CN110578012A - Kit for rapidly detecting pseudomonas aeruginosa - Google Patents
Kit for rapidly detecting pseudomonas aeruginosa Download PDFInfo
- Publication number
- CN110578012A CN110578012A CN201910960360.9A CN201910960360A CN110578012A CN 110578012 A CN110578012 A CN 110578012A CN 201910960360 A CN201910960360 A CN 201910960360A CN 110578012 A CN110578012 A CN 110578012A
- Authority
- CN
- China
- Prior art keywords
- reagent
- kit
- indicator
- detection
- pseudomonas aeruginosa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12Q1/6876—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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for rapidly detecting pseudomonas aeruginosa, which comprises a specific primer pair and a kit containing the primer pair, wherein the kit comprises a first reagent, a second reagent and a sealing agent; the first reagent comprises Ft, Bt, IF, IB, dNTP, BstDNA polymerase and an indicator; reagent two comprises (NH)4)2SO4、KCl、MgSO4Tween20 and betaine, pH 8.0. The kit provided by the invention adopts a unique buffer system, has the advantage of strong amplification compatibility, can carry out high-efficiency specific amplification without thoroughly removing impurities such as protein and the like, is suitable for the field quarantine inspection and detection requirements under the background of mass flow and quick customs clearance, and is also suitable for quick detection in a laboratory.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit for rapidly detecting pseudomonas aeruginosa.
Background
Pseudomonas aeruginosa (pseudomonas aeruginosa) is commonly known as pseudomonas aeruginosa and is ubiquitous in the environment, particularly in humid environments. Pseudomonas aeruginosa is a common pathogenic bacterium under the condition, is one of main pathogenic bacteria for nosocomial infection, often causes postoperative wound infection, and can also cause bedsore, abscess, suppurative otitis media and the like. The infection focus caused by the bacterium can cause blood spreading, and then bacteremia and septicemia occur, so that pseudomonas aeruginosa infection after burn can cause death. The susceptible to this bacterium is a patient suffering from metabolic diseases, hematologic diseases and malignancies as well as after surgery or after certain treatments. The bacterium can be delivered to patients through medical personnel and is the second most common pathogenic bacterium of intensive care unit infection; the bacterium is also a common pathogenic bacterium of urinary tract infection and is also a common pathogenic bacterium of otitis externa; infection of the bacteria by contamination of the contact lens or lens fluid can cause corneal ulceration in the eye; after foot trauma or deep puncture wound, the bacteria can be found in the draining sinus, which causes cellulitis and osteomyelitis; infection rarely occurs in prosthetic valves used in cardiac surgery or in the natural valves of phlebotomists, and this bacteria can cause endocarditis.
According to the national standard, pathogenic bacteria such as pseudomonas aeruginosa and the like cannot be detected in cosmetics and sanitary products. Currently, the detection of pseudomonas aeruginosa is mostly bacteria isolation culture and biochemical identification, the time is long, and the operation is complex. With the development of pathogenic bacteria nucleic acid detection technology, PCR and fluorescence real-time PCR detection methods are also developed in succession. Particularly, with the rapid development of isothermal amplification technology, isothermal amplification technologies based on various principles, such as transcription dependent amplification system TAS, self-sustained sequence replication 3SR, nucleic acid sequence dependent amplification NASBA, etc., have been developed in foreign countries, and these technologies can only amplify RNA at present, require continuous addition of enzymes during the reaction process, and still require complicated subsequent procedures for product detection. The strand displacement amplification SDA still needs a denaturation process, the amplified target sequence cannot exceed 200bp, and the subsequent detection is complex. The products of rolling circle amplification RCA amplification are complex. Depending on helicase gene to amplify the HAD, three enzymes are needed for amplification, and the application range of the three enzymes is limited. The loop-mediated isothermal amplification LAMP technology has the most advantages in the existing isothermal amplification technology, only one enzyme is needed, the amplification efficiency is high, and the method is the method which is most applied and has the largest thesis publication amount in the existing INAT technology. However, LAMP requires at least 6 primers and has complex design, so that the false positive rate is high due to the excessively complex product, the recovery and sequencing of LAMP products are difficult, and the limitation of China on transformation application is strong due to the protection of Japanese intellectual property rights.
Meanwhile, commonly used methods for judging the nucleic acid detection result comprise methods such as an agarose gel electrophoresis method, a fluorescence imaging method, a nucleic acid thin film chromatography test strip method, a real-time turbidity method and the like, but all of the methods have the common characteristic of needing professional instruments and have high requirements on the professional level of operators.
The extraction of nucleic acids from a biological sample to be tested is the first step in the detection of nucleic acids. The nucleic acid extraction process generally adopts a kit based on various principles such as a guanidinium cracking method, a silicon membrane method, a magnetic bead method and the like, or adopts an automatic nucleic acid extractor, but the extraction of nucleic acid by using the kit wastes time and labor, is complex in operation, usually needs more than 2 hours, cannot meet the requirements of rapid and simple instant detection, and the automatic nucleic acid extractor can realize high throughput, is simple and convenient to operate, but is heavy and high in maintenance cost.
therefore, based on the defects of insufficient sensitivity, high requirements on experimental conditions and the like of the existing means for detecting pseudomonas aeruginosa, the provided kit for sensitively, simply and rapidly detecting pseudomonas aeruginosa is a problem to be solved by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a kit for rapidly detecting pseudomonas aeruginosa, which adopts a unique buffer system, has the advantage of strong amplification compatibility, can carry out efficient specific amplification without thoroughly removing impurities such as protein and the like, has the characteristics of sensitivity, rapidness, simplicity, convenience and real-time detection of pseudomonas aeruginosa, is suitable for the field quarantine inspection detection requirements under the background of mass flow and rapid clearance, and is also suitable for rapid detection in laboratories.
