CN101949929A - Method for using stranded DNA aptamer to detect Lysteria monocytogenes - Google Patents
Method for using stranded DNA aptamer to detect Lysteria monocytogenes Download PDFInfo
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- CN101949929A CN101949929A CN2010102748165A CN201010274816A CN101949929A CN 101949929 A CN101949929 A CN 101949929A CN 2010102748165 A CN2010102748165 A CN 2010102748165A CN 201010274816 A CN201010274816 A CN 201010274816A CN 101949929 A CN101949929 A CN 101949929A
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Abstract
The invention relates to a method for using a stranded DNA aptamer to detect Lysteria monocytogenes. An experimental group, a blank control group, a negative control group and a positive control group are set at the same time; after each group is subject to measurement of denaturation, centrifugal layering, coloration and absorbance, the result of the experimental group is judged to obtain the final detection result. The invention has the advantages of simple steps and explicit criterions, short time consumption, high efficiency and relatively lower cost.
Description
[technical field]
The present invention relates to the detection method of a kind of pathogen, relate in particular to a kind of method of using the fit detection monokaryon of single stranded DNA hyperplasia listeria spp.
[background technology]
Monokaryon hyperplasia listeria spp (Listeria monocytogenes) is a kind of pathogen of zoonosis, mainly shows as septicemia, meningitis and monocytosis after the people infects.It is widespread in nature, be difficult for by freeze thawing, can tolerate higher osmotic pressure, but growth and breeding still in 4 ℃ environment, the monokaryon hyperplasia listeria spp that exists in the food has danger to human beings'health safety, and China classifies it as 21 century and Chinese's hygiene and health had one of 12 kinds of pathogenic microorganisms of significant impact.Traditional detection method need increase bacterium, bacterium colony separation and biochemistry and serology identification experiment repeatedly, cost height, step complexity, length consuming time.(a kind of technological means of new energy specific detection target molecule has become possibility for systematic evolution of ligandsby exponential enrichment, the SELEX) rise of technology along with the phyletic evolution of index concentration aglucon.
Fit (aptamer) etymology is to fit in Latin language aptus, mean " being fit to ", be called adaptive son again, it is small molecule DNA that combines with the target molecule specificity or the RNA fragment that screens by the SELEX process from an external synthetic random oligonucleotide library.It has, and storage capacity is big, advantages such as the target molecule scope is wide, affinity is high, high specificity.At present, this technology has been successfully applied to the screening of many target molecules, as: protein, nucleotide, amino acid, organism, even metallic ion.But it is pathogenic microorganism is very few as the relevant report of target molecule screening.
For these reasons, the applicant has published the article of " screening that monokaryon hyperplasia listeria spp is fit and structure analysis " by name on " Food Science ", it has disclosed and has adopted the SELEX technology that the random dna library of external synthetic 78 bases is screened and obtain to have with monokaryon hyperplasia listeria spp the fit group's of single stranded DNA that the high-affinity specificity combines method, and disclose and form wherein 32 base sequences that single stranded DNA is fit of this fit group, for certain basis is established in fit follow-up study.
[summary of the invention]
Technical matters to be solved by this invention is to provide a kind of method of using the fit detection monokaryon of single stranded DNA hyperplasia listeria spp, and not only step is simple, criterion is clear and definite, and weak point consuming time, efficient height, cost are relatively low.
