CN113444822A - Primer for detecting pseudomonas fluorescens and alkaline protease in dairy products and corresponding detection method - Google Patents
Primer for detecting pseudomonas fluorescens and alkaline protease in dairy products and corresponding detection method Download PDFInfo
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Abstract
The invention is applicable to the technical field of biological engineering, and discloses a primer for detecting pseudomonas fluorescens and alkaline protease in dairy products and a corresponding detection method thereof, wherein the primer is based onaprX gene andgyrb gene, through establishing the phylogenetic tree of pseudomonas, analyze the fluorescent pseudomonas bacterial characteristic of producing alkaline protease, design the specificity primer; the designed primer is applied to a real-time fluorescent ring-mediated isothermal amplification (RealAmp) technology to realize the detection of pseudomonas fluorescens and alkaline protease in dairy products. The primer of the invention has strong specificity and high sensitivity; the method of the invention can specifically, sensitively and rapidly detect the fluorescent pseudomonas in the dairy productsBacteria and alkaline protease, especially can specifically detect the pseudomonas fluorescens which produces the alkaline protease.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and relates to a method for detecting pseudomonas fluorescens and alkaline protease, in particular to primers for detecting the pseudomonas fluorescens and the alkaline protease in dairy products and a corresponding detection method.
Background
Pseudomonas fluorescens (Pseudomonas fluorescens) belongs to the genus Pseudomonas, is a gram-negative bacterium, widely exists in water and soil, and can grow and reproduce in a low-temperature environment. Pseudomonas fluorescens is a common contaminant in dairy products and can be rapidly and massively transmitted in refrigerated milk and other dairy products. Although pseudomonas fluorescens has no infection and pathogenicity to healthy people in vitro, after pseudomonas fluorescens enters blood, the pseudomonas fluorescens can cause severe consequences such as septicemia, septic shock, intravascular coagulation and the like, is harmful to human health and belongs to opportunistic pathogens.
The thermostable alkaline protease secreted by Pseudomonas fluorescens is a major factor in the degradation and even deterioration of dairy products during storage. Pseudomonas fluorescens will lose its activity during pasteurization, while the secreted thermostable alkaline protease will still be 10% active under ultra high temperature sterilization. These residual enzymes decompose the proteins in the milk during storage, resulting in a series of quality defects such as increased milk viscosity, coagulation and whey precipitation, severely deteriorating the organoleptic quality of the milk and significantly shortening the shelf life of the milk.
Currently, for the detection of Pseudomonas fluorescens and alkaline protease, the conventional detection methods include: sample pretreatment, selective enrichment, selective plate separation, purification, analysis, physiological and biochemical analysis and detection and the like. The detection and identification period for these assays is typically about 4-7 days. The detection time is long, and the repeatability is relatively low. In addition, general physiological and biochemical assays often rely on phenotypic expression of P.fluorescens, which is susceptible to environmental influences, and are prone to misjudgment.
With the development of the food industry and the pursuit of high-throughput rapid monitoring technology, the traditional detection method is not suitable for the scientific research and production practice at present, so that the development of a rapid, accurate and effective pseudomonas fluorescens and alkaline protease detection method is very important.
