CN111534615A - TaqMan probe quantitative detection method for detecting pseudomonas fluorescens and corresponding kit - Google Patents
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Abstract
The invention discloses a TaqMan probe quantitative detection method for detecting pseudomonas fluorescens and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the pseudomonas fluorescens from strains or insect species of other species, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting pseudomonas fluorescens has the advantages of high sensitivity, rapidness, convenience, good specificity, rigorous and accurate judgment and the like, and has good application prospect and market value.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for carrying out TaqMan probe quantitative detection on pseudomonas fluorescens by using a specific gene and a corresponding detection kit.
Background
Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas (Pseudomonas adacea), Pseudomonas (Pseudomonas) and Pseudomonas. Pseudomonas fluorescens is widely present in water, sewage and soil, is a pathogenic bacterium of fish red skin disease, and is saprophytic bacteria causing aquatic product putrefaction.
The strain is rod-shaped, has two blunt ends, and has a size of 0.7-0.8 μm × 2.3-2.8 μm. The aged culture is short and delicate. Individually or in rows. Can move, has 1-3 extreme flagella, and individual bacteria sometimes lose flagella. No spores. The thallus is evenly stained and gram-negative.
The bacterium can mainly infect grass carp and black carp, and also can infect other fishes such as porgy, carp, pike, grouper and weever. Bacteria invade skin tissues through wounds to cause bleeding, inflammation, erosion and ulcer of the skin on the body surface. The affected parts are mostly on the two sides of the trunk and the abdomen, and the fins and gills. The tissues among the fins are decayed to form fin-eaten pieces. Sometimes the fish intestinal tract is also congested and inflamed. Fish of different sizes can be infected and attack. There is no obvious season of prevalence.
After pseudomonas fluorescens is infected, no effective method is available for treatment at present, and the probability of preventing and controlling outbreak of the pseudomonas fluorescens can be achieved only by enhancing epidemic disease monitoring and quarantine. Through a large amount of research, scholars at home and abroad establish a series of detection methods aiming at pseudomonas fluorescens, such as a histopathological anatomy method, an electron microscope observation method, a Leeb's liquid thioglycolate culture method, a PCR detection method and a LAMP method. However, the first three methods are complex to operate, long in time consumption and low in detection sensitivity, and only can diagnose fishes and shrimps with obvious diseases; although the PCR detection method has accurate result, the detection time is long, the operation is complex, quantification cannot be realized, and the method is difficult to popularize and apply in production; although the LAMP method has short detection time, the false positive is high, and the requirement of accurate detection cannot be met.
Disclosure of Invention
One of the purposes of the invention is to provide a TaqMan probe quantitative detection method for detecting pseudomonas fluorescens, which can quantitatively, rapidly and real-timely detect the pseudomonas fluorescens and enable the detection of the pseudomonas fluorescens to be more accurate, sensitive, rapid and safe.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, quantitatively detecting and detecting a pseudomonas fluorescens specific gene lpd gene by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene lpd of the pseudomonas fluorescens is less than 35, the detection result of the pseudomonas fluorescens is positive; when the Ct value of the specific gene lpd of the pseudomonas fluorescens is more than 35, the detection result of the pseudomonas fluorescens is negative.
As a preferred technical scheme, the step S2 further comprises the step of designing a detection primer of lpd gene and a TaqMan probe.
As a preferred technical scheme, a detection primer of the pseudomonas fluorescens specific gene lpd gene is shown as SEQID NO 1-2;
SEQ ID NO:1(5’-CAAGGCGCAGATGAACTACGA-3’)；
SEQ ID NO:2(5’-TTGAGGGTCTGCTCGGTCTT-3’)。
the probe sequence is shown as SEQ ID NO. 3;
SEQ ID NO:3(5’-FAM-TCGGTCATCTACACCCACCCGGAAAT-BHQ1-3’)。
as a preferred technical scheme, the reaction system for the fluorescent quantitative PCR detection is 25 mu l, and comprises 12.5 mu l of 2 × TaqPCR Mix, 1 mu l of total10uM Primers Mix, 0.5 mu l of TaqMan probe, 2 mu l of DNA input, H2O 9μl。
As a preferred technical scheme, the reaction conditions of the fluorescent quantitative PCR detection are denaturation at 95 ℃ for 2min30s, annealing at 94 ℃ for 15s, annealing at 60 ℃ for 30s, collecting fluorescent signals and performing 40 cycles.
