AU2020205292B2 - Biosensor for detection of salmonella typhimurium and its application - Google Patents
Biosensor for detection of salmonella typhimurium and its application Download PDFInfo
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- AU2020205292B2 AU2020205292B2 AU2020205292A AU2020205292A AU2020205292B2 AU 2020205292 B2 AU2020205292 B2 AU 2020205292B2 AU 2020205292 A AU2020205292 A AU 2020205292A AU 2020205292 A AU2020205292 A AU 2020205292A AU 2020205292 B2 AU2020205292 B2 AU 2020205292B2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present disclosure provides a biosensor for the detection of Salmonella
typhimurium, belonging to the technical field of harmful microorganism detection. The
biosensor is composed of a carbon quantum dot (Td-CD) modified by the aptamer of
Salmonella typhimurium and a magnetic bead (Td-MB) modified by the aptamer of
Salmonella typhimurium. Among them, the aptamer of Salmonella typhimurium is
assembled with other nucleic acids to form a DNA tetrahedral structure through
sequence design, and then used to modify carbon quantum dots and magnetic beads,
respectively. The method of detecting Salmonella typhimurium by the biosensor of the
present invention is simple to operate, short in time, and does not require complex
pretreatment of the tested sample, with low detection cost.
2/2
300
2250 y 46.69x - 41.396
R2= 0.9987
200
150
100
P 50
0
0 1 2 3 4 5 6 7
Bacterial Concentration(log CFU-mL 1 )
Figure 2
60
50
40 Staphylococcus aureus
30 Listeria monocytogenes
Escherichia coli
20
E Salmonella typhimurium
10
0
Figure 3
Description
2/2
300
2250 y 46.69x - 41.396 R 2 = 0.9987 200
150
100
P 50
0 0 1 2 3 4 5 6 7 Bacterial Concentration(log CFU-mL 1
) Figure 2
60
50
40 Staphylococcus aureus
30 Listeria monocytogenes
Escherichia coli 20 E Salmonella typhimurium 10
0
Figure 3
[0001] Technical field
[0002] The present disclosure belongs to the technical field of detecting harmful
microorganisms, in particular to a biosensor for detecting Salmonella typhimurium
and its application.
[0003] Background
[0004] Salmonella typhimurium, a commonly used foodborne pathogen, is a
Gram-negative anaerobic bacterium that is widely used for food, water, and plants,
but the contaminated food and water matches gastroenteritis in mammals and humans.
Common symptoms of Salmonella typhimurium infection include high fever, cough,
diarrhea, abdominal cramps, and vomiting. Severe illness can lead to death, resulting
in significant losses in food safety and human health. So, it is very important to
accurately identify and detect Salmonella typhimurium.
[0005] At present, the detection methods of Salmonella typhimurium include
biological isolation and culture, enzyme-linked immunoassay, PCR molecular
detection and so on. Traditional biological isolation and culture method can obtain
accurate detection results, but the detection process takes a long time, taking four to
seven days. Enzyme linked immunoassay (ELISA) has high specificity and is
convenient and rapid, but the detection process is easily affected by pH change and
environmental temperature, etc., and the sensitivity is low, so it is difficult to meet the
sensitivity requirements of Salmonella typhimurium detection. PCR molecular detection method is expensive and has high professional requirements for operation, which is difficult to meet the actual detection requirements. Therefore, it is of great significance to provide a sensitive, rapid, and accurate detection method for
Salmonella typhimurium.
[0006] The composition of food samples is complex, so the detection of Salmonella
typhimurium in food is easily affected by the interference of food substrate. For this
purpose, magnetic separation technology is a good method. It uses magnetic
microspheres to identify specific molecules that bind to pathogenic bacteria in food,
and the complex formed is separated from other impurities under the effect of
magnetic force to rapidly enrich pathogenic bacteria.
[0007] Aptamer is a DNA or RNA sequences by screening, also known as "chemical
antibodies," as a new generation of recognition molecules, its low molecular weight
(15 to 50 base), aptamer has a fairly even higher affinity with protein antibody, and
compared with the traditional biological methods to obtain antibody, has the
advantages of easy synthesis and storage because of its strong specificity, high
affinity and good stability of biological molecules, no immunogenicity and toxicity
and is widely applied in the recognition and detection.
[0008] The DNA tetrahedral structure is an excellent DNA nanostructure formed by
assembling four different nucleic acids. The aptamer can be formed by assembling the
aptamer and the other three nucleic acid strands through sequence design. This
structure can overcome the shortcoming of the aptamer that is not easy to be fixed.
Quantum dots are superior to other luminescent materials in terms of small size, high
fluorescence intensity, stokes large displacement, anti-photobleaching, long fluorescence life and good biocompatibility. Since they are used as bioluminescent markers for living cells 20 years ago, they have attracted much attention. The accuracy and sensitivity can be improved by modifying the DNA tetrahedral structure on the surface of quantum dots.
[0009] Contents of the invention
[0010] Aiming at the existing problems in the existing technology, the first purpose of the invention is to design the synthetic DNA tetrahedral aptamer nanostructure for the specific and efficient identification of Salmonella typhimurium;
[0011] The second purpose of the invention is to synthesize non-toxic carbon nano-quantum dots, establish a signal amplification method based on self-assembled carbon quantum dots microspheres, and improve the detection sensitivity of Salmonella typhimurium;
[0012] The third purpose of this invention is the preparation of a magnetic separation quantum dot sandwich biosensor, in which regular tetrahedron DNA aptamer nanostructures serves as signal recognizer, self-assembled quantum dots carbon microspheres serves as fluorescent signal amplifier, and the method of the biosensor to detect Salmonella typhimurium, in order to solve the existing detection of Salmonella typhimurium technology of low sensitivity, detection methods, instruments and equipment requirements of higher complicated issues.
[0013] To achieve the above purposes, the invention adopts the following technical
scheme:
[0014] Abiosensor for detecting Salmonella typhimurium is composed of carbon
quantum dot Td-CD modified by aptamer of Salmonella typhimurium and magnetic
bead Td-MB modified by aptamer of Salmonella typhimurium;
[0015] DNA regular tetrahedron structure is formed through design Salmonella
typhimurium aptamer and other nucleic acid sequence assembly of DNA, and then
used to modify carbon quantum dots and magnetic beads respectively. The aptamer
DNA tetrahedron used to modify carbon quantum dots is Td-Aptamerl, and the
aptamer DNA tetrahedron used to modify magnetic beads is Td-Aptamer2.
[0016] Based on the above scheme, the nucleic acid sequence of Td-Aptamerl
described above is:
Alstrand: 5'-ACATTC CTAAGT CTG AAA CAT TACAGC TTG CTA CAC
CTT CTG CTT GCG AA-3' (SEQ ID NO:1)
B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT
AAT AGA TGC GAG GGT CCAATA C-3' (SEQ ID NO:3);
Cstrand: 5'-biotin-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG
GAA TCT ACT ATG GCG GCT CTT C-3' (SEQ ID NO:4);
D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT
CGT TTG TAT TGG ACC CTC GCA T-3' (SEQ ID NO:5) .
[0017] The nucleic acid sequence of Td-Aptamerl described above is:
A2strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC
GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3' (SEQ ID NO:2) ;
Bstrand: 5'-biotin-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGT
AATAGATGCGAGGGTCCAATAC-3' (SEQIDNO:3) ;
Cstrand: 5'-TCAACT GCC TGG TGA TAAAAC GAC ACT ACG TGG GAA
TCTACTATGGCGGCTCTTC-3' (SEQIDNO:4) ;
Dstrand: 5'-TTC AGA CTTAGGAAT GTG CTT CCC ACG TAG TGT CGT
TTGTATTGGACCCTCGCAT-3' (SEQIDNO:5)
[0018] Based on the above scheme, the preparation methods of Td-Aptamerl and
Td-Aptamer2 are as follows: the four nucleic acid strands of DNA tetrahedron
structure are mixed and dissolved in TE buffer solution at the same molarity,
respectively. After eddy mixing, the solution is heated to 95 °C for 5 min, and then
rapidly cooled in ice water for 10 min to 4 °C.
[0019] Based on the above scheme, the preparation methods of Salmonella
typhimurium aptamer modified carbon quantum dots, Td-CD, is:
Td-aptamerl and streptavidin -carbon quantum dot SA-CDs are added to PBS
buffer at a molal ratio of 4:1, violently shaken for 1 h at room temperature, rotated at
,000 rpm, centrifuged for 10 min, and the supernatant is discarded to obtain Td-CD
microspheres, which are then dispersed in PBS buffer to obtain Td-CD solution.
