CN109884296B

CN109884296B - Kit for rapidly detecting African swine fever by directly labeling primers with nanogold

Info

Publication number: CN109884296B
Application number: CN201910140489.5A
Authority: CN
Inventors: 陈爱亮; 王之莹; 李婷婷
Original assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Current assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Priority date: 2019-02-26
Filing date: 2019-02-26
Publication date: 2022-02-18
Anticipated expiration: 2039-02-26
Also published as: CN109884296A

Abstract

The invention discloses a kit for rapidly detecting African swine fever by directly labeling a primer with nanogold. The test paper comprises a test paper strip for rapidly detecting African swine fever, a primer pair and a buffer solution; the test strip comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate; along the flow direction of the sample, the sample absorption pad, the reaction membrane and the water absorption pad are sequentially fixed on the bottom plate; the reaction membrane of the test strip comprises a detection area and a quality control area; the detection area is coated with streptavidin; the quality control region is coated with a quality control probe connected with streptavidin or biotin; the primer pair is an upstream primer of which the 5' end is marked with nanogold and a downstream primer of which biotin is modified. The invention utilizes the nanogold to directly mark the specific PCR primer, reduces the cost of the kit by means of the principle of double-strand complementation and common PCR, and realizes simple, rapid and low-cost field detection.

Description

Kit for rapidly detecting African swine fever by directly labeling primers with nanogold

Technical Field

The invention relates to a detection kit in the field of biotechnology, in particular to a kit for detecting diseases, and especially relates to a kit for rapidly detecting African swine fever by using a nanogold direct-labeled primer.

Background

African Swine Fever (ASF) is a virulent, highly contagious infectious disease of pigs caused by African Swine fever virus. African Swine Fever Virus (ASFV) is the only member of the African Swine fever virus family, genus African Swine fever virus. Because of the high morbidity and mortality of the disease, the swine industry can cause huge economic loss, the African swine fever is specified as a type of animal infectious disease in China, and the animal health organization of the world lists the disease as the animal epidemic disease which is reported by the legal system. The establishment of a rapid, sensitive and specific detection method is important for the prevention and control of ASFV.

At present, the ASF diagnosis methods include erythrocyte adsorption tests, direct immunofluorescence tests, indirect immunofluorescence tests, virus nucleic acid detection methods and the like. Among them, the nucleic acid detection method is widely used because of its simple operation and high sensitivity. The method for detecting ASFV nucleic acid includes common polymerase chain reaction, real-time polymerase chain reaction, and the like.

The test strip is a simple, convenient and sensitive on-site rapid detection product, and is widely applied to the detection industry. At present, most of methods using antigen and antibody are used for carrying out test strip color reaction, the cost is high, and the reaction is not fast enough.

Disclosure of Invention

The invention aims to provide a kit for rapidly detecting African swine fever by directly labeling a primer with nanogold.

The invention designs a specific PCR primer of African swine fever, utilizes nanogold to directly mark the primer, and develops an African swine fever identification kit based on a PCR test strip technology; compared with a method using an antigen-antibody to carry out test strip color reaction, the method reduces the cost of the kit by means of a double-chain complementary principle and common PCR and realizes simple, rapid and low-cost field detection.

The invention provides a test strip for rapidly detecting African swine fever, which comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate;

along the flow direction of the sample, the sample absorption pad, the reaction membrane and the water absorption pad are sequentially fixed on the bottom plate;

the reaction membrane of the test strip comprises a detection area and a quality control area; the detection area is coated with streptavidin; the quality control region is coated and connected with a quality control probe of streptavidin or biotin; the nucleotide sequence of the quality control probe is shown as SEQ ID NO. 1.

In the test strip, the concentration of the streptavidin forming the detection area can be 4 mg/mL-10 mg/mL, and specifically can be 5mg/mL or 4 mg/mL-8 mg/mL;

the concentration of the streptavidin or biotin forming the quality control region can be 1 mg/mL;

the concentration of the quality control probe can be 100 mu M;

the quality control probe with the streptavidin or the biotin coated on the quality control area is obtained on a reaction membrane according to the following steps: and reacting the streptavidin or the biotin with the quality control probe on the reaction membrane for 1-2 hours at room temperature.

