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CN117327817A - Molecular targets for identification of Pseudomonas aeruginosa and their quantitative detection methods - Google Patents

Molecular targets for identification of Pseudomonas aeruginosa and their quantitative detection methods Download PDF

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CN117327817A
CN117327817A CN202311329300.XA CN202311329300A CN117327817A CN 117327817 A CN117327817 A CN 117327817A CN 202311329300 A CN202311329300 A CN 202311329300A CN 117327817 A CN117327817 A CN 117327817A
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pseudomonas aeruginosa
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primer
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吴清平
王楚芳
叶青华
杨宁
陈惠元
刘振杰
张菊梅
吴诗
王涓
丁郁
薛亮
陈谋通
庞锐
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Institute of Microbiology of Guangdong Academy of Sciences
Guangdong Huankai Biotechnology Co Ltd
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Guangdong Huankai Biotechnology Co Ltd
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Abstract

本发明公开了用于铜绿假单胞菌鉴定的分子靶标及其检测方法，属于微生物技术领域。本发明的用于铜绿假单胞菌鉴定的分子靶标，所述分子靶标的序列如SEQ ID NO.1～SEQ ID NO.4任一项所示。本发明的分子靶标可检测到更多的铜绿假单胞菌，铜绿假单胞菌覆盖率更大，增强了实用性；本发明的检测方法针对铜绿假单胞菌检测具有操作简单、结果判定易、检测时间短、特异性强、成本低、稳定性好的优点。

The invention discloses molecular targets for identification of Pseudomonas aeruginosa and detection methods thereof, and belongs to the technical field of microorganisms. For the molecular target used for identification of Pseudomonas aeruginosa of the present invention, the sequence of the molecular target is as shown in any one of SEQ ID NO. 1 to SEQ ID NO. 4. The molecular target of the present invention can detect more Pseudomonas aeruginosa, and the coverage rate of Pseudomonas aeruginosa is greater, which enhances practicality; the detection method of the present invention has the advantages of simple operation and result determination for the detection of Pseudomonas aeruginosa. It has the advantages of ease, short detection time, strong specificity, low cost and good stability.

Description

Molecular target for identifying pseudomonas aeruginosa and quantitative detection method thereof
The present application is a divisional application of CN114107532a (application date 2021, 12, 20, 2021115607002, and the invention and creation name are molecular targets for identifying pseudomonas aeruginosa and quantitative detection methods thereof).
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a molecular target for pseudomonas aeruginosa identification and a detection method thereof.
Background
Pseudomonas aeruginosa is commonly known as Pseudomonas aeruginosa and is widely distributed in nature, one of the most common bacterial species in fresh water and soil, and the bacteria can exist in the air, animal skin, intestinal tract, respiratory tract and the like. The bacterium can produce various pathogenic factors such as exotoxins, endotoxins and the like, can be transmitted through various ways such as water sources, soil, tools, contact and the like, has extremely strong resistance to physical and chemical factors such as disinfectant, dryness, ultraviolet rays and the like and bad environments, and is a complete pathogenic bacterium causing acute intestinal diseases and skin inflammation. In recent years, the report quantity of the bacteria on the pollution of packaged drinking water is obviously increased, and the pollution condition is serious. Early researches show that the detection rate of the pseudomonas aeruginosa of the natural mineral water sold in China can reach 23.6 percent, and the detection rate of the pseudomonas aeruginosa of bottled purified water for drinking can reach 11.6 percent; the water sample of the inner sampling examination of Guangdong province in 2018 shows that the pollution rate of pseudomonas aeruginosa to the packaged drinking water and natural mineral water reaches 8.36 percent (23/275); foreign pollution conditions such as detection of pseudomonas aeruginosa positive in 1.2% -10.2% of packaged drinking water by Germany, greek found that the pseudomonas aeruginosa pollution rate reached 5.9% (90/1527) in sampling mineral water samples, and the pollution rate level of pseudomonas aeruginosa under one examination in Australia reached 10 5 CFU/mL。
