CN103233078A - Multi-gene detection method of Listeria monocytogenes based on quantum dot/graphene oxide nanometer platform - Google Patents
Multi-gene detection method of Listeria monocytogenes based on quantum dot/graphene oxide nanometer platform Download PDFInfo
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Abstract
The invention discloses a multi-gene detection method of Listeria monocytogenes based on a quantum dot/graphene oxide nanometer platform and belongs to the technical field of gene nanometer detection. The multi-gene detection method of Listeria monocytogenes comprises the following steps of: selecting two or more than two genes of the Listeria monocytogenes as target points of genetic detection and designing two pairs or more than two pairs of genetic PCR (Polymerase Chain Reaction) amplimers according to the analysis result of a gene conserved area; amplifying two or more than two genetic special sequences at the same time according to the principle of LATE-PCR to obtain a corresponding single stranded amplification product; then, hybridizing the single stranded amplification product with a quantum dot fluorescence probe; and finally, quenching and removing the non-hybridized quantum dot probe by using graphene oxide. When the target gene does not exist, all quantum dot fluorescence probes are quenched. On the contrary, when the target gene exists, corresponding fluorescent signals are obtained. The detection method has high detection reliability to Listeria monocytogenes.
Description
Technical field
The invention belongs to gene nanometer detection technical field, particularly based on the polygenic detection method of Listeria monocytogenes of quantum dot/stannic oxide/graphene nano platform.
Background technology
Food-borne pathogens is one of major reason that influences food safety, the serious threat human health, and bring enormous economic loss to human society.According to U.S. CDC, the case that the U.S. is caused by food pathogenic every year has 7600 examples, wherein dead 5000 examples.Therefore, perfect testing food pathogenic method system has become the important measures that food safety is supervised in countries in the world.Food-borne pathogens source is various, can breeding fast in food, can in severe environment, survive, this has brought huge challenge for detection, prevention and treatment of food-borne pathogens, and food safety is constituted directly threat.Therefore, establishing and improve the food-borne pathogens detection architecture is significant for food safety.
Traditional food-borne pathogens detection method has: conventional detection method, immune partition method, the Microscopic inspection etc. cultivated, these methods are with low cost, can provide quantitatively and sxemiquantitative information.But there are shortcomings such as length consuming time, sensitivity are not high, specificity is bad, plant and instrument costliness.In recent years, along with molecular biological develop rapidly, a series of gene Fast Detection Technique based on nucleic acid level emerge.At present, the gene method for quick mainly includes polymerase chain reaction (PCR) method, nucleic acid constant-temperature amplification technology, Taqman probe technique, LightCycler probe technique, combined probe technology, gene chip detecting technique, biosensor detection technique etc.These technology have changed original food-borne pathogens detection technique system completely, but there is deficiency separately in above method, exist signal to noise ratio lower, exist background signal to disturb, use shortcomings such as poisonous and harmful reagent, complex steps, and these technology are based on monogenic detection technique mostly, food-borne pathogens for highly infective, highly pathogenic and wide-scale distribution, only be conceived to monogenic detection technique and obviously can not obtain enough reliabilities, problems such as false negative and omission take place easily.
Summary of the invention
In order to overcome the deficiency that existing method exists, the object of the present invention is to provide a kind of Listeria monocytogenes polygene detection method based on quantum dot/stannic oxide/graphene nano platform.This detection method is by selecting two or more gene of Listeria monocytogenes as the target spot of gene test, to the detecting reliability height of Listeria monocytogenes.
