CN103592167B - Based on the Detection Methods of Fluoroquinolones Residue of luciferase mark engineering bacteria - Google Patents
Based on the Detection Methods of Fluoroquinolones Residue of luciferase mark engineering bacteria Download PDFInfo
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- CN103592167B CN103592167B CN201310578673.0A CN201310578673A CN103592167B CN 103592167 B CN103592167 B CN 103592167B CN 201310578673 A CN201310578673 A CN 201310578673A CN 103592167 B CN103592167 B CN 103592167B
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Abstract
The invention belongs to wild animal resources field, be specifically related to a kind of detection method and application of the fluo quinolone drug residual based on luciferase marker gene engineering bacteria.The present invention comprises a kind of construction and application that simultaneously can detect the luciferase marker gene engineering bacteria of 11 kinds of fluo quinolone drug residuals.This project bacterium is Escherichia coli (Escherichia coli) pK12, and preserving number is CCTCC NO:M2013385, by the plasmid pRecAlux3 transformation of E. coli K12 gained containing recA promoter and luciferase reporter gene luxCDABE.Compared with prior art, the genetic engineering bacterium that the present invention builds can identify multiple fluoroquinolones simultaneously, has widened the detected object of prior art, has had the outstanding advantages such as simple, quick, sensitive, accurate and sxemiquantitative function.
Description
Technical field
This method belongs to wild animal resources and gene engineering technology field.Be specifically related to the structure of the luciferase marker gene engineering bacteria that simultaneously can detect 11 kinds of fluoroquinolones, detect the foundation of the detection method of 11 kinds of fluoroquinolones simultaneously.
Background technology
Fluoroquinolones is the important chemosynthesis antimicrobial of a class.This type of medicine antibacterial spectrum is wide, antibacterial activity is strong, is veterinary clinic common drug.Owing to not using fluoroquinolones for animals according to off-drug period regulation or being used on animal by people by fluoroquinolones, cause it residual serious in livestock and poultry and aquatic products.Fluoroquinolones remaining except having direct damaging effect to human body in animal derived food, also have impact on China's foreign trade.The Ministry of Agriculture of China (2002), the World Health Organization (WHO), European Union and countries in the world all define the maximum residue limit(MRL) of fluoroquinolones in animal derived food (Maximum Residue Limit, MRL).
Current method rationalization analytic approach, immunoassay and microbiological analysis etc. for detecting fluo quinolone drug residual.Physico-chemical analysis method is mainly used in confirmation, also can be used for screening, but needs expensive instrument as the guarantee detected, and its pre-treatment program is more complicated and be not suitable for a large amount of detection.Immune analysis method is highly sensitive, high specificity, but it can not detect multiple fluoroquinolones simultaneously, and antibody preparation process is complicated.Traditional microbiological analysis is easy and simple to handle, sample pre-treatments is simple, can realize the multi-residue determination of fluoroquinolones, but the method is difficult to quantitatively and sensitivity is low.Fluorescence labeling engineering bacteria technology, also known as making bioluminescence microbiological sensor technology, a kind of higher than traditional micro-biological process sensitivity, and a class even multiclass material can be detected, have in field of biological detection and apply extremely widely, as environmental contaminants heavy metal ion, oxidative stress material, nitrate, DNA damage agent etc.
There is the application of some bibliographical informations fluorescence labeling engineering bacteria technology in antibacterial medicine residue detects at present, such as Tetracyclines [Korpela et al., Anal.Chem., 1998,70 (21), pp4457 – 4462; Pellinen et al., J.Agric.Food Chem., 2002,50 (17), pp4812 – 4815] and beta-lactam antibiotic (Valtonen et al., J.Biomol.Screen, 2002,7 (2), pp127-134) etc.This technology is recently also for the drug test of chloromycetