阅读说明：本技术 一种基于信号级联双重放大系统以高灵敏且快速检测食源性致病菌的方法 (A method of based on signal cascade dual amplification system with highly sensitive and quick detection food-borne pathogens ) 是由 叶邦策 左鹏 李傲 于 2019-07-30 设计创作，主要内容包括：本发明公开了一种基于信号级联双重放大系统以高灵敏且快速检测食源性致病菌的方法,该检测方法主要分为以下步骤：(1)将致病菌的多/单克隆抗体偶联至磁珠表面,获得免疫磁珠；(2)将该免疫磁珠与含有食源性致病菌的样品混合,通过抗原抗体反应特异性捕获致病菌；(3)利用引发序列修饰的致病菌适配体及DNA发卡结构探针以引发杂交链式反应,获得HCR产物(带有切口的双链DNA)；(4)向HCR产物中加入拉曼信号分子和拉曼基底,进行相应的SERS测定,实现信号的级联放大及输出。本发明对保障食品安全并预防食源性疾病的发生具有重大意义,同时对于其他生物分析也展现出了巨大的应用潜力。(The invention discloses a kind of based on signal cascade dual amplification system in the method for highly sensitive and quick detection food-borne pathogens, the detection method is broadly divided into following steps: (1) more/monoclonal antibody of pathogenic bacteria being coupled to magnetic bead surfaces, adaptive immune magnetic bead；(2) immunomagnetic beads are mixed with the sample containing food-borne pathogens, pathogenic bacteria is captured by antigen-antibody reaction specificity；(3) it is obtained HCR product (double-stranded DNA with notch) using the pathogenic bacteria aptamers and DNA hairpin structure probe that cause sequence modification with causing hybridization chain reaction；(4) Raman signal molecule and Raman substrate are added into HCR product, carries out corresponding SERS measurement, realizes Cascaded amplification and the output of signal.The present invention is of great significance to ensuring food safety and preventing food origin disease, has also shown huge application potential simultaneously for other biological analysis.)

1. it is a kind of based on signal cascade dual amplification system in the method for highly sensitive and quick detection food-borne pathogens, be to be based on The signal cascade dual amplification system of immuning hybridization chain reaction joint Surface enhanced Raman scattering is eaten with high-sensitivity rapid detection The method of borne pathogen, which is characterized in that (1) more/monoclonal antibody of pathogenic bacteria is coupled to magnetic bead surfaces, adaptive immune Magnetic bead；(2) immunomagnetic beads are mixed with the sample containing food-borne pathogens, is captured and is caused by antigen-antibody reaction specificity Germ；(3) it is obtained using the pathogenic bacteria aptamers and DNA hairpin structure probe for causing sequence modification with causing hybridization chain reaction HCR product is obtained, that is, has the double-stranded DNA of notch；(4) Raman signal molecule and Raman substrate are added into HCR product, carries out phase The SERS measurement answered, realizes Cascaded amplification and the output of signal.

2. detection method according to claim 1, which is characterized in that the immuning hybridization chain reaction is immunomagnetic beads By the specific reaction of antigen-antibody to capture target bacterium, target bacterium extends with corresponding aptamer sensor response again HCR product out, System forming one immunomagnetic beads-target bacterium-DNA double chain structural composites.

3. detection method according to claim 1, which is characterized in that the Surface enhanced Raman scattering is, used Raman signatures spectral peak signal is quenched in Raman signal molecule intercalation of DNA double-strand；On the contrary, the Raman signal point in free state It is sub then form hot spot in conjunction with nano-Ag particles by electrostatic interaction and generate Raman signatures spectral peak, and signal strength and target bacterium Quantity there is correlativity in a certain range.

4. detection method according to claim 1, which is characterized in that the magnetic bead is surface modification active group Magnetic bead, the antibody are anti-target bacterium polyclonal antibody or monoclonal antibody.