The present invention solves the problems of the technologies described above by the following technical programs: a kind of method of using the fit detection monokaryon of single stranded DNA hyperplasia listeria spp, a kind of method of using the fit detection monokaryon of single stranded DNA hyperplasia listeria spp, it is characterized in that: set experimental group, blank group, negative control group and positive controls simultaneously, concrete operations are as follows:
Step 100:
Experimental group:
A. in containing the first fit test tube of single stranded DNA, add 500 μ L and select damping fluid to be placed on heat denatured under 95 ℃ of temperature, in ice cube, carry out ice bath 10min behind the heat denatured 5min, and the amount that wherein single stranded DNA is fit is defined as 20-35 μ L;
B. get enrichment liquid to be checked in first centrifuge tube, and be placed on and carry out centrifugal layering on the centrifugation apparatus, and the amount of this enrichment liquid to be checked is defined as 500-1000 μ L;
C. will through the beds of precipitation in second centrifuge tube of B step process with evenly mix through the solution in first test tube of A step process, and this mixed liquor placed under the room temperature place 30min;
Blank group: after the repeated experiments group A step first test tube is placed 30min under room temperature;
Negative control group: the A of repeated experiments group, B, C step, and in the B step, enrichment liquid to be checked is changed into the bacterium liquid of non-purpose bacterium;
Positive controls: the A of repeated experiments group, B, C step, and in the B step change enrichment liquid to be checked into the bacterium liquid of purpose bacterium, i.e. monokaryon hyperplasia listeria spp bacterium liquid;
Step 200: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The mixed liquor of gained in the step 100 is moved in second centrifuge tube, and second centrifuge tube placed carry out high speed centrifugation on the centrifugation apparatus and make the mixed liquor layering; Add 100 μ L again and react with the alkaline phosphatase of anti digoxin antibody mark, and should dilute through selecting damping fluid with alkaline phosphatase of anti digoxin antibody mark, extension rate is 15000 times;
Step 300: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
After 10min is carried out in the reaction for the treatment of step 200, second centrifuge tube is carried out the high speed centrifugation layering once more; Centrifugal end adds 500 μ L selection damping fluid after abandoning supernatant again, stirs by concussion and makes mixed liquor suspension in second centrifuge tube;
Step 400: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The high speed centrifugation layering is carried out in the suspension of step 300 gained night; Abandon supernatant after the centrifugal end, and will precipitate immigration second test tube, repeated washing second centrifuge tube three times, and each cleansing solution moves into second test tube; Then add 100 μ L para-nitro-pheneye phosphate solution in second test tube and carry out chromogenic reaction, adding 100 μ L 2mol/L NaOH make this reaction terminating behind the chromogenic reaction 30min;
Step 500: adopt microplate reader in carrying out absorbance measurement under the 405nm wavelength respectively the solution after each group chromogenic reaction termination: measure between the absorbance of gained between blank group and negative control group if experimental group is measured the absorbance of gained, then do not contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group; If the absorbance that experimental group is measured gained is measured between negative control group and positive controls between the absorbance of gained or measure the absorbance of gained greater than positive controls, then contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group.
Further, the prescription of described selection damping fluid is: add the sterilized PBS of 1000mL in the solution of 1mL Tween-20, and the pH value of this PBS is 7.4.
Further, described non-purpose bacterium is a salmonella.
The beneficial effect that the present invention uses the method for the fit detection monokaryon of single stranded DNA hyperplasia listeria spp is: not only step is simple, criterion is clear and definite, and weak point consuming time, efficient height, is a kind of detection method quickly and easily
[description of drawings]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the process flow diagram that the present invention uses experimental group in the method for the fit detection monokaryon of single stranded DNA hyperplasia listeria spp.