Purpose of the present invention is achieved through the following technical solutions:
A kind of Listeria monocytogenes polygene detection method based on quantum dot/stannic oxide/graphene nano platform may further comprise the steps:
1) design of primers
Target gene is analyzed, determined the conserved regions of target gene; Utilize primer-design software respectively target gene to be carried out design of primers; During the design primer, keep the annealing temperature of restriction primer to be higher than 5～10 ℃ of excessive primers;
2) genome extracts
Bacterial classification added in the liquid nutrient medium cultivate; The Listeria monocytogenes of cultivating is carried out genome DNA extraction;
3) checking primer feasibility
By the PCR experiment, the amplification target gene; Amplified production is carried out electrophoresis detection, and the primer that the selection electrophoretic band is clear and specificity is good is as the amplimer of LATE-PCR;
4) optimize LATE-PCR
The many groups primer that the primer concentration ratio is positioned at the 1:50-100 scope carries out the PCR reaction, and product dyes detection with native polyacrylamide gel electrophoresis and silver, and the primer that the selection specificity is better, amplification efficiency is high is as the primer of LATE-PCR;
5) preparation of quantum dot fluorescence probe
Select the different quantum dot of two or more fluorescence emission peak for use, its surface is labelled streptavidin respectively; The quantum dot that surface markers is had a Streptavidin respectively with 5' DNA probe labeled with biotin mixing, lucifuge is hatched, and makes surface markers that quantum dot and the abundant combination of 5' DNA probe labeled with biotin of Streptavidin be arranged, and forms the quantum dot fluorescence probe; Make free dna probe and quantum dot fluorescence probe separates by the molecular sieve ultrafiltration again, the quantum dot fluorescence probe after separating is dissolved in the phosphate buffered saline buffer;
6) polynary LATE-PCR experiment
Add primer according to the primer ratio that step 4) is optimized, set up the LATE-PCR reaction system, amplifying polygenic single stranded product simultaneously, and suitably adjust the primer proportioning between different genes, make heterogeneic amplification efficiency consistent;
7) hybrid experiment
Get the polynary LATE-PCR amplified production phase mixing in the quantum dot fluorescence probe for preparing in the step 5) and the step 6), hybridize; Then, get the quantum dot fluorescence probe that an amount of graphene oxide cancellation is not hybridized, dissociated, use the spectrophotometer detection signal at last.
Further, adopt TIANamp Bacteria DNA kit test kit that Listeria monocytogenes is carried out genome DNA extraction step 2).
Further, the quantum dot that surface markers has a Streptavidin in the step 5) respectively with the ratio mixing of 5' DNA probe labeled with biotin in 1:30, lucifuge is hatched 30min.
Further, the concentration of the quantum dot fluorescence probe after the separation is 15nM in the step 5).
Further, quantum dot fluorescence probe and polynary LATE-PCR amplified production are hybridized 30min in 37 ℃ in the step 7).
With respect to prior art, beneficial effect of the present invention is as follows:
1. utilize graphene oxide absorption single-chain nucleic acid and can cancellation the principle of the fluorescent marker of mark on it, organically principle and the quantum dot/stannic oxide/graphene nano platform with polynary LATE-PCR combines, and detects the polygenic purpose of Listeria monocytogenes to reach.
2. the efficient of LATE-PCR amplification used in the present invention and quantum dot detection technique is all very high, thereby the sensitivity of measuring is very high.
3. the present invention has avoided conventional P CR detection technique after amplification procedure is finished, and also needs to utilize means such as electrophoresis detection consuming time, and testing process is simpler, fast.
4. the present invention has realized that the polygene of Listeria monocytogenes detects, and has overcome when detecting the Listeria monocytogenes of highly infective, highly pathogenic and wide-scale distribution the shortcoming of reliability deficiency.
Description of drawings
Describe the present invention below in conjunction with accompanying drawing.
Fig. 1 is polynary LATE-PCR amplified production electrophorogram.
Fig. 2 is the hlyA gene test sensitivity figure as a result of Listeria monocytogenes.
Fig. 3 is the iap gene test sensitivity figure as a result of Listeria monocytogenes.
Fig. 4 is Listeria monocytogenes detection specificity figure as a result.
Embodiment
The Listeria monocytogenes polygene detection method that the present invention is based on quantum dot/stannic oxide/graphene nano platform may further comprise the steps:
1) design of primers
Utilize the conserved regions of the target gene of DNAman to analyze, determine the conserved regions of target gene.Utilize primer-design software Primer Premier respectively hlyA and iap gene to be carried out design of primers, the sequence of designing is bought from Shanghai English fine horse, and the purifying mode is selected iDSL.Primer sequence sees the following form:
Table 1.hlyA and iap gene LATE-PCR primer sequence
| The primer title | Primer sequence | Theoretical annealing temperature |
| Hly A limits primer | GCTGCCGTAAGTGGGAAATCTGTCTCAG | 70.4°C |
| The excessive primer of hly A | ATGATTTGAACTTCATCTTTTGC | 59.9°C |
| Iap1 limits primer | AACAAGCTGCACCTGCTGCAGA | 70.3°C |
| The excessive primer of iap2 | CTTTTGACAGCGTGTGTAGT | 61.9°C |
2) genome extracts
(1) increases bacterium
Experimental wares such as the beaker that the laboratory is used, Erlenmeyer flask, culture dish are with the ultrasonic 20min of ultrasonic machine, and are with the tap water flushing, clean with distilled water flushing more then, put into baking oven and dry.