The invention also aims to provide a TaqMan probe quantitative detection kit for detecting pseudomonas fluorescens, which comprises a detection primer of the pseudomonas fluorescens specific gene lpd gene and a TaqMan primer probe.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the orphan gene capable of distinguishing the species and the strain specificity of the pseudomonas fluorescens and other species is mined out from a large amount of early-stage research data on the pseudomonas fluorescens and other species of insect species and strains, whether the pseudomonas fluorescens is contained in other strains or positive samples of the insect species and environmental samples or not is accurately judged by detecting the specific gene, the early-stage large data mining and the comparison between different species are based, and the selected specific gene has the species and the genus specificity.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primer which is optimized for lpd gene detection amplification conditions is selected as the detection primer, so that the amplification efficiency of the primer is improved, the detection primer with stability, high efficiency, high sensitivity and reproducibility can be achieved, and the correct detection of a trace sample is realized.
(3) Compared with other detection methods in the market, the detection strategy of the invention can intuitively judge the infection of the pseudomonas fluorescens and the infection of other insect species or strains, and the result is more rigorous and accurate.
(4) The detection method can be applied to screening and detecting samples from various sources such as culture water samples or oyster samples and the like, accurately judges whether the culture water samples contain pseudomonas fluorescens or not, and is convenient, rapid and sensitive to operate. The detection kit can be applied to the rapid detection of various culture samples without additionally carrying out microbial culture, so that the gene detection is applied to the detection of culture water and daily life, and has the advantages of high sensitivity, strong operability, rapidness, high efficiency and the like.
Drawings
FIG. 1lpd Gene detection primer standard curve.
FIG. 2 shows the lpd gene amplification curve in example 1.
FIG. 3 is an lpd gene amplification curve in example 2.
FIG. 4 shows the amplification curve of lpd gene in example 3.
FIG. 5 is an amplification curve of lpd gene in sample 1 of the breeding ground in example 4.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
The following reagents used in the present invention can be purchased from conventional sources.
TABLE 1
| Name (R) | Brand | Model number | Specification of |
| Healthy weever | Certain cultivation farm of pearl sea | - | 100g |
| Pseudomonas fluorescens | Certain aquatic product promotion station | - | Freezing |
| Cryptocaryon irritans | Certain aquatic product promotion station | - | Freezing |
| Vibrio vulnificus | Certain aquatic product promotion station | - | Freezing |
| Vibrio parahaemolyticus | Certain aquatic product promotion station | 17802 | Freezing |
| Streptococcus agalactiae | ATCC | 244793 | Freezing |
| Edwardsiella tarda | ATCC | 8117 | Freezing |
| QIAamp DNA Mini Kit | Qiagen | 51304 | 50 reactions |
| QIAGEN Plasmid Midi Kit | QIAGEN | 12145 | 100 reactions |
| Taq PCR Mix fluorescent quantitative PCR premixed reaction liquid | TaKaRa | RR391A | 200 reactions |
| Peptone yeast extract agar | Sigma | 77196-500 | 500 g |
In some embodiments, positive standard plasmids are constructed using the amplification products, and the amplification standard curve of the primers is detected and plotted to obtain the amplification efficiency of the detection primers, and the amplification efficiency and the confidence degree of the primer sequences provided by the invention are optimized.
In some embodiments, by detecting specific genes for different species or strains of insects, samples infected by pseudomonas fluorescens can be correctly distinguished from samples infected by other species and strains of bacteria, so that pseudomonas fluorescens can be accurately detected and judged.
In some embodiments, the number of the channels is 10, 100, 1000, 10000, 105And 106The extraction and detection of each positive bacterium can accurately distinguish the pseudomonas fluorescens and microorganisms of other genera, thereby accurately detecting and judging the pseudomonas fluorescens. And can guarantee that as low as 10 positive bacteria can still be accurately detected, and a microbial sample with higher titer can be also accurately detected by diluting to a detection interval.
In some embodiments, pure water, a pseudomonas fluorescens sample, a culture base sample and a culture water sample are collected, and whether the pseudomonas fluorescens is contained in the sample can be rapidly and accurately detected through detecting the specific genes, so that the method is convenient and rapid.
It will be understood by those skilled in the art that the genes to be detected and the specific primers to be designed in the method can be designed to achieve the corresponding detection purpose through the design of other sections of the same detection object.
The method and the kit for detecting the quantitative detection of the pseudomonas fluorescens TaqMan probe can be widely applied to a plurality of inspection and quarantine categories such as culture water sample detection, culture base detection and the like, and can provide a convenient, quick, accurate and efficient gene detection-based monitoring product for breeding and the like.
Example 1 Standard Curve for primer amplification detection and plasmid sensitivity test
1. Cultivation of microorganisms
LB medium was used as the medium for the Pseudomonas fluorescens plasmid, and the growth was good in the plate with pH7.0-7.4. The colony diameter on the plate is 2mm, round, smooth and transparent. And selecting a microorganism sample which is in vigorous growth to perform subsequent experiments.
2. Genomic DNA extraction
Extraction of genomic DNA was performed according to the QIAamp DNA Mini Kit instructions from Qiagen.
3. Standard quality particle preparation of amplification product
DNA extracted from Pseudomonas fluorescens was used, amplification was performed with primers for lpd gene, the amplification product was treated with A, ligated into T vector using T4 ligase, and after transduction of competent cells by the plasmid was amplified on a large scale. The primer sequence and TaqMan probe sequence of lpd gene are shown in SEQ ID NO 1-3, and are specifically shown in Table 2.
TABLE 2
4. Plasmid extraction and Standard Curve gradient configuration
Respectively coating the standard clone bacteria of the primer amplification products corresponding to the lpd gene, collecting the monoclonal bacterial plaques, and introducingCulturing the shake bacteria in LB culture overnight, amplifying to obtain 5ml plasmid positive strain in exponential growth phase, extracting plasmid DNA according to QIAGEN plasmid Midi Kit instruction, detecting plasmid DNA concentration after extraction, and preparing 7 standard samples with concentration gradients of 1fg/μ L and 1 × 10 by 10-fold dilution method, wherein the concentration gradients are respectively 1fg/μ L and 1 ×1fg/μL，1×102fg/μL，1×103fg/μL，1×104fg/μL，1×105fg/μL，1×106fg/μL。
5. Drawing a standard curve of the detection primer
And performing q-PCR amplification of a concentration gradient standard curve according to the instruction of TaKaRa Taq PCR Mix instruction, and drawing a corresponding amplification curve to obtain the amplification efficiency of the primer. 3 biological replicates and 3 technical replicates were set for each q-PCR reaction, the q-PCR reaction system and amplification reaction conditions are shown in Table 3, and the standard curve is plotted as shown in FIG. 1.
TABLE 3
6. Analysis of results
The result of the standard curve for detecting primer amplification shows that the primers corresponding to the lpd gene are optimized primer design and a reaction system. Linear standard curve confidence R2>0.990, the primer design and the reaction system were proved to be optimal, the standard deviation and the coefficient of variation of the amplification curve of lpd gene and the amplification efficiency under 7 concentration gradients were both satisfied with the optimal conditions, the primers could satisfy the detection requirements under the reaction conditions, which indicated that the method had high plasmid sensitivity, and the coefficient of variation between CT values measured by 3 technical repeat experiments was less than 5.0%, which proved that the method had high reproducibility, the detection results are shown in table 4 and fig. 2, fig. 2 is the amplification curve of lpd gene at different plasmid concentrations, the concentration of the amplification curve from left to right was 1 × 10 in sequence6fg/μL，1×105fg/μL，1×104fg/μL，1×103fg/μL，1×102fg/μL，1×101fg/μL，1×100fg/μL。
TABLE 4
Example 2 plasmid reproducibility test
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
3. q-PCR detection of target genes
The q-PCR amplification of the sample was performed according to the TaKaRa Taq PCR Mix instructions and the Ct reading corresponding to the primers was obtained the q-PCR reaction of this experiment set 2 concentrations, 1 × 10 respectively6fg/. mu.L and 1 × 105fg/. mu.L, 4 biological replicates and 5 technical replicates per concentration, respectively, were set, and the q-PCR reaction system and amplification reaction conditions are shown in Table 5.
TABLE 5
4. Analysis of results
In this example, it is analyzed that the correlation coefficient and reliability of the lpd gene amplification curve and the amplification efficiency at 2 concentrations both meet the requirements of the optimal state, and the primers can satisfy the detection requirements under the reaction conditions. The detection results are shown in table 6 and fig. 3, 4 groups are set for each concentration condition, each group is repeatedly measured for 5 times, and the measurement results show that the coefficient of variation is less than 5 percent, which proves that the method has good repeatability and high stability.
TABLE 6
EXAMPLE 3 Positive and negative sample detection
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
3. q-PCR detection of target genes
q-PCR amplification of the samples was performed as indicated in TaKaRa Taq PCR Mix instructions and Ct value readings were obtained for the primers. The q-PCR reaction set 3 biological and 3 technical repeats, and the q-PCR reaction system and amplification reaction conditions were the same as in example 2.
4. Analysis of results
As shown in Table 7 and FIG. 4, the results of q-PCR amplification of the primers showed that P.fluorescens showed a positive result for the lpd gene, while all other insect species or genera showed negative results. Therefore, the detection of the lpd gene can be carried out on the pseudomonas fluorescens and other insect species or genera, so that the pseudomonas fluorescens and other insect species or genera can be better distinguished. In conclusion, the corresponding primers and probes of lpd gene can satisfy the detection judgment of Pseudomonas fluorescens.
TABLE 7
Example 4 detection of environmental samples
1. Environmental sample collection and extraction of genomic DNA
And respectively collecting a pure water sample, a pseudomonas fluorescens sample, a culture base sample and a plurality of culture water samples, and extracting the genome DNA according to the instruction of the QIAamp DNA Mini Kit instruction of Qiagen.
2. q-PCR detection of target genes
The same as in example 2.
3. Analysis of results
As shown in Table 8, the q-PCR amplification results of the primers showed that lpd gene was detected in all samples except for P.fluorescens samples and a sample from a breeding ground, which confirmed that the corresponding sample sources contained P.fluorescens. The final criterion for judging the pseudomonas fluorescens is that the Ct value obtained by lpd gene detection is less than 35 cycles. If the Ct value obtained by all lpd genes is more than 35 cycles, the Pseudomonas fluorescens is judged to be negative and not detected.
TABLE 8
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangdong Meige Gene science and technology Co., Ltd
<120> TaqMan probe quantitative detection method for detecting pseudomonas fluorescens and corresponding kit
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Claims (6)
1. The TaqMan probe quantitative detection method for detecting the pseudomonas fluorescens is characterized by comprising the following steps of:
s1, collecting a sample;
s2, extracting genome DNA;
s3, quantitatively detecting and detecting a pseudomonas fluorescens specific gene lpd gene by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene lpd of the pseudomonas fluorescens is less than 35, the detection result of the pseudomonas fluorescens is positive; when the Ct value of the specific gene lpd of the pseudomonas fluorescens is more than 35, the detection result of the pseudomonas fluorescens is negative.
2. The TaqMan probe quantitative detection method for Pseudomonas fluorescens according to claim 1, wherein the step S2 further comprises a step of designing a detection primer of lpd gene and TaqMan probe.
3. The TaqMan probe quantitative detection method for detecting pseudomonas fluorescens according to claim 1, wherein a detection primer of a specific gene lpd gene of the pseudomonas fluorescens is shown as SEQ ID NO 1-2, and a sequence of the TaqMan probe is shown as SEQ ID NO 3.
4. The TaqMan probe quantitative detection method for detecting Pseudomonas fluorescens according to claim 1, wherein the TaqMan probe quantitative detection method isThe reaction system for the fluorescent quantitative PCR detection is 25 mu l, and comprises 2 × Taq PCR Mix 12.5 mu l, total10 uMPrimers Mix 1 mu l, TaqMan probe 0.5 mu l, DNA input 2 mu l, H2O 9μl。
5. The TaqMan probe quantitative detection method for Pseudomonas fluorescens according to claim 1, wherein the reaction conditions of the fluorescence quantitative PCR detection are denaturation at 95 ℃ for 2min30s, annealing at 94 ℃ for 15s, annealing at 60 ℃ for 30s, collecting fluorescence signals, and 40 cycles.
6. A TaqMan probe quantitative detection kit for detecting pseudomonas fluorescens is characterized by comprising a detection primer of a pseudomonas fluorescens specific gene lpd gene and a TaqMan probe.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397099B (en) * | 2013-08-16 | 2015-03-04 | 江苏省农业科学院 | Method for detecting quantity of pseudomonas fluorescent in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) |
| AU2010200871B2 (en) * | 2010-03-08 | 2016-02-25 | Monash University | Detection of pseudomonas strains |
| CN106702008A (en) * | 2017-03-03 | 2017-05-24 | 中南大学 | Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR |
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Patent Citations (3)
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| AU2010200871B2 (en) * | 2010-03-08 | 2016-02-25 | Monash University | Detection of pseudomonas strains |
| CN103397099B (en) * | 2013-08-16 | 2015-03-04 | 江苏省农业科学院 | Method for detecting quantity of pseudomonas fluorescent in rhizospheric soil during growth period of transgenic wheat by virtue of fluorescent quantitative PCR (Polymerase Chain Reaction) |
| CN106702008A (en) * | 2017-03-03 | 2017-05-24 | 中南大学 | Method for detecting six functional genes of Cr(VI) reducing complex microorganisms through multiple real-time fluorescent PCR |
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| Title |
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| H-J BACH等: "Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification", 《J MICROBIOL METHODS.》 * |
| OLGA V MAVRODI等: "Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR", 《APPL ENVIRON MICROBIOL.》 * |
| RATUL SAHA等: "Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments", 《J FOOD SCI.》 * |
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