CN110607354A - Rapid detection method of pathogenic vibrio cholerae - Google Patents
Rapid detection method of pathogenic vibrio cholerae Download PDFInfo
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- CN110607354A CN110607354A CN201911055118.3A CN201911055118A CN110607354A CN 110607354 A CN110607354 A CN 110607354A CN 201911055118 A CN201911055118 A CN 201911055118A CN 110607354 A CN110607354 A CN 110607354A
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Classifications
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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
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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
- C12Q1/6876—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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a method for quickly detecting pathogenic vibrio cholerae. The invention firstly provides a primer and a probe for detecting pathogenic vibrio cholerae. The primers and probes have high sensitivity and specificity. The invention also provides a method for rapidly detecting pathogenic vibrio cholerae, which adopts specific primers and probes and utilizes recombinase-mediated isothermal nucleic acid amplification combined with a lateral chromatography technology to realize rapid, sensitive and specific detection of the pathogenic vibrio cholerae, has simple and convenient operation and short time consumption, and solves the problems of long time consumption, complicated steps and the like of the traditional detection technology. The technology of the invention does not need special instruments and equipment, reduces the detection cost, enlarges the application range of the technology, can be applied to special environments such as field sites and the like, and has great significance for the early detection and diagnosis of vibrio cholerae.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a rapid detection method of pathogenic vibrio cholerae.
Background
Vibrio cholerae belongs to the family Vibrionaceae, gram-negative bacteria. O1 and O139 are the two most prominent pathogenic serotypes of Vibrio cholerae and are closely related to the large-scale outbreak of cholera, and the major virulence factors of Vibrio cholerae include Cholera Toxin (CT) encoded by the ctxAB gene and virulence coordinated regulatory pili (TCP) encoded by the tcpA gene. People are infected by ingesting water or food contaminated with vibrio cholerae, resulting in acute watery diarrhea. Despite the current improvements in water quality, hygiene and clinical treatment of cholera, the disease still causes about 10 million deaths worldwide each year. One analysis showed that in countries with an epidemic of cholera, approximately 2900 ten thousand cases and 95000 cases of death occur each year, with africa accounting for 60% and 68%, respectively, and almost all deaths occurring in developing countries.
The disclosed patent technology for detecting vibrio cholerae comprises a multiplex fluorescence PCR detection kit (CN 105603091B), wherein a PCR primer is designed aiming at the hemolysin gene of the vibrio cholerae, and the detection limit is 103copy/ml, high specificity, but has the problems of needing special instruments and equipment, needing professional personnel, consuming long time and the like. The loop-mediated isothermal amplification is combined with a lateral chromatography technology (CN 106957917A) to realize 100The detection of the copied Vibrio cholerae target gene has high specificity and sensitivity, but the primer design and screening are complex. The colloidal gold method detection kit (CN 106290842A) adopts a test strip which takes colloidal gold as a marker to realize the specific detection of the vibrio cholerae O139 serotype, but has lower sensitivity and mainly takes qualitative and semi-quantitative detection as main points.
The standard of the domestic entry and exit industry adopts a microbiological method for identification and detection, and separation identification is carried out through enrichment, separation culture, biochemical reaction, hemolytic test and the like. The method has long inspection period and complicated steps, and cannot meet the requirement of rapid detection. Therefore, it is necessary to research a simple and rapid vibrio cholerae detection method, so that pathogenic bacteria can be detected at the first time, measures can be taken to remove and reduce the risk of the consumer eating products polluted by vibrio cholerae in time, firm guarantee is provided for the front-line food safety, and technical support is provided for detection departments.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for rapidly detecting pathogenic vibrio cholerae, which adopts recombinase-mediated isothermal nucleic acid amplification combined with a lateral chromatography technology to rapidly detect the pathogenic vibrio cholerae, thereby making up the defects of the prior art.