The invention also provides a kit for rapidly detecting the African swine fever, which comprises a test strip for rapidly detecting the African swine fever, a primer pair and a buffer solution;

the test strip comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate; along the flow direction of the sample, the sample absorption pad, the reaction membrane and the water absorption pad are sequentially fixed on the bottom plate; the reaction membrane of the test strip comprises a detection area and a quality control area; the detection area is coated with streptavidin; the quality control region is coated and connected with a quality control probe of streptavidin or biotin;

the primer pair is an upstream primer of which the 5' end is marked with nanogold and a downstream primer of which biotin is modified.

In the invention, the primer pair is used for amplifying the African swine fever virus.

In the kit, the nucleotide sequence of the quality control probe is shown as SEQ ID NO. 1;

the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3;

the concentration of the streptavidin forming the detection area can be 4 mg/mL-10 mg/mL, and specifically can be 5mg/mL or 4 mg/mL-8 mg/mL;

the concentration of the quality control probe can be 100 mu M;

the quality control probe with the streptavidin or the biotin coated on the quality control area is obtained on a reaction membrane according to the following steps: reacting streptavidin or biotin with a quality control probe on a reaction membrane at room temperature for 1-2 hours;

the Buffer solution is TE Buffer.

The invention also provides a method for rapidly detecting the African swine fever virus by using the kit, which comprises the following steps: (1) placing a sample to be detected in a PCR amplification system with the primer pair for PCR reaction to obtain an amplification product;

(2) and mixing the amplification product with a buffer solution, and then dropwise adding the mixture to a sample absorption pad of the test strip, wherein the detection result is as follows:

1) when the detection area is colored and the quality control area is also colored, the detection result of the sample to be detected is positive;

2) when the detection area does not develop color and the quality control area develops color, the detection result of the sample to be detected is negative;

3) when the quality control area does not develop color, the test strip is invalid, and the test strip needs to be used for re-determination.

In the invention, the PCR reaction process is as follows: 95 ℃ for 4 min; at 95 ℃ for 30 s; 30s at 55 ℃; at 72 ℃, 1min, 30 cycles; 72 ℃ for 10 min.

In the step (1), the volume ratio of the sample to be detected to the primer pair is 1: 2; specifically, 1 mu L of primer to be detected is used, and the using amount of the primer is 2 mu L;

the volume ratio of the amplification product to the buffer solution is 1: 5-9, and specifically can be 1: 6.

The invention also provides a primer pair for detecting the African swine fever virus, wherein the primer pair is an upstream primer of which the 5' end is marked with nanogold and a downstream primer of which the biotin is modified.

In the primer pair, the preparation of the upstream primer of the 5' end labeled nanogold comprises the following steps:

placing the nano gold colloidal solution and the upstream primer for reaction under dark; and then adding a PB buffer solution, adding a NaCl aqueous solution for dilution, and standing for reaction to obtain the upstream primer of the 5' end labeled nanogold.

In the primer pair, the particle size of the nano gold colloid in the nano gold colloid solution is 13-25 nm, specifically 13nm, and the concentration of the upstream primer is 100 mu M;

the volume ratio of the nano gold colloidal solution to the upstream primer is 25-50: 1, specifically 25: 1;

placing the mixture under dark place for reaction for 16-24 h, specifically 6 h;

the concentration of the PB buffer solution is 10mM, and the volume ratio of the upstream primer to the PB buffer solution is 1: 2.8;

adding 2M NaCl aqueous solution to dilute the system to 0.3M, and then reacting for 8-12 h, specifically 8 h;

the post-treatment step of obtaining the upstream primer of the 5' end labeled nano-gold comprises the following steps: centrifuging and removing supernatant; then, the suspension was resuspended in NaCl aqueous solution and PB buffer solution for use.

In the invention, the preparation of the upstream primer of the 5' end labeled nano-gold specifically comprises the following steps:

placing 500 μ L of nanogold (colloidal gold solution, 13 nm) and 20 μ L of 100 μ M upstream primer in dark for 16h, adding 56 μ L of 10mM PB buffer solution, adding 2M NaCl to 0.3M, and standing for 8 h; centrifuging at 16100 Xg for 30min at 4 deg.C, and removing supernatant; finally, the suspension was resuspended in 0.3M NaCl and 10mM PB buffer and kept at 4 ℃ until use.