Disclosure of Invention
The invention aims to provide a primer for detecting pseudomonas fluorescens and alkaline protease in dairy products, which is based on the characteristics of gyrB genes, aprX genes and pseudomonas fluorescens producing alkaline protease, designs a specific primer and achieves the aims of being applied to a real-time fluorescent ring-mediated isothermal amplification (RealAmp) technology, and specifically, sensitively and rapidly detecting the pseudomonas fluorescens and the alkaline protease in the dairy products;
the invention also aims to provide a method for detecting pseudomonas fluorescens and alkaline protease in dairy products, which achieves the aim of specifically, sensitively and rapidly detecting the pseudomonas fluorescens and the alkaline protease in the dairy products by applying the primers to a real-time fluorescence ring-mediated isothermal amplification (RealAmp) technology.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a primer for detecting pseudomonas fluorescens and alkaline protease in dairy products comprises a primer based on aprX gene and a primer based on gyrB gene, wherein:
the primer based on the aprX gene and the sequence thereof are as follows:
primer A-FOP: 5'-GCCCGCTGATCGACGACAT-3',
Primer A-BOP: 5'-AGTCCAGGGTGTCGTTGCC-3',
Primer A-FIP: 5'-AGGTGGTGTCCGTGGCGCGATCCAGAAGCTCTA-3',
Primer A-BIP: 5'-GGTTCAACTCCAACACCGCCGTCCCATACCGAGAACA-3',
Primer A-FLP: 5'-GCTGAGGTTGGCACCG-3' and
primer A-BLP: 5'-GCTACTTCCAATGCCGACA-3', respectively;
primer based on gyrB gene and its sequence are:
primer G-FOP: 5' -GGTATCGTCCTC primer A-3
Primer G-BOP: 5'-AAGCACAACAGGTTCT-3',
Primer G-FIP: 5'-GGTATTCAACGAACGCGCGCGGCAAGGAAGAAC-3',
Primer G-BIP: 5'-GTCAACCAGGTGTTACTGCAGGGCGATTTCCACG-3',
Primer G-FLP: 5'-CCGCCTTCGTACTTGAACA-3' and
primer G-BLP: 5'-CCACTTCAACAT-3' are provided.
The invention also provides a method for detecting pseudomonas fluorescens and alkaline protease in dairy products, which is sequentially carried out according to the following steps:
s1, extracting DNA of a sample to be detected as a template;
s2, mixing a RealAmp reaction system by adopting the aprX gene-based primer in claim 1 to obtain a system alpha; mixing a RealAmp reaction system by using the primer based on the gyrB gene as the claim 1 to obtain a system beta;
s3, respectively carrying out RealAmp reaction on the system alpha and the system beta, and monitoring the fluorescence intensity in real time to obtain reaction results;
s4, judging a result:
【1】 Judging that the sample to be detected with the system alpha showing a positive result through reaction contains alkaline protease;
and if the result is negative, the sample to be detected does not contain the alkaline protease.
【2】 Judging that the sample to be detected with the system beta showing a positive result after reaction contains pseudomonas fluorescens;
and if the result is negative, the sample to be detected does not contain the pseudomonas fluorescens.
As another limitation, the system α, in parts by volume, comprises: 1.8-2.5 parts of 10X reaction buffer solution, 0.8-1.2 parts of magnesium sulfate solution, 0.8-1.2 parts of dNTPs, 2.5-4 parts of primer A-FIP, 2.5-4 parts of primer A-BIP, 0.45-0.9 part of primer A-FOP, 0.45-0.9 part of primer A-BOP, 0.45-0.9 part of primer A-FLP, 0.45-0.9 part of primer A-BLP, 0.6-1.2 parts of polymerase, 0.1-0.4 part of SYBRgreenl, 0.5-1.5 parts of template and 5-10 parts of sterilized distilled water.
As a further limitation, the concentration of the magnesium sulfate solution is 35-65mmol/L, the concentration of dNTPs is 7-13mmol/L, and the concentration of each primer is 7-13 mu mol/L.
As a third definition, the system β, in parts by volume, comprises: 2.3-2.7 parts of 10X reaction buffer solution, 0.8-1.2 parts of magnesium sulfate solution, 0.8-1.2 parts of dNTPs, 3.2-3.7 parts of primer G-FIP, 3.2-3.7 parts of primer G-BIP, 0.3-0.8 part of primer G-FOP, 0.3-0.8 part of primer G-BOP, 3.2-3.8 parts of primer G-FLP, 3.2-3.8 parts of primer G-BLP, 0.8-1.2 parts of polymerase, 0.2-0.4 part of SYBRgreenl, 0.5-1.5 parts of template and 1-3.5 parts of sterilized distilled water.
As a further limitation, the concentration of the magnesium sulfate solution is 35-65mmol/L, the concentration of dNTPs is 8-10mmol/L, and the concentration of each primer is 7-13 mu mol/L.