The invention firstly provides a primer for detecting pathogenic vibrio cholerae, and the sequence information of the primer is as follows:
forward primer F-RAA: 5'-AGTCAGGTGGTCTTATGCCAAGAGGACAGAG-3', respectively;
reverse primer R-RAA: 5'-TGGAAACATATCCATCATCGTGCCTAACAAA-3', respectively;
wherein, Biotin Biotin labeling is carried out at the 5' end of the reverse primer R.
The invention also provides a probe for detecting pathogenic vibrio cholerae, and the sequence information of the probe is as follows:
and (3) probe P: 5'-GTCAGGTGGTCTTATGCCAAGAGGACAGAGGAGACTTTGACCGAGGTA-3'
A30 th base from the 5 'end of the probe P is labeled with an FITC (FITC fluorescent group), Tetrahydrofuran (THF) is inserted behind the 30 th base to serve as a abasic site, a fluorescence quenching group BHQ is inserted behind the THF, and the 3' end of the probe P is labeled with C3-Spacer. The modified probe information is as follows:
and (3) probe P: 5 '-GTCAGGTGGTCTTATGCCAAGAGGACAGAG (FITC-dt) G (THF) AG (BHQ-dt) ACTTTGACCGAGGTA (C3-spacer) -3'.
The primer and the probe are used for detecting pathogenic vibrio cholerae.
The invention also provides a method for rapidly detecting pathogenic vibrio cholerae, which specifically comprises the following steps:
RAA amplification
Preparing a DNA solution to be detected, preparing a reaction system according to a commercially available RAA kit (test strip method), wherein the reaction system comprises the primer and the probe for detecting the pathogenic vibrio cholerae, and carrying out amplification reaction on the prepared basic reaction unit at 39-42 ℃ for 25-30 minutes. The RAA kit is preferably a kit manufactured by Jiangsu Qitian gene biotechnology limited, and the model is T00001A.
b. Detection of RAA amplification products using lateral chromatography test strips
Adding 8-10 mu L of nucleic acid amplification product into buffer solution, uniformly mixing with 3-5 mu L of magnetic bead-streptavidin conjugate and anti-FITC rabbit antibody with the same volume as the nucleic acid amplification product, and then dropwise adding into a sample pad area of a lateral chromatography test strip.
Preferably, the concentration of the anti-FITC rabbit antibody in the step (b) is 0.02-0.04 mg/L.
Preferably, the lateral chromatography test strip comprises a sample pad, a binding pad, an NC membrane, a back plate and a labeling pad which are sequentially arranged from a sample adding end, wherein a T line and a C line are coated on the NC membrane, a goat anti-rabbit secondary antibody capable of being bound with an anti-FITC antibody is coated on the T line, biotin-BSA bound with magnetic bead-streptavidin is coated on the C line, and the T line and the C line form an interpretation zone.
Preferably, the preparation method of the magnetic bead-streptavidin conjugate comprises the following steps:
(1) activating magnetic beads: washing the magnetic beads in a centrifugal tube for 2-5 times by using MEST buffer solution, adding an activating agent into the centrifugal tube, uniformly mixing, and carrying out a rotary reaction for 25-35 min;
(2) coupling antibody: after activation of the magnetic beads is finished, washing the magnetic beads for 1-3 times by using MEST buffer solution, then washing the magnetic beads by using BST buffer solution, sequentially adding streptavidin and BST buffer solution after washing, uniformly mixing, performing rotary reaction for 2-4h, placing the magnetic beads on a magnetic frame for 4-6min after coupling is finished, and removing supernatant to obtain the magnetic beads after coupling the antibody;
(3) sealing the immunomagnetic beads: washing the magnetic beads coupled with the antibody with BST buffer solution, adding confining liquid after washing, fully and uniformly mixing, and reacting for 25-35 min;
(4) and (3) storage: and washing the blocked immunomagnetic beads with BST buffer solution to obtain magnetic bead-streptavidin conjugate, storing the magnetic bead-streptavidin conjugate in a storage solution, and storing the magnetic bead-streptavidin conjugate at 3-5 ℃ for later use.