The invention further provides an amplification system for detecting African swine fever virus, which comprises the primer pair, one or more DNA polymerases, dNTPs, a buffer solution and ddH₂O。

In the above-mentioned amplification system, the Buffer solution comprises TE Buffer.

The invention has the following advantages:

the invention designs a specific PCR primer of African swine fever, utilizes nanogold to directly mark the primer, and develops an African swine fever identification kit based on a PCR test strip technology. The using method is simple to operate, the cost of the kit is greatly reduced by using the common PCR Mix, the amplification is completed by using a PCR amplification instrument, the result can be observed by naked eyes within 10min, and a special detection instrument is not needed; compared with a method using an antigen-antibody to carry out test strip color reaction, the method reduces the cost of the kit by means of a double-chain complementary principle and common PCR and realizes simple, rapid and low-cost field detection.

Drawings

FIG. 1 shows the detection principle of the kit of the present invention.

FIG. 2 shows the results of gel electrophoresis using different primer pairs according to the present invention.

FIG. 3 is a detection result of the specificity test of the kit of the present invention, wherein FIG. 3(a) is an electrophoresis result of a primer on plasmid DNA templates of different viruses, and FIG. 3(b) is a detection result of a primer on plasmid DNA templates of different viruses in the kit of the present invention.

FIG. 4 is a drawing showingThe detection results of the sensitivity test of the reagent of the present invention are 10 from left to right in FIG. 4⁶、10⁵、10⁴、10³、10²、10¹1 copy/. mu.L.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, 2 XTAQQ Master Mix for PAGE was purchased from Biotech Inc. of Bainovei, Beijing; the primers were synthesized by Shanghai Bioengineering technology, Inc. The nucleic acid test strip is purchased from Beijing Yan Qin Bay Biotechnology GmbH; the electrophoresis apparatus was purchased from Bio-Rad; gel imaging system (Alphalmager EP) was purchased from UVP corporation, usa; ultraviolet spectrophotometer was purchased from Thermo limited bio; the microcentrifuge was purchased from Thermo Fisher Scientific, Inc. Colloidal gold solution (13 nm); streptavidin, sodium chloride, sodium azide, PBS buffer, PB buffer and TE buffer are purchased from Beijing Bainovei Biotech Co., Ltd; a water bath kettle; nanodrop-2000 (Gene Co.); centrifuging the tube: 0.1mL, 1.5mL, 2 mL; a timer; a liquid transferring gun: the measuring range is 0.5-10 muL, 10-100 muL and 100-1000 muL; sterile deionized water.

The ASFV plasmid is originated from Shanghai Biotechnology engineering service, Inc.

Examples of the following,

1. Preparation of test paper strip

Coating streptavidin on the reaction membrane according to the coating concentration of 5mg/ml to form a detection zone; reacting 1mg/ml of streptavidin or biotin with 100 mu M of quality control probes at room temperature for two hours, and then taking the reaction membrane as a quality control area to obtain the reaction membrane coating the detection area and the quality control area. The test strip is prepared by sequentially adhering a sample absorption pad (Shanghainej-Biotechnology Co., Ltd., JY-BX 111), the prepared reaction film coating the detection area and the quality control area, and a water absorption pad on a bottom plate. The nucleotide sequence of the quality Control probe is shown as SEQ ID NO.1 and as shown in Table 1, Control.

2. Selecting a primer: according to the invention, different primer pairs ZF 1-ZF 3 shown in Table 1 are designed, and as can be seen from comparison of gel electrophoresis shown in figure 2, positive bands of ZF1 and ZR1 are most clear and bright, and ZF1 and ZR1 are selected as the optimal primer pair.

The 5' end of the ZF1 upstream primer is marked with nanogold, and the ZR1 downstream primer is modified with biotin. Primer design Primer Premier 5.0 software was used, and the Primer sequences are shown in table 1. The nucleotide sequences of ZF1, ZR1, ZF2, ZR2, ZF3 and ZR3 are respectively shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4-7.