5. detection method according to claim 1, which is characterized in that the preparation method of anti-target bacterial immunity magnetic bead is such as Under:

The magnetic bead for having modified active group in right amount is taken, appropriate amount of buffer solution cleaning is added and saves liquid several times to remove original, is then added The activated solution shaken at room temperature half an hour of Fresh is with the active group on activated magnetic beads surface；Be added anti-target bacterium it is more/Dan Ke Grand antibody, shaken at room temperature are incubated for a period of time, remaining antibody-solutions are removed, then cleaned twice with PBST, finally by acquisition Immunomagnetic beads are stored in BSA confining liquid and save backup.

6. detection method according to claim 1, which is characterized in that the method for immunomagnetic ca pture target bacterium is as follows:

Appropriate immunomagnetic beads storage liquid is taken, clean 2~3 times in removing extra solution on magnetic frame, then with PBST, removing is extra BSA, be then added the sample containing target bacterium, be incubated at room temperature certain time, finally clean 1~2 time with PBST, adaptive immune Magnetic bead-target bacterium compound.

7. detection method according to claim 1, which is characterized in that the preparation HCR method is as follows:

The aptamers for causing sequence modification are added into above-mentioned immunomagnetic beads-target bacterium compound, are incubated for a period of time, remove not In conjunction with aptamers after add the probe H1 and H2 of hairpin structure, be incubated for a period of time to cause hybridization chain reaction, remove After unreacted probe, final adaptive immune magnetic bead-target bacterium-aptamers HCR extension products compound.

8. detection method according to claim 1, which is characterized in that Surface enhanced Raman scattering detection method is as follows:

Set the determination condition of Raman；Into above-mentioned immunomagnetic beads-target bacterium-aptamers HCR extension products compound Excessive Raman signal molecule is added, mixes, is protected from light incubation a period of time；Proper amount of nano silver colloid is added, mixes, is protected from light incubation For a period of time；Appropriate system is taken to measure after background correction under the conditions of being protected from light on glass plate.

9. based on the method for salmonella typhimurium in immunomagnetic ca pture sample, catching method the following steps are included:

(1) anti-salmonella typhimurium antibody and magnetic bead are coupled, obtain anti-Immunized With Salmonella. Typhimurium magnetic bead；

(2) anti-Immunized With Salmonella. Typhimurium magnetic bead is mixed with the sample containing target bacterium, it is anti-by antigen and antibody specific The target bacterium in sample should be captured；

The magnetic bead is the surface modification magnetic bead of carboxylic group；

The anti-salmonella typhimurium antibody is anti-salmonella typhimurium polyclonal antibody or monoclonal antibody；

The preparation method of step (1) the anti-Immunized With Salmonella. Typhimurium magnetic bead is as follows:

Appropriate carboxyl magnetic bead is taken, MES buffer (pH6) is added and cleans 2 times to remove former preservation liquid, Fresh is then added EDC solution shaken at room temperature half an hour is with the carboxylic group of activated carboxyl magnetic bead surfaces；Anti- mouse wound after PBS buffer solution dilution is added Cold salmonella is more/monoclonal antibody, shaken at room temperature is incubated for for a period of time, removes remaining antibody-solutions, then cleaned with PBST Twice, finally the immunomagnetic beads of acquisition are stored in BSA confining liquid and be stored in 4 DEG C it is spare；

The method of step (2) the immunomagnetic ca pture salmonella typhimurium is as follows:

Step (1) resulting immunomagnetic beads are mixed to incubation a period of time with the sample containing salmonella typhimurium, formation is exempted from Epidemic disease magnetic bead-salmonella typhimurium compound.