Preferably, the activator comprises the following components in percentage by weight: 97% MEST buffer, 1% l0.5 g/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 2% 0.25g/mL N-hydroxysuccinimide (NHS).
Preferably, the solid-to-liquid ratio of the magnetic beads to the activator in step (1) is 1 mg: and (15-20) mu L.
Preferably, the washing of the magnetic beads with the MEST buffer in the step (1) or (2) comprises the following steps: and (3) mixing the magnetic beads and the MEST buffer solution according to the solid-to-liquid ratio of 1 mg: (400) mu L of the mixture is uniformly mixed, the mixture is placed into a magnetic rack for standing, and when the magnetic beads are completely adsorbed on the wall of the centrifugal tube and the solution is clarified, the supernatant is discarded, namely the washing is carried out once.
Preferably, the volume ratio of the streptavidin to the BST buffer in the step (2) is (1-5): (1-5), wherein the solid-to-liquid ratio of the magnetic beads, the streptavidin and the total volume of the BST buffer solution is 1: (400-600) μ L.
Preferably, the blocking solution is obtained by dissolving BSA in BST buffer solution, wherein the mass fraction of the BSA is 0.8-1.2%.
The solid-to-liquid ratio of the magnetic beads to the confining liquid is 1 mg: (400-600) μ L.
The solid-to-liquid ratio of the magnetic beads to the preservation solution is 1 mg: (400-600) μ L.
Preferably, the preservation solution is NaN3And BSA in BST buffer, wherein NaN is3And BSA in the amount of 0.8-1.2% by mass.
The diameter of the magnetic bead is 150-200 nm.
The invention also provides a lateral chromatography test strip for detecting pathogenic vibrio cholerae, which comprises a sample pad, a binding pad, an NC membrane, a back plate and a marking pad which are sequentially arranged from a sample adding end, wherein the NC membrane is coated with a T line and a C line, the T line is coated with a goat anti-rabbit secondary antibody capable of being combined with an anti-FITC antibody, the C line is coated with biotin-BSA combined with magnetic bead-streptavidin, and the T line and the C line form an interpretation area.
The invention also provides a kit for rapidly detecting the pathogenic vibrio cholerae, which comprises the primer and the probe for detecting the pathogenic vibrio cholerae.
The technical solution of the present invention is further explained and illustrated below: the recombinase-mediated isothermal nucleic acid amplification kit can amplify DNA of vibrio cholerae at 39-42 ℃, and a reverse primer and a probe are respectively marked with a biotin group and a FITC group during amplification, so that a DNA amplification product simultaneously contains the biotin group and the FITC group, and the structural schematic diagram of the product is shown in FIG. 2. The amplified product is incubated with an anti-FITC rabbit antibody and a magnetic bead-streptavidin conjugate, after full reaction, a biotin group and a FITC group on the product can be respectively combined with the anti-FITC rabbit antibody and the magnetic bead-streptavidin conjugate to form a nucleic acid antibody complex, the nucleic acid antibody complex is dripped on a sample pad of a lateral chromatography test strip, the nucleic acid antibody complex is chromatographed on a nitrocellulose membrane under the action of chromatography, and the complex can be combined with a goat anti-rabbit secondary antibody on the detection line due to the anti-FITC rabbit antibody when passing through the detection line, and the detection line is brown due to the colored complex carrying the marker (magnetic nanoparticles are brown); when the nucleic acid-antibody complex or the separate magnetic bead labeled with streptavidin passes through the quality control line, the quality control line contains biotin capable of binding to the streptavidin, so that as long as the labeled magnetic bead labeled with the streptavidin is chromatographed to the quality control line, no matter whether the antibody is bound with the labeled nucleic acid, the complex can be formed, and the quality control line is brown due to the fact that the complex carries the colored label (the magnetic nanoparticles are brown). The invention has the beneficial technical effects that:
