CN110846423A - Fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, kit and application - Google Patents
Fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, kit and application Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, a kit and application. The method and the kit carry out fluorescent quantitative PCR detection on pseudomonas fluorescens in a sample by mixing genome DNA obtained by enriching the sample to be detected with a primer and a corresponding probe, and have strong detection specificity and wide adaptability; the detection method is rapid, high in sensitivity and suitable for high-throughput screening; can be applied to high-throughput screening of pseudomonas fluorescens in samples, particularly food.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, a kit and application.
Background
Pseudomonas fluorescens (Pseudomonas fluorescens) is a psychrophilic microorganism and is the dominant bacterial species causing putrefaction in high-protein and high-fat foods such as refrigerated raw meat and raw milk. The pseudomonas fluorescens can secrete a large amount of heat-resistant lipase and protease, and after heating sterilization, the residual heat-resistant protease and lipase still have partial activity, so that the flavor quality of the food can be damaged, and the shelf life of the food can be shortened. With the improvement of living standard and the change of eating habits of people, the potential threat of pseudomonas fluorescens to human health is increasingly expanded. The establishment of a rapid and accurate pseudomonas fluorescens detection method has important significance for perfecting a food safety monitoring system.
At present, the detection of pseudomonas fluorescens still uses a traditional culture method as a 'gold standard', comprises the steps of sample pretreatment, selective enrichment, selective plate separation and physiological and biochemical characteristic experiment, the experiment period at least needs 4 days, and is not suitable for large-scale rapid detection. The fluorescent quantitative PCR technology has high detection speed, high specificity and sensitivity and realizes quantitative detection. In recent years, the fluorescent quantitative PCR method has been widely applied to the rapid detection of various pathogenic bacteria. However, no researchers developed a TaqMan probe-based fluorescent quantitative PCR detection system for Pseudomonas fluorescens so far.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative PCR rapid detection method for pseudomonas fluorescens.
Another purpose of the invention is to provide a fluorescent quantitative PCR rapid detection kit for pseudomonas fluorescens.
The third purpose of the invention is to provide the application of the detection method and the kit in detection.
In order to achieve the first object, the fluorescent quantitative PCR rapid detection method for pseudomonas fluorescens provided by the invention comprises the following steps:
(1) enriching the sample to be detected in the selective enrichment solution, and extracting the bacterial genome DNA by adopting a phenol-chloroform-isoamylol method or a boiling method;
(2) mixing the genomic DNA obtained in the step (1) with primers and corresponding probes, and carrying out fluorescent quantitative PCR detection, wherein the primers and the probes are as follows:
specific detection primers and probes are designed by using a gyrB gene of pseudomonas fluorescens pf SBW25 as a template and using software primer express3.0, wherein the sequence of the detection primer gyrB-F is 5 'TGCGGTCAACCAGGTGTTCC 3', the sequence of the gyrB-R is 5 'CGAGATAATCGCGGTCAGG 3', and the sequence of the probe gyrB-taqmen is 5 'CATCCAGCGTGAAGACGGCATCG 3'.
In the present invention, the sample to be tested is an ex vivo sample potentially containing Pseudomonas fluorescens. Since Pseudomonas fluorescens is a major spoilage bacterium in food, it is preferred that the sample is derived from food, such as if the sample is food, or if the sample is a bacterial plate culture of food, or the like.
The concentration of the probe in the fluorescent quantitative PCR reaction system has a great influence on the PCR reaction efficiency, and according to the research of the inventor of the invention, the final concentration of the taqman probe is 500 nmol/L.
The reaction process of the fluorescent quantitative PCR is well known to those skilled in the art, and each cycle generally comprises denaturation at 92-96 ℃, low-temperature annealing and extension at about 72 ℃, wherein the most variable is the annealing temperature. According to the inventors' study, the most preferred real-time RT-PCR process is 36 cycles, where each cycle is denaturation 94 ℃/45s, annealing 57 ℃/45s, and extension 72 ℃/45 s.
In order to realize the second purpose, the invention provides a fluorescent quantitative PCR rapid detection kit for pseudomonas fluorescens. The kit comprises PCR reaction liquid, wherein the PCR reaction liquid comprises detection primers and probes, wherein the sequence of the detection primers gyrB-F is 5 'TGCGGTCAACCAGGTGTTCC 3', the sequence of the detection primers gyrB-R is 5 'CGAGATAATCGCGGTCAGG 3', and the sequence of the probes gyrB-taqmen is 5 'CATCCAGCGTGAAGACGGCATCG 3'.
In order to achieve the third object, the invention also provides the application of the detection method and the kit in detecting pseudomonas fluorescens in a sample.
Through long-term research, the inventor designs a pair of primers and corresponding probes aiming at gyrB gene, establishes a fluorescent quantitative PCR detection system of pseudomonas fluorescens, has strong specificity, high sensitivity, wide detection adaptability and strong anti-interference capability, and can be applied to high-throughput screening of pseudomonas fluorescens in samples, especially food.
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FIG. 1 is a graph of pure culture level detection amplification (AS 1.55). Wherein Blank: ddH2O; a to g, the final concentration of the template in each system is 3.0 multiplied by 10 respectively5，3.0×104，3.0×103，3.0×102， 3.0×101，3.0，3.0×10-1，3.0×10-2CFU/mL。
Detailed Description
For the purpose of facilitating understanding, the invention will hereinafter be described in detail by way of the accompanying drawings and specific embodiments. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.
Unless otherwise specified, the method can be performed according to the methods listed in the handbook of experiments such as "molecular cloning laboratory Manual" (third edition), which is familiar to those skilled in the art (Cold Spring Harbor laboratory Press), "cell laboratory Manual" (scientific Press, Beijing, China, 2001), and the like, and the references cited in the present invention.
1.1 Primary reagents and instruments
Nutrient broth medium (NB medium), nutrient broth agar medium (NA medium), and selective enrichment broth (buffered peptone water) were purchased from Beijing Luqiao technology corporation; premix Ex TaqTMPurchased from Dalibao bioengineering, Inc.; the primer sequence gyrBF/R, taqman probe for detecting the specificity of the pseudomonas fluorescens used in the experiment is synthesized by Dalibao bioengineering GmbH; applied Biosystems Step-One Real-Time PCR system, ABI, USA; ultraviolet nucleic acid and protein analyzer DU8Model 00, Beckman Coulter, Inc., USA; MS-2 vortex shaker, IKA, Germany; 5415D high speed centrifuge, Eppendorf, Germany; HZ-8211K constant temperature shaking table, Taicang scientific and educational equipment factory; DHP-9162 constant temperature incubator, Shanghai-constant technologies, Inc.; CA-1480-2 vertical laminar flow clean bench, Shanghai clean and purify Equipment Limited; ES-315 autoclave, TOMY, Japan.
1.2 extraction of the Strain and genomic DNA
The strains are purchased from the strain preservation center of the institute of microbiology of China academy of sciences, the preservation center of the microbial strains of China medical science, the China industrial microbial strain bank and the preservation management center of the food safety microbial strains of China academy of sciences, and the serial numbers of the strains are shown in Table 1. The culture method can be carried out according to the method recommended by the supplier of the above strains, and the bacterial genomic DNA can be extracted by the phenol-chloroform-isoamyl alcohol method or the boiling method according to the manufacturer's instructions.
TABLE 1 detection results of the specificity of the strain number
Tab.1 Strains used in the test of PCR specificity
1.3 design and Synthesis of detection primers and taqman probes
A gyrB gene of pseudomonas fluorescens pf SBW25 is taken as a template, a software primer express3.0 is used for designing a detection primer and a probe, the sequence of the detection primer gyrB-F is 5 'TGCGGTCAACCAGGTGTTCC 3', the sequence of the gyrB-R is 5 'CGAGATAATCGCGGTCAGG 3', and the sequence of the probe gyrB-taqmen is 5 'CATCCAGCGTGAAGACGGCATCG 3'.
1.4 Probe concentration optimization experiment
A series of probe concentration gradients were set so that the probe concentration in each reaction system was 100nmol/L, 200nmol/L, 300nmol/L, 400nmol/L, and 500nmol/L, respectively. PCR amplification (using P.fluorescens AS1.55 genomic DNA AS a template) was performed on these 5 gradients, with three parallel values for each concentration, and the optimal probe concentration was determined based on the Ct value.
The relation between the probe concentration and the Ct value is shown in Table 2, and it can be seen that the Ct value is the smallest when the probe concentration is 500nmol/L, and the data of 400nmol/L group and 600nmol/L group are calculated by excel software to find that there is a significant difference (P < 0.05), so the probe concentration is selected to be 500nmol/L for subsequent experiments.