In order to achieve the purpose, the invention adopts the following technical scheme:
According to the gene Sequence of pseudomonas aeruginosa toxA disclosed by NCBI (Reference Sequence: NC-002516.2), a specific primer pair for rapidly detecting pseudomonas aeruginosa by using a double indicator is designed, and the nucleotide Sequence of the primer pair is as follows:
Ft：5’-gcatcgtcttcggcggggtGGAGCGCGGCTATGTGT-3’；SEQ ID NO.1；
Bt：5’-gcatcgtcttcggcggggtTCGGGTTCCTGGTCCTG-3’；SEQ ID NO.2；
IF：5’-AAGGTGCCGTGGTAGCC-3’；SEQ ID NO.3；
IB：5’-TCTATATCGCCGGCGATCC-3’；SEQ ID NO.4。
wherein, IF and IB are used as accelerating primers, the reaction rate is greatly improved after the introduction, and the Ct value is advanced to within 20min when turbidity detection is carried out at the later stage.
a kit comprising the above-mentioned specific primer pair.
Further, the kit comprises a reagent I, a reagent II and a blocking agent; wherein the first reagent comprises Bst DNA polymerase, Ft and Bt primers, IF and IB primers, dNTP and an indicator;
Reagent two comprises (NH)4)2SO4、KCl、MgSO40.2% Tween20 and betaine, pH 8.0.
further, the reagent-10U Bst DNA polymerase, 50. mu.M each of Ft and Bt primers, 50. mu.M each of IF and IB primers, 10mM dNTP and indicator;
Reagent two contains 20mM (NH)4)2SO4、100mM KCl、16mM MgSO40.2% Tween20 and 16M betaine, pH 8.0.
Among them, Bst DNA polymerase does not contain glycerol.
further, the indicator is a cresol red-phenol red indicator and/or a fluorescent dye; the cresol red-phenol red indicator includes cresol red at a final concentration of 0.08mM and phenol red at a final concentration of 0.02 mM. The fluorescent dye was SYBR Green I or EveGreen (working concentration) in 1 Xaqueous solution.
Further, the blocking agent is paraffin.
The reagent in the kit is stored by using a unitary packaged diagnostic reagent bottle, wherein the reagent bottle is provided with a detection tube, a detection tube cover, a placing groove and a covering film; wherein, the detection tube is a hollow reaction tube with one end open and the other end closed; the detection tube cover is matched with the detection tube, covers the opening end of the detection tube and closes the detection tube, wherein the detection tube and the detection tube cover are integrally formed; the inner side of the detection tube cover is provided with a placing groove, and the placing groove and the detection tube cover are integrally formed; the covering film is a PPE film and covers the placing groove.
According to the invention, the first reagent is placed in the placing groove on the detection tube cover, then the placing groove is sealed by the covering film, the second reagent is placed at the bottom of the detection tube, then the sealing agent is added, and finally the detection tube cover is covered, so that the assembly of the kit is completed.
the aerosol pollution is a difficult problem which troubles the whole nucleic acid amplification field, the polymerase spiral reaction has high amplification efficiency and violent reaction, nucleic acid with a large amount of amplification production can not be generated, the aerosol pollution sealant is added into a reaction tube before the reaction, the aerosol pollution sealant is melted into liquid to cover reaction liquid in the reaction process, the volatilization of the aerosol is prevented, the amplification reaction is not influenced, the aerosol pollution sealant can be condensed into solid again after the reaction is finished, the reaction liquid is sealed permanently, and the pollution is avoided.
In the present invention, the purpose of halving the amounts of the accelerated primers IF and IB compared with the amounts of the main primers Ft and Bt is to allow the main primers to bind to the target sequence first and to evaluate the influence of the number of accelerated primers on the reaction.
The use method of the kit is characterized by comprising the following steps:
(1) Carrying out heating pyrolysis on a sample to be detected by using a pyrolysis solution to extract DNA;
(2) Adding the sample solution cracked in the step (1) into the reagent I of the kit, mixing with the reagent II, and adding a confining liquid to prevent pollution; setting positive and negative controls;
(3) Amplifying at constant temperature of 63-67 deg.C for 30-60min, and observing the judgment result by color development method or detecting by turbidity method.
when the kit is used, only one drop of sample lysate needs to be added to the reagent I, and the sample lysate, the reagent II and the sealing agent are mixed, and the pH value is just 8.8 after the mixture is mixed; wherein the pH of the sample lysate is about 11, and the pH of the mixture is about 8.8 after mixing, wherein the enzyme, the primer and the dNTP are acidic substances. The kit is convenient to use and rapid in detection, and can interpret the amplification result by adopting a color development method, so that the problems of complicated and complicated judgment of the nucleic acid amplification result are solved.
