阅读说明：本技术 一种致病性霍乱弧菌的快速检测方法 (Rapid detection method of pathogenic vibrio cholerae ) 是由 卢瑛 方微微 蔡杨杨 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种致病性霍乱弧菌的快速检测方法。本发明首先提供了一种检测致病性霍乱弧菌的引物和探针。所述引物和探针具有高灵敏性和特异性。本发明还提供一种致病性霍乱弧菌的快速检测方法,采用特异性引物和探针,利用重组酶介导等温核酸扩增结合侧向层析技术,实现了致病性霍乱弧菌的快速、灵敏、特异性检测,且操作简便、耗时短,解决了传统检测技术耗时长、步骤繁琐等问题。本发明技术不需要特殊的仪器设备,降低了检测成本,扩大了该技术的应用范围,可应用于野外现场等特殊环境,对霍乱弧菌的早期检测与诊断具有重大意义。(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.)

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.

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.