In addition, the problem of contamination of ready-to-eat vegetables and fruits by pseudomonas aeruginosa has been disturbing to consumer health. Instant vegetables have gained consumer favor for their healthy public praise and, in recent years, however, instant vegetables are often a source of food-borne diseases. Jamaica researchers have found that vegetables in retail markets and supermarkets are commonly contaminated with pseudomonas aeruginosa, agricultural products from the markets are more frequently contaminated, and research on pseudomonas aeruginosa resistance and virulence factors in fresh vegetable substrates has been conducted, so that the pseudomonas aeruginosa is also a main contaminant of fresh vegetables, and also can be an infectious source of susceptible people in communities. Early researches show that the pollution rate of the pseudomonas aeruginosa to the vegetable salad can be realizedUp to 64.5%, with improved hygiene levels, researchers have found in 2015 that pseudomonas aeruginosa has a 5% incidence of contaminating tomato or green pepper products. Pseudomonas aeruginosa can contaminate ready-to-eat vegetables and fruits in many links, for example, during the pre-harvest stage, the Pseudomonas aeruginosa population can colonise the growing crop and after harvest, the risk may be amplified by further direct contamination or spread of the existing bacterial population during processing and post-harvest treatments. The source of water contaminated by pseudomonas aeruginosa may be in the form of chains into the field, including runoff from nearby animal pastures and irrigation of the source of pollution. The pseudomonas aeruginosa has low requirement on organic nutrition, is a dominant bacterial colony for spoiling vegetables and fruits such as lettuce tomatoes and cucumbers, and seriously influences the transportation and storage of instant vegetables and fruits. If the infection is caused by taking pseudomonas aeruginosa, the healthy people need to be more than 10 5 The concentration of cfu/g of bacteria, but even less than 10 3 The concentration of cfu/g bacteria may also colonise the gut of susceptible people and may lead to gastrointestinal infections, bacteremia and blood borne transmission. Infants may also develop pseudomonas sepsis, manifested by necrotic intestinal lesions with a history of diarrhea. Therefore, in the food safety, drinking water safety and clinical treatment process, the rapid identification and accurate quantification of pseudomonas aeruginosa is particularly important for preventing the pollution thereof.
The development of molecular biology provides convenience for rapid identification of pseudomonas aeruginosa. Current standard SN/T2206.12-2014, cosmetic microbiological test method section 12: the PCR method of pseudomonas aeruginosa is to design PCR amplification primers by taking the conserved fragment of the exotoxin A gene of pseudomonas aeruginosa as a target fragment, and rapidly detect the pseudomonas aeruginosa in cosmetics. In addition, some reports on pseudomonas aeruginosa molecular detection targets and primers exist in the PCR detection method at home and abroad, and common specific genes include SyrB, toxA, I6S-23S, l6SrDNA, oprl, fliC, ecfX, ecfX + gyrB, ETA, opr, exoU, exoS and the like. In the detection method of pseudomonas aeruginosa, the traditional culture method is a gold standard. However, the biochemical identification in the traditional method needs about 48 hours for enrichment, then the culture of a color development medium and further biochemical identification are carried out for 24-48 hours, the test period needs about one week or more, and the test steps are tedious and time-consuming. Meanwhile, the traditional method judges whether the bacterial strain generates the copper green pigment or not, and the false detection or false detection may exist in the method of judging whether the bacterial strain is pseudomonas aeruginosa or not. For example, in the current national standard GB 8538-2016 of food safety national standard drinking natural mineral water inspection method, the bacterial colony is judged to be pseudomonas aeruginosa according to the generation of blue/green, and in practice pseudomonas putida also generates blue/green under the same condition, so that false positive can occur when the suspicious bacterial colony is judged and counted to generate pigment; in this standard, other colonies except blue/green, fluorescent and reddish brown were judged as non-pseudomonas aeruginosa, but studies showed that little pseudomonas aeruginosa did not produce pigment, which may appear to be inaccurate in positive results. In addition, the biochemical test is used for identifying the pseudomonas aeruginosa, the biochemical reaction is unstable, the repeatability of the identification result is poor, and misjudgment is easy to cause.