the invention provides a method for rapidly detecting pathogenic vibrio cholerae, which adopts recombinase-mediated isothermal nucleic acid amplification combined with a lateral chromatography technology to detect the vibrio cholerae, greatly shortens the detection time compared with a PCR kit, can complete the detection within 1h, does not need special instruments and equipment, and has lower cost. The lateral chromatography technology adopted by the technology takes the magnetic nano-microspheres as a marker, the specificity problem is solved by designing the RAA primer and the probe of the ctxA gene of the vibrio cholerae, the accurate quantification is realized while the fast qualitative is realized by utilizing the three-dimensional magnetic signal acquisition of the magnetic nano-material, and the 10 can be detected by simple visual observation2CFU/mL Vibrio cholerae, which is much lower than 10 of the current industry standard8-103CFU/mL pathogenic range (SN/T1022-2010), and the detection signal is very stable. In conclusion, this documentThe technology of the invention realizes the rapid detection of pathogenicity vibrio cholerae with high sensitivity and specificity, makes up the defects of the prior detection technology, does not need special instruments and equipment, reduces the detection cost, enlarges the application range of the technology, can be applied to special environments such as field sites and the like, has simple operation and short time, and improves the detection technical level of pathogenic bacteria.
Drawings
FIG. 1 is a schematic structural diagram of a lateral chromatography test strip;
FIG. 2 is a schematic diagram of the structure of a recombinase-mediated isothermal nucleic acid amplification (RAA) product;
FIG. 3 is a sensitivity detection graph (DNA concentration representation) in which: n is negative control, and double distilled water is used for replacing a DNA template; other Vibrio cholerae DNAs with different concentrations are respectively, and the DNA content in the reaction system is respectively 40 fg/muL, 400 fg/muL, 4 pg/muL, 40 pg/muL, 400 pg/muL, 4 ng/muL and 40 ng/muL;
FIG. 4 is a sensitivity measurement chart (shown by the concentration of bacterial liquid) of 10 each9CFU/mL，108CFU/mL，107CFU/mL，106CFU/mL，105CFU/mL，104CFU/mL，103CFU/mL，102CFU/mL，101CFU/mL；
FIG. 5 is a diagram showing the results of specificity detection, which are: 1-VPF, 2-VPF54, 3-VP1201, 4-VP1203, 5-VP1204, 6-VP1205, 7-VP1206, 8-VP6, 9-VCMY45, 10-VCMY 5311-Salmonella, 12-Klebsiella pneumoniae, 13-Hawei ATCC33842, 14-Escherichia coli, 15-VC13Y3, 16-Staphylococcus aureus, 17-Listeria monocytogenes, 18-Klebsiella oxytoca, 19-ATCC33847, 20-Hawei ATCCBA1117, 21-ATCC17802, 22-VP1215, 23-VP1209, 24-VP1207, 25-cholera LK, 26-positive control.
Detailed Description
On the basis of fully analyzing virulence gene ctxA of vibrio cholerae, the invention designs a primer and a probe with high sensitivity and specificity, and combines a Recombinase-mediated isothermal nucleic acid amplification technology (recombination Aide amplification RAA) and a Lateral flow test strip (Lateral flow dipstick) technology to detect pathogenic vibrio cholerae.
The present invention will be described in detail with reference to examples.
Example 1: design and optimization of primers and probes
Designing primers and probes according to a main virulence gene ctxA coded by vibrio cholerae:
6 groups of primer pairs are designed for optimal primer screening, and the primer groups and sequences are as follows:
group 1:
forward primer F1-RAA: 5'-GGTCTTATGCCAAGAGGACAGAGTGAGTAC-3'
Reverse primer R1-RAA: 5'-AAACATATCCATCATCGTGCCTAACAAATC-3'
Group 2:
forward primer F2-RAA: TCAGGTGGTCTTATGCCAAGAGGACAGAGT
Reverse primer R2-RAA: GGAAACATATCCATCATCGTGCCTAACAAA
Group 3:
forward primer F3-RAA: 5'-GGTCTTATGCCAAGAGGACAGAGTGAGTAC-3'
Reverse primer R3-RAA: 5'-GGAAACATATCCATCATCGTGCCTAACAAA-3'
Group 4:
forward primer F4-RAA: 5'-AGTCAGGTGGTCTTATGCCAAGAGGACAGAG-3'
Reverse primer R4-RAA: 5'-TGGAAACATATCCATCATCGTGCCTAACAAA-3'
Group 5:
forward primer F5-RAA: 5'-ACCTCCTGATGAAATAAAGCAGTCAGGTGGTC-3'
Reverse primer R5-RAA: 5'-GGAAACATATCCATCATCGTGCCTAACAAATC-3'
Group 6:
forward primer F6-RAA: 5'-AGTCAGGTGGTCTTATGCCAAGAGGACAGAGTG-3'
Reverse primer R6-RAA: 5'-TGGAAACATATCCATCATCGTGCCTAACAAATC-3'
Designing a probe: a ctxA gene sequence is used as a template to design a probe, and the sequence information of the probe is as follows:
and (3) probe P:
5′-GTCAGGTGGTCTTATGCCAAGAGGACAGAGGAGACTTTGACCGAGGTA-3′。
a30 th base from the 5 'end of the probe P is labeled with an FITC (FITC fluorescent group), Tetrahydrofuran (THF) is inserted behind the 30 th base to serve as a abasic site, a fluorescence quenching group BHQ is inserted behind the THF, and the 3' end of the probe P is labeled with C3-Spacer. The modified probe information is as follows:
and (3) probe P: 5 '-GTCAGGTGGTCTTATGCCAAGAGGACAGAG (FITC-dT) G (THF) AG (BHQ-dT) ACTTTGACCGAGGTA (C3-spacer) -3'.
And (2) primer screening: the reaction system was prepared by RAA nucleic acid amplification (fluorescence method) using 6 sets of designed primer pairs. The reaction tube is placed into a fluorescent gene detector to react for 20min at 39 ℃, and the result is observed in real time. The experimental result shows that the amplification efficiency of the 4 th primer is better than that of the other 5 primer groups under the same reaction condition, so the 4 th primer is taken as the optimal primer, and Biotin Biotin labeling is carried out on the 5' end of the reverse primer R4-RAA. The sequence of the RAA primers was as follows:
forward primer F-RAA: 5'-AGTCAGGTGGTCTTATGCCAAGAGGACAGAG-3'
Reverse primer R-RAA: 5 '-biotin-TGGAAACATATCCATCATCGTGCCTAACAAA-3'
Example 2: detection of pathogenic Vibrio cholerae
A method for rapidly detecting pathogenic vibrio cholerae adopts recombinase-mediated isothermal amplification (fluorescence method), and comprises the following steps:
s1, preparing a lateral chromatography test strip: the lateral chromatography test strip comprises a sample pad, a combination pad, an NC membrane, a back plate and a marking pad which are sequentially arranged from a sample adding end. The NC membrane is coated with a T line and a C line, the T line is coated with a goat anti-rabbit secondary antibody capable of being combined with an anti-FITC antibody, the C line is coated with biotin-BSA combined with magnetic bead-streptavidin, and the T line and the C line form an interpretation zone. The specific structure is shown in figure 1.