As a limitation, in the system alpha, the final concentration ratio of the primer A-FOP, the primer A-BOP, the primer A-FIP, the primer A-BIP, the primer A-FLP and the primer A-BLP is 1:1:5:5:1: 1;
in the system beta, the final concentration ratio of the primer G-FOP, the primer G-BOP, the primer G-FIP, the primer G-BIP, the primer G-FLP and the primer G-BLP is 1:1:7:7:7: 7.
As still further defined, the Pseudomonas fluorescens is an alkaline protease-producing Pseudomonas fluorescens.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the primers are based on aprX genes and gyrB genes, the characteristics of pseudomonas fluorescens strains for producing alkaline protease are analyzed by establishing a phylogenetic tree of pseudomonas, the obtained specific primers are designed, and the designed primers are applied to a real-time fluorescent ring-mediated isothermal amplification (RealAmp) technology, so that the detection of the pseudomonas fluorescens and the alkaline protease in dairy products can be realized; the primer has strong specificity and high sensitivity, and the method can specifically, sensitively and quickly detect the pseudomonas fluorescens and the alkaline protease in the dairy products, not only identifies the species of the pseudomonas fluorescens, but also can specifically detect the pseudomonas fluorescens producing the alkaline protease.
The quality influence of the pseudomonas fluorescens on the dairy products is mainly realized by the alkaline protease, if the milk source is abandoned only by identifying the existence of the pseudomonas fluorescens, part of the milk source polluted by the pseudomonas fluorescens which does not produce the protease is wasted, and the part of the milk source can still be used after being sterilized by a sterilization means such as pasteurization, so the technology can improve the utilization rate of the milk source and reduce the production cost.
For the sterilized finished milk product, the taste of the milk product is deteriorated under the condition of no microbial pollution, probably because the milk source is polluted by pseudomonas fluorescens which can produce alkaline protease at the early stage, 10 percent of alkaline protease exists after sterilization, and the quality of the milk product is changed.
Compared with the 1-1.5h amplification procedure of the fluorescent quantitative PCR and the 0.5-0.6h reaction time of the realAMP, the method of the invention is more efficient.
In conclusion, the primer is suitable for the real-time fluorescence ring-mediated isothermal amplification (RealAmp) technology, and the method is suitable for industrial production and can be used for preparing pseudomonas fluorescens and alkaline protease detection kits.
The invention is described in further detail below with reference to the figures and the embodiments.
Drawings
FIG. 1 is a phylogenetic tree diagram of Pseudomonas in example 1 based on the aprX gene;
FIG. 2 is a phylogenetic tree diagram of Pseudomonas in example 1 based on the gyrB gene;
FIG. 3 is an enlarged graph of example 2;
FIG. 4 is a graph of dissociation curves in example 8;
FIG. 5 is a graph showing the detection limit of Pseudomonas fluorescens in pure culture in example 9;
FIG. 6 is a graph showing the limit of detection of Pseudomonas fluorescens in 10% skim milk powder solution in example 9.
Detailed Description
Example 1 primer for detecting Pseudomonas fluorescens and alkaline protease in dairy products
Principle of primer design
Two phylogenetic trees are respectively constructed according to the amplification region sequences of aprX and gyrB genes of the pseudomonas fluorescens and the sequence of the non-pseudomonas fluorescens with the identity value of more than 92 percent in GenBank. 28 sequences were selected by aligning aprX sequences from Pseudomonas in GenBank and similar species using Clustalx1.81 software. After aligning the 28 sequences using the DAMBE software, the same sequences were deleted. Finally, 13 unique sequences were retained for the construction of phylogenetic trees.
41 sequences were determined by aligning gyrB sequences of Pseudomonas in GenBank using Clustalx1.81 software. After aligning the 41 sequences using the DAMBE software, the same sequences were deleted. While the remaining 21 unique sequences were used to construct phylogenetic trees.
A phylogenetic tree of Pseudomonas was constructed by the neighbor joining method based on the aprX gene (FIG. 1). The phylogenetic tree can be seen in FIG. 1 as being divided into three main branches: a first branch, a second branch, and an outer group. Branch I comprises 2 species, i.e. Pseudomonas fluorescens (p. fluoroscens) and Pseudomonas liliaceae (p. lurida). The secondary strains consist of 3 species, namely Pseudomonas azotoform (Pseudomonas azotoform), Pseudomonas oblata (Pseudomonas panacis) and Pseudomonas trivialis (Pseudomonas trivialis).