10. a kind of combine SERS based on immune HCR in the method for highly sensitive detection salmonella typhimurium, detecting step is as follows:

(1) aptamers for causing sequence modification are added in Xiang Shangshu immunomagnetic beads-salmonella typhimurium compound, are incubated for one After the section time, immunomagnetic beads-salmonella typhimurium-aptamers compound is formed, while exposing initiation sequence；Then plus Enter the probe H1 and H2 of hairpin structure, is incubated for a period of time, extends the double-stranded DNA with notch of different length, most end form At the compound of " immunomagnetic beads-salmonella typhimurium-aptamers HCR extension products "；

(2) appropriate immunomagnetic beads-salmonella typhimurium-aptamers HCR extension products compound is taken, excessive Raman signal is added Molecule is protected from light after being incubated for a period of time, and nano silver colloid is added, and is protected from light after being incubated for a period of time, and Raman measurement is carried out.

Technical field

The present invention relates to a kind of based on immune HCR joint SERS with the side of highly sensitive and quick detection food-borne pathogens Method, belongs to that food-borne pathogens are highly sensitive and rapid detection technical field.

Background technique

In recent years, the food-safety problem that salmonella causes remains unchanged severe.Due to by salmonella-polluted food simultaneously Apparent sensory properties will not be shown, therefore is easy to be eaten by mistake by people and animals, causes the generation of food origin disease.In general, intake Amount is more than 10⁵The salmonella of CFU can be such that ordinary people infects, and for hypoimmunity or sensitive group, 15~ 20CFU can cause a disease, therefore develop novel detection method to realize that detecting salmonella with sensitivity seems most important.

Although traditional detection method is considered as goldstandard, but time-consuming effort, entire detection process needs 4~7 days left sides The right side is not able to satisfy instant and sensitive testing goal；Detection method based on immunology or molecular biology such as ELISA, PCR, Although the methods of LAMP has obtained very big improvement in sensitivity, it is easy to appear false positive issue, therefore specificity needs It improves.Aptamer is one section of oligonucleotide sequence obtained through in-vitro screening, can be tied with the strong specificity of target high-affinity It closes, thus can be used for being designed to aptamer sensor.Nucleic acid amplification technologies of the HCR as a kind of constant temperature without enzyme, due to having Low cost is easily-synthesized the advantage high with stability, is expected to the means for combining aptamer sensor substitution PCR to amplify as signal； On the other hand, SERS has significant fingerprint capacity and powerful signal amplifying power, and complicated without carrying out to sample Pre-treatment, be applied in various analysis at present, if the mode signal output as other amplifying techniques such as HCR, It is then expected to further increase the sensitivity of detection, while shortening detection duration.

Summary of the invention

The object of the present invention is to provide one kind based on immune HCR joint SERS carry out signal cascade amplification with highly sensitive inspection and Quickly the method for detection food-borne pathogens, specific technical solution are as follows:

A method of it is base based on signal cascade dual amplification system with highly sensitive and quick detection food-borne pathogens Combine the signal cascade dual amplification system of Surface enhanced Raman scattering in immuning hybridization chain reaction with high-sensitivity rapid detection The method of food-borne pathogens, which is characterized in that the immuning hybridization chain reaction is spy of the immunomagnetic beads by antigen-antibody To capture target bacterium, target bacterium extends HCR product, final body with corresponding aptamer sensor response again for opposite sex reaction System forms the sandwich structural composites of one " immunomagnetic beads-target bacterium-DNA double chain ".

The Surface enhanced Raman scattering is that Raman signatures are quenched in Raman signal molecule intercalation of DNA double-strand used Spectral peak signal；On the contrary, the Raman signal molecule in free state then passes through electrostatic interaction and forms heat in conjunction with nano-Ag particles It puts and generates Raman signatures spectral peak, and signal strength and the quantity of target bacterium have correlativity in a certain range.

The magnetic bead is the surface modification magnetic bead of active group, and the antibody is anti-target bacterium polyclonal antibody or Dan Ke Grand antibody.