According to beef-protein medium prescription (as table 2) configuration liquid nutrient medium, mix up the pH value respectively after, packing, 30min then sterilizes.In Bechtop, the bacterial classification of getting 1mL adds in the liquid nutrient medium mixing, 37 ℃ of constant-temperature shaking culture 12h.
Table 2. beef-protein medium prescription
| The Pepton(peptone) | 0.5g |
| Beef extract(extractum carnis) | 0.3g |
| NaCl | 0.5g |
| Distilled water(distilled water) | 100mL |
| pH | 7.0-7.2 |
(2) extraction of Listeria monocytogenes genomic dna
Adopt TIANamp Bacteria DNA kit test kit that Listeria monocytogenes is carried out genome DNA extraction.
Operation steps is as follows: get inoculum 1ml, and centrifugal one minute of 1000rpm, supernatant exhausts as far as possible; Add 200 μ l damping fluid GA in the bacterial sediment, vibrating to thalline thoroughly suspends; Xiang Guanzhong adds 20 μ l Proteinase K solution, mixing; Add 220 μ l damping fluid GB (improvement phosphate buffered saline buffer), vibration 15s places 10min for 70 ℃, and the solution strain is limpid, and is brief centrifugal to remove the globule of cap wall; Add 220 μ l dehydrated alcohols, the mixing 15s that fully vibrates, flocks may appear in this moment, and is brief centrifugal to remove the globule of cap wall; Previous step gained solution and flocks are all added among the adsorption column CB3 (adsorption column is put into collection tube), and the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; In adsorption column CB3, add please check to have added dehydrated alcohol whether earlier before 500 μ l damping fluid GD(use), the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; In adsorption column CB3, add please check to have added dehydrated alcohol whether earlier before 700 μ l rinsing liquid PW(use), the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; In adsorption column CB3, add please check to have added dehydrated alcohol whether earlier before 500 μ l rinsing liquid PW(use), the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; Adsorption column CB3 is put back in the collection tube, and the centrifugal 2min of 12000rpm outwells waste liquid; Place room temperature to place several minutes adsorption column CB3, thoroughly to dry rinsing liquid remaining in the sorbing material; Adsorption column CB3 is changed in the clean centrifuge tube, and to the middle part of adsorption film unsettled Dropwise 5 0-200 μ l elution buffer TE, room temperature is placed 2-5min, and the centrifugal 2min of 12000rpm collects solution in the centrifuge tube.
3) checking primer feasibility
By conventional PCR experiment, the amplification target gene, PCR reaction system and consumption thereof are as shown in table 3, and wherein, the TapDNA polysaccharase is bought from Bao Bio-Engineering Company; Amplified production detects by agarose gel electrophoresis, verifies its feasibility.The primer that the selection electrophoretic band is clear and specificity is good is as the amplimer of LATE-PCR.
Table 3.PCR reaction system
| TaKaRa?Taq（5U/uL） | 0.25uL |
| 10ⅩPCR?buffer（Mg 2+plus） | 5uL |
| Each 2.5mM of dNTP Mixture() | 4uL |
| Template DNA | 1uL |
| Restriction primer (20uM) | 1uL |
| Excessive primer (20uM) | 1uL |
| Sterile purified water | 50uL |
4) optimize the LATE-PCR reaction system
Asymmetric pcr need be optimized the concentration difference between primer, generally controls between 1:50-100.The primer concentration ratio adopts 1:40,1:60,1:80,1:100,1:120 respectively, carries out the PCR reaction respectively.Product dyes detection with native polyacrylamide gel electrophoresis and silver, and the primer that the selection specificity is better, amplification efficiency is high is as the primer of LATE-PCR.