In practical application, along with continuous discovery of pseudomonas aeruginosa variant strains and drug-resistant strains in the clinical treatment process, the established molecular detection method based on the original detection target points by taking virulence factors is inaccurate due to easy deletion and limited quantity of the target points. In addition, with the rapid development of whole genome sequencing technology and continuous perfection of gene libraries, the coverage rate of the existing targets on pseudomonas aeruginosa strains is incomplete, so that the specificity of pseudomonas aeruginosa molecular targets is poor. The problems of small target quantity and poor specificity lead to the fact that the rapid and accurate identification of the pseudomonas aeruginosa is very challenging. Therefore, the search of a novel specific target molecule for the rapid and accurate detection of pseudomonas aeruginosa has important significance. Through literature search of the prior art, no report on a novel molecular target specific to pseudomonas aeruginosa and corresponding PCR and qPCR methods is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a molecular target for pseudomonas aeruginosa identification and a detection method thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the molecular target is used for identifying pseudomonas aeruginosa, and the sequence of the molecular target is shown in any one of SEQ ID NO.1-SEQ ID NO. 4.
The inventor of the application finally obtains the molecular target for pseudomonas aeruginosa identification through extensive screening and design verification by using 1000 pseudomonas aeruginosa and 1017 non-pseudomonas aeruginosa whole genome data in the NCBI database. Through a large number of experiments, the molecular target provided by the invention is used for identifying pseudomonas aeruginosa, has good specificity and larger coverage rate, and has a detection rate of 100% on pseudomonas aeruginosa, which is far higher than the positive detection rate of toxA genes on pseudomonas aeruginosa in the prior art.
The invention also provides a primer group for identifying pseudomonas aeruginosa, which is used for detecting the molecular target of any one of SEQ ID NO.1-SEQ ID NO. 4; the primer group for detecting the molecular target shown in SEQ ID NO.1 has a sequence shown in SEQ ID NO.5 and SEQ ID NO. 6; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.2 are shown in SEQ ID NO.7 and SEQ ID NO. 8; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.3 are shown in SEQ ID NO.9 and SEQ ID NO. 10; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.4 are shown as SEQ ID NO.11 and SEQ ID NO. 12.
The invention also provides a primer group for identifying pseudomonas aeruginosa, which is used for detecting the molecular target of any one of SEQ ID NO.1-SEQ ID NO. 4; the sequence of the primer group for detecting the molecular target shown in SEQ ID NO.1 is shown as SEQ ID NO.13 and SEQ ID NO. 14; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.2 are shown in SEQ ID NO.15 and SEQ ID NO. 16; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.3 are shown in SEQ ID NO.17 and SEQ ID NO. 18; the sequences of the primer group for detecting the molecular target shown in SEQ ID NO.4 are shown as SEQ ID NO.19 and SEQ ID NO.20.
According to the invention, a series of primer groups are designed according to the molecular targets for pseudomonas aeruginosa identification, and a large number of screening and verification prove that the finally obtained primer groups have good specificity when being used for pseudomonas aeruginosa identification, and the coverage rate of the primer groups to pseudomonas aeruginosa is 100%.
The invention also provides a method for identifying pseudomonas aeruginosa, which comprises the following steps:
(1) Performing PCR amplification on the DNA of the sample to be detected by using at least one group of the primer groups;
(2) Detecting the amplified product by gel electrophoresis;
(3) And observing whether the amplified product meets the expectations.
The amplification product of the method for identifying the pseudomonas aeruginosa has good specificity, and whether the pseudomonas aeruginosa exists can be judged by observing whether the amplification product is at an expected position.
As a preferred embodiment of the method for identifying Pseudomonas aeruginosa according to the present invention, the PCR amplification system is: 10 XPCR reaction buffer 2.5. Mu.L, 25mmol/L, mgCl 2 2. Mu.L, 2.5mmol/LdNTP 1. Mu.L, template DNA100ng, 10. Mu. Mol/L primers 1. Mu.L each, tag enzyme 1U, sterilized double distilled water make up to 25. Mu.L; the PCR amplification procedure is as follows: pre-denaturation at 98℃for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 72 ℃ for 45s; carrying out denaturation, annealing and extension for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
The invention also provides a method for quantitatively detecting pseudomonas aeruginosa, which adopts at least one group of the primer groups to carry out qPCR amplification on the DNA of the sample to be detected and analyzes the amplification result.