[embodiment]
See also Fig. 1, the present invention uses the method for the fit detection monokaryon of single stranded DNA hyperplasia listeria spp and sets experimental group, blank group, negative control group and positive controls simultaneously, and its concrete operations are as follows:
Step 100:
Experimental group:
A. in containing the first fit test tube of single stranded DNA, add 500 μ L and select damping fluid to be placed on heat denatured under 95 ℃ of temperature, in ice cube, carry out ice bath 10min behind the heat denatured 5min, and the amount that wherein single stranded DNA is fit is defined as 20-35 μ L;
B. get enrichment liquid to be checked in first centrifuge tube, and be placed on and carry out centrifugal layering on the centrifugation apparatus, its centrifugal speed can be made as 12000r/min, and centrifugation time is made as 10min, and the amount of described enrichment liquid to be checked is defined as 500-1000 μ L;
C. will through the beds of precipitation in second centrifuge tube of B step process with evenly mix through the solution in first test tube of A step process, and this mixed liquor placed under the room temperature place 30min;
Blank group: after the repeated experiments group A step first test tube is placed 30min under room temperature;
Negative control group: the A of repeated experiments group, B, C step, and in the B step, change enrichment liquid to be checked the bacterium liquid of non-purpose bacterium into, adopt salmonella as non-purpose bacterium in the present embodiment;
Positive controls: the A of repeated experiments group, B, C step, and in the B step change enrichment liquid to be checked into the bacterium liquid of purpose bacterium, i.e. monokaryon hyperplasia listeria spp bacterium liquid;
Step 200: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The mixed liquor of gained in the step 100 is moved in second centrifuge tube, and second centrifuge tube placed carry out high speed centrifugation on the centrifugation apparatus and make the mixed liquor layering; Add 100 μ L again and react with the alkaline phosphatase of anti digoxin antibody mark, and should dilute through selecting damping fluid with alkaline phosphatase of anti digoxin antibody mark, extension rate is 15000 times;
Step 300: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
After 10min is carried out in the reaction for the treatment of step 200, second centrifuge tube is carried out the high speed centrifugation layering once more; Centrifugal end adds 500 μ L selection damping fluid after abandoning supernatant again, stirs by concussion and makes mixed liquor suspension in second centrifuge tube;
Step 400: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The high speed centrifugation layering is carried out in the suspension of step 300 gained night; Abandon supernatant after the centrifugal end, and will precipitate immigration second test tube, repeated washing second centrifuge tube three times, and each cleansing solution moves into second test tube; Then add 100 μ L para-nitro-pheneye phosphate solution in second test tube and carry out chromogenic reaction, adding 100 μ L 2mol/L NaOH make this reaction terminating behind the chromogenic reaction 30min;
Step 500: adopt microplate reader in carrying out absorbance measurement under the 405nm wavelength respectively the solution after each group chromogenic reaction termination: measure between the absorbance of gained between blank group and negative control group if experimental group is measured the absorbance of gained, then do not contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group; If the absorbance that experimental group is measured gained is measured between negative control group and positive controls between the absorbance of gained or measure the absorbance of gained greater than positive controls, then contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group.
Wherein, related high speed centrifugation in step 200, step 300, the step 400, its centrifugal speed all can be made as 13000r/min, and centrifugation time all can be made as 10min; The prescription of the selection damping fluid in this method is to add the sterilized PBS of 1000mL in the solution of 1mL Tween-20, and the pH value of this PBS is 7.4.
The present invention uses the method for the fit detection Listeria monocytogenes of single stranded DNA and compares with traditional detection method, and not only step is simple, criterion is clear and definite, and weak point consuming time, efficient height, cost are relatively low, are a kind of quickly and easily detection methods.