5) preparation of quantum dot fluorescence probe
Select the different quantum dot of two or more fluorescence emission peak for use, its surface is labelled streptavidin respectively, dna probe adopts the 5' biotin labeling, the quantum dot that surface markers is had a Streptavidin respectively with the ratio mixing of 5' DNA probe labeled with biotin in 1:30, lucifuge is hatched 30min, make surface markers that quantum dot and the abundant combination of 5' DNA probe labeled with biotin of Streptavidin be arranged, form the quantum dot fluorescence probe, make free dna probe and quantum dot fluorescence probe separates by the molecular sieve ultrafiltration again, obtain purer quantum dot fluorescence probe.The quantum dot fluorescence probe is dissolved in the phosphate buffered saline buffer (PBs), and it is standby to be stored in 4 ℃ of refrigerators.
6) polynary LATE-PCR experiment
Add primer according to the primer ratio that step 4) is optimized, set up the LATE-PCR reaction system, amplifying polygenic single stranded product simultaneously, and suitably adjust the primer proportioning between different genes, make heterogeneic amplification efficiency consistent, it is standby that the product of amplification is put-20 degrees centigrade of refrigerators.Experimental result as shown in Figure 1.
7) hybrid experiment
Get the polynary LATE-PCR amplified production phase mixing in the quantum dot fluorescence probe (final concentration is 15nM) for preparing in the step 5) and the step 6), 37 ℃ of hybridization 30min, then, get the quantum dot fluorescence probe that an amount of graphene oxide cancellation is not hybridized, dissociated, use phosphorescence spectrophotometer detection signal at last.The genome concentration gradient is set respectively is: 1ng/uL, 100pg/uL, 10pg/uL, 1pg/uL, 0.1pg/uL do sensitivity experiment, and experimental result as shown in Figures 2 and 3.
Extract Listeria monocytogenes (Listeria monocytogenes respectively, CMCC54007) genome, intestinal bacteria (E.coli O157:H7, GW1.2020) genome, Salmonellas (Salmonella enterica, CMCC50040) genome records fluorescent signal as shown in Figure 4 according to above method.As shown in Figure 4, when the intestinal bacteria that have equal in quality concentration in the sample respectively, Salmonellas and Listeria monocytogenes genome, when having only the goal gene of existence group, just can obtain higher fluorescent signal, can judge that thus method of the present invention detects Listeria monocytogenes and has extraordinary specificity.
The above embodiment has only expressed one embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. the Listeria monocytogenes polygene detection method based on quantum dot/stannic oxide/graphene nano platform is characterized in that, may further comprise the steps:
1) design of primers
Target gene is analyzed, determined the conserved regions of target gene; Utilize primer-design software respectively target gene to be carried out design of primers; During the design primer, keep the annealing temperature of restriction primer to be higher than 5～10 ℃ of excessive primers;
2) genome extracts
Bacterial classification added in the liquid nutrient medium cultivate; The Listeria monocytogenes of cultivating is carried out genome DNA extraction;
3) checking primer feasibility
By the PCR experiment, the amplification target gene; Amplified production is carried out electrophoresis detection, and the primer that the selection electrophoretic band is clear and specificity is good is as the amplimer of LATE-PCR;
4) optimize LATE-PCR
The many groups primer that the primer concentration ratio is positioned at the 1:50-100 scope carries out the PCR reaction, and product dyes detection with native polyacrylamide gel electrophoresis and silver, and the primer that the selection specificity is better, amplification efficiency is high is as the primer of LATE-PCR;
5) preparation of quantum dot fluorescence probe
Select the different quantum dot of two or more fluorescence emission peak for use, its surface is labelled streptavidin respectively; The quantum dot that surface markers is had a Streptavidin respectively with 5' DNA probe labeled with biotin mixing, lucifuge is hatched, and makes surface markers that quantum dot and the abundant combination of 5' DNA probe labeled with biotin of Streptavidin be arranged, and forms the quantum dot fluorescence probe; Make free dna probe and quantum dot fluorescence probe separates by the molecular sieve ultrafiltration again, the quantum dot fluorescence probe after separating is dissolved in the phosphate buffered saline buffer;
6) polynary LATE-PCR experiment
Add primer according to the primer ratio that step 4) is optimized, set up the LATE-PCR reaction system, amplifying polygenic single stranded product simultaneously, and suitably adjust the primer proportioning between different genes, make heterogeneic amplification efficiency consistent;
7) hybrid experiment
Get the polynary LATE-PCR amplified production phase mixing in the quantum dot fluorescence probe for preparing in the step 5) and the step 6), hybridize; Then, get the quantum dot fluorescence probe that an amount of graphene oxide cancellation is not hybridized, dissociated, use the spectrophotometer detection signal at last.