The amplification product of the method for quantitatively detecting the pseudomonas aeruginosa has good specificity, and the concentration of the pseudomonas aeruginosa in the system is quantitatively detected by judging the fluorescent signal intensity in the system. The qPCR method established by the invention has ideal linearity, and the minimum detection limit for pure bacteria can be 10 2 The detection method established by the invention has good sensitivity, which is equivalent to or better than the prior literature report method by 1-2 orders of magnitude. In addition, the method has strong anti-interference capability and is concentrated in coliformThe degree reaches 10 8 CFU/mL, the detection concentration was 10 4 Nor does it cause interference.
As a preferred embodiment of the method for quantitatively detecting pseudomonas aeruginosa according to the present invention, the qPCR amplification system of the qPCR amplification is: 10. Mu.L of 2 XTBGreenPremix reaction, 100ng of template DNA, 1. Mu.L of each of 10. Mu. Mol/L primers, and the volume of sterilized double distilled water was made up to 20. Mu.L; the qPCR amplification procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, the denaturation and annealing were performed for 40 cycles in total.
The invention also provides application of the novel molecular target for identifying pseudomonas aeruginosa or the primer group in identifying pseudomonas aeruginosa.
The invention also provides application of the novel molecular target for pseudomonas aeruginosa identification or the primer group in quantitative detection of pseudomonas aeruginosa.
The invention also provides a kit for identifying pseudomonas aeruginosa, which comprises the primer set.
The invention has the beneficial effects that: the invention provides a group of novel molecular targets for pseudomonas aeruginosa identification and a detection method thereof. (1) The detection method can detect more pseudomonas aeruginosa, the pseudomonas aeruginosa coverage rate is larger, the pseudomonas aeruginosa coverage rate is 100%, and the practicability is enhanced; (2) The method is used for identifying that the detection rate of the novel molecular target of the pseudomonas aeruginosa on 95 strains of pseudomonas aeruginosa is 100%, and the positive detection rate of the toxA gene reported in the literature on the strains is 82.1%, which indicates that the specificity of the novel molecular target of the pseudomonas aeruginosa is good; (3) The detection method provided by the invention has the advantages of simplicity in operation, easiness in result judgment, short detection time, strong specificity, low cost and good stability aiming at pseudomonas aeruginosa, the whole detection process of the method established by the invention needs about 15 hours, and the traditional method needs about 3-5 days to complete, so that the detection time is greatly saved, and the detection time is improved.
Drawings
FIG. 1 shows the result of specific evaluation electrophoresis of Pseudomonas aeruginosa by PCR identification method;
FIG. 2 shows the results established by the qPCR quantitative detection method for the pure culture solution of Pseudomonas aeruginosa;
FIG. 3 shows the results established by the qPCR quantitative detection method for pseudomonas aeruginosa in artificially contaminated samples;
FIG. 4 shows the results of the qPCR quantitative determination of Pseudomonas aeruginosa in the actual samples of example 7.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1 design of screening for specific novel molecular targets for Pseudomonas aeruginosa identification
And performing genome-wide analysis according to genome data of 1000 pseudomonas aeruginosa strains and 1017 non-pseudomonas aeruginosa strains in NCBI website, and finally obtaining a molecular target for pseudomonas aeruginosa identification through a large number of screening and design verification, wherein the nucleotide sequence of the molecular target is shown as SEQ ID NO.1-SEQ ID NO. 4.