Wherein, the color development method comprises the following steps: and after the constant-temperature amplification reaction is finished, cooling to room temperature, observing the result, wherein the yellowing of the detection tube indicates that the pseudomonas aeruginosa genes (positive) exist in the sample to be detected, and the color of the detection tube is changed into red, and indicates that the pseudomonas aeruginosa genes (negative) do not exist in the sample to be detected. If the negative control tube is changed into yellow or the color of the positive control tube is red within 50 minutes, the detection result is invalid, and the detection is required to be carried out again.
The detection principle of the color development method is as follows: when a DNA polymerase binds a deoxyribonucleotide molecule to a newly synthesized DNA double strand, a hydrogen ion is generated as a by-product, and as the PSR reaction proceeds, a large amount of hydrogen ions are generated, and the pH of the reaction system decreases as the concentration of the hydrogen ion increases. When the pH indicator solution was added to 25. mu.l of the PSR reaction system, the positive reaction became yellow and the negative control solution became red.
The turbidity method detection principle is as follows: mg formation during the PSR reaction2P2O7I.e. magnesium pyrophosphate, as a white precipitate, the reaction formula occurs as follows:
(DNA)n-1+dNTP→(DNA)n+P2O7 4-
P2O7 4-+2Mg2+-→Mg2P2O7↓。
According to the reaction principle, the real-time turbidimeter measures the turbidity of the reaction tube every 6 seconds and draws a curve to judge whether the reaction is positive or negative. Forming a reaction curve-S-shaped broken line, and then the reaction curve is positive; no obvious reaction curve is formed, and no S-shaped broken line is formed, the reaction is negative.
The fluorescence real-time detection method comprises the following steps: setting the absorption wavelength to be about 497nm and the maximum emission wavelength to be about 520nm, wherein the wavelength is the same as SYBR Green I and is Green fluorescence; the reaction system was placed in a fluorescence quantitative instrument and the reaction was started.
The positive reaction appeared as: an S-shaped amplification curve appears in the reaction stage, and the dissolution curve is a single peak;
The appearance of negative reactions was: there was no amplification curve during the reaction phase and no peak in the dissolution curve.
and (4) result judgment standard: under the premise that the positive control has a positive reaction and the negative control has a negative reaction, the sample has an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the position of the positive control single peak (the Tm change range is within 1-3 ℃), and the sample can be judged to be positive. Otherwise, the result is negative. If a negative reaction occurs in the positive sample or a positive reaction occurs in the negative sample, the experiment should be repeated.
The principle of the fluorescence real-time detection method is as follows: the nucleic acid fluorescent dye can be embedded into a DNA double strand, and can emit fluorescence under the excitation of fluorescence with a specific excitation wavelength so as to be detected by an instrument.
further, the working system of the kit is as follows: mu.L of the second reagent at pH8.0, 1. mu.L of the first reagent, 9. mu.L of the DNA template, and 25. mu.L of purified water were filled.
Further, the solvent of the first reagent is water and/or a vitrification liquid. (all solvents not specifically mentioned in the present invention are water).
The vitrification liquid is selected from any 2 or more of dimethyl sulfoxide, formamide, Ethylene Glycol (EG), propylene glycol, glycerol and other reagents; or selected from EFS30 glass liquid, EFS40 glass liquid, EDFS30 glass liquid, EDFS40 glass liquid, EDFSF30 glass liquid or EDFSF40 glass liquid.
the EFS30 vitrification solution contains 30% v/vEG and 70% v/vFS solution;
The EFS40 vitrification solution contains 40% v/vEG and 60% v/vFS solution;
The EDFS30 vitrification solution contains 15% v/vEG, 15% v/vDMSO and 70% v/vFS solution;
The EDFS40 vitrification solution contains 20% v/vEG, 20% v/vDMSO and 60% v/vFS solution;
FS solution: 0.5mol/L sucrose was added to 300g/L ficoll, and dissolved in PBS buffer to prepare an FS solution.
The EDFSF30 vitrification solution contains 15% v/vEG, 15% v/vDMSO and 70% v/vFSF solution;
The EDFSF40 vitrification solution contains 20% v/vEG, 20% v/vDMSO and 60% v/vFSF solution;
FSF solution: 0.5mol/L sucrose is added into 300g/L polysucrose, and after being dissolved by PBS buffer solution, 20% fetal calf serum is added to prepare FSF solution.
Further, the reagent I is placed in a placing groove on the detection tube cover and then dried or baked.