TABLE 2 relationship of Probe concentration and Ct value
Tab.2 The correlation of probe concentration and Ct value
1.5 real-time fluorescent quantitative PCR reaction
PCR reaction system (in the examples of the present invention, the PCR system is referred to as follows): premix Ex Taq TM10. mu.L, 1-10 ng of DNA template, 0.5. mu.L of each of the upstream and downstream primers (10. mu. mol/L), 1. mu.L of probe (5. mu.M), and up to 20. mu.L of ddH 2O. Performing PCR amplification and fluorescence detection on an ABI7500 fluorescence quantitative PCR instrument, wherein the amplification conditions are as follows: pre-denaturation at 94 ℃/2 min; denaturation at 94 ℃/45s, annealing at 57 ℃/45s, extension at 72 ℃/45s, fluorescence reading, and 36 cycles.
1.6 evaluation of specificity
The strains listed in Table 1 were subjected to fluorescent quantitative PCR amplification with sterile distilled water as a blank. If the Ct value of the amplification is less than 35, the result is a positive detection result. The results showed that the Ct values were all < 35 for the amplification using genomic DNA from P.fluorescens as template, whereas the Ct values could not be detected using genomic DNA from non-P.fluorescens as template.
1.7 evaluation of sensitivity
Counting the plates of Pseudomonas fluorescens AS1.55 cultured for 12h to obtain initial bacteria liquid with concentration of 3.0 × 109CFU/mL, followed by evaluation of the pure culture sensitivity of the system as described above. The results are shown in FIG. 1 at a dilution of 10-7Ct values were read in all three replicates and at a dilution of 10-8Time threeIf Ct values cannot be read out from all the parallel cells, the sensitivity of the pure culture of the fluorescent quantitative PCR detection system is 3.0 multiplied by 102CFU/mL。
1.8 evaluation of anti-interference ability
Selecting four strains of interference bacteria of pseudomonas putida (AS1.1130), pseudomonas aeruginosa (IQCC12625), pseudomonas alcaligenes (IQCC12604) and pseudomonas cepacia (CICC21624), respectively culturing with pseudomonas fluorescens (AS1.55) to a stationary phase, diluting with 10 times of gradient, and respectively counting plates. Mixing four kinds of interference bacteria, and setting three interference concentrations (Nx 10)4，N×106，N×108) Gradient dilution (10) different from Pseudomonas fluorescens-4～10-10) Mixing, culturing at 28 deg.C and 150r/min, sampling for 0h and 15h (selected according to artificial pollution result), boiling to extract genome DNA, performing PCR amplification, and detecting anti-interference ability with mixed solution of interference bacteria without Pseudomonas fluorescens as blank control.
Respectively culturing pure cultures of pseudomonas fluorescens and four kinds of interference bacteria to a stationary phase, counting plates, and calculating to obtain initial bacteria liquid with the concentration of 1.2 multiplied by 109CFU/mL，2.6×108CFU/mL， 6.4×108CFU/mL，1.3×109CFU/mL，1.2×109CFU/mL, and then carrying out the anti-interference capability experiment according to the method. The results are shown in Table 3: addition of about 104The sensitivity of the PCR detection system is hardly affected by the CFU/mL of pure culture of the interfering bacteria, and the addition of about 108The pseudomonas fluorescens can be accurately detected even after full enrichment culture (15h) when the pure culture of the interference bacteria is CFU/mL.
TABLE 3 Effect of the Presence of interfering bacteria on the sensitivity of pure culture PCR detection
Tab.3 Influence of background non-Pseudomonas fluorescens bacteria onthe detection sensitivity of Pseudomonas fluorescens in pure culture
Note: ND: not detection, not detected.
1.9 Artificial contaminated Soy milk sample experiment
Activated pseudomonas fluorescens (AS1.55) bacterial liquid is inoculated into NB medium for shake cultivation to a stationary phase, the solution is diluted by 10 times of gradient, and plate counting is carried out to calculate the number of original colonies. The method comprises the steps of inoculating 25mL of soymilk into 225mL of selective enrichment solution, respectively inoculating 0.1-1 CFU/mL, 1-10 CFU/mL and 10-100 CFU/mL of three gradient bacteria solutions, independently repeating for three times, culturing for 24h under the conditions of 28 ℃ and 150r/min, sampling once for each of 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h, extracting genomic DNA by a boiling method, respectively carrying out PCR amplification, and taking sterilized distilled water as blank control. The selected food samples were confirmed to be free of Pseudomonas fluorescens by culture.