2. Listeria monocytogenes polygene detection method according to claim 1 is characterized in that: step 2) in adopt TIANamp Bacteria DNA kit test kit that Listeria monocytogenes is carried out genome DNA extraction.
3. Listeria monocytogenes polygene detection method according to claim 1 is characterized in that: the quantum dot that surface markers has a Streptavidin in the step 5) respectively with the ratio mixing of 5' DNA probe labeled with biotin in 1:30, lucifuge is hatched 30min.
4. Listeria monocytogenes polygene detection method according to claim 3 is characterized in that: the concentration of the quantum dot fluorescence probe after separating in the step 5) is 15nM.
5. according to each described Listeria monocytogenes polygene detection method of claim 1 to 4, it is characterized in that: quantum dot fluorescence probe and polynary LATE-PCR amplified production are in 37 ℃ of hybridization 30min in the step 7).
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| CN103642924A (en) * | 2013-12-10 | 2014-03-19 | 华南师范大学 | Method for quickly identifying food pathogenic bacteria subtype based on asymmetric polymerase chain reaction (PCR) combined test strip platform and kit |
| CN103834727A (en) * | 2014-01-26 | 2014-06-04 | 浙江师范大学 | Preparation method of fluorescence probe and applications of the fluorescence probe |
| CN104328113A (en) * | 2014-10-12 | 2015-02-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence composition and application thereof in Listeria monocytogenes identification |
| CN105603055A (en) * | 2014-11-19 | 2016-05-25 | 中国科学院上海应用物理研究所 | Multiple polymerase chain reaction method and application thereof |
| CN104328113B (en) * | 2014-10-12 | 2017-01-04 | 中国疾病预防控制中心传染病预防控制所 | One group of nucleotide sequence and the application in Listeria monocytogenes is identified |
| RU2614111C1 (en) * | 2015-11-25 | 2017-03-22 | Федеральное государственное автономное образовательное учреждение высшего образования "Северо-Восточный федеральный университет имени М. К. Аммосова" | Method for point mutation diagnosing in native dna using graphene oxide |
| CN108949910A (en) * | 2018-06-08 | 2018-12-07 | 武汉博杰生物医学科技有限公司 | A kind of detection method and biosensor of high throughput miRNAs express spectra |
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| CN103642924A (en) * | 2013-12-10 | 2014-03-19 | 华南师范大学 | Method for quickly identifying food pathogenic bacteria subtype based on asymmetric polymerase chain reaction (PCR) combined test strip platform and kit |
| CN103642924B (en) * | 2013-12-10 | 2016-03-02 | 华南师范大学 | Differentiate method and the test kit of food pathogenic hypotype fast in conjunction with test strip platform based on asymmetric pcr |
| CN103834727A (en) * | 2014-01-26 | 2014-06-04 | 浙江师范大学 | Preparation method of fluorescence probe and applications of the fluorescence probe |
| CN104328113A (en) * | 2014-10-12 | 2015-02-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence composition and application thereof in Listeria monocytogenes identification |
| CN104328113B (en) * | 2014-10-12 | 2017-01-04 | 中国疾病预防控制中心传染病预防控制所 | One group of nucleotide sequence and the application in Listeria monocytogenes is identified |
| CN105603055A (en) * | 2014-11-19 | 2016-05-25 | 中国科学院上海应用物理研究所 | Multiple polymerase chain reaction method and application thereof |
| CN105603055B (en) * | 2014-11-19 | 2020-06-05 | 中国科学院上海应用物理研究所 | Multiplex polymerase chain reaction method and application thereof |
| RU2614111C1 (en) * | 2015-11-25 | 2017-03-22 | Федеральное государственное автономное образовательное учреждение высшего образования "Северо-Восточный федеральный университет имени М. К. Аммосова" | Method for point mutation diagnosing in native dna using graphene oxide |
| CN108949910A (en) * | 2018-06-08 | 2018-12-07 | 武汉博杰生物医学科技有限公司 | A kind of detection method and biosensor of high throughput miRNAs express spectra |
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