Example 2PCR method for identifying Pseudomonas aeruginosa
The embodiment provides 4 rapid detection methods, wherein primers are designed according to any one of 4 specific new molecular targets of pseudomonas aeruginosa, the rapid detection methods can be formed, and the method comprises the following steps:
(1) Primer design: designing a specific PCR amplification primer set according to the sequences SEQ ID NO.1-SEQ ID NO.4 in example 1, wherein the sequences of the primer sets are shown in the following table 1;
(2) Preparing a DNA template: respectively culturing the strains to be detected in LB liquid culture medium, and respectively extracting bacterial genome DNA of the strains to be detected by using a commercial bacterial genome DNA extraction kit to serve as templates to be detected;
(3) Performing PCR amplification on the DNA of the sample to be detected by using the primer group; PCR detection system: 10 XPCR reaction buffer 2.5. Mu.L, 25mmol/L, mgCl 2 2. Mu.L, 2.5mmol/LdNTP 1. Mu.L, template DNA100ng, 10. Mu. Mol/L primers 1. Mu.L each, tag enzyme 1UThe double distilled water was sterilized to make up the volume to 25. Mu.L. The PCR amplification procedure is that the PCR amplification is performed for 5min at 95 ℃; denaturation at 95℃for 30s; annealing at 58 ℃ for 30s; extending at 72 ℃ for 45s; carrying out denaturation, annealing and extension for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
(4) Performing gel electrophoresis on the PCR amplification product;
(5) The positions of each primer set corresponding to the size of the product were observed for the presence of a single amplified band. If present, indicating that the sample contains Pseudomonas aeruginosa; if no corresponding single amplified band is present, the sample does not contain the corresponding P.aeruginosa.
TABLE 1 specific PCR detection primer set
Example 3 evaluation of specificity of Pseudomonas aeruginosa PCR detection method
The evaluation method comprises the following steps: the PCR was performed as described in example 2, except that Pseudomonas aeruginosa 95 was used as the non-target strain 39. Wherein, the S1DNA template is prepared to extract genome DNA of each bacterium respectively; in the S2PCR amplification, the primer used is a primer set. A blank is set, and the template of the blank is an aqueous solution without genome.
Evaluation results: the strains of each bacterium used and the results of the tests are shown in Table 2 below, in which "+" in the column of the results of the tests indicates positive and "-" indicates negative. The electrophoresis result of the PCR products is shown in figure 1; in the figure, the numbers 1 to 5 are pseudomonas aeruginosa; numbers 6-44 are non-target strains; m is 2000Maker. As can be seen from FIGS. 1 and 2, the PCR primer sets showed specific amplified bands only for Pseudomonas aeruginosa, and no specific bands for non-target bacterial strains, indicating high specificity of the identification method of the present invention.
TABLE 2 evaluation test results of Pseudomonas aeruginosa identification specificity
Example 4 method for quantitative detection of Pseudomonas aeruginosa
The qPCR method for quantitatively detecting pseudomonas aeruginosa comprises the following steps:
(1) Primer design: designing a specific qPCR amplification primer set according to the sequences SEQ ID NO.1-SEQ ID NO.4 in example 1, the primer set sequence being as follows in Table 3;
(2) Preparing a DNA template: culturing Pseudomonas aeruginosa in LB liquid culture medium, and respectively extracting bacterial genome DNA of the Pseudomonas aeruginosa by using commercial bacterial genome DNA extraction kit as a template to be detected;
(3) And (3) PCR amplification: PCR amplification is carried out on the DNA of the sample to be detected by using the primer group 5, and then96 fluorescent quantitative amplification instrument. qPCR detection System: 10. Mu.L of 2 XTBGreenPremix reaction, 100ng of template DNA, 1. Mu.L of each of 10. Mu. Mol/L primers, and the volume of sterilized double distilled water was made up to 20. Mu.L; the qpcr amplification procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 60℃for 30s, the denaturation and annealing being carried out for 40 cycles in total;
(4) By means of software96SW1.1 it is analyzed whether the amplification result meets the expectations. If the fluorescent signal is generated on the premise that the blank control does not exist, the sample contains pseudomonas aeruginosa; if no fluorescent signal is generated, the sample does not contain Pseudomonas aeruginosa.
TABLE 3 specific qPCR detection primer set
EXAMPLE 5 Pseudomonas aeruginosa qPCR method for identification of specific evaluation
The evaluation method comprises the following steps: taking 63 strains of pseudomonas aeruginosa, taking other species as 32 strains of non-target bacteria, respectively extracting genome DNA of each bacteria, 1 blank control, designing a primer group sequence by using the primer, setting 1 blank control, and setting a template of the blank control as an aqueous solution without genome.