[0020] Based on the above scheme, the preparation method of streptavidin -carbon
quantum dot SA-CDs is:
5 mg amino-functionalized carbon quantum dots are dissolved in 1.5ml anhydrous ethanol and evenly dispersed by ultrasound. Streptavidin solution is added to the dots. After 15 min of ultrasound, the dots are shaken violently at room temperature for 3 h at a speed of 12,000 rpm, centrifuged for 20min, and the supernatant is discarded to obtain streptavidin modified carbon quantum dots.
[0021] Based on the above scheme, the preparation method of amino-functionalized carbon quantum dots is: 0.4g p-cyclodextrin is dissolved in 20 mL ultra-pure water and evenly dispersed by ultrasonic. It is transferred into a stainless-steel high-pressure reactor lined with Teflon and placed in an air-blast heating drying oven. The hydrothermal reaction is conducted at 200 °C for 6 h. After the reaction, the liquid is naturally cooled to room temperature and dialyzed in ultrafine water for 24 h in a dialysis bag. Finally, the product is rotated-steamed at 45 °C on a rotary evaporator, and the solvent is removed. The light-yellow powder is the amino functionalized carbon quantum dots prepared.
[0022] Based on the above scheme, the preparation method of the carbon quantum dot Td-MB modified by aptamer of Salmonella typhimurium is as follows:: 1 mg streptavidin functionalized magnetic beads and 1.6 M Td-Aptamer2 are mixed and dispersed in 500 mL Tris-HCl buffer solution for 30 min. Then the magnetic beads are separated from the system with a magnet, the supernatant is discarded, the magnetic beads are washed three times with 1 mL PBS buffer, and finally the magnetic beads are dispersed in PBS buffer, namely Td-MB solution.
[0023] The biosensor of Salmonella typhimurium is used to detect Salmonella typhimurium.
[0024] A detection kit for Salmonella typhimurium containing the biosensor.
[0025] The biosensor is used to detect Salmonella typhimurium through the
following steps:
(1) The Td-MB solution and Td-CD solution are incubated with the sample
solution to be tested at room temperature for 20 minutes, and the reaction is fully
combined by slow shaking. The first magnetic separation is performed and the
supernatant is discarded to obtain the MB-Salmonella typhimurium -CD complex.
(2) Re-suspension of MB-Salmonella typhimurium -CD complex with PBS,
Td-MB and Td-CD are disintegrated by mixing Biotin with 4 M concentration for 30
minutes. Then, the second magnetic separation is carried out to discard the magnetic
beads and retain the supernatant solution.
(3) The superfine solution obtained by fluorescence spectrophotometer scanning
step (2) and fluorescence emission spectrum at 470nm excitation wavelength are used
to measure the fluorescence intensity. The measured fluorescence intensity is
introduced into the standard curve and the concentration of Salmonella typhimurium
in the sample to be measured is calculated.
[0026] Advantages of the technical scheme of the invention:
[0027] The method of detecting Salmonella typhimurium by the biosensor of the
present invention is simple to operate and does not require complex pretreatment of
the tested sample, and the detection cost is low;
[0028] The biosensor of the invention can effectively amplify the signal of Salmonella typhimurium and can be used for detection of Salmonella typhimurium at low concentration. The sensitivity of the detection method of the invention is 6 CFU/mL;
[0029] The method of detecting Salmonella typhimurium with the biosensor of the present invention is short in time, the whole detection process is not more than one hour, and the detection speed is fast; and
[0030] The biosensor can be used for the detection of Salmonella typhimurium in food and the qualitative and quantitative analysis of Salmonella typhimurium can be realized.
[0031] Brief Description of the Drawings
[0032] Similar parts in the attached drawings are represented by the same attached drawing marks.
[0033] FIG. 1 is a schematic diagram of the detection principle of the present invention method;
[0034] FIG. 2 is detection results of Salmonella typhimurium at different concentrations; and
[0035] FIG. 3 is FIG. of detection results of different foodborne pathogenic bacteria.
[0036] Specific implementation method
[0037] In order to explain the invention more clearly, the following is combined with specific embodiments and further describes the invention in detail with reference to data. The following embodiments are intended only to exemplify the invention and not to limit the scope of the invention in any way.
[0038] Unless otherwise stated, terms used in the present invention generally have meanings normally understood by ordinary technicians in the field.
