.sakazakii, CMCC 45401); 19 is enterococcus faecalis (e.faecalis, ATCC 29212); 20-21 are 2 strains of bacillus: bacillus cereus (b.cereus, CMCC63301) and bacillus subtilis (b.subtilis, CMCC 63501); 22 is listeria monocytogenes (l.monocytogenes, GIM 1.347); 23 is vibrio parahaemolyticus (v. parahaemolyticus, ATCC 17802); and 24 is a clear water control. .
FIG. 6 shows the result of the multiplex PCR detection sensitivity experiment of the present invention, wherein 1 is a mixed sample of DNA of four target bacteria at 15 ng/. mu.L concentration; 2-6 are DNA samples diluted by 10 times of ratio gradient respectively; 7 is ddH 2 And (4) O negative control.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.
The pathogenic bacteria used in the present example were purchased from the bacterial collection centers of the China Industrial microbiology institute, the China veterinary medical bacterial collection center and the Guangzhou microbiology institute, respectively: salmonella enterica (s. enterica): cic 21510, salmonella typhimurium (s.typhimurium): CMCC50115, salmonella choleraesuis (s. cholereas): ATCC 13312; pseudomonas aeruginosa (p. aeruginosa): ATCC27853, ATCC 15442; staphylococcus aureus (s.aureus) ATCC26003, ATCC26113, ATCC 29213; coli (e.coli): CMCC44102, CVCC1562, CVCC248, CVCC1490, ATCC 35401.
The main reagents used in this example:
taq DNA polymerase, dNTPs, 2000bp DNA Marker [ Bao bioengineering (Dalian) Co., Ltd ], primer (Beijing Hua Dai Gen technology Co., Ltd.), LB (Qingdao Haibo Biotechnology Co., Ltd.).
The main instruments used in this example:
life Touch TC-XP gene amplification instrument (Hangzhou Bori science and technology Co., Ltd.);
DYCP-31DN horizontal electrophoresis apparatus (Beijing Liuyi instruments Co., Ltd.);
Tanon-2500R full-automatic digital gel image analysis system (Shanghai Tianneng science and technology Co., Ltd.).
Example 1 multiplex PCR Rapid detection kit and method for specific bacteria in hydrophilic cosmetics
This example is used to illustrate the method for detecting specific bacteria in hydrophilic cosmetics by using the multiple PCR rapid detection kit for specific bacteria in cosmetics according to the present invention. A multiple PCR rapid detection kit for specific bacteria in cosmetics comprises: the kit comprises specific primers of four target bacteria of pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and salmonella, a positive quality control product, a negative quality control product and a PCR reaction mixed solution, wherein the PCR reaction mixed solution comprises a PCR buffer solution, Taq DNA polymerase and dNTPs, specific genes of the salmonella, the pseudomonas aeruginosa, the staphylococcus aureus and the escherichia coli are amplified at one time by adopting a multiple PCR technology according to the primers and a PCR reaction system in the kit, and then PCR amplification products are detected by agarose gel electrophoresis.
The specific primers of the pseudomonas aeruginosa, the staphylococcus aureus, the escherichia coli and the salmonella comprise specific primers ETA-F1 and ETA-R1 for amplifying the pseudomonas aeruginosa, specific primers nuc-F1 and nuc-R1 for amplifying the staphylococcus aureus, specific primers LacZ-F1 and LacZ-R1 for amplifying the escherichia coli, and specific primers invA-F1 and invA-R1 for amplifying the salmonella, and the nucleotide sequences of the primers are as follows:
ETA-F1：5’-GCGTGCTGCACTACTCCATG-3’；
ETA-R1：5’-CGATGACTGATGACCGTGGG-3’；
nuc-F1：5’-AGCGATTGATGGTGATACGG-3’；
nuc-R1：5’-TAGCCAAGCCTTGACGAACT-3’；
LacZ-F1：5’-TTTACAGGGCGGCTTCGT-3’；
LacZ-R1：5’-CGTTCGGTTGCACTACGC-3’；
invA-F1：5’-GGGTCGTTCTACATTGACAG-3’；
invA-R1：5’-AAGGGTCGTCGTTAGGACTG-3’；
adding four kinds of cosmetic detection specific bacteria mixed bacteria liquid into sterile toning lotion or emulsion, then carrying out enrichment culture, extracting total DNA in a sample, carrying out multiple PCR amplification by using primers and a reaction system in the kit, and then detecting a PCR amplification product by agarose gel electrophoresis, wherein the specific steps are as follows:
(1) preparation of DNA template of hydrophilic cosmetic simulated pollution sample
Mixing 1ml of sterile cosmetic water or lotion sample with 99ml of 1.5% sodium salt culture medium sterilized at high temperature and high pressure, adding four kinds of target bacteria mixed bacteria liquid into 1ml of the mixed liquid, and making the final concentration of each bacteria 10 6 cfu/ml, sequentially diluted to 10 times 0 cfu/ml, and a blank medium was set as a control. Enrichment was carried out at 37 ℃ and 250rpm overnight. Then, 500. mu.L of enrichment fluid is taken to prepare nucleic acid by a chemical lysis method.