According to the technical scheme, compared with the prior art, the kit for rapidly detecting the pseudomonas aeruginosa by using the double indicators provided by the invention has the following technical advantages:
the invention provides a kit for rapidly detecting pseudomonas aeruginosa, which adopts a unique buffer system, has the advantage of stronger amplification compatibility, can carry out efficient and specific amplification without thoroughly removing impurities such as protein and the like, has the characteristics of sensitivity, rapidness, simplicity, convenience and real-time detection of the pseudomonas aeruginosa, is suitable for the field quarantine inspection detection requirements under the background of mass flow and rapid clearance, and is also suitable for rapid detection in a laboratory. Part of the reagent can be stored and transported at room temperature after vitrification, and does not depend on cold chain transportation and low-temperature storage.
drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a unitary packaged diagnostic reagent bottle according to the present invention;
FIG. 2 is a schematic diagram showing the composition of a primer pair, a main primer (composite primer) and an accelerated primer, according to the present invention;
FIG. 3 is a graph showing the effect of the accelerated primers of the present invention on the amplification efficiency of PSR;
Wherein curve 1 is obtained using both IF and IB; 2 is only IF; 3 is IB alone; 4 is no accelerating primer;
FIG. 4 is a graph showing the effect of betaine concentration on assay;
1-7 are respectively the working concentration of 0.5-1.1M betaine in the reaction system;
FIG. 5 is a graph illustrating the effect of the addition of a color indicator on nephelometric detection provided by the present invention;
Wherein the positive group takes pseudomonas aeruginosa DNA as a template, and the negative control group takes pure water as a template; 1 is the positive group without added indicator; 2 is the positive group for adding indicator; 3 is a negative control group without the addition of an indicator; 4 is a negative control group added with a pH indicator;
FIG. 6 is a diagram showing the results of the chromogenic assay for the added indicator according to the present invention;
wherein the positive group takes pseudomonas aeruginosa DNA as a template, and the negative control group takes pure water as a template; 1 is the positive group without added indicator; 2 is the positive group for adding indicator; 3 is a negative control group without the addition of an indicator; 4 is a negative control group added with a pH indicator;
FIG. 7 is a graph showing the effect of different pH samples on chromogenic detection according to the present invention;
Wherein 1 is pseudomonas aeruginosa liquid; 2-5: is the pseudomonas aeruginosa liquid with the pH values of 4, 6, 8 and 10 respectively; 6, Chelex lysate replaces pseudomonas aeruginosa lysate;
FIG. 8 is a diagram showing the results of a color development test for the detection kit according to the present invention;
FIG. 9 is a diagram showing the result of an experiment for detecting the applicability of a sample by a fluorescence method using the detection kit according to the present invention;
The red curve is a positive group, and the purple curve is a negative group;
FIG. 10 is a diagram showing the results of a fluorescence-specific assay performed by the detection kit according to the present invention;
A and B, positive group, the pseudomonas aeruginosa bacterial liquid is detected to be in S-shaped curve, and the result is positive;
C.D.E.F.G, which are respectively other bacteria detection (escherichia coli, salmonella, shigella, listeria monocytogenes and staphylococcus aureus), no amplification curve exists, and the result is negative;
H, negative control group, water detection has no amplification curve, and the result is negative.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
example 1
primer design
According to the gene Sequence of pseudomonas aeruginosa toxA disclosed by NCBI (Reference Sequence: NC-002516.2), based on the principle of strand displacement activity of enzyme, a specific primer pair for rapidly detecting pseudomonas aeruginosa is designed, and the nucleotide Sequence of the primer pair is as follows:
Ft：5’-gcatcgtcttcggcggggtGGAGCGCGGCTATGTGT-3’；SEQ ID NO.1；
Bt：5’-gcatcgtcttcggcggggtTCGGGTTCCTGGTCCTG-3’；SEQ ID NO.2。
In this example, Chelex-100, betaine, Sigma; manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, ammonium sulfate, national drug group chemical reagents, Inc.; Tris-HCl, Shanghai Biotech limited; TritonX-100, Beijing Meilaibo medical science and technology, Inc.; dNTP, Pharmacia company; NP-40, Fluka corporation; 2 × TapMIX kit, tiangen biochemistry technology (beijing) limited; agarose, Amresco inc.
the reagent in the kit is stored by using a unitary packaged diagnostic reagent bottle, as shown in figure 1, wherein the reagent bottle is provided with a detection tube 1, a detection tube cover 3, a placing groove 4 and a covering film 2; wherein, the detection tube 1 is a hollow reaction tube with one end open and the other end closed; the detection tube cover 3 is matched with the detection tube 1, covers the opening end of the detection tube 1 and closes the detection tube 1, wherein the detection tube 1 and the detection tube cover 3 are integrally formed; a placing groove 4 is formed in the inner side of the detection tube cover 3, and the placing groove 4 and the detection tube cover 3 are integrally formed; the cover film 2 is a PPE film and covers the placement groove 4.
Wherein, 1 microliter of the reagent of the invention is placed in a placing groove 4 on a detection tube cover 3, then the placing groove 4 is sealed by a covering film 2, 12.5 microliter of the reagent without heat-sensitive components is placed at the bottom of the detection tube 1, 2.5 microliter of pure water and one drop of sealing agent paraffin are added, and finally the detection tube cover 3 is covered to finish the assembly. Wherein reagent one comprises 10U of Bst DNA polymerase without glycerol, 50 μ M of Ft, Bt primer, 10mM dNTP and indicator; wherein the second reagent is 12.5 mul of a reaction system comprising (NH)4)2SO420 mM；KCl 100mM；MgSO416 mM; 200.2% of Tween; betaine 1.6M; finally, the pH was adjusted to 8.0 with 1% KOH solution.