[0039] The detection principle of the biosensor of the invention is shown in FIG. 1, specifically as follows: (1) The four DNA strands Al, B, C and D are mixed and assembled into Td-Aptamerl, and thenTd-Aptamerl is self-assembled with SA-CD to form Td-CD microspheres; (2) The four DNA strands A2, B, C and D are mixed and assembled into Td-Aptamerl, and thenTd-Aptamer2 is self-assembled with SA-MB to form Td-MB; (3) Detection process
(O)Td-MB and Td-CD are added to the samples to make Td-MB and Td-CD bind with Salmonella typhimurium to form MB-Salmonella typhimurium -CD complex.
®Magnetic separation, MB-Salmonella typhimurium -CD complex is obtained by discarding supernatant;
®Adding Biotin, the free Biotin disintegrated Td-MB and Td-CD in the
MB-Salmonella typhimurium -CD complex;
®Magnetic separation, discard the magnetic beads and keep the supernatant
solution (a mixture of bacteria, quantum dots and DNA tetrahedral structure); and
®After the reaction, the solution is scanned by fluorescence spectrophotometer,
and the fluorescence emission spectrum at the excitation wavelength of 470 nm is
used to measure the fluorescence intensity. The measured fluorescence intensity is
introduced into the standard curve, and the concentration of Salmonella typhimurium
in the sample to be measured is calculated.
[0040] Description of the preferred embodiment
[0041] The aptamer specific of the invention acts on Salmonella typhimurium.
[0042] Aptamerl strand: 5'-CTGACA GAT TAAAAG TGG TTG GGCAAC TTC
TGC TTG CGA A -3'
[0043] Aptamerl strand: 5'-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC
ATT ATG ACA G -3'
[0044] Abiosensor for detecting Salmonella typhimurium is composed of carbon
quantum dot Td-CD modified by aptamer of Salmonella typhimurium and magnetic bead Td-MB modified by aptamer of Salmonella typhimurium;
[0045] DNA regular tetrahedron structure is formed through design Salmonella
typhimurium aptamer and other nucleic acid sequence assembly of DNA, and then
used to modify carbon quantum dots and magnetic beads respectively. The aptamer
DNA tetrahedron used to modify carbon quantum dots is Td-Aptamerl, and the
aptamer DNA tetrahedron used to modify magnetic beads is Td-Aptamer2.
[0046] 1.1 The preparation method of Td-Aptamer:
Al strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC
CTT CTG CTT GCG AA-3' (SEQ ID NO:1)
A2 strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC
GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3' (SEQ ID NO:2)
B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT
AAT AGA TGC GAG GGT CCAATA C-3' (SEQ ID NO:3) ;
C strand: 5'-biotin-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG
GAA TCT ACT ATG GCG GCT CTT C-3' (SEQ ID NO:4) ;
D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT
CGT TTG TAT TGG ACC CTC GCA T-3' (SEQ ID NO:5) .
OTake two 1.5mL EP tubes, Al, B, C, D, and A2, B, C, D DNA strands are
mixed and dissolved in 1 mL TE buffer solution (10 mM Tris, 1 mM EDTA, pH 8.0)
at 2 M concentration;
OThe solution is transferred to a metal bath and heated at 95 °C for 5 min;
OThe solution is rapidly cooled in ice water for 10 min until the solution
temperature dropped to 4 °C. The resulting solutions Td-Aptamerl and Td-Aptamer2 are stored at 4 °C for standby use respectively.
[0047] 1.2 The preparation method of SA-CD:
OThe preparation method of amino functionalization carbon quantum dots: 0.4g p-cyclodextrin is dissolved in 20 mL ultra-pure water and evenly dispersed
by ultrasonic. It is transferred into a stainless-steel high-pressure reactor lined with
Teflon and placed in an air-blast heating drying oven. The hydrothermal reaction is
conducted at 200 °C for 6 h. After the reaction, the liquid is naturally cooled to room
temperature and dialyzed in ultrafine water for 24 h in a dialysis bag. Finally, the
product is rotated-steamed at 45 °C on a rotary evaporator, and the solvent is removed.
The light-yellow powder is the amino functionalized carbon quantum dots prepared.