Claims (3)
1. method of using the fit detection monokaryon of single stranded DNA hyperplasia listeria spp, it is characterized in that: set experimental group, blank group, negative control group and positive controls simultaneously, concrete operations are as follows:
Step 100:
Experimental group:
A. in containing the first fit test tube of single stranded DNA, add 500 μ L and select damping fluid to be placed on heat denatured under 95 ℃ of temperature, in ice cube, carry out ice bath 10min behind the heat denatured 5min, and the amount that wherein single stranded DNA is fit is defined as 20-35 μ L;
B. get enrichment liquid to be checked in first centrifuge tube, and be placed on and carry out centrifugal layering on the centrifugation apparatus, and the amount of this enrichment liquid to be checked is defined as 500-1000 μ L;
C. will through the beds of precipitation in second centrifuge tube of B step process with evenly mix through the solution in first test tube of A step process, and this mixed liquor placed under the room temperature place 30min;
Blank group: after the repeated experiments group A step first test tube is placed 30min under room temperature;
Negative control group: the A of repeated experiments group, B, C step, and in the B step, enrichment liquid to be checked is changed into the bacterium liquid of non-purpose bacterium;
Positive controls: the A of repeated experiments group, B, C step, and in the B step change enrichment liquid to be checked into the bacterium liquid of purpose bacterium, i.e. monokaryon hyperplasia listeria spp bacterium liquid;
Step 200: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The mixed liquor of gained in the step 100 is moved in second centrifuge tube, and second centrifuge tube placed carry out high speed centrifugation on the centrifugation apparatus and make the mixed liquor layering; Add 100 μ L again and react with the alkaline phosphatase of anti digoxin antibody mark, and should dilute through selecting damping fluid with alkaline phosphatase of anti digoxin antibody mark, extension rate is 15000 times;
Step 300: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
After 10min is carried out in the reaction for the treatment of step 200, second centrifuge tube is carried out the high speed centrifugation layering once more; Centrifugal end adds 500 μ L selection damping fluid after abandoning supernatant again, stirs by concussion and makes mixed liquor suspension in second centrifuge tube;
Step 400: described experimental group, blank group, negative control group and positive controls are carried out following operation respectively:
The high speed centrifugation layering is carried out in the suspension of step 300 gained night; Abandon supernatant after the centrifugal end, and will precipitate immigration second test tube, repeated washing second centrifuge tube three times, and each cleansing solution moves into second test tube; Then add 100 μ L para-nitro-pheneye phosphate solution in second test tube and carry out chromogenic reaction, adding 100 μ L 2mol/L NaOH make this reaction terminating behind the chromogenic reaction 30min;
Step 500: adopt microplate reader in carrying out absorbance measurement under the 405nm wavelength respectively the solution after each group chromogenic reaction termination: measure between the absorbance of gained between blank group and negative control group if experimental group is measured the absorbance of gained, then do not contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group; If the absorbance that experimental group is measured gained is measured between negative control group and positive controls between the absorbance of gained or measure the absorbance of gained greater than positive controls, then contain monokaryon hyperplasia listeria spp in the enrichment liquid to be checked of experimental group.
2. the method for the fit detection monokaryon of application single stranded DNA as claimed in claim 1 hyperplasia listeria spp, it is characterized in that: the prescription of described selection damping fluid is: add the sterilized PBS of 1000mL in the solution of 1mL Tween-20, and the pH value of this PBS is 7.4.
3. the method for the fit detection monokaryon of application single stranded DNA as claimed in claim 1 hyperplasia listeria spp, it is characterized in that: described non-purpose bacterium is a salmonella.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676506A (en) * | 2012-05-30 | 2012-09-19 | 曹际娟 | Mononuclear cell proliferation listeria monocytogenes nucleic acid standard sample and establishing method and application thereof |
| WO2013094741A1 (en) * | 2011-12-21 | 2013-06-27 | Ｎｅｃソフト株式会社 | Dna molecules binding to listeria monocytogenes and use of same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665821A (en) * | 2008-09-01 | 2010-03-10 | 中国人民解放军军事医学科学院基础医学研究所 | Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665821A (en) * | 2008-09-01 | 2010-03-10 | 中国人民解放军军事医学科学院基础医学研究所 | Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘丰伟: "体外筛选铜绿假单胞菌适体的研究及初步应用", 《中国优秀硕士学位论文全文数据库》, no. 1, 15 January 2008 (2008-01-15), pages 13 - 17 * |
| 陈敏: "单核增生李斯特氏菌适体的筛选与结构分析", 《食品科学》, vol. 30, no. 19, 1 October 2009 (2009-10-01), pages 197 - 199 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013094741A1 (en) * | 2011-12-21 | 2013-06-27 | Ｎｅｃソフト株式会社 | Dna molecules binding to listeria monocytogenes and use of same |
| CN102676506A (en) * | 2012-05-30 | 2012-09-19 | 曹际娟 | Mononuclear cell proliferation listeria monocytogenes nucleic acid standard sample and establishing method and application thereof |
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Application publication date: 20110119 |