The results are shown in Table 4: when the initial inoculation amount is 23CFU/mL, a positive result can be detected after the enrichment culture is carried out for 12 h; when the inoculation amount is 2.3CFU/mL, a positive result can be detected after the enrichment culture is carried out for 15 h; after 24h of culture, the detection rate of the sample with the initial inoculation amount of 0.23CFU/mL is 1/3. Therefore, the system can accurately detect the existence of the pseudomonas fluorescens by increasing the bacteria in a proper time under the condition that the bacteria content of the sample is low.
TABLE 4 PCR test results for artificially contaminated samples
Tab.4 Detective results of the artificial contamination
Note: after 24 hours of enrichment, only one of the three replicates with an initial inoculum size of 0.23CFU/mL was positive.
1.10 food sample testing
Food samples (56 parts in total, table 5) were collected from commercial food samples susceptible to infection by pseudomonas fluorescens and the samples were processed according to the national standard GB/T4789.7-2008. 25g (mL) of each sample is taken and added into 225mL of selective enrichment medium for shake culture (28 ℃, 150rpm), and the enrichment time is determined according to the artificial pollution result. After enrichment, the genome DNA is extracted by a phenol-chloroform-isoamylol method or a boiling method, and a fluorescence quantitative PCR detection system established by the research is used for detecting the sample. After the selective enrichment culture is carried out for 24h, the samples are detected by adopting the traditional culture method, and each sample is 2 parts in parallel to prevent detection omission.
The culture method has the detection results that pseudomonas fluorescens is separated from 3 parts of meat samples and 3 parts of water products, and a fluorescent quantitative PCR system detects 13 parts of positive results in milk products, meat products and water products respectively. The detection rate of the fluorescent quantitative PCR system in the actual sample is 23.2 percent and is 10.7 percent higher than that of the culture method.
TABLE 5 food sample test results
Tab.5 Detective results of all the natural food samples
The kit and the use thereof according to the present invention can be carried out by those skilled in the art based on the above-mentioned examples in combination with common general knowledge.
Sequence listing
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Claims (6)
1. A fluorescent quantitative PCR rapid detection method for pseudomonas fluorescens is characterized in that: the method comprises the following steps:
(1) enriching the sample to be detected in the selective enrichment solution, and extracting the bacterial genome DNA by adopting a phenol-chloroform-isoamylol method or a boiling method;
(2) mixing the genomic DNA obtained in the step (1) with primers and corresponding probes, and carrying out fluorescent quantitative PCR detection, wherein the primers and the probes are as follows:
specific detection primers and probes are designed by using a software primer express3.0 by taking a gyrB gene of pseudomonas fluorescens pf SBW25 as a template, wherein the sequence of the detection primer gyrB-F is 5 'TGCGGTCAACCAGGTGTTCC 3', the sequence of the gyrB-R is 5 'CGAGATAATCGCGGTCAGG 3', and the sequence of the probe gyrB-taqmen is 5 'CATCCAGCGTGAAGACGGCATCG 3'.
2. The fluorescent quantitative PCR rapid detection method for Pseudomonas fluorescens according to claim 1, characterized in that:
the sample to be detected is food or bacterial plate culture of food.
3. The fluorescent quantitative PCR rapid detection method for Pseudomonas fluorescens according to claim 1 or 2, characterized in that: the final concentration of the taqman probe was 500 nmol/L.
4. Use of the method of any one of claims 1 to 3 for detecting pseudomonas fluorescens in a sample.
5. A fluorescent quantitative PCR rapid detection kit for pseudomonas fluorescens is characterized in that:
the kit comprises PCR reaction liquid, wherein the PCR reaction liquid comprises a detection primer and a probe, wherein the sequence of the detection primer gyrB-F is 5 'TGCGGTCAACCAGGTGTTCC 3', the sequence of the detection primer gyrB-R is 5 'CGAGATAATCGCGGTCAGG 3', and the sequence of the probe gyrB-taqmen is 5 'CATCCAGCGTGAAGACGGCATCG 3'.
6. The fluorescent quantitative PCR rapid detection kit for pseudomonas fluorescens as claimed in claim 5, which is applied to the detection of pseudomonas fluorescens in a sample.
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| CN113512600A (en) * | 2021-06-18 | 2021-10-19 | 广西壮族自治区水牛研究所 | Primer and method for detecting pseudomonas putida and application thereof |
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| CN113512600A (en) * | 2021-06-18 | 2021-10-19 | 广西壮族自治区水牛研究所 | Primer and method for detecting pseudomonas putida and application thereof |
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Application publication date: 20200228 |