OThe preparation method of SA-CD: A 5 mg amino-functionalized carbon quantum dot is dissolved in 1.5 mL
anhydrous ethanol and evenly dispersed by ultrasonic. Streptavidin solution is added
to it. After 15 min of ultrasonic, it is shaken violently at room temperature for 3 h at a
speed of 12,000 rpm, centrifuged for 20min, and the supernatant is discarded to obtain
streptavidin modified carbon quantum dot. 1.0 mg of carbon quantum dots are
weighed and dispersed in 1 mL HEPES (10 mM, pH7.0) buffer solution, and the
SA-CD solution with a concentration of 1 mg- ml-1 is obtained, which is used
uniformly by ultrasound
[0048] 1.3 Td-Aptamerl and SA-CD self-assembly to form Td-CD microspheres:
0Td-Aptamerl 200 L and SA-CD 100 L are added into 1 mL PBS buffer at a molar ratio of 4:1, respectively;
©The mixture is placed on a vortex oscillator at room temperature and shaken
violently for 1 h;
©The high speed centrifuge is centrifuged at 5,000 rpm for 10 min. The supernatant is discarded to obtain Td-CD microspheres, which are dispersed in PBS buffer to obtain Td-CD solution for standby use.
[0049] 1.4 Td-Aptamer2 and SA-MB self-assembly to form Td-CD microspheres:
1 mg streptavidin functionalized magnetic beads and 1.6 M Td-Aptamer2 200 L are mixed and dispersed in 500 mL tris-HCl buffer (10m M Tris-Hcl, 1mM EDTA, IM NaCl, 0.01%-0.1% Tween-20), vortex for 30 min; ©After that, magnetic beads are separated from the system with a magnet, and the supernatant is discarded to obtain Td-MB magnetic beads; ©The Td-MB is washed with1 mL PBS buffer solution for three times, and finally the Td-MB is dispersed in PBS buffer solution, namely Td-MB solution.
[0050] Description of the preferred embodiment2
[0051] A method for the detection of Salmonella typhimurium by:
[0052] 2.1 Standard curve drawing: Six 1.5ml EP tubes are added with 1 mL of salmonella typhimurium free drinking water, and the concentrations of salmonella typhimurium solution are respectively added 10 CFU/mL, 102 CFU/mL, 10 CFU/mL, 104CFU/mL, 105 CFU/mL, 106 CFU/mL. Another 6 1.5mL EP tubes are added into 100 L bacterial solution, 1 mL Td-MB solution and 1 mL Td-CD solution for co-incubation at room temperature for 20 minutes, followed by slow shaking and full binding reaction; Magnetic frame separation, MB-Salmonella typhimurium -CD complex is obtained by discarding supernatant. The complex is suspended in 1.5 mL PBS and then mixed with 2 mL biotin molecule solution at a certain concentration for 30 minutes. Then, the magnetic beads are separated for the second time, and the supernatant solution is retained. Finally, the reaction solution is scanned with a fluorescence spectrophotometer and the fluorescence emission spectrum at the excitation wavelength of 470 nm is used to measure the fluorescence intensity, and the standard curve 2 is obtained. The detection limit of the biosensor is 6 CFU/mL.
[0053] 2.2 Sample testing
A 1.5 mL EP tube is added with 100 L of the sample to be tested. 1 mL of
Td-MB solution prepared by example 1 and 1 mL of Td-CD solution prepared by
example 1 are taken and incubated at room temperature for 20 minutes. The mixture
is shaken slowly and fully combined; Magnetic frame separation, MB-Salmonella
typhimurium -CD complex is obtained by discarding supernatant. The complex is
suspended in 1.5 mL PBS and then mixed with 2 mL biotin molecule solution at a
certain concentration for 30 minutes. Then, the magnetic beads are separated for the
second time, and the supernatant solution is retained. Finally, the reaction solution is
scanned with a fluorescence spectrophotometer and the fluorescence emission
spectrum at the excitation wavelength of 470 nm is used to measure the fluorescence
intensity, the fluorescence intensity is introduced into the standard curve and the
concentration of Salmonella typhimurium is calculated.
[0054] The specific detection of the biosensor described in Example 1
[0055] The three interfering foodborne pathogens of Staphylococcus aureus, Listeria
monocytogenes and Escherichia coli are tested together with Salmonella typhimurium
to verify the specificity of the aptamer biosensor, and the concentration of each bacterium is 102 CFU/mL. The specific results are shown in FIG. 3: The fluorescence value showed that the value of Salmonella typhimurium is significantly higher than that of other bacteria. Therefore, the sensor is highly specific to Salmonella typhimurium.