And preparing a reaction detection system by using the Ft and Bt primer pair. Two positive reaction amplification products (9 μ l) are respectively taken, 9 μ l of the positive reaction amplification product is dripped on the reagent I, the detection tube cover 3 is buckled, and the reaction tube is inverted for about 10 seconds. A negative control (double distilled water) was set. Respectively picking up the test tubes 1 of the experimental group and the negative control group, and forcibly throwing the liquid on the test tube cover 3 into the tube bottom. The detection tube is placed in a real-time turbidimeter to react for 60min at 65 ℃, and meanwhile, the DNA concentration is measured by using the NanoDrop 1000, and the DNA concentration reaches 200 ng/mul.
in this example, the DNA was purified using a general PCR product purification kit from Tiangen Biochemical technology (Beijing) Ltd, the specific steps are described in the specification, and finally, the DNA was eluted with 50. mu.l of deionized water, and the DNA concentration was measured using NanoDrop 1000, which was 165 ng/. mu.l.
the amplification product used for purification was 20. mu.l, the final eluate was 50. mu.l, and the recovery loss rate was about 1.1-1.2, so the actual amplification concentration of PSR was (165+200)/2 × 50/20 × (1.1-1.2) — 500-.
Example 2
accelerated primer introduction
IF only two primers are adopted, the peak-off time is generally 50min later, the requirements of rapid and sensitive field detection cannot be met, the introduced accelerating primers comprise IF and IB, the IF and the IB are respectively arranged between F, N, B and N, the directions from the 5 'end to the 3' end are respectively opposite to Ft and Bt, and the schematic position diagram of the IF and the IB on a target sequence is shown in FIG. 2.
in this example, the accelerating primer is IF: 5'-AAGGTGCCGTGGTAGCC-3', respectively; SEQ ID No. 3;
IB：5’-TCTATATCGCCGGCGATCC-3’；SEQ ID NO.4。
The reagent in the kit is stored by using a unitary packaged diagnostic reagent bottle, wherein the reagent bottle is provided with a detection tube 1, a detection tube cover 3, a placing groove 4 and a coating film 2; wherein, the detection tube 1 is a hollow reaction tube with one end open and the other end closed; the detection tube cover 3 is matched with the detection tube 1, covers the opening end of the detection tube 1 and closes the detection tube 1, wherein the detection tube 1 and the detection tube cover 3 are integrally formed; a placing groove 4 is formed in the inner side of the detection tube cover 3, and the placing groove 4 and the detection tube cover 3 are integrally formed; the cover film 2 is a PPE film and covers the placement groove 4.
wherein, 1 mul of the reagent of the invention is arranged in a placing groove 4 on a detecting tube cover 3, then the placing groove 4 is sealed by a covering film 2, a reagent II without heat-sensitive components is placed at the bottom of the detecting tube 1, then 2.5 mul of pure water and a sealing agent paraffin one drop are added, and finally the detecting tube cover 3 is covered to finish the assembly. Wherein reagent one contains 10U of BstDNA polymerase without glycerol, 50 muft and Bt primers for M, IF and IB primers for 50. mu.M, 10mM dNTP and an indicator; wherein the second reagent is 12.5 mul of a reaction system comprising (NH)4)2SO420mM；KCl 100mM；MgSO416 mM; twen200.2%; betaine 1.6M; finally, the pH was adjusted to 8.0 with 1% KOH solution.
And preparing a reaction detection system by using the primer pair of Ft, Bt, IF and IB, wherein 9 mu l of positive reaction amplification product is taken and dripped on the reagent I by 9 mu l of positive reaction amplification product. The test tube cap 3 is closed and inverted for about 10 seconds. Control groups were set without IF, IB and accelerating primers. Respectively picking up each group of the detection tubes 1, and forcibly throwing the liquid on the detection tube cover 3 into the tube bottom. The detection tube is placed in a real-time turbidimeter to react for 60min at 65 ℃, and the detection result is shown in figure 3. As can be seen from FIG. 3, when two accelerating primers IF and IB are used in the reaction system at the same time, the reaction rate is greatly increased, the Ct value is advanced to within 20min, and the effect is not as obvious as that of the two accelerating primers when only one accelerating primer is used, but the reaction rate is also increased.
Example 3
The betaine concentration was verified on the basis of example 2.
Seven betaine concentrations (working concentrations) of 0.5M to 1.1M were set at an interval of 0.1, and the effect of different betaine concentrations on the Ct value of the reaction was examined.
The results of the turbidity assay are shown in FIG. 4, where curve No.3 (0.7M) and curve No. 4 (0.8M) showed the fastest peak rate, but the peak of the betaine concentration at 0.8M was slightly higher than 0.7M, and 0.8M was finally used as the optimum concentration.
Example 4
in this example, the indicator is a cresol red-phenol red indicator, including cresol red at a final concentration of 0.08mM and phenol red at a final concentration of 0.02 mM. Wherein the indication characteristics of phenol red and cresol red are shown in Table 1, the color change range meets the reduction of pH value from 8.8 to 6.0-6.5 before and after PSR reaction, and the pH change ranges of phenol red and cresol red are slightly different.