The outer group comprises 7 species, i.e. Pseudomonas chrysogenum (Pseudomonas synxanthona), Pseudomonas torra (Pseudomonas tolasaii), Pseudomonas orientalis (Pseudomonas orientalis), Pseudomonas libanoensis (Pseudomonas libanensis), Pseudomonas similis (Pseudomonas simiae), Pseudomonas poae (Pseudomonas poae) and Pseudomonas rhodesiae (Pseudomonas rhodesiae).
Therefore, the aprX gene can serve as a reliable molecular marker for identifying Pseudomonas fluorescens thermostable alkaline proteases other than Pseudomonas liliicola (P.lurida).
Based on the gyrB gene, a phylogenetic tree of Pseudomonas was established by the neighbor joining method (FIG. 2). From FIG. 2, it can be seen that the phylogenetic tree is divided into five main branches: one, two, three, four and outer groups.
Only one of the branches is pseudomonas fluorescens. The second branch consists of 3 species, namely Pseudomonas simians (Pseudomonas simiae), Pseudomonas Torasis (Pseudomonas tolasaii) and Pseudomonas marginalis (Pseudomonas marginalis). The third branch has 8 species in common, namely Pseudomonas rhodesiae (Pseudomonas rhodesiae), Pseudomonas azotoforma (Pseudomonas azotoform), Pseudomonas pseudomonads (Pseudomonas poae), Pseudomonas reactiori (Pseudomonas reactionans), Pseudomonas trilobata (Pseudomonas salomonii), Pseudomonas orientalis (Pseudomonas orientalis), Pseudomonas albuginea (Pseudomonas brenneri) and Pseudomonas mixugula (Pseudomonas migula). The fourth branch consists of 6 species, i.e., Pseudomonas lilacina (P.lurida), Pseudomonas moraxei (Pseudomonas moraviens), Pseudomonas parvus (Pseudomonas trivialis), Pseudomonas xanthogenes (Pseudomonas synxanthogena), Pseudomonas saponaria (Pseudomonas saponariphi) and Pseudomonas korea (Pseudomonas koreanensis). The outer population consists of one species, Pseudomonas stutzeri.
In a phylogenetic tree, pseudomonas fluorescens alone clustered on one shoot with 95% confidence and pseudomonas lilyturf (p.lurida) clustered on a different shoot; pseudomonas fluorescens can be identified from different species of Pseudomonas based on the gyrB gene.
Thus, specific primers can be designed based on the specific regions of the aprX and gyrB genes.
(II) primer design
Species-specific primers were designed by adjusting similar gyrB gene sequences using the intraspecific conserved variation region and the inter-specific variation region of the gyrB gene of Pseudomonas fluorescens. RealAmp primers were developed by targeting the gyrB gene of Pseudomonas fluorescens ATCC13525(genebank Acc. No: LT907842.1) using the online primer design software primeredorer V5(https:// primereplorer. jp/elamp 5.0.0/index. html.) including the anterior outer primer G-FOP and the posterior outer primer G-BOP, the anterior inner primer G-FIP and the posterior inner primer G-BIP, the anterior loop primer G-FLP and the posterior loop primer G-BLP, with specific sequences as shown in Table 1.
Species-specific primers were designed by adjusting similar aprX gene sequences using the intra-species conserved variation region and inter-specific variation region of the aprX gene of Pseudomonas fluorescens. RealAmp primers were developed from aprX gene targeting Pseudomonas fluorescens P.fluoescens F (genebank Acc.No: DQ146945.2), including the front outer primer A-FOP and the rear outer primer A-BOP, the front inner primer A-FIP and the rear inner primer A-BIP, the front loop primer A-FLP and the rear loop primer A-BLP, and the specific sequences are shown in Table 1. The above primers were synthesized by Shanghai Biotechnology Ltd.