[0056] The above is only a better embodiment of the invention and is not a
restriction of any other form of the invention, and any technical person familiar with
the profession may make use of the technical content disclosed above to make
changes or modifications as equivalent embodiments of changes. However, any
simple modifications, equivalent changes and modifications made to the above
embodiments in accordance with the technical essence of the invention without
leaving the content of the technical scheme of the invention shall still be within the
scope of protection of the technical scheme of the invention.
SEQUENCE LISTING Jul 2020
<110> QINGDAO AGRICULTURAL UNIVERSITY
<120> Biosensor for detection of Salmonella typhimurium and its application
<160> 5
<170> SIPOSequenceListing 1.0 2020205292
<210> 1 <211> 95 <212> DNA <213> Artificial sequence(Salmonella typhimurium)
<400> 1 acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtactgac 60 agattaaaag tggttgggca acttctgctt gcgaa 95
<210> 2 <211> 95 <212> DNA <213> Artificial sequence(Salmonella typhimurium)
<400> 2 acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtatatgg 60 cggcgtcacc cgacggggac ttgacattat gacag 95
<210> 3 <211> 55 <212> DNA <213> Artificial sequence(Salmonella typhimurium)
<400> 3 tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatac 55
<210> 4 <211> 55 <212> DNA
<213> Artificial sequence(Salmonella typhimurium) Jul 2020
<400> 4 tcaactgcct ggtgataaaa cgacactacg tgggaatcta ctatggcggc tcttc 55
<210> 5 <211> 55 <212> DNA <213> Artificial sequence(Salmonella typhimurium) 2020205292
<400> 5 ttcagactta ggaatgtgct tcccacgtag tgtcgtttgt attggaccct cgcat
Claims (9)
1. A biosensor for use in rapid detection of Salmonella typhimurium in food applications is characterized by the composition of Td-CD, carbon quantum dot modified by nucleic acid aptamer of Salmonella typhimurium, and Td-MB, magnetic bead modified by nucleic acid aptamer of Salmonella typhimurium, DNA regular tetrahedron structure is formed through design Salmonella typhimurium nucleic acid aptamer and other nucleic acid sequence assembly of DNA, and then used to modify carbon quantum dots and magnetic beads respectively, the aptamer DNA tetrahedron used to modify carbon quantum dots is Td-Aptamerl, and the aptamer DNA tetrahedron used to modify magnetic beads is Td-Aptamer2; characterized in that the nucleic acid Td-Aptamerl is Al strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ACT GAC AGA TTA AAA GTG GTT GGG CAA CTT CTG CTT GCG AA-3'; B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'; C strand: 5'-biotin-TCAACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3'; D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3'; and the nucleic acid sequence Td-Aptamer2 is: A2 strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3'; B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'; C strand: 5'-biotin-TCAACT GCC TGG TGA TAA AAC GAC ACT ACG TGG
GAA TCT ACT ATG GCG GCT CTT C-3'; D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT
CGT TTG TAT TGG ACC CTC GCA T-3'.
2. According to claim 1 detection of Salmonella typhimurium biosensor in food
applications, its characteristics is described in the Td-Aptamerl and Td - Aptamer2
preparation method is: the four nucleic acid strands of DNA tetrahedron structure are
mixed and dissolved in TE buffer solution at the same molarity, respectively; after
eddy mixing, the solution is heated to 95 °C for 5 min, and then rapidly cooled in ice
water for 10 min to 4 °C.
3. According to the biosensor for detection of Salmonella typhimurium
described in claim 1, the characteristic of which is that the preparation method of the
carbon quantum dot Td-CD modified by the aptamer of the nucleic acid of Salmonella
typhimurium is:
Td-aptamerl and streptavidin-carbon quantum dot SA-CD are added to PBS
buffer at a molar ratio of 4:1, violently shaken for 1 hour at room temperature, rotated
at 5,000 rpm, centrifuged for 10 min, and the supernatant is discarded to obtain
Td-CD microspheres, which are dispersed in PBS buffer to obtain Td-CD solution.
4. The biosensor for the detection of Salmonella typhimurium described in
claim 3 is characterized by the preparation method of the streptavidin-carbon quantum
dot SA-CD described in claim 3:
a 5 mg amino-functionalized carbon quantum dot is dissolved in 1.5 mL
anhydrous ethanol and evenly dispersed by ultrasonic, streptavidin solution is added
to it. After 15 min of ultrasonic, it is shaken violently at room temperature for 3 h at a
speed of 12,000 rpm, centrifuged for 20min, and the supernatant is discarded to obtain
streptavidin modified carbon quantum dot.
5. According to claim 4 detection of Salmonella typhimurium biosensor, its characteristics is described in the preparation methods of amino functionalization carbon quantum dots are as follows: 0.4g p-cyclodextrin is dissolved in 20 mL ultra-pure water and evenly dispersed by ultrasonic, it is transferred into a stainless-steel high-pressure reactor lined with Teflon and placed in an air-blast heating drying oven, the hydrothermal reaction is conducted at 200 °C for 6 h, after the reaction, the liquid is naturally cooled to room temperature and dialyzed in ultrapure water for 24 h in a dialysis bag, finally, the product is rotated-steamed at 45 °C on a rotary evaporator, and the solvent is removed, the light-yellow powder is the amino functionalized carbon quantum dots prepared.
6. According to the biosensor for the detection of Salmonella typhimurium described in claim 1, the characteristic of which is that the preparation method of the carbon quantum dot Td-MB modified by nucleic acid aptamer of Salmonella typhimurium is as follows: 1 mg streptavidin functionalized magnetic beads and 1.6 M Td-Aptamer2 are mixed and dispersed in 500 mL Tris-HCl buffer solution for 30 min, then the magnetic beads are separated from the system with a magnet, the supernatant is discarded, the magnetic beads are washed three times with 1 mL PBS buffer, and finally the magnetic beads are dispersed in PBS buffer, namely Td-MB solution.
7. Claims 1-6 application of any of the biosensor for detection of Salmonella typhimurium in detection of Salmonella typhimurium.
8. A Salmonella typhimurium detection kit for food applications containing the biosensor of claim 1; characterized in that said biosensor contains Td-CD, carbon quantum dot modified by nucleic acid aptamer of Salmonella typhimurium, and Td-MB, magnetic bead modified by nucleic acid aptamer of
Salmonella typhimurium, DNA regular tetrahedron structure is formed through design Salmonella typhimurium nucleic acid aptamer and other nucleic acid sequence assembly of DNA, and then used to modify carbon quantum dots and magnetic beads respectively, the aptamer DNA tetrahedron used to modify carbon quantum dots is Td-Aptamerl, and the aptamer DNA tetrahedron used to modify magnetic beads is Td-Aptamer2; characterized in that the nucleic acid Td-Aptamerl is Al strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ACT GAC AGA TTA AAA GTG GTT GGG CAA CTT CTG CTT GCG AA-3'; B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'; C strand: 5'-biotin-TCAACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3'; D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3'; and the nucleic acid sequence Td-Aptamer2 is: A2 strand: 5'-ACA TTC CTAAGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT ATA TGG CGG CGT CAC CCG ACG GGG ACT TGA CAT TAT GAC AG-3'; B strand: 5'-biotin-TAT CAC CAG GCA GTT GAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'; C strand: 5'-biotin-TCAACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3'; D strand: 5'-biotin-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3'.
9. A method of rapid detection of Salmonella typhimurium in food applications by any of the biosensor of claims 1 through 6 is characterized by the following steps:
(1) the Td-MB solution and Td-CD solution are incubated with the sample
solution to be tested at room temperature for 20 minutes, and the reaction is fully
combined by slow shaking, the first magnetic separation is performed and the
supernatant is discarded to obtain the MB-Salmonella typhimurium - CD complex;
(2) with PBS buffer to suspended MB - Salmonella typhimurium - CD
complexes, join after 4 M Biotin molecule density mixture combined with 30
minutes; then, the second magnetic separation is carried out to discard the magnetic
beads and retain the supernatant solution;
(3) the superpure solution obtained by fluorescence spectrophotometer scanning
step (2) and fluorescence emission spectrum at 470nm excitation wavelength are used
to measure the fluorescence intensity, the measured fluorescence intensity is
introduced into the standard curve and the concentration of Salmonella typhimurium
in the sample to be measured is calculated.
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| CN115236050B (en) * | 2022-07-21 | 2024-04-16 | 合肥工业大学 | Method for detecting salmonella typhimurium by using fluorescent probe based on nitrogen-sulfur co-doped graphene quantum dots |
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