S2, preparing immunomagnetic beads, which specifically comprises the following steps:
(1) activating magnetic beads: taking 1mg of magnetic beads (the diameter is 180nm), washing the magnetic beads for 3 times in a centrifugal tube by using 500 mu LMEST buffer solution, adding 18 mu L of activating agent into the centrifugal tube, uniformly mixing, placing on a vertical mixer, and carrying out rotary reaction for 30min at room temperature;
(2) coupling antibody: after activation of the magnetic beads is finished, washing the magnetic beads for 2 times by using 500 mu LMEST buffer solution to remove the residual activating agent, washing the magnetic beads for 2 times by using 500 mu LBST buffer solution, adding streptavidin, adding BST buffer solution, mixing uniformly, placing on a vertical mixer, carrying out room-temperature rotation reaction for 3h, after coupling is finished, placing on a magnetic frame for 5min, and removing supernatant to obtain magnetic beads coupled with antibodies;
(3) sealing the immunomagnetic beads: washing the magnetic beads coupled with the antibody for 2 times by using 500 mu LBST buffer solution, adding 500 mu L of blocking solution (BSA is dissolved in BST buffer solution to obtain the antibody, wherein the mass fraction of BSA is 1%), fully mixing on a vortex mixer, placing on a vertical mixer, and reacting for 30min at room temperature;
(4) and (3) storage: the blocked immunomagnetic beads were washed 3 times with 500. mu. LBST buffer, and the immunomagnetic beads were stored in 500. mu.L of a storage solution (NaN was added)3And BSA in BST buffer, wherein NaN is3And BSA (bovine serum albumin) in 1 percent by mass), and storing the mixture at 4 ℃ for later use to obtain the magnetic bead-streptavidin conjugate.
S3, preparation and detection of a detection sample, which specifically comprises the following steps:
RAA amplification
Preparing a DNA solution to be detected, and preparing a reaction system according to a commercially available RAA kit (test strip method), wherein the reaction system comprises the primers and the probes for detecting the pathogenic vibrio cholerae, and the RAA kit is specifically a kit produced by Jiangsu Qitian gene biotechnology limited and has the model of T00001A. The prepared basic reaction unit is subjected to amplification reaction at 39 ℃ for 30 minutes.
b. Detection of RAA amplification products using lateral chromatography test strips
And adding 10 mu L of nucleic acid amplification product into a buffer solution, uniformly mixing with 3 mu L of magnetic bead-streptavidin conjugate and anti-FITC rabbit antibody with the same volume as the nucleic acid amplification product, and dropwise adding into a sample pad area of the test strip. And judging the amplification result by the test strip. The detection line and the control line of the test strip are brown, which indicates that the result is positive; only the control line is brown, and the position of the detection line is colorless, which indicates that the result is negative; the control line did not develop color and the results were invalid.
And the concentration of the FITC-resistant rabbit antibody in the step b is 0.03 mg/L.
Washing the magnetic beads with the MEST buffer solution in the step (1) or (2) comprises the following steps: and uniformly mixing the magnetic beads and the MEST buffer solution, placing the mixture into a magnetic rack for standing, and discarding the supernatant when the magnetic beads are completely adsorbed on the wall of the centrifugal tube and the solution is clarified, namely washing once.
The volume ratio of the streptavidin to the BST buffer solution in the step (2) is 1: and 5, the total volume of the streptavidin and the BST buffer solution is 500 mu L.
The activating agent comprises the following components in percentage by weight: 97% MEST buffer, 1% l0.5 g/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 2% 0.25g/mL N-hydroxysuccinimide (NHS).
Sensitivity of detection
Vibrio cholerae DNA with different concentrations (DNA concentration is 40 ng/mu L,4 ng/mu L, 400 pg/mu L,40 pg/mu L,4 pg/mu L, 400 fg/mu L and 40 fg/mu L) and bacterial liquid with different concentrations (10 ng/mu L respectively)9CFU/mL，108CFU/mL，107CFU/mL，106CFU/mL，105CFU/mL，104CFU/mL，103CFU/mL，102CFU/mL，101CFU/mL) as template RAA nucleic acid amplification was performed according to the aforementioned loading method, and the amplification product was detected by LFD: the results are shown in FIGS. 3 and 4.
The results showed that LFD was minimally detectable 40pg DNA and 10 pg DNA after RAA amplification2The result of the nucleic acid amplification product obtained by using DNA extracted from CFU/mL bacterial liquid as a template shows that the primer set disclosed by the invention can ensure the sensitivity in detection.