TABLE 1 primer sequence Listing
Example 2 method for detecting Pseudomonas fluorescens and alkaline protease in dairy products
One) preparation of sample to be tested
1) 10% skimmed milk powder solution.
Adding 10g skimmed milk powder into a triangular bottle, adding 90g 50-60 deg.C water, stirring to dissolve, sterilizing with high pressure steam at 115 deg.C for 15min, and packaging the 10% skimmed milk powder solution into 9mL sterile test tubes.
2) And (5) culturing the bacteria to be tested.
16 P. fluorescens strains and 34 similar strains were taken and cultured in the above 10% skim milk powder solution for 18 hours under the corresponding optimum conditions as shown in Table 2.
TABLE 2 information table of strains to be tested
aATCC represents american type culture collection;
bCICC stands for China center for Industrial culture Collection;
cCMCC stands for the national medical culture Collection;
other strains are deposited in the food science and engineering laboratories of Shijiazhuang institute of food science and engineering, and all the strains can be obtained by the public.
3) And preparing a sample to be tested.
1.5mL of the suspension obtained by culturing the above 50 strains (16P. fluorescens and 34 similar strains) was used as a sample to be tested for DNA extraction.
II) method for detecting pseudomonas fluorescens and alkaline protease in dairy products
The method comprises the following steps:
s1, extracting DNA of a sample to be detected as a template:
DNA in a sample to be detected is extracted by adopting a DNA kit of Tiannanpu bacteria (TIANGEN biotechnology (Beijing) Co., Ltd.). The DNA was collected and resuspended in 100. mu.L of TE (Tris EDTA) buffer. DNA samples were quantified using a nanodrop 2000 spectrophotometer. The resulting DNA was stored at-20 ℃ until use.
S2, mixing a RealAmp reaction system, wherein the ratio of a system alpha to a system beta is as follows:
RealAmp reaction system for amplification of aprX gene in a volume of 25. mu.L, containing 2.5. mu.L of 10 × Thermopol reaction buffer (New England Biolabs Inc., USA), 1.0. mu.L of magnesium sulfate solution (50mmol/L), 1.0. mu.L of dNTPs (10mmol/L) (Sigm primer A-Aldrich, St. Louis, MO, USA), 3.5. mu.L of primer A-FIP and primer A-BIP (10. mu. mol/L), 0.7. mu.L of primer A-FOP and primer A-BOP (10. mu. mol/L), 0.7. mu.L of primer A-FLP and primer A-BLP (10. mu. mol/L), 0.9. mu.L of 8U of Bst polymerase (New England Biolabs Inc., USA), 0.3. mu.L of 1/300 diluted 10000 × SYBRnI, 1. mu. LDeee and the balance of distilled water;
in the system alpha, the final concentration ratio of the primer A-FOP, the primer A-BOP, the primer A-FIP, the primer A-BIP, the primer A-FLP and the primer A-BLP is 1:1:5:5:1: 1.
RealAmp reaction system for amplification of the gyrB gene in a volume of 25. mu.L, containing 2.5. mu.L of 10 × Thermopol reaction buffer (New England Biolabs Inc., USA), 1.0. mu.L of magnesium sulfate solution (50mmol/L), 1.0. mu.L of dNTPs (10mmol/L) (Sigm primer A-Aldrich, St. Louis, MO, USA), 3.5. mu.L of primer G-FIP and primer G-BIP (10. mu. mol/L), 0.5. mu.L of each of primer G-FOP and primer G-BOP (10. mu. mol/L), 3.5. mu.L of each of primer G-FLP and primer G-BLP (10. mu. mol/L), 0.9. mu.L of 8U Bst polymerase (New England Biolabs laboratories, 0.3. mu.L of 1/300 diluted 10000 × SYBRnI, 1. mu. LDeenA template and the balance of distilled water;
in the system beta, the final concentration ratio of the primer G-FOP, the primer G-BOP, the primer G-FIP, the primer G-BIP, the primer G-FLP and the primer G-BLP is 1:1:7:7:7: 7.