Preparing an upstream primer ZF1 of which the 5' end is marked with nanogold: the upstream primer is complementary with the nucleotide sequence of the quality control probe of the test strip, and after the nanogold is marked on the upstream primer, the reaction product and the loading Buffer TE Buffer are dripped on the test strip. If the product has a target sequence, streptavidin on a detection area of the test strip grasps biotin of the downstream primer, and nanogold of the upstream primer develops color in the detection area. If no target sequence exists, the detection area can not develop color, and the quality control probe sequence in the quality control area is complementary with the gold-labeled primer for quality control. Preparing a gold-labeled primer: mu.L of nanogold (colloidal gold solution, 13 nm) and 20. mu.L of 100. mu.M forward primer were left for 16h in the dark, 56. mu.L of 10mM PB buffer, 2M NaCl to 0.3M and left for 8 h. Centrifuged at 16100 Xg for 30min at 4 ℃ and the supernatant removed. Finally, the suspension was resuspended in 0.3M NaCl and 10mM PB buffer and kept at 4 ℃ until use.

TABLE 1 primer and Probe sequences

Primer	Sequences（5'-3'）
		ZF1	HS-C6-CCTACTCACCACGCAGAGAT
ZR1	Bio-TTGCATTGCCTCCGTAGTGG
		ZF2	AGCTCTTCCAGACGCATGTT
ZR2	CGTGGCTTCAAAGCAAAGGT
		ZF3	GATGATCCGGGTGCGATGAT
ZR3	CCACGTAATCCGTGTCCCAA
		Control	Bio-ATCTCTGCGTGGTGAGTAGG

Note: in Table 1, the 5' -end of the nucleotide sequence of ZF1 is linked to C6 (6 methylene groups, CH)₂) And a thiol group (SH), wherein the nanogold is linked to the sulfur in the thiol group; the 5 'end of the nucleotide sequence of ZR1 is connected with Bio to represent biotin, and the 5' end of the nucleotide sequence Control of the quality Control probe is connected with Bio to represent streptavidin or biotin.

3. The kit for rapidly detecting African swine fever consists of the test strip prepared in the step 1, the primer pair in the step 2 and a Buffer solution (TE Buffer).

4. The use method of the kit for rapidly detecting African swine fever comprises the following steps: (1) plasmid DNA from African swine fever was amplified by 2 XTAQA Master Mix for PAGE and the amplified products were detected by electrophoresis. The PCR reaction system is as follows: 25. mu.L of reaction buffer, 2. mu.L of forward primer and 2. mu.L of reverse primer, 1. mu.L of DNA template, and 20. mu.L of ddH₂And O. The reaction process is as follows: 95 ℃ for 4 min. At 95 ℃ for 30 s; at a temperature of 55 c,30 s; 72 ℃, 1min, 30 cycles. 72 ℃ for 10 min.

(2) Mixing 10 mu L of the amplification product with 60 mu L of TE Buffer, and then dropwise adding the mixture to the detection area of the test strip, wherein the detection result is as follows:

3) when the quality control area does not develop color, the test strip is ineffective, and a new test strip is needed for re-determination.

The detection principle of the kit of the invention is as follows:

the principle of the nucleic acid test strip for detecting African swine fever plasmid DNA is shown in figure 1. A pair of specific primers is used for amplifying DNA obtained from African swine fever plasmids, an upstream primer is marked with nanogold, and the 5' end of a downstream primer is modified with biotin. After the amplification product is purified, the amplification product and the developing solution are dripped on a test strip, and the mixed solution flows forwards through capillary action. When the amplification product exists, the amplification product flows forwards along with the liquid phase to reach the detection line, the streptavidin fixed on the detection line captures the amplification product with biotin modified at one end, so that the colloidal gold combined with one end of the amplification product stays at the detection line to generate a red strip. And (4) continuously carrying out chromatography on the redundant colloidal gold, and finally combining with a quality control probe sequence on the quality control line to ensure that the quality control line is colored as well. When the amplification product does not exist, the colloidal gold particles can not stay at the detection line and are combined with the quality control probe sequence on the quality control line, so that the color development condition of the quality control line can be used for quality control of the test strip.