Detection specificity
Selecting a plurality of strains of vibrio parahaemolyticus, vibrio harveyi, vibrio cholerae without ctxA gene, salmonella, escherichia coli, vibrio cholerae, klebsiella, staphylococcus aureus and the like as specificity determination experimental strains. The amount of the DNA template of each test bacterium was 40ng according to the aforementioned sample addition method. The nucleic acid amplification product is detected by LFD.
As a result, as shown in FIG. 5, only the detection line and the control line in which the DNA template was the positive group of Vibrio cholerae containing ctxA showed a band, while the detection lines in the other groups were blank and only the band appeared at the control line. The result shows that the method can realize the specific detection of pathogenic vibrio cholerae and does not have cross reaction with other related pathogenic bacteria.
In summary, the rapid detection method of pathogenic vibrio cholerae provided by the invention, namely the RAA-LFD detection method, has the following advantages:
firstly, the sensitivity is high, the lowest line for detecting the pathogenic vibrio cholerae can reach 40 pg/mu L, and the detection sensitivity is 1000 times higher than that of the traditional agarose gel electrophoresis detection of nucleic acid amplification products.
Secondly, the specificity is strong, no cross reaction with other food-borne pathogenic bacteria exists, and the detection specificity is strong.
And thirdly, the detection time is short, the detection result can be obtained within 1h, and the method has great advantages compared with the conventional detection method.
And fourthly, the requirements of instruments and equipment are low, a temperature circulator, a PCR (polymerase chain reaction) instrument, an electrophoresis apparatus or a gel imaging system is not needed, the detection can be completed only by one constant temperature heater, and even the artificial heating by using the body temperature can be carried out.
Fifthly, the operation is simple and convenient, the result is obvious, the whole detection process does not involve complex and expensive instruments and equipment, the sample pretreatment is simple, DNA purification is not needed, only sterile double distilled water is needed for cracking, and personnel with little molecular biology basis can complete the whole operation; the detection result is clear and obvious, and can be judged directly through visual observation.
And sixthly, the method is safer to the experimenters and the environment, and toxic reagents such as EB (Electron beam) and the like are not used because gel electrophoresis is not needed in the detection process. Compared with the molecular biological detection method with stronger instrument dependence such as PCR detection in the prior art, the method for detecting pathogenic vibrio cholerae has higher specificity, sensitivity, practicability and convenience, can be applied in actual field, is favorable for preventing and controlling the occurrence of the epidemic situation of the vibrio cholerae, and can be used for preventing in advance. The method for on-site detection of the pathogenic bacterium vibrio cholerae can prevent the pathogenic bacterium from harming food and human health in time, guarantee food safety and establish a more complete food safety supervision system.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Sequence listing
<110> Shanghai ocean university
<120> a rapid detection method of pathogenic vibrio cholerae
<141> 2019-10-29
<160> 3
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tggaaacata tccatcatcg tgcctaacaa a 31
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<212> DNA
<213> Artificial Sequence
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gtcaggtggt cttatgccaa gaggacagag gagactttga ccgaggta 48
Claims (10)
1. A primer and a probe for detecting pathogenic vibrio cholerae are characterized in that the sequence information of the primer is as follows:
forward primer F-RAA: 5'-AGTCAGGTGGTCTTATGCCAAGAGGACAGAG-3', respectively;
reverse primer R-RAA: 5'-TGGAAACATATCCATCATCGTGCCTAACAAA-3', respectively;
the probe sequence information is as follows:
5′-GTCAGGTGGTCTTATGCCAAGAGGACAGAGGAGACTTTGACCGAGGTA-3′。
2. the primers and the probe for detecting pathogenic vibrio cholerae of claim 1, wherein the 5 ' end of the reverse primer is labeled with Biotin, the 30 th base from the 5 ' end of the probe is labeled with FITC luminophore, tetrahydrofuran THF is inserted after the 30 th base as abasic site, a fluorescence quencher BHQ is inserted thereafter, and the 3 ' end of the probe is labeled with C3-Spacer.