S3, respectively carrying out RealAmp reaction on the system alpha and the system beta, and monitoring the fluorescence intensity in real time to obtain a reaction result:
both reaction systems were covered and insulated with 20 μ L of mineral oil, respectively, to prevent contamination that could lead to false positive problems. The two reaction systems were reacted at 62 ℃ using Applied Biosystems QuantStaudio 3, and the fluorescence amplification curve was monitored in real time for 40min, as shown in FIG. 3.
S4, judging a result: the sample to be tested, in which the judgment system alpha shows a positive result through reaction, contains alkaline protease, and the sample to be tested, in which the judgment system beta shows a positive result through reaction, contains pseudomonas fluorescens, and the results are shown in table 3.
With reference to fig. 3 and table 3, in the method for detecting pseudomonas fluorescens and alkaline protease in dairy products of the present invention, the system α result is consistent with the theoretical result, and the system β result shows: the result of detecting DNA of 16 P.fluorescens was positive (+), and the other 34 similar P.fluorescens strains and blanks were negative (-). Experimental results show that the primer designed based on the gyrB gene has species specificity to pseudomonas fluorescens (P. fluorescens), and the primer disclosed by the invention is high in sensitivity and accurate in result.
Table 3 table of test results of example 1
Therefore, the specificity of the primer designed by the invention is verified; the designed primers can reliably distinguish between Pseudomonas fluorescens (P. fluorescens) and similar species of Pseudomonas fluorescens.
Examples 3-7 methods for detecting Pseudomonas fluorescens and alkaline protease in Dairy products
Examples 3-7 are a method for detecting Pseudomonas fluorescens and alkaline protease in dairy products, respectively, the steps are similar to example 2, except that the raw material parameters for the mixed RealAmp reaction system in S2 are different, see Table 4 specifically, and the other process parameters are the same as example 1.
TABLE 4 RealAmp reaction System parameters as mixed in examples 3-7S2
In the above system α and system β, 8U Bst polymerase (new uk biosciences, usa) was used as the polymerase, and 10000 × SYBRgreenI was used as 1/300 dilution, and the other steps and parameters were the same as in example 2.
Example 8 dissociation curves for the method for detecting Pseudomonas fluorescens and alkaline protease in Dairy products
In the gyrB gene-based RealAmp assay for system β, the dissociation temperature of the pseudomonas fluorescens (p. fluoroscens) amplification product was almost always maintained at 88.0 ℃, indicating that there was no non-specific amplification reaction in the RealAmp assay (fig. 4). Pseudomonas liliflora (p. lurida) strain cic 22026 and the blank control group had no dissociation temperature. The dissociation temperatures of the forward amplification products of pseudomonas fluorescens (p. fluorochens) ATCC13525 and pseudomonas fluorescens (p. fluorochens) cic 23250 in the RealAmp assay were 88.27 ℃ and 88.38 ℃, respectively.
In the system α aprX gene-based RearlAmp assay, the dissociation temperature of the amplification product was almost always maintained at 90 ℃ (fig. 4).
The dissociation temperatures of the amplification products of Pseudomonas fluorescens (P.fluorescens) ATCC13525, Pseudomonas fluorescens (P.fluorescens) CICC23250 and Pseudomonas fluorescens (P.fluorescens) CICC22026 in the RealAmp assay were approximately 90.42 ℃, 89.80 ℃ and 90.11 ℃, respectively. The dissociation temperatures of the cross amplification product and the forward amplification product are not different.
The results show that the primer designed in the experiment can specifically identify the pseudomonas fluorescens producing the alkaline protease without non-specific amplification.
Example 9 method for detecting Pseudomonas fluorescens and alkaline protease in Dairy products detection Limit
(1) Determination of detection Limit in 10% skim milk powder solution
Pseudomonas fluorescens is inoculated in BHI meat liquid and cultured for 18h at 30 ℃. 3mL of the bacterial suspension was centrifuged at 12000rpm for 2 min. The supernatant was discarded, resuspended in 1mL sterile water, and added to 9mL 10% sterile skim milk powder solution. 1mL of the bacterial solution taken from 10mL of the inoculated 10% skim milk powder solution was serially diluted 10-fold and counted on the medium plate. The bacteria concentration of the inoculated 10% skimmed milk powder solution is from 2.2X 10-1CFU/mL to 2.2X 108CFU/mL varied. The cells were collected by centrifugation in 1mL of inoculated 10% skim milk powder solution. DNA was extracted and dissolved in 100. mu.LTE buffer.