The anti-target bacterial immunity magnetic bead the preparation method is as follows:

The magnetic bead for having modified active group in right amount is taken, appropriate amount of buffer solution cleaning is added and saves liquid several times to remove original, then The activated solution shaken at room temperature half an hour of Fresh is added with the active group on activated magnetic beads surface；Be added anti-target bacterium it is more/ Monoclonal antibody, shaken at room temperature are incubated for a period of time, remove remaining antibody-solutions, then cleaned twice with PBST, will finally obtain The immunomagnetic beads obtained are stored in BSA confining liquid and save backup.

The method of the immunomagnetic ca pture target bacterium is as follows:

Appropriate immunomagnetic beads storage liquid is taken, clean 2~3 times in removing extra solution on magnetic frame, then with PBST, removing Then the sample containing target bacterium is added in extra BSA, be incubated at room temperature certain time, finally cleaned 1~2 time with PBST, obtains Immunomagnetic beads-target bacterium compound.

DNA sequence dna used is as follows:

The preparation HCR method is as follows:

The aptamers for causing sequence modification are added into above-mentioned immunomagnetic beads-target bacterium compound, is incubated for a period of time, removes It goes after unbonded aptamers to add the probe H1 and H2 of hairpin structure, is incubated for a period of time to cause hybridization chain reaction, After removing unreacted probe, final adaptive immune magnetic bead-target bacterium-aptamers HCR extension products compound.

The Surface enhanced Raman scattering detection method is as follows:

Set the determination condition of Raman；It is compound to above-mentioned immunomagnetic beads-target bacterium-aptamers HCR extension products Excessive Raman signal molecule is added in object, mixes, is protected from light incubation a period of time；Proper amount of nano silver colloid is added, mixes, is protected from light It is incubated for a period of time；Appropriate system is taken to measure after background correction under the conditions of being protected from light on glass plate.

The present invention is for detecting salmonella typhimurium, based on salmonella typhimurium in immunomagnetic ca pture sample Method, catching method the following steps are included:

(1) anti-salmonella typhimurium antibody and magnetic bead are coupled, obtain anti-Immunized With Salmonella. Typhimurium magnetic bead；

(2) anti-Immunized With Salmonella. Typhimurium magnetic bead is mixed with the sample containing target bacterium, it is special by antigen-antibody Property reaction capture sample in target bacterium；

Further, the magnetic bead is the surface modification magnetic bead of carboxylic group.

Further, the anti-salmonella typhimurium antibody is anti-salmonella typhimurium polyclonal antibody or Dan Ke Grand antibody.

Further, the preparation method of step (1) the anti-Immunized With Salmonella. Typhimurium magnetic bead is as follows:

Appropriate carboxyl magnetic bead is taken, MES buffer (pH 6) is added and cleans 2 times to remove former preservation liquid, is then added fresh The EDC solution shaken at room temperature half an hour of preparation is with the carboxylic group of activated carboxyl magnetic bead surfaces；After PBS buffer solution dilution is added Anti- salmonella typhimurium is more/monoclonal antibody, shaken at room temperature is incubated for a period of time, removes remaining antibody-solutions, then use PBST clean twice, finally the immunomagnetic beads of acquisition are stored in BSA confining liquid and be stored in 4 DEG C it is spare.

The method of step (2) the immunomagnetic ca pture salmonella typhimurium is as follows:

Step (1) resulting immunomagnetic beads are mixed to incubation a period of time, shape with the sample containing salmonella typhimurium At immunomagnetic beads-salmonella typhimurium compound.

SERS is combined with the side of highly sensitive detection salmonella typhimurium based on immune HCR the present invention also provides a kind of Method, detecting step are as follows:

(1) aptamers for causing sequence modification are added in Xiang Shangshu immunomagnetic beads-salmonella typhimurium compound, incubate After educating a period of time, immunomagnetic beads-salmonella typhimurium-aptamers compound is formed, while exposing initiation sequence；So The probe H1 and H2 of hairpin structure are added afterwards, is incubated for a period of time, extends the double-stranded DNA with notch of different length, most End form at " immunomagnetic beads-salmonella typhimurium-aptamers HCR extension products " compound；

(2) appropriate immunomagnetic beads-salmonella typhimurium-aptamers HCR extension products compound is taken, excess DAPI is added (Raman signal molecule) is protected from light after being incubated for a period of time, nano silver colloid is added, and is protected from light after being incubated for a period of time, carries out Raman Measurement.