Evaluation results: the strains of each bacterium used and the results of the tests are shown in Table 4 below, in which "+" in the column of the results of the tests indicates positive and "-" indicates negative. qPCR fluorescence results are shown in FIG. 2; in the figure, the numbers 1 to 5 are pseudomonas aeruginosa; no.6 to 37 are non-target strains. As can be seen from FIG. 2 and Table 4, the detection results of the qPCR primer set only show fluorescence signals of Pseudomonas aeruginosa, and neither the non-target strain nor the blank show fluorescence signals, which indicates that the qPCR identification method of the invention has high specificity.
TABLE 4 Pseudomonas aeruginosa qPCR method identification specificity evaluation test results
Example 6 sensitivity evaluation of pure Pseudomonas aeruginosa culture solution qPCR quantitative detection method
The evaluation method comprises the following steps: culturing to a concentration of 10 8 The standard strain Pseudomonas aeruginosa ATCC15442 of CFU/mL is diluted by 10 times of sterile normal saline with the concentration of 0.85 percent to obtain the concentration of 10 1 ，10 2 ，10 3 ，10 4 ，10 5 ，10 6 ，10 7 ，10 8 CFU/mL pure culture of strain, extraction of DNA template was performed as in example 4Namely, a qPCR detection standard for pseudomonas aeruginosa, was performed according to example 4, and three parallel experiments were performed for each template. Drawing a standard curve: taking the logarithm of the concentration of the pure culture of the strain of the standard substance as an abscissa, taking the real-time Ct value of the corresponding qPCR as an ordinate, and fitting the obtained curve to obtain the standard curve of the pseudomonas aeruginosa.
Evaluation results: the standard curve is shown in FIG. 3, and the detection limit of the primer group 5 shown in FIG. 3.A to pure bacteria is 10 3 CFU/mL, the fitted standard curve of the pseudomonas aeruginosa is y= -3.0677x+40.259, and the correlation coefficient R 2 = 0.9901; FIG. 3.b shows that the detection limit of the primer set 6 against pure bacteria is 10 2 CFU/mL, the fitted standard curve of the pseudomonas aeruginosa is y= -2.3429x+33.717, and the correlation coefficient R is the same as that of the pseudomonas aeruginosa 2 = 0.9915; FIG. 3.c shows that the detection limit of the primer set 7 against pure bacteria is 10 2 CFU/mL, the fitting standard curve of the pseudomonas aeruginosa is y= -1.3954x+35.83, and the correlation coefficient R is 2 = 0.9924; the detection limit of the primer group 8 shown in FIG. 3.D on pure bacteria is 10 3 CFU/mL, the fitted standard curve of the pseudomonas aeruginosa is y= -3.2347x+40.592, and the correlation coefficient R 2 ＝0.9935。
Example 7 sensitivity evaluation of Pseudomonas aeruginosa qPCR quantitative detection method by artificially adding a labeled sample
The evaluation method comprises the following steps: the method is characterized in that a sterile sample is prepared by adopting a method of cutting off the surface layer of the sample after the fresh tomatoes are sterilized by alcohol, and the results after the NA nutrient agar plate culture show that no microorganism exists in the treated fresh vegetable sample, so that the microorganisms in the fresh vegetable sample in the follow-up experiment are all derived from artificial pollution. NA counting plate results show that the initial bacterial liquid concentration of the pseudomonas aeruginosa of the artificially contaminated sample is 1.33 multiplied by 10 8 CFU/mL. 10-fold gradient dilution of the homogenized solution of the artificially contaminated sample was performed using 0.85% sterile physiological saline to prepare a solution containing Pseudomonas aeruginosa at a concentration of 10 1 CFU/mL～10 8 CFU/mL of artificial contaminated simulated sample. qPCR was performed as in example 4 with bacterial genomic DNA extracted from each gradient homogenate as template and sterile distilled water as blank, and three replicates were performed for each template. According to examplesAnd 5, establishing a corresponding manual standard adding sample standard curve in a curve fitting mode.