DNA was serially diluted 10-fold in TE buffer. DNA dilutionThe concentration of the solution ranged from 0.289 fg/. mu.L to 28.9 ng/. mu.L. The number of CFU/reaction was calculated using the following formula: number of CFU/reaction ═ number of CFU/mL × 1mL)/(100 μ L × 10-3)]×1μL×10-3。
The detection limit of the RealAmp assay was obtained by testing serial dilutions of 10% skim milk powder solution inoculated with pseudomonas fluorescens.
(2) Determination of detection limits in pure cultures
Pseudomonas fluorescens was inoculated in BHI broth and cultured for 18h at 30 ℃. 1mL of the bacterial solution was serially diluted 10-fold in 0.85% physiological saline and counted on a plate medium. Ten-fold serial dilutions were prepared with bacterial concentrations ranging from 4.9X 100CFU/mL to 4.9X 108 CFU/mL. The cells were collected from 1mL of the bacterial solution by centrifugation, and the subsequent operation was the same as in (1).
As a result, as shown in fig. 5 and 6, the lower the detection limit, the higher the detection accuracy. The limit of detection of P.fluorescens in the pure culture medium by the method of the invention was 4.9X 102CFU/mL (50.2 fg/. mu.L), i.e.4.9 CFU/reaction (FIG. 5). The limit of detection of Pseudomonas fluorescens in 10% skim milk powder solution by the method of the present invention was 2.2X 102CFU/mL (28.9 fg/. mu.L), i.e., about 2.2 CFU/reaction (FIG. 6). The detection time is about 40 min. The detection limit of RealAmp for detecting alkaline protease secreted by Pseudomonas fluorescens in 10% skim milk powder culture solution is almost the same as that in pure culture solution or even lower than that in pure culture, which indicates that the food substrate has no influence on the detection of RealAmp.
Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
SEQUENCE LISTING
<110> Shijiazhuang Junle Baoru Co Ltd
<120> primer for detecting pseudomonas fluorescens and alkaline protease in dairy products and corresponding detection method
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Claims (8)
1. A primer for detecting pseudomonas fluorescens and alkaline protease in dairy products is characterized by comprising a primer based on pseudomonas fluorescens and alkaline proteaseaprPrimers and genes for the X geneIn thatgyrA primer for gene B, wherein:
primer A-FOP: 5'-GCCCGCTGATCGACGACAT-3',
Primer A-BOP: 5'-AGTCCAGGGTGTCGTTGCC-3',
Primer A-FIP: 5'-AGGTGGTGTCCGTGGCGCGATCCAGAAGCTCTA-3',
Primer A-BIP: 5'-GGTTCAACTCCAACACCGCCGTCCCATACCGAGAACA-3',
Primer A-FLP: 5'-GCTGAGGTTGGCACCG-3' and
primer A-BLP: 5'-GCTACTTCCAATGCCGACA-3', respectively;
primer G-FOP: 5' -GGTATCGTCCTC primer A-3
Primer G-BOP: 5'-AAGCACAACAGGTTCT-3',
Primer G-FIP: 5'-GGTATTCAACGAACGCGCGCGGCAAGGAAGAAC-3',
Primer G-BIP: 5'-GTCAACCAGGTGTTACTGCAGGGCGATTTCCACG-3',
Primer G-FLP: 5'-CCGCCTTCGTACTTGAACA-3' and
primer G-BLP: 5'-CCACTTCAACAT-3' are provided.
2. A method for detecting pseudomonas fluorescens and alkaline protease in dairy products is characterized by comprising the following steps in sequence:
s1, extracting DNA of a sample to be detected as a template;
s2, the method based on claim 1 is adoptedaprMixing a primer of the X gene with a RealAmp reaction system to obtain a system alpha;
use of the base of claim 1gyrMixing a primer of the gene B with a RealAmp reaction system to obtain a system beta;
s3, respectively carrying out RealAmp reaction on the system alpha and the system beta, and monitoring the fluorescence intensity in real time to obtain reaction results;
s4, judging a result: the sample to be detected with the positive result of the determination system alpha after reaction contains alkaline protease, and the sample to be detected with the positive result of the determination system beta after reaction contains pseudomonas fluorescens.