The above-mentioned sample containing salmonella typhimurium may include food liquid sample such as milk, meat soup etc., solid-state food Sample such as meat, birds, beasts and eggs etc..

Innovation of the invention is, modifies one section of initiation Sequence composition aptamer sensor to aptamers, is allowed to both Can specific recognition and combine target bacterium, and can expose cause sequence to cause hybridization chain reaction, put as a signal Greatly；Combine surface enhanced Raman scattering techniques simultaneously, realizes that signal is further amplified and exports；It finally can be highly sensitive and rapidly The salmonella typhimurium in food samples is detected, for ensuring food safety and preventing the generation of food origin disease with great Meaning.

Detailed description of the invention

Fig. 1 is the characterization result of the immunomagnetic beads of preparation.

Fig. 2 is the result of embodiment immunomagnetic ca pture salmonella typhimurium.

Fig. 3 is the ultraviolet-visible absorption spectroscopy and grain size distribution of the nano silver colloid of preparation.

Fig. 4 is the result of HCR condition optimizing.

The raman spectra and the standard curve between corresponding SERS intensity that Fig. 5 is each gradient target bacterium.

Fig. 6 is the specificity verification result of this method.

Table 1 is this method compared with colony counting method.

Specific embodiment

Based on immune HCR joint SERS with the method for highly sensitive and quick detection salmonella typhimurium, including following step It is rapid:

(1) take appropriate carboxyl magnetic bead, first with MES solution clean 2~3 times with remove it is former save liquid, then with Fresh EDC solution shaken at room temperature half an hour, with activated carboxyl group.

(2) the carboxyl magnetic bead after taking appropriate activation, the anti-salmonella typhimurium after PBS buffer solution dilution is added are polyclonal Antibody, shaken at room temperature are incubated for a period of time.

(3) extra antibody-solutions are removed, then are cleaned 2 times with PBST, immunomagnetic beads obtained are stored in BSA confining liquid In, 4 DEG C save backup.

(4) gradient dilution salmonella typhimurium by the immunomagnetic beads after closing and contains the sample of salmonella typhimurium Product mixing, incubation at room temperature a period of time, adaptive immune magnetic bead-target bacterium compound.

(5) aptamers for causing sequence modification are added into immunomagnetic beads-target bacterium compound, are incubated under the conditions of 37 DEG C For a period of time, unbonded aptamers are washed away with PBST.

(6) the probe H1 and H2 of hairpin structure, 37 DEG C of items are added into immunomagnetic beads-target bacterium-aptamers compound It is incubated for a period of time under part, washes away unreacted probe with PBST.

(7) excess DAPI Raman signal is added into immunomagnetic beads-target bacterium-aptamers HCR extension products compound Molecule is incubated for a period of time under the conditions of being protected from light.

(8) proper amount of nano silver colloid system is added, after being incubated for a few minutes under the conditions of being protected from light, takes appropriate amount of sample in glass plate On, SERS measurement is carried out after background correction, obtains standard curve.

(9) other gram-positive bacterias, Gram-negative bacteria, actinomyces and bacteria control group is separately taken to make comparisons, detection step Suddenly with above-mentioned salmonella typhimurium, to determine the special implementations of this method.

(10) certain density salmonella typhimurium is added into food samples, obtains mark-on sample, determines this method With the rate of recovery of colony counting method.

Following embodiment further illustrates the contents of the present invention, but is not limitation of the invention.Without departing substantially from of the invention real In the case where matter, step of the present invention or condition are modified, all belonged to the scope of the present invention.