CN107177686A - Based on FRET technology for detection Escherichia coli O 157s:H7 method - Google Patents

Based on FRET technology for detection Escherichia coli O 157s:H7 method Download PDF

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CN107177686A
CN107177686A CN201710476924.2A CN201710476924A CN107177686A CN 107177686 A CN107177686 A CN 107177686A CN 201710476924 A CN201710476924 A CN 201710476924A CN 107177686 A CN107177686 A CN 107177686A
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cell
xxl
escherichia coli
fret
growth
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李延斌
王灵
王蓉晖
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Zhejiang University ZJU
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

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Abstract

FRET technology for detection Escherichia coli O 157s are based on the invention discloses one kind:H7 method, comprises the following steps successively：1) B cell, is cultivated：By B cell culture to exponential phase of growth；2), genetic coding calcium ion fluorescence indicator plasmid TN XXL electricity is transformed into the B cell of exponential phase of growth, TN XXL B cell must be expressed；Expression TN XXL B cell detects Escherichia coli O 157 as based on FRET:H7 cell biological sensor；3) when, the B cell for expressing TN XXL is contacted with determinand, FRET efficiency is determined by Photobleaching；Work as Escherichia coli O 157:When H7 bacterial concentrations are high, detected FRET efficiency is of a relatively high.