3. The method for detecting pseudomonas fluorescens and alkaline protease in dairy products according to claim 2, wherein the system α comprises, in parts by volume: 1.8-2.5 parts of 10X reaction buffer solution, 0.8-1.2 parts of magnesium sulfate solution, 0.8-1.2 parts of dNTPs, 2.5-4 parts of primer A-FIP, 2.5-4 parts of primer A-BIP, 0.45-0.9 part of primer A-FOP, 0.45-0.9 part of primer A-BOP, 0.45-0.9 part of primer A-FLP, 0.45-0.9 part of primer A-BLP, 0.6-1.2 parts of polymerase, 0.1-0.4 part of SYBRgreenl, 0.5-1.5 parts of template and 5-10 parts of sterilized distilled water.
4. The method for detecting pseudomonas fluorescens and alkaline protease in dairy products according to claim 3, wherein the concentration of the magnesium sulfate solution is 35-65mmol/L, the concentration of dNTPs is 7-13mmol/L, and the concentration of each primer is 7-13 μmol/L.
5. The method for detecting pseudomonas fluorescens and alkaline protease in dairy products according to claim 2 or 3, wherein the system β comprises, in parts by volume: 2.3-2.7 parts of 10X reaction buffer solution, 0.8-1.2 parts of magnesium sulfate solution, 0.8-1.2 parts of dNTPs, 3.2-3.7 parts of primer G-FIP, 3.2-3.7 parts of primer G-BIP, 0.3-0.8 part of primer G-FOP, 0.3-0.8 part of primer G-BOP, 3.2-3.8 parts of primer G-FLP, 3.2-3.8 parts of primer G-BLP, 0.8-1.2 parts of polymerase, 0.2-0.4 part of SYBRgreenl, 0.5-1.5 parts of template and 1-3.5 parts of sterilized distilled water.
6. The method for detecting pseudomonas fluorescens and alkaline protease in dairy products according to claim 5, wherein the concentration of the magnesium sulfate solution is 35-65mmol/L, the concentration of dNTPs is 8-10mmol/L, and the concentration of each primer is 7-13 μmol/L.
7. The method for detecting pseudomonas fluorescens and alkaline protease in dairy products according to claim 2, wherein in the system alpha, the final concentration ratio of the primer A-FOP, the primer A-BOP, the primer A-FIP, the primer A-BIP, the primer A-FLP and the primer A-BLP is 1:1:5:5:1: 1;
in the system beta, the final concentration ratio of the primer G-FOP, the primer G-BOP, the primer G-FIP, the primer G-BIP, the primer G-FLP and the primer G-BLP is 1:1:7:7:7: 7.
8. The method for detecting Pseudomonas fluorescens and alkaline protease in dairy products according to claim 7, wherein the Pseudomonas fluorescens is an alkaline protease-producing Pseudomonas fluorescens.
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| US20190345535A1 (en) * | 2016-11-14 | 2019-11-14 | Commonwealth Scientific And Industrial Research Organisation | Protease sensor molecules |
| CN108048582A (en) * | 2017-11-29 | 2018-05-18 | 中国海洋大学 | The Pseudomonas fluorescens method of thermophilic protease is produced in a kind of quantitative quick detection raw milk using LAMP primer group |
| CN110846423A (en) * | 2019-08-14 | 2020-02-28 | 武汉轻工大学 | Fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, kit and application |
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Address after: 050000 No. 68 stone Copper Road, Hebei, Shijiazhuang Applicant after: JUNLEBAO Dairy Group Co.,Ltd. Address before: 050000 No. 68 stone Copper Road, Hebei, Shijiazhuang Applicant before: THE SHIJIAZHUANG JUNLEBAO DAIRY Co.,Ltd. |
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Application publication date: 20210928 |