TABLE 1 pH indicating characteristics of phenol Red and cresol Red
The first reagent 1 microliter of the invention is placed in a placing groove 4 on a detection tube cover 3, then the placing groove 4 is sealed by a covering film 2, the second reagent without heat-sensitive components is placed at the bottom of the detection tube 1, then 2.5 microliter of pure water and a sealant paraffin one drop are added, and finally the detection tube cover 3 is covered to finish the assembly. Wherein reagent one comprises 10U of Bst DNA polymerase without glycerol, 50 μ M of Ft and Bt primers, 50 μ M of IF and IB primers, 10mM dNTP and indicator; wherein the second reagent is 12.5 mul of a reaction system comprising (NH)4)2SO420mM；KCl 100mM；MgSO416 mM; 200.2% of Tween; betaine 1.6M; finally, the pH was adjusted to 8.0 with 1% KOH solution. A control group without indicator was also set.
the positive reaction amplification products were subjected to chromogenic and nephelometric detection using the method of example 2 and the system described above in this example, respectively, and negative controls were set, with the results shown in FIGS. 5-6.
As can be seen from FIGS. 5 to 6, the positive reaction turned yellow because a large amount of hydrogen ions were generated in the solution due to the PSR reaction, and the pH was lowered, while the pH of the negative control was unchanged, and the solution was red. After the cresol red-phenol red indicator is added, the Ct value of the PSR reaction is not obviously influenced, which indicates that the pH indicator can not inhibit the PSR reaction, and the negative control is the same as the conventional PSR and has no false positive phenomenon.
Example 5
5.0g of Chelex-100(Bio-rad) and 1.0ml of TritonX-100 were dissolved in 100ml of TE buffer (Tris10mM, EDTA 1mM) to prepare a Chelex lysate.
Taking culture solution of pseudomonas aeruginosa, adjusting bacteria solution to different pH values with dilute hydrochloric acid and KOH solution, wherein the pH values are 4, 6, 8 and 10 respectively, and taking 200 culture solutions with different pH values respectivelyμlPlacing in a test tube containing 2mL of Chelex lysate, shaking, mixing uniformly, placing in a constant temperature amplification detector, and heating at 90 ℃ for 10min to obtain the lysate.
and respectively taking 9 mu l of lysate, dropwise adding the lysate onto the reagent I, mixing the mixture with the reagent II, pure water and the sealant, placing the mixture into a detector, reacting at 65 ℃ for 40min, taking out the mixture, and observing color change, wherein the result is shown in figure 7. The components of the reagent I, the reagent II, the pure water and the sealant are used in the same amount as in example 4.
It can be seen from fig. 7 that the color development effect of samples with different pH values after lysis and loading are consistent, which indicates that when cresol red-phenol red is used as the color development agent, the color development agent is not affected by the pH value of the sample, because the pH of the total system does not change significantly when the sample is added into the lysis solution in a ratio of 1: 10.
Example 6
The reagent in the kit is stored by using a unitary packaged diagnostic reagent bottle, wherein the reagent bottle is provided with a detection tube 1, a detection tube cover 3, a placing groove 4 and a coating film 2; wherein, the detection tube 1 is a hollow reaction tube with one end open and the other end closed; the detection tube cover 3 is matched with the detection tube 1, covers the opening end of the detection tube 1 and closes the detection tube 1, wherein the detection tube 1 and the detection tube cover 3 are integrally formed; a placing groove 4 is formed in the inner side of the detection tube cover 3, and the placing groove 4 and the detection tube cover 3 are integrally formed; the cover film 2 is a PPE film and covers the placement groove 4.
The method comprises the steps of placing 1 mu L of a first reagent in a placing groove 4 on a detection tube cover 3, sealing the placing groove 4 by using a covering film 2 after airing, placing a second reagent without heat-sensitive components at the bottom of the detection tube 1, adding 2.5 mu L of pure water and one drop of sealing agent paraffin, and finally covering the detection tube cover 3 to finish assembly. Wherein the first reagent comprises 10U of BstDNA polymerase without glycerol, 50 mu M of each of Ft and Bt primers, 50 mu M of each of IF and IB primers, 10mM dNTP and an indicator, and the solvent is EFS30 vitrification solution; wherein the second reagent is 12.5 mul of a reaction system comprising (NH)4)2SO420mM；KCl 100mM；MgSO416 mM; 200.2% of Tween; betaine 1.6M; finally, the pH was adjusted to 8.0 with 1% KOH solution. The indicators were cresol red-phenol red indicator (0.08mM cresol red +0.02mM phenol red) and SYBR GreenI (working concentration) in 1 × aqueous solution.
The detection sample mainly comprises sputum, feces, urine, blood, saliva and the like, wherein the blood and the feces have complex components and colors. In the embodiment, the feces, urine and fingertip blood of healthy volunteers are dipped by sterile cotton swabs, wherein the positive groups are feces + pseudomonas aeruginosa, urine + pseudomonas aeruginosa and blood + pseudomonas aeruginosa respectively. And (3) respectively dropwise adding 1mL of a lysis solution into 100 mu L of each group of detection samples, and heating at 90 ℃ for 10min to obtain a sample lysis solution. And respectively taking 9 mu l of sample lysate to the first reagent on the detection tube cover, standing for 1min, mixing with the second reagent, pure water and the sealing agent uniformly, putting into a detector, reacting at 65 ℃ for 40min, taking out, and observing color change, wherein the result is shown in figure 8. The color of the negative group of the feces and the urine is consistent to be red, the positive group of the feces turns yellow, the positive group of the feces turns slightly red, and impurities in the feces have certain influence on the reaction, but the contrast of the positive and negative is not influenced. After the blood is added, the color of the negative group is still consistent with that of the pure lysate group, the positive group turns yellow, and the negative and positive groups are distinguished obviously, which indicates that the detector can be suitable for the detection of complex samples.