Evaluation results: the standard curve is shown in FIG. 4, and the detection limit of the primer set 5 on artificial contamination is 1.33X10 as shown in FIG. 4.A 4 CFU/g, the fitted standard curve of the pseudomonas aeruginosa is y= -1.00701x+36.621, and the correlation coefficient R 2 0.9944; as shown in FIG. 4.B, the detection limit of the primer set 6 on artificial contamination was 1.33X10 3 CFU/g, the fitted standard curve of the pseudomonas aeruginosa is y= -2.1233x+35.354, and the correlation coefficient R 2 0.9851; as shown in FIG. 4.C, the detection limit of the primer set 7 on artificial contamination was 1.33X10 3 CFU/g, the fitted standard curve of the pseudomonas aeruginosa is y= -2.82390 x+39.775, and the correlation coefficient R is 2 0.9814; as shown in FIG. 4.D, the detection limit of the primer set 8 on artificial contamination was 1.33X10 4 CFU/g, the fitted standard curve of the pseudomonas aeruginosa is y= -1.1851x+27.745, and the correlation coefficient R 2 0.9853.
Example 8 quantitative detection results of Pseudomonas aeruginosa PCR and qPCR in actual samples
The experimental method comprises the following steps: fresh lettuce, cucumber, lettuce and tomato were collected from the local vegetable farm in Guangzhou and a total of 29 samples were placed in sterile homogenization bags and returned to the laboratory. In a clean bench, vegetable samples are further divided and weighed under aseptic operation, 25g of each vegetable sample is placed into 225ml of pseudomonas aeruginosa enrichment liquid, after 18 hours of enrichment, genome DNA of each sample is extracted respectively for detection by the qPCR method in example 4, and experiments are carried out by using primer groups 5-8. And control detection was performed using conventional culture-mass spectrometry, as well as the PCR method described above.
The experimental results are shown in Table 5, and can be obtained from Table 5, compared with the traditional gold-culture standard method, the PCR and qPCR method of the invention is used for detecting pseudomonas aeruginosa in 29 detected instant vegetable books, and all positive and negative samples can be accurately detected. The embodiment shows that the qualitative and quantitative detection method of the pseudomonas aeruginosa has high reliability and can effectively identify the target pseudomonas aeruginosa in the actual sample.
TABLE 5 results of Pseudomonas aeruginosa detection in actual samples
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims (6)

1. The molecular marker for identifying pseudomonas aeruginosa is characterized in that the sequence of the molecular marker is shown as SEQ ID NO. 4.
2. A primer set for identifying pseudomonas aeruginosa, characterized in that the sequence of the primer set for detecting the molecular marker shown in SEQ ID No.4 is shown as any one of the following primer combinations:
combining: SEQ ID NO.11 and SEQ ID NO.12,
and (2) combining two: SEQ ID NO.19 and SEQ ID NO.20.
3.A method for identifying pseudomonas aeruginosa for non-disease diagnostic and therapeutic purposes comprising the steps of:
(1) Performing PCR amplification of the sample DNA to be detected using at least one of the primer sets of claim 2;
(2) Detecting the amplified product by gel electrophoresis;
(3) And observing whether the amplified product meets the expectations.
4.A non-disease for the identification of pseudomonas aeruginosa according to claim 3A method for diagnostic and therapeutic purposes, characterized in that the amplification system of the PCR amplification is: 10 XPCR reaction buffer 2.5. Mu.L, 25mmol/L, mgCl 2 2. Mu.L, 2.5mmol/LdNTP 1. Mu.L, template DNA100ng, 10. Mu. Mol/L primers 1. Mu.L each, tag enzyme 1U, sterilized double distilled water make up to 25. Mu.L; the PCR amplification procedure is as follows: pre-denaturation at 98℃for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 72 ℃ for 45s; carrying out denaturation, annealing and extension for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
5. Use of a molecular marker for pseudomonas aeruginosa identification according to claim 1 or of a primer set according to claim 2 for identifying pseudomonas aeruginosa for non-disease diagnosis and treatment purposes.
6. A kit for identifying pseudomonas aeruginosa, comprising the primer set of claim 2.
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