(2) Multiplex PCR reaction system configuration
The amplification system comprises 5 mu L of PCR reaction mixed solution, 2 mu L of four target bacteria specific primer mixed solution, 1.0 mu L of DNA template extracted in the step (1) and ddH 2 O make up 25. mu.L.
(3) Multiplex PCR amplification reaction
The PCR amplification conditions were: 94 ℃ for 4 min; 30s at 94 ℃, 30s at 64 ℃ and 45s at 72 ℃ for 32 cycles; finally, extension is carried out for 5min at 72 ℃. The PCR reaction can be completed on a Hangzhou Bori Life Touch PCR instrument. The 5. mu.L PCR product was then loaded with 1.2% agarose gel for electrophoresis, viewed under UV light, and photographed using a gel imaging analysis system.
(4) Result detection
mu.L of the PCR product was separated by 1% agarose electrophoresis, and the results are shown in FIG. 1 and FIG. 2. The results show that: the four target bacteria amplified by the multiplex PCR system can generate amplification bands of about 750bp, 600bp, 400bp and 300bp, the amplification bands are consistent with the positive quality control fragments in size, and no amplification band is generated in blank control and PCR negative control without bacteria and cosmetics, and no false positive or false negative result is generated. The results show that the four target bacteria in the cosmetic water or the emulsion can be detected 10 by overnight enrichment culture 0 cfu/mL, has better detection sensitivity, reaches the detection standard that specific bacteria in cosmetics cannot be detected, and can be used for detecting specific bacteria in hydrophilic cosmetics such as astringent or emulsion.
Example 2 multiple PCR Rapid detection kit and method for specific bacteria of hydrophobic cosmetic variety
The main reagents and apparatus used in this example were the same as those used in example 1.
This example is provided to illustrate the method for detecting specific bacteria in hydrophobic cosmetics by using the multiple PCR rapid detection kit for specific bacteria in cosmetics according to the present invention. Adding a mixed solution of four target bacteria into a sterile lipstick, performing enrichment culture, extracting total DNA of a sample, performing multiplex PCR amplification by using the kit in the embodiment 1, and detecting a PCR amplification product through agarose gel electrophoresis, wherein the method for rapidly detecting the multiplex PCR of specific bacteria in the hydrophobic cosmetics specifically comprises the following steps:
(1) preparation of hydrophobic cosmetic simulated pollution sample DNA template
100mg of lipstick was added to 1ml of sterilized mineral oil and 1ml of sterilized tween80 for homogenization, then 8ml of sterilized normal saline was added thereto, and after thorough mixing, 1ml was added to 99ml of 1.5% sodium salt medium. Adding four bacteria mixed bacteria solution into 1ml of the above mixed solution to make the final concentration of each bacteria 10 6 cfu/ml, sequentially diluted to 10 times 0 cfu/ml, and a blank medium was set as a control. Enrichment was carried out at 37 ℃ and 250rpm overnight. Then, 500. mu.L of enrichment fluid is taken to prepare nucleic acid by a chemical lysis method.
(2) The multiplex PCR system was prepared as in example 1.
(3) The multiplex PCR reaction conditions were the same as in example 1.
(4) And (4) detecting a result: the PCR amplification reaction product is subjected to agarose electrophoresis detection, the method is the same as the example 1, the electrophoresis result is shown in the attached figure 3, and the amplification result shows that: adding the same concentration of 10 6 -10 0 cfu/mL mixed cosmetics special bacteria lipstick sample, using chemical cracking prepared DNA sample multiple PCR amplification can generate about 750bp, 600bp, 400bp and 300bp four amplification bands; neither the medium blank control nor the PCR negative control produced amplified bands, and no false positive or false negative results appeared.
The results show that the four target bacteria in the lipstick sample can be detected 10 by overnight enrichment culture 0 cfu/mL, has better detection sensitivity, reaches the detection standard that specific bacteria in cosmetics cannot be detected, and can be used for detecting specific bacteria in hydrophobic cosmetics such as lipstick and foundation liquid.