Description

Based on FRET technology for detection Escherichia coli O 157s:H7 method
Technical field
The invention belongs to field of biosensors, it is related to food-borne pathogens Escherichia coli O 157s:H7(Escherichia coli O157:H7 specific detection) is a kind of based on FRET (Fluorescence resonance energy Transfer, FRET) technology and intracellular calcium signal path bone-marrow-derived lymphocyte biology sensor structure Build.
Background technology
In the biology sensor CBBs based on cell, full cell is often used as a primary sensor, and two grades pass The structure of sensor often relies on the type of cell signal detection.Because the remarkable characteristic of lymphocyte and its huge application are dived Power, increasing lymphocyte has been applied in CBBs and has been detected.Wherein, B cell is due to its own energy specificity Identification antigen and the characteristic for needing to T cell progress antigen to offer by BCR (B cell receptor, B-cell receptor), Superior detection speed and detection specificity are shown in CBBs.B cell is known natural most quick pathogenic bacteria identification Element, its intrinsic reaction time is less than 1 second.After B cell recognizes pathogenic bacteria, BCR meeting mediate calcium ion refluxes, the reaction is B Cell tackles a key reaction of antigenic stimulus.After occurring the crosslinking of bivalent antigen or polyvalent antigen and BCR, it will cause Offering after transmembrane signal transduction and antigen internalization.Because calcium ion important role when regulating and controlling different cellular activities, Therefore there is work important all the more using Physiologic Studies of the Calcium imaging of fluorescence indicator in primary cell and tissue With.Calcium ion indicator (the Genetically encoded Ca of genetic coding2+Indicators, GECIs) it is green fluorescence One of biggest beneficiary of albumen (green fluorescent protein, GFP) technology, it is by fluorescin and biology Conductor Calcium ion sensor series connection is obtained.GECIs is had become after aequorin and chemical calcium ion indicator, detects born of the same parents A kind of very valuable instrument during interior calcium ion concentration.It can be overcome well based on the GFP calcium ion indicator built The deficiency of aequorin and chemical synthesis indicator.The GECI of one of them efficient self assembly is TN-XXL.TN-XXL calcium from Sub- biology sensor is that origin comes from bone and the calcium ion-binding protein troponin (troponin C, TnC) of cardiac muscle is derivative And come, it has been successfully applied in numerous studies, such as from the nerve cell of drosophila and mouse until the T of mouse is thin Born of the same parents.As far as we know, also no TN-XXL is used in B cell detection food-borne microorganism.
The content of the invention
FRET technology for detection Escherichia coli O 157s are based on the technical problem to be solved in the present invention is to provide one kind:H7 side Method.
In order to solve the above-mentioned technical problem, the present invention provides a kind of based on FRET technology for detection Escherichia coli O 157s:H7's Method, comprises the following steps successively：
1) B cell, is cultivated：By B cell culture to exponential phase of growth；
2), genetic coding calcium ion fluorescence indicator plasmid TN-XXL electricity is transformed into the B cell of exponential phase of growth, table is obtained Up to TN-XXL B cell；
Expression TN-XXL B cell detects Escherichia coli O 157 as based on FRET:H7 cell biological sensor；
3) when, the B cell for expressing TN-XXL is contacted with determinand, FRET efficiency is determined by Photobleaching；
Work as Escherichia coli O 157:When H7 bacterial concentrations are high, detected FRET efficiency is of a relatively high.
As the present invention based on FRET technology for detection Escherichia coli O 157s:The improvement of H7 method：
Step 1) in the B cell culture medium that uses for DMEM culture mediums with HIFBS (heat-inactivated fetal bovine serum) according to 9:1 Volume ratio is obtained by mixing.
The DMEM culture mediums be the sodium acid carbonate containing 4mM Glus and 1.5g/L, 4.5g/L glucose, The DMEM culture mediums of 1vol% nonessential amino acid (MEM NAA).
As the present invention based on FRET technology for detection Escherichia coli O 157s:H7 further improvements in methods：Step 2) In, plasmid TN-XXL is transfected into B cell (Bio-Rad by the B cell for taking exponential phase of growth to grow using electric transformation technology Gene-Pulser, Bio-Rad, Hercules, CA, USA), it is 1.43 × 10 that 0.35mL concentration is used during electricity conversion6cells/ The TN-XXL DNAs of mL B cell and 3 μ g, electricity conversion technological parameter be：0.4cm electricity conversion cups, 500v (Bio-rad Gene Pulser XcellTM Electroporation Systems)。
The present invention builds a kind of new cell biological sensor, and it utilizes TN-XXL pairs of genetic coding calcium ion indicator The rapidity that the sensitivity and B cell of intracellular calcium ion are reacted pathogenic bacteria, food-borne is detected with reference to FRET technologies there is provided one kind Pathogenic bacteria E.coli O157:H7 new detection method.This method is a kind of construction method of brand-new cell biological sensor (Fig. 1).
The present invention is according to B cell to E.coli O157:H7 specificity and TN-XXL is special to the sensitiveness of intracellular calcium ion Property, construct the B cell biology sensor containing calcium ion fluorescence indicator.The present invention is by expression TN-XXL B cell and not With concentration E.coli O157:After the mixing of H7 somatic cells, B cell will capture bacterial cell, so as to trigger B cell surface The intracellular calcium ion torrent of BCR mediations, TN-XXL conformation changes to have triggered FRET efficiency to change, by altogether Focusing microscope is to the measure of FRET efficiency, so that it is determined that whether containing target pathogenic bacteria in testing sample.
Brief description of the drawings
The embodiment to the present invention is described in further detail below in conjunction with the accompanying drawings.