Specific experiments:

the specificity test was performed according to the established reaction system, and the primers designed in the present invention were used to test plasmid DNA templates of different viruses (ASFV positive plasmid, and porcine circovirus type 2 (PCV-2), Classical Swine Fever Virus (CSFV), transmissible gastroenteritis virus (TGEV), Porcine Epidemic Diarrhea Virus (PEDV) positive DNA or cDNA), and the test results are shown in fig. 3, and the results in fig. 3(a) show that the plasmids of several viruses other than the ASFV positive plasmid were not amplified, thus demonstrating that the specificity of the present invention is good. The electrophoresis result was consistent with the test strip result, as shown in fig. 3 (b).

Sensitivity test: to determine the minimum detectable amount of the present method, the synthetic plasmid standards were diluted to 10⁶、10⁵、10⁴、10³、10²、10¹And 1 copy/. mu.L as a template. The result is shown in FIG. 4, the method has a wide detection range, and the minimum detection limit is 10¹Copies/. mu.L (FIG. 4).

Sequence listing

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Claims

1. a test kit for rapid detection of African swine fever, characterized in that: the test kit comprises test strips, primer pairs and buffer solution for rapid detection of African swine fever;

The test strip includes a sample absorption pad, a reaction film, a water absorption pad and a bottom plate; along the flow direction of the sample, the sample absorption pad, the reaction film, and the water absorption pad are sequentially fixed on the bottom plate; the test paper The reaction membrane of the strip includes a detection area and a quality control area; the detection area is coated with streptavidin; the quality control area is coated with a quality control probe connected with streptavidin or biotin; the The nucleotide sequence of the quality control probe is shown in SEQ ID NO.1;

The primer pair is an upstream primer labeled with gold nanoparticles at the 5' end and a downstream primer modified with biotin;

The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. test kit according to claim 1 is characterized in that: the concentration of the described streptavidin that forms described detection zone is 4mg/mL～10mg/mL;

The concentration of the streptavidin or biotin forming the quality control region is 1 mg/mL;

The concentration of the quality control probe is 100 μM;

The quality control probe coated with streptavidin or biotin is obtained on the reaction membrane according to the following steps: the streptavidin or biotin and the quality control probe are reacted on the reaction membrane at room temperature 1 ~2 hours;

The buffer solution shown is TE Buffer.

3. The test kit according to claim 2, wherein the concentration of the streptavidin forming the detection zone is 4mg/mL～8mg/mL.

4. The kit according to claim 3, wherein the concentration of the streptavidin forming the detection zone is 5 mg/mL.

5. kit according to claim 1, is characterized in that: preparing the upstream primer of described 5' end labeling nano-gold comprises the following steps:

The nano-gold colloidal solution and the upstream primer are placed to react in the dark; then PB buffer is added, and NaCl aqueous solution is added to dilute, and the reaction is left to obtain the upstream primer labeled with nano-gold at the 5' end.

6. The kit according to claim 5, characterized in that: the particle diameter of the gold nanoparticles colloid in the nano gold colloid solution is 13-25 nm, and the concentration of the upstream primer is 100 μM;

The volume ratio of the nano-gold colloid solution to the upstream primer is 25-50:1;

The reaction time is 16~24h when placed in the dark place;

The concentration of the PB buffer is 10 mM, and the volume ratio of the upstream primer to the PB buffer is 1:2.8;

Add 2M NaCl aqueous solution to dilute the system to 0.3M, and then the reaction time is 8~12h;

The post-processing steps for obtaining the upstream primers labeled with the 5'-end gold nanoparticles are as follows: centrifugation, removing the supernatant; then resuspending in NaCl aqueous solution and PB buffer for use.

7. A method for rapidly detecting African swine fever virus using the kit according to any one of claims 1 to 6, comprising the steps of: (1) placing the plasmid DNA of African swine fever in a primer pair having the primer pair. PCR amplification system carries out PCR reaction to obtain amplification products;

(2) The amplification product is mixed with the buffer solution, and then added dropwise to the sample absorption pad of the test strip. The detection results are as follows:

1) When the detection area develops color and the quality control area also develops color, the test result is positive;

2) When the quality control area does not develop color, the test strip is invalid, and a new test strip needs to be used for re-measurement.

8. The method according to claim 7, wherein in step (1), the volume ratio of the African swine fever plasmid DNA to the primer pair is 1:2;

The volume ratio of the amplification product to the buffer solution is 1:5 to 9.