Example 3 specific detection of primers in the kit
This example illustrates the specificity of primers designed in the kit, including the specificity of a single primer pair and the specificity of a primer mixture in the kit combined by four target bacteria specific primers. The primer specificity verification method comprises the following steps:
(1) firstly, the primers designed and synthesized in the example 1 are aligned on a BLAST (http:// www.ncbi.nlm.nih.gov/BLAST) network, and the similarity of the primer pair ETA-F1/ETA-R1 and Pseudomonas aeruginosa is 100 percent and the similarity of other strains is less than 50 percent; nuc-F1/nuc-R1 has 100 percent of similarity with staphylococcus aureus only and has lower similarity with other strains; the primer pair LacZ-F1/LacZ-R1 and different serotype strains of Escherichia coli, Klebsiella sp.CP010574, Enterobacter albuginea (E.asburia, CP011591) and Enterobacter cloacae (E.cloacae, CP011584) are found to have coverage of more than 97 percent and similarity of 100 percent, and the similarity with other non-target strains is lower; the invA-F1/invA-R1 only has 100 percent of similarity with the salmonella, and has lower similarity with other strains.
(2) Single primer pair specificity verification
4 pairs of primers ETA-F1/ETA-R1, LacZ-F1/LacZ-R1, nuc-F1/nuc-R1 and invA-F308/invA-R1128 which are described in the example 1 are respectively subjected to PCR amplification, and non-target bacterium genome DNA consisting of 3 strains of salmonella, 2 strains of pseudomonas aeruginosa, 4 strains of staphylococcus aureus, 5 strains of different serotypes of escherichia coli and 9 strains of other common intestinal pathogens is taken as a template to verify the specificity of a single primer pair.
(3) Specificity verification of primer mixed liquor in kit
Combining 4 pairs of primers in the step (2) together to carry out multiplex PCR amplification so as to verify the specificity of the primer mixture. Selecting factors having great influence on the multiplex PCR according to the result of the single PCR: and (3) carrying out single-factor optimization experiments on the annealing temperature, the primer concentration and the template amount to grope and determine the optimal multiplex PCR reaction system. The reaction components and reaction conditions were optimized as follows: the annealing temperature was 55 ℃ to 68 ℃, the extension time for amplification was 30s, 60s and 90s, respectively, the primer concentration was 0.02mM to 0.4mM, and the dNTP concentration was 0.02mM to 0.5 mM. After optimization, a PCR reaction system is configured according to the following system: 5 mu L of PCR reaction mixture, 2 mu L of four target bacteria specific primer mixture, 1.0 mu L of DNA template in the step (2), and 25 mu L of ddH 2O.
The PCR conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s and elongation at 72 ℃ for 45s for 30 cycles; final extension at 72 ℃ for 5 min.
5 mu L of PCR products are separated by 1 percent agarose electrophoresis, the electrophoresis result of a single primer pair is shown in the attached figure 4, the result shows that the primer pair LacZ-F1/LacZ-R1 amplifies different serotype strains of escherichia coli to generate about 400bp bands, and no bands are generated in amplified salmonella and other non-target strains; the primer pair ETA-F1/ETA-R1 can obtain bands with the size of about 600bp when different strains of the pseudomonas aeruginosa are amplified, and no band is generated when other bacteria are amplified; the primer pair nuc-F1/nuc-R1 can amplify different strains of staphylococcus aureus to obtain a band with the size of about 300bp, and no band is generated when other bacteria are amplified. The primer pair invA-F1/invA-R1 can amplify different strains of salmonella to obtain a band with the size of about 800bp, and no band is generated when other bacteria are amplified, so that the result shows that the specific bacteria primer for the designed cosmetics has high specificity. The specific amplification bands of the specific bacteria of the cosmetics are recycled and sequenced, the actual size of the specific amplification band of the escherichia coli is 431bp, the size of the specific amplification band of the pseudomonas aeruginosa is 619bp, the size of the specific amplification band of the staphylococcus aureus is 281bp, and the specific amplification band is consistent with the design expectation of the primers, so that the designed primers are good in specificity.
The electrophoresis result of the specific detection of the primer mixed liquid in the kit is shown in figure 5, and the result shows that four target bacteria respectively amplify to generate bands of about 750bp, 600bp, 400bp and 300 bp; amplifying mixed DNA samples formed by equivalently and randomly combining genomic DNAs of four different target bacteria to obtain target bands corresponding to the added samples respectively; 8 other control bacterial strains and the negative control had no amplified bands. In the mixed DNA samples of different combinations, each target band is clear, the molecular weight is consistent with the expectation, and the amplification result is consistent with the expectation. This result indicates that the multiplex PCR detection system has excellent amplification specificity under optimized reaction conditions and reaction systems.
Example 4 multiplex PCR sensitivity detection assay
This example illustrates the detection sensitivity of the kit of the invention. The multiplex PCR reaction system in step (2) of example 1 is subjected to a sensitivity detection experiment under optimized reaction conditions. The genomic DNA of four target bacteria is diluted by 10 times of gradient and then is subjected to multiple PCR amplification, each treatment is repeated for 3 times, and the electrophoresis result is shown in figure 3.
The results show that: the amplification of the multiplex PCR system can generate amplification bands of about 750bp, 600bp, 400bp and 300bp by amplifying 15 ng/muL-1.5 ng/muL mixed DNA; amplification of 150 pg/mu L of mixed DNA can generate amplification bands of about 750bp, 400bp and 300bp, a specific band of about 600bp of the pseudomonas aeruginosa is extremely weak and hardly visible with naked eyes, and the amplification sensitivity of the system to the pseudomonas aeruginosa is 1.5 ng; amplifying a 15 pg/mu L mixed DNA sample by a multiplex PCR system to generate amplified bands of about 750bp and 300bp without a specific band of about 400bp of escherichia coli, and indicating that the detection sensitivity of the system to the escherichia coli is 150 pg; the multiplex PCR system amplification 1.5 pg/mu L mixed DNA sample only generates a weaker specific amplification band of about 750bp staphylococcus aureus, which indicates that the detection sensitivity of the staphylococcus aureus amplified by the multiplex PCR system is 15 pg; the multiplex PCR system amplifies 0.15 pg/mu L mixed DNA sample and has no amplified band, which shows that the detection sensitivity of the system to salmonella is 1.5 pg; negative controls produced no amplified bands.
In conclusion, the detection limits of the multiplex PCR system established by the invention on salmonella, pseudomonas aeruginosa, escherichia coli and staphylococcus aureus are 1.5pg, 1.5ng, 150pg and 15pg respectively, which indicates that the established multiplex amplification system has better detection sensitivity.
The above description is only an example of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can make many variations and modifications of the present invention without departing from the scope of the present invention by using the method disclosed above, and the present invention is covered by the claims.
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<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Claims (6)
1. A multiple PCR rapid detection kit for specific bacteria in cosmetics is characterized in that: the kit comprises specific primers of four target bacteria of pseudomonas aeruginosa, staphylococcus aureus, escherichia coli and salmonella, a positive quality control product, a negative quality control product and a PCR reaction mixed solution; the specific primers of the pseudomonas aeruginosa, the staphylococcus aureus, the escherichia coli and the salmonella comprise specific primers ETA-F1 and ETA-R1 for amplifying the pseudomonas aeruginosa, specific primers nuc-F1 and nuc-R1 for amplifying the staphylococcus aureus, specific primers LacZ-F1 and LacZ-R1 for amplifying the escherichia coli, and specific primers invA-F1 and invA-R1 for amplifying the salmonella; the nucleotide sequence of the specific primer is as follows in sequence: 1, 2, 3, 4, 5, 6, 7 and 8.
2. The multiple PCR rapid detection kit for specific bacteria in cosmetics according to claim 1, wherein: the PCR reaction mixed solution comprises PCR buffer solution, Taq DNA polymerase and dNTPs.
3. A rapid detection method using the kit according to any one of claims 1 to 2, characterized in that: the method comprises the following steps:
(1) preparing DNA templates of simulated pollution samples of cosmetics in different formulations, which comprises the following steps:
(a) hydrophilic sample: mixing sterile cosmetic water or emulsion sample in sodium salt culture medium, adding mixed bacteria liquid of four target bacteria, diluting in multiple proportion, increasing bacteria overnight, preparing nucleic acid by bacteria increasing liquid chemical cracking method,
(b) hydrophobic sample: adding a lipstick or foundation liquid sample into sterilized mineral oil and sterilized tween80 for homogenizing, adding sterilized normal saline, fully and uniformly mixing and adding into a sodium salt culture medium to obtain a mixed solution, adding mixed bacteria liquid of four target bacteria into the mixed solution, sequentially diluting in a multiple ratio, enriching overnight, and preparing nucleic acid by a bacteria enriching liquid chemical cracking method;
(2) configuring a multiple PCR reaction system, wherein the reaction system comprises 5 mu L of PCR reaction mixed solution, 2 mu L of four target bacteria specific primer mixed solution, 1.0 mu L of DNA template extracted in the step (1) and ddH 2 O complement 25 μL；
(3) Performing amplification by adopting a multiplex PCR method;
(4) and (3) carrying out agarose electrophoresis detection on the PCR amplification reaction product, separating 5 mu L of the amplification reaction product by using 1% agarose electrophoresis, and judging the result according to the band absence and the size.
4. The rapid detection method according to claim 3, wherein: and (3) configuring a multiple reaction system in the step (2) to add a positive quality control substance or a negative quality control substance into each batch of test to replace a template as a test control.
5. The rapid detection method according to claim 3, wherein: the amplification condition of the multiple PCR method in the step (3) is 94 ℃ and 4 min; 30s at 94 ℃, 30s at 64 ℃ and 45s at 72 ℃ for 32 cycles; finally, extension is carried out for 5min at 72 ℃.
6. The rapid detection method according to claim 3, wherein: the detection method has the detection limit of 10 after the bacteria increase of the simulated pollution samples of the cosmetics of different formulations 0 cfu/mL。
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