Cell biological sensor quick detection food-borne pathogens E.coli O157s of the Fig. 1 based on FRET:H7 principle is shown It is intended to.
Fig. 2 various concentrations E.coli O157:H7 FRET efficiency comparatives figure.
The ROC curve figure of Fig. 3 cell biological sensors.
Non-target bacteria E.coli O157 in Fig. 4 drinking water:H7 measure comparison diagram.
Embodiment
Embodiment 1, one kind are based on FRET technology for detection food-borne pathogens Escherichia coli O157:H7 side Method, is followed the steps below successively：
1) B cell culture：
B cell culture medium is the addition 100mL HIFBS (heat-inactivated fetal bovine serum) in 900mL DMEM culture mediums .The DMEM culture mediums be the sodium acid carbonate containing 4mM Glus and 1.5g/L, 4.5g/L glucose, 1vol%'s The DMEM culture mediums of nonessential amino acid (MEM NAA).Cell is at 37 DEG C, and gas concentration lwevel is 7% carbon dioxide culture Cultivated in case.
Every time with 1:B cell is seeded in B cell culture medium by 10 ratio (volume ratio), in T25 or T75 cell culture Cultivated (Falcon, Oxnard, USA) in bottle, after culture 72h；Using TC10 automated cells calculating instrument (Bio-Rad, Hercules, CA, USA) carry out the measure of cell count and cell survival rate.The cell of exponential phase of growth is taken subsequently to be tried Test.B cell is passaged to B cell culture medium according to above-mentioned condition of culture；From algebraically liquid nitrogen is stored in for the B cell in 5-10 generations In.
2) electricity conversion
The B cell of above-mentioned exponential phase of growth growth is taken, plasmid TN-XXL is transfected into B cell using electric transformation technology (Bio-Rad Gene-Pulser,Bio-Rad,Hercules,CA,USA).Electricity conversion when using 0.35mL concentration be 1.43 × 106The TN-XXL DNAs of cells/mL B cell and 3 μ g, electricity conversion technological parameter be：0.4cm electricity conversion cups, 500v (Bio-rad Gene Pulser XcellTM Electroporation Systems)；Final volume is about 0.4mL.Turn in electricity After change, cell after conversion is placed in T25 Tissue Culture Flasks at once, addition 10mL B cells culture medium progress incubated overnight ( 37 DEG C, gas concentration lwevel for 7% CO2gas incubator in cultivated).
3) photobleaching detection FRET efficiency
Using Laser Scanning Confocal Microscope Zeiss LSM780 (Carl Zeiss Inc., Thornwood, NY), YFP passages are used In 514nm laser channelings using 100% laser energy to ROI carry out photobleaching.When collecting data, same detection is obtained Image before being bleached under device compensation condition and after bleaching is analyzed.Before photobleaching, scan the imaging before three bleachings and make For baseline, then photobleaching is carried out until 50% YFP fluorescence intensities are bleached.
FRET efficiency values are calculated with equation below：
FRETEfficiency(%)=[(CFPpost-CFPpre)/CFPpost]×100；
Wherein, CFPpreCFP signals before being photobleaching, CFPpostIt is the CFP signals after photobleaching.Use Zeiss Software obtains CFP and YFP after ROI signal value, and ROI FRET efficiency is calculated by above equation.In photobleaching The reduction of YFP signals and the increase of FRET efficiency of ROI before is all 0；After photobleaching, YFP signals are reduced to 50%, this When donor CFP signal enhancing, this has signified the enhancing of FRET efficiency.
4) sample detection
After electricity conversion 48h, the concentration for expressing TN-XXL B cell reaches 5.4 × 105Detected during cells/mL. By E.coli O157:H7, as target pathogenic bacteria, is 6.1 × 10 to its gradient dilution using HBSS1~6.1 × 108CFU/mL。
Detection test operation step is summarized as follows：Using HBSS blank control group is used as instead of pathogenic bacteria；Use E.coli O157:H7 is used as target pathogenic bacteria group；B cell is washed after three times with HBSS, in blank control group, the B after 1mL is washed Cell and 1mL HBSS are gently mixed；In target pathogenic bacteria group, the pathogenic bacteria of B cell and 1mL after 1mL is washed mix. The mixture for taking out 20 μ L is added dropwise on slide and uses nail oil seal, is placed in room temperature dark medium to be detected (according to above-mentioned step It is rapid 3) described).All sample preparations are all that fresh preparation, i.e. system are surveyed before testing.
5) data analysis
Test data is the repetition experiment of 20 samples, as various concentrations E.coli O157:In the presence of H7, measure FRET has very big difference.According to Fig. 2, we learn：When bacterial concentration is higher, detected FRET efficiency is of a relatively high.
The corresponding FRET efficiency (%) of blank control group is about 7.33092.
In order to assess the accuracy of the biology sensor, we depict ROC curve.ROC curve feature can be utilized, just Step is estimated the concentration range of bacterium：As AUR≤0.7, bacterial concentration is less than 103cells/mL；As 0.7 ＜ AUR ＜ 0.9 When, bacterial concentration is between 104-105cells/mL；As AUR >=0.9, bacterial concentration is more than 106Cells/mL (Fig. 3).
Test 1, utilize the cell biological sensor obtained by cell biological sensor, carry out detection Escherichia coli O 157:H7 Method, follow the steps below successively：
1) sample preparation：
Escherichia coli O 157 is added in drinking water:H7, it is respectively 5.6 × 10 to make its final concentration2, 5.6 × 105With 5.6 × 108CFU/mL, i.e., respectively sample A, B and C.
2) sample detection：
It is 4.9 × 10 by the concentration for having expressed TN-XXL plasmids5Cells/mL B cell is pressed with testing sample respectively According to volume ratio 1：1 is well mixed, and is placed at once under Laser Scanning Confocal Microscope Zeiss LSM780 and carries out FRET Efficiency testings respectively.Institute Result is obtained as shown in figure 4, when non-target bacteria concentration is 5.6 × 102, 5.6 × 105With 5.6 × 108During CFU/mL, sample A, B and C Measured FRET efficiency (%) is respectively 9.851 ± 0.8582,13.96 ± 0.8712 and 18.31 ± 0.7875.With blank control (BC) comparing has significant difference (P ﹤ 0.0001).
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (3)

1. based on FRET technology for detection Escherichia coli O 157s:H7 method, it is characterized in that comprising the following steps successively：
1) B cell, is cultivated：By B cell culture to exponential phase of growth；
2), genetic coding calcium ion fluorescence indicator plasmid TN-XXL electricity is transformed into the B cell of exponential phase of growth, must be expressed TN-XXL B cell；
Expression TN-XXL B cell detects Escherichia coli O 157 as based on FRET:H7 cell biological sensor；
3) when, the B cell for expressing TN-XXL is contacted with determinand, FRET efficiency is determined by Photobleaching；
Work as Escherichia coli O 157:When H7 bacterial concentrations are high, detected FRET efficiency is of a relatively high.
2. according to claim 1 be based on FRET technology for detection Escherichia coli O 157s:H7 method, it is characterized in that：
Step 1) in the B cell culture medium that uses for DMEM culture mediums with HIFBS according to 9:1 volume ratio is obtained by mixing.
3. according to claim 1 be based on FRET technology for detection Escherichia coli O 157s:H7 method, it is characterized in that：
Step 2) in, plasmid TN-XXL is transfected into B cell by the B cell for taking exponential phase of growth to grow using electric transformation technology, It is 1.43 × 10 that 0.35mL concentration is used during electricity conversion6The TN-XXL DNAs of cells/mL B cell and 3 μ g, electricity conversion Technological parameter is：0.4cm electricity conversion cups, 500v.
CN201710476924.2A 2017-06-21 2017-06-21 Based on FRET technology for detection Escherichia coli O 157s:H7 method Pending CN107177686A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110086649A (en) * 2010-01-22 2011-07-29 서울대학교산학협력단 Spectroscopy of aquatic pathogenic bacteria by using protein chip
CN103468797A (en) * 2013-08-13 2013-12-25 无锡中德伯尔生物技术有限公司 Kit for rapid and accurate detection of Escherichia coli O157:H7 live bacterium and its detection method
CN106841011A (en) * 2016-12-23 2017-06-13 浙江大学 Flow cytometry quick detection Escherichia coli O 157:The method of H7

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110086649A (en) * 2010-01-22 2011-07-29 서울대학교산학협력단 Spectroscopy of aquatic pathogenic bacteria by using protein chip
CN103468797A (en) * 2013-08-13 2013-12-25 无锡中德伯尔生物技术有限公司 Kit for rapid and accurate detection of Escherichia coli O157:H7 live bacterium and its detection method
CN106841011A (en) * 2016-12-23 2017-06-13 浙江大学 Flow cytometry quick detection Escherichia coli O 157:The method of H7

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王灵: "快速特异检测大肠杆菌0157:H7的B细胞生物传感器研究", 《中国博士论文全文数据库 信息科技辑》 *

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Application publication date: 20170919