3. Use of the primers and probes according to claim 1 or 2 for the detection of pathogenic vibrio cholerae.
4. A method for rapidly detecting pathogenic vibrio cholerae is characterized by comprising the following steps:
raa amplification: preparing a DNA solution to be detected, preparing a reaction system according to a commercially available RAA kit, wherein the reaction system comprises the primer and the probe for detecting the pathogenic vibrio cholerae of the claim 1 or the claim 2, and carrying out amplification reaction on the prepared basic reaction unit at 39-42 ℃ for 25-30 minutes;
b. detecting the RAA amplification product by using a lateral chromatography test strip: and adding the nucleic acid amplification product into a buffer solution, uniformly mixing with the magnetic bead-streptavidin conjugate and the anti-FITC rabbit antibody, and dropwise adding the mixture into a sample pad area of a lateral chromatography test strip.
5. The method for rapidly detecting the pathogenic vibrio cholerae of claim 4, wherein the lateral chromatography test strip comprises a sample pad, a binding pad, an NC membrane, a back plate and a labeling pad which are sequentially arranged from a sample adding end, wherein the NC membrane is coated with a T line and a C line, the T line is coated with a goat anti-rabbit secondary antibody capable of being combined with an anti-FITC antibody, and the C line is coated with biotin-BSA capable of being combined with magnetic bead-streptavidin.
6. The method for rapidly detecting pathogenic vibrio cholerae according to claim 4, wherein the magnetic bead-streptavidin conjugate is prepared by the following steps:
(1) activating magnetic beads: washing the magnetic beads in a centrifugal tube for 2-5 times by using MEST buffer solution, adding an activating agent into the centrifugal tube, uniformly mixing, and carrying out a rotary reaction for 25-35 min;
(2) coupling antibody: after activation of the magnetic beads is finished, washing the magnetic beads for 1-3 times by using MEST buffer solution, then washing the magnetic beads by using BST buffer solution, sequentially adding streptavidin and BST buffer solution after washing, uniformly mixing, performing rotary reaction for 2-4h, placing the magnetic beads on a magnetic frame for 4-6min after coupling is finished, and removing supernatant to obtain the magnetic beads after coupling the antibody;
(3) sealing the immunomagnetic beads: washing the magnetic beads coupled with the antibody with BST buffer solution, adding confining liquid after washing, fully and uniformly mixing, and reacting for 25-35 min;
(4) and (3) storage: and washing the blocked immunomagnetic beads with BST buffer solution to obtain magnetic bead-streptavidin conjugate, storing the magnetic bead-streptavidin conjugate in a storage solution, and storing the magnetic bead-streptavidin conjugate at 3-5 ℃ for later use.
7. The method for rapidly detecting pathogenic Vibrio cholerae according to claim 6, wherein the activator in step (1) comprises the following components in percentage by weight: 97% MEST buffer, 1% l0.5 g/mL 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 2% 0.25g/mL N-hydroxysuccinimide (NHS).
8. The method for rapidly detecting pathogenic Vibrio cholerae of claim 6, wherein the blocking solution is BST buffer solution containing BSAObtaining the BSA, wherein the mass fraction of the BSA is 0.8-1.2%; the preservation solution is prepared by adding NaN3And BSA in BST buffer, wherein NaN is3And BSA in the amount of 0.8-1.2% by mass.
9. The method for rapidly detecting pathogenic Vibrio cholerae according to claim 6, wherein the volume ratio of the streptavidin to the BST buffer in step (2) is (1-5): (1-5), wherein the solid-to-liquid ratio of the magnetic beads, the streptavidin and the total volume of the BST buffer solution is 1: (400-600) μ L.
10. A kit for rapidly detecting pathogenic Vibrio cholerae, comprising the primer and probe of claim 1 or 2.
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