FIG. 9 shows the result of a fluorescence-based sample suitability test in which Pseudomonas aeruginosa is added to urine and feces and detected by a kit, wherein the S-shaped curve of the fluorescence method is positive; urine and feces without the addition of positive control bacteria, negative control, no amplification curve, and negative.
Example 7
the specificity of the kit fluorescence detection in example 6 was verified.
The positive sample is pseudomonas aeruginosa, the negative sample is escherichia coli, salmonella, shigella, listeria monocytogenes and staphylococcus aureus, and water is used as a negative control. The results are shown in table 2 and fig. 10.
TABLE 2 detection results of PSR amplification fluorescence method
| Numbering | Curve line | Sample (I) | S-shaped curve | CT value | Determination of results |
| 1 | A | Pseudomonas aeruginosa 1 | Is provided with | 7.02 | positive for |
| 2 | B | Pseudomonas aeruginosa 2 | Is provided with | 6.84 | positive for |
| 3 | C | escherichia coli | Is free of | / | Negative of |
| 4 | D | Salmonella | Is free of | / | negative of |
| 5 | E | Shigella | Is free of | / | Negative of |
| 6 | F | listeria monocytogenes | Is free of | / | Negative of |
| 7 | G | Staphylococcus aureus | Is free of | / | negative of |
| 8 | H | Negative control | Is free of | / | Negative of |
The invention provides a kit for rapidly detecting pseudomonas aeruginosa, which adopts a unique buffer system, has the advantage of stronger amplification compatibility, can carry out efficient and specific amplification without thoroughly removing impurities such as protein and the like, has the characteristics of sensitivity, rapidness, simplicity, convenience and real-time detection of the pseudomonas aeruginosa, is suitable for the field monitoring of the border ports, the field quarantine inspection, examination and detection requirements under the background of mass flow and rapid clearance, and is also suitable for rapid detection in laboratories.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
the previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> scientific research institute of Chinese inspection and quarantine
<120> kit for rapidly detecting pseudomonas aeruginosa
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial
<400> 1
gcatcgtctt cggcggggtg gagcgcggct atgtgt 36
<210> 2
<211> 36
<212> DNA
<213> Artificial
<400> 2
gcatcgtctt cggcggggtt cgggttcctg gtcctg 36
<210> 3
<211> 17
<212> DNA
<213> Artificial
<400> 3
aaggtgccgt ggtagcc 17
<210> 4
<211> 19
<212> DNA
<213> Artificial
<400> 4
tctatatcgc cggcgatcc 19
Claims (7)
1. A specific primer pair for rapidly detecting pseudomonas aeruginosa is characterized in that the nucleotide sequence of the primer pair is as follows:
Ft：5’-gcatcgtcttcggcggggtGGAGCGCGGCTATGTGT-3’；SEQ ID NO.1；
Bt：5’-gcatcgtcttcggcggggtTCGGGTTCCTGGTCCTG-3’；SEQ ID NO.2；
IF：5’-AAGGTGCCGTGGTAGCC-3’；SEQ ID NO.3；
IB：5’-TCTATATCGCCGGCGATCC-3’；SEQ ID NO.4。
2. A kit comprising the specific primer set according to claim 1.
3. The kit of claim 2, comprising a first reagent, a second reagent, and a blocking agent; wherein the first reagent comprises Bst DNA polymerase, Ft and Bt primers, IF and IB primers, dNTP and an indicator;
reagent two comprises (NH)4)2SO4、KCl、MgSO4Tween20 and betaine, pH 8.0.
4. The kit of claim 3, wherein reagent one comprises 10U Bst DNA polymerase, 50 μ M each of Ft and Bt primers, 50 μ M each of IF and IB primers, 10mM dNTP and indicator;
Reagent two contains 20mM (NH)4)2SO4、100mMKCl、16mM MgSO40.2% Tween20 and 1.6M betaine, pH 8.0.
5. The kit of claim 3, wherein the indicator system comprises a pH indicator and/or a fluorescent dye; the pH indicator is a cresol red-phenol red indicator; the indicator comprises cresol red at a final concentration of 0.08mM and phenol red at a final concentration of 0.02 mM; the fluorescent dye is SYBR Green I or EveGreen in 1 x aqueous solution.
6. The method of using the kit according to claim 3, comprising the steps of:
(1) Heating and cracking a sample to be detected by using a cracking solution to release DNA;
(2) Adding the sample solution cracked in the step (1) into the reagent I of the kit, mixing with the reagent II, and adding a confining liquid to prevent pollution; setting positive and negative controls;
(3) Amplifying at constant temperature of 63-67 deg.C for 30-60min, and observing the judgment result by color development method or detecting by fluorescence method or turbidity method.
7. The use method of the kit according to claim 6, wherein the kit is a 25 μ L working system comprising: mu.L of the second reagent at pH8.0, 1. mu.L of the first reagent, 9. mu.L of the DNA template, and 25. mu.L of purified water were filled.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910960360.9A CN110578012A (en) | 2019-10-10 | 2019-10-10 | Kit for rapidly detecting pseudomonas aeruginosa |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910960360.9A CN110578012A (en) | 2019-10-10 | 2019-10-10 | Kit for rapidly detecting pseudomonas aeruginosa |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110578012A true CN110578012A (en) | 2019-12-17 |
Family
ID=68814398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910960360.9A Pending CN110578012A (en) | 2019-10-10 | 2019-10-10 | Kit for rapidly detecting pseudomonas aeruginosa |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110578012A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337965A (en) * | 2018-09-04 | 2019-02-15 | 江苏中济万泰生物医药有限公司 | A kind of fluorescent dye and electrophoresis PCR double-purpose buffer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219874A (en) * | 2015-11-04 | 2016-01-06 | 中国人民解放军疾病预防控制所 | The PSR detection method of Pseudomonas aeruginosa and primer special thereof and test kit |
| CN109355407A (en) * | 2018-09-26 | 2019-02-19 | 华南理工大学 | A kind of primer, kit and its method of PSR isothermal amplification detection pseudomonas aeruginosa |
-
2019
- 2019-10-10 CN CN201910960360.9A patent/CN110578012A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219874A (en) * | 2015-11-04 | 2016-01-06 | 中国人民解放军疾病预防控制所 | The PSR detection method of Pseudomonas aeruginosa and primer special thereof and test kit |
| CN109355407A (en) * | 2018-09-26 | 2019-02-19 | 华南理工大学 | A kind of primer, kit and its method of PSR isothermal amplification detection pseudomonas aeruginosa |
Non-Patent Citations (1)
| Title |
|---|
| 董德荣: "一种新型核酸恒温扩增方法的研究及其在现场检测中的应用", 《万方学位论文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337965A (en) * | 2018-09-04 | 2019-02-15 | 江苏中济万泰生物医药有限公司 | A kind of fluorescent dye and electrophoresis PCR double-purpose buffer |
| CN109337965B (en) * | 2018-09-04 | 2022-01-25 | 江苏中济万泰生物医药有限公司 | Fluorescent dye and electrophoresis PCR (polymerase chain reaction) dual-purpose buffer solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2430188B1 (en) | A method and kit for detecting antibiotic resistant bacteria | |
| CN113322338B (en) | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit | |
| CN106916906B (en) | Primer composition for detecting infectious diarrhea pathogens and kit thereof | |
| CN111286559B (en) | Primer, probe and kit for detecting African swine fever virus | |
| CN104946749A (en) | Universal primers and probe for on-site rapid detection of Brucella and kit | |
| CN104263838B (en) | Listeria monocytogenes LAMP-LFD detection kit and detection method thereof | |
| WO2021179469A1 (en) | Composition for detecting pathogens, and kit and method therefor | |
| CN110564879A (en) | Kit for rapidly detecting vibrio cholerae | |
| CN113637781B (en) | LAMP primer group for detecting swine susceptibility related pathogenic bacteria, kit and LAMP chip based on LAMP primer group and application | |
| CN110157837B (en) | Primer and method for detecting peste des petits ruminants virus and bluetongue virus | |
| CN110578012A (en) | Kit for rapidly detecting pseudomonas aeruginosa | |
| CN111020042B (en) | Compositions and methods for detecting group A streptococci | |
| CN115873979A (en) | Constant-temperature nucleic acid amplification RAA primer probe combination for detecting candida auricula and application thereof | |
| CN110029179B (en) | Nucleotide molecules and application thereof in identification of corynebacterium striatum | |
| CN109628621B (en) | Real-time quantitative LAMP primer group and kit for detecting Klebsiella pneumoniae | |
| CN107557456B (en) | LAMP (loop-mediated isothermal amplification) detection primer group and kit for ureaplasma urealyticum | |
| CN112195257A (en) | Primer group, reagent, kit and detection method for detecting vibrio parahaemolyticus | |
| CN113512598A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis, and special primer and probe thereof | |
| CN112063759A (en) | RT-LAMP primer, kit and detection method for simultaneously detecting multiple viruses of banana | |
| CN112063734A (en) | Primer, probe and method for quantitatively detecting brucella in human blood by adopting real-time fluorescent quantitative PCR technology | |
| CN111424073A (en) | Closed-tube nucleic acid amplification detection method and device | |
| CN110923341B (en) | LAMP (loop-mediated isothermal amplification) detection method for Shewanella alga pathogenic bacteria of cultured fishes and application | |
| CN114214441B (en) | CDA primer group and kit for detecting Listeria and application of CDA primer group and kit | |
| CN114427005A (en) | CDA primer group and kit for detecting candida albicans and application of CDA primer group and kit | |
| CN116287478B (en) | Primer probe composition and kit for detecting multiple respiratory pathogens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |