CN111518927A - TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit - Google Patents
TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit Download PDFInfo
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- CN111518927A CN111518927A CN201911235469.2A CN201911235469A CN111518927A CN 111518927 A CN111518927 A CN 111518927A CN 201911235469 A CN201911235469 A CN 201911235469A CN 111518927 A CN111518927 A CN 111518927A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a TaqMan probe quantitative detection method for detecting pseudomonas putida and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the pseudomonas putida 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 putida 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 putida through a specific gene and a corresponding detection kit.
Background
Pseudomonas putida (Pseudomonas putida), belonging to the family Pseudomonas (Pseudomonas adaceae), the genus Pseudomonas (Pseudomonas). Is a common pathogenic bacterium of aquaculture animal diseases and can cause diseases of marine and freshwater aquaculture fishes.
The bacterium is gram-negative brevibacterium, with round ends, single or multiple flagellate, dynamic, no spore, and thallus size of 0.6-1.0 μm × 1.5-3.0 μm. A wet colony with a neat, round, flat, white and transparent edge grows on a common nutrient agar plate, and grows slowly on a TCBS culture medium to be a green and transparent colony. The peptone water does not grow in salt-free peptone water, can grow in peptone water with 6% NaCl concentration, can grow at the temperature of 15-30 ℃ and at the temperature of 4 ℃ and does not grow at the temperature of 41 ℃.
The pseudomonas putida has biological type A and biological type B, is pathogenic and highly pathogenic, and oral infection can not only cause diarrhea and even death of birds, but also cause food poisoning of human beings. In aquatic animals, it can cause yellow and black gill diseases of macrobrachium rosenbergii, gill rot disease of European eel and fester disease of rainbow trout. In addition, toxic proteins such as adhesin of Pseudomonas putida and hemolysin of extracellular product have a pathological effect.
After pseudomonas putida is infected, no effective method is available for treatment at present, and the probability of outbreak of the disease can be prevented and controlled only by strengthening 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.
The invention provides a method for quantitatively, quickly and real-timely monitoring pseudomonas putida, which overcomes the defects of the prior art and enables the pseudomonas putida to be detected more accurately, sensitively, quickly and conveniently.
Disclosure of Invention
One of the purposes of the invention is to provide a TaqMan probe quantitative detection method for detecting pseudomonas putida, which can quantitatively, rapidly and real-timely detect pseudomonas putida, so that the detection of pseudomonas putida is 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 the pseudomonas putida specific gene 16sRNA gene by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the pseudomonas putida specific gene 16sRNA gene is less than 35, the detection result of the pseudomonas putida is positive; and when the Ct value of the pseudomonas putida specific gene 16sRNA gene is more than 35, the detection result of the pseudomonas putida is negative.
As a preferred technical scheme, the step S2 further comprises the step of designing a detection primer and a TaqMan probe of the 16sRNA gene.
As a preferred technical scheme, a detection primer of the pseudomonas putida specific gene 16sRNA gene is shown as SEQ ID NO. 1-2;
SEQ ID NO:1(5’-GCGGTGGAGCATGTGGTT-3’)；
SEQ ID NO:2(5’-CTGGAAAGTTCTCTGCATGTCAA-3’)。
the probe sequence is shown as SEQ ID NO. 3;
SEQ ID NO:3(5’-FAM-TCGAAGCAACGCGAAGAACCTTACCA-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 total 10uM 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 the pseudomonas putida, which comprises a detection primer of the pseudomonas putida specific gene 16sRNA 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 specificity of the pseudomonas putida and other species of strains is mined out from a large amount of early-stage research data on the pseudomonas putida and other species of strains, whether other strain positive samples and environment samples contain the pseudomonas putida is accurately judged by detecting the specific gene, the early-stage big data mining and the comparison between different species are based, and the selected specific gene has the specificity of species and genus.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primers which are optimized for the 16sRNA gene detection amplification conditions are selected as the detection primers, so that the amplification efficiency of the primers is improved, the detection primers with stability, high efficiency, high sensitivity and reproducibility can be achieved, and the trace samples can be correctly detected.
(3) Compared with other detection methods in the market, the detection strategy of the invention can intuitively judge the infection of the pseudomonas putida and the infection of other strains, and the result is more rigorous and accurate.
(4) The invention can be implemented from 101Diluting to 10 times6The detection range of the dilution is doubled, the sensitivity is high, the application range is wide, a sample with higher concentration can be detected through dilution conversion, the detection result is stable, and the reproducibility is good.
(5) The detection method can be applied to screening and detecting samples from various sources such as culture base samples or culture water samples and the like, accurately judges whether the culture water samples contain the pseudomonas putida, 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. 116 sRNA gene detection primer standard curve.
FIG. 2 is a 16sRNA gene amplification curve in example 1.
FIG. 3 is a 16sRNA gene amplification curve in example 2.
FIG. 4 is an amplification curve of the 16sRNA gene in the specificity test in example 3.
FIG. 5 is an amplification curve of the 16sRNA gene of sample 1 at the breeding site in example 5.
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
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 putida can be correctly distinguished from samples infected by other species of fungi and insects, thereby accurately detecting and judging pseudomonas putida.
In some embodiments, the number of the pairs is 10, 1021, 1031, 104、105And 106The positive bacteria are extracted and detected, and the pseudomonas putida and microorganisms of other genera can be accurately distinguished, so that the pseudomonas putida can be accurately detected and judged. 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, pseudomonas putida samples, culture base samples and culture water samples are collected, and whether the samples contain pseudomonas putida can be quickly and accurately detected through detecting specific genes, so that the method is convenient and quick.
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 putida 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 aquaculture, breeding and the like of aquatic animals.
Example 1 Standard Curve for primer amplification detection and plasmid sensitivity test
1. Cultivation of microorganisms
LB medium was used as the medium for Pseudomonas putida 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 putida was used, amplification was performed with primers for the 16sRNA 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 the 16sRNA 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 plating standard clone bacteria of primer amplification products corresponding to 16sRNA genes, collecting monoclonal bacterial plaques, performing LB culture and shake bacteria overnight for amplification, finally obtaining plasmid positive strains in 5ml exponential growth phase, extracting plasmid DNA according to the instruction of QIAGEN plasmid Midi Kit, detecting the concentration of the plasmid DNA after extraction, and configuring 7 standard products with concentration gradients of 1 fg/mu L and 1 × 10 times through a 10-time dilution method1fg/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 results of the standard curve for detecting primer amplification show that the primers corresponding to the 16sRNA 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 the optimal state, the standard deviation and the coefficient of variation of the amplification curve of the 16sRNA gene and the amplification efficiency under 7 concentration gradients were both satisfied with the requirements of the optimal state, 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 the 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 the 16sRNA gene at different plasmid concentrations, and the concentration of the amplification curve from left to right was 1 × 106fg/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
Performing q-PCR amplification of the sample according to the instruction of TaKaRa Taq PCR Mix instruction, and obtaining Ct value reading corresponding to the primerThe q-PCR reaction of this experiment was set to 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, the correlation coefficient and reliability of the amplification curve of the 16sRNA gene and the amplification efficiency at 2 concentrations were analyzed, and both meet the requirements of the optimal state, and the primers can meet 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 Pseudomonas putida showed a positive result for the 16sRNA gene, while all other insect species or genera showed negative results. Therefore, the 16sRNA gene can be detected for the Pseudomonas putida and other insect species or genera, so that the Pseudomonas putida and other insect species or genera can be better distinguished. In summary, the corresponding primers and probes of the 16sRNA gene can meet the detection and judgment of Pseudomonas putida.
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 putida sample, a culture base sample and multiple culture water samples, and extracting the genome DNA according to the instruction of a QIAamp DNA Mini Kit 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 results of the q-PCR amplification with the primers revealed that 16sRNA genes were not detected in any of the samples except for the Ppu sample and the two samples from any of the breeding sites, and it was confirmed that the Ppu sample and the two samples from any of the breeding sites contained Pseudomonas putida. The final criterion for judging the pseudomonas putida is that the Ct value obtained by 16sRNA gene detection is less than 35 cycles. If the Ct value obtained by detecting all the 16sRNA genes is more than 35 cycles, the Pseudomonas putida 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
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<120> TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit
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Claims (6)
1. The TaqMan probe quantitative detection method for detecting the pseudomonas putida is characterized by comprising the following steps of:
s1, collecting a sample;
s2, extracting genome DNA;
s3, quantitatively detecting and detecting the pseudomonas putida specific gene 16sRNA gene by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the pseudomonas putida specific gene 16sRNA gene is less than 35, the detection result of the pseudomonas putida is positive; and when the Ct value of the pseudomonas putida specific gene 16sRNA gene is more than 35, the detection result of the pseudomonas putida is negative.
2. The TaqMan probe quantitative detection method for detecting Pseudomonas putida according to claim 1, wherein the step S2 further comprises a step of designing a detection primer of 16sRNA gene and a TaqMan probe.
3. The TaqMan probe quantitative detection method for detecting Pseudomonas putida according to claim 1, wherein a detection primer of the Pseudomonas putida specific gene 16sRNA gene is shown as SEQ ID NO 1-2, and a TaqMan probe sequence is shown as SEQ ID NO 3.
4. The method for quantitative detection of TaqMan probe for detection of Pseudomonas putida according to claim 1, wherein the reaction system of the fluorescence quantitative PCR detection is 25 μ l, comprising 2 × Taq PCR Mix 12.5 μ l, total 10uMPrimers Mix 1 μ l, TaqMan probe 0.5 μ l, DNA input 2 μ l, H2O 9μl。
5. The TaqMan probe quantitative detection method for detecting Pseudomonas putida 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 putida is characterized by comprising a detection primer of the pseudomonas putida specific gene 16sRNA gene and a TaqMan probe.
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| CN113549671A (en) * | 2021-07-21 | 2021-10-26 | 连云港市第一人民医院 | Intestinal flora for diagnosing sarcopenia of maintenance hemodialysis patient |
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| CN113549671A (en) * | 2021-07-21 | 2021-10-26 | 连云港市第一人民医院 | Intestinal flora for diagnosing sarcopenia of maintenance hemodialysis patient |
| CN113549671B (en) * | 2021-07-21 | 2023-09-08 | 连云港市第一人民医院 | Intestinal flora for diagnosing sarcopenia of maintenance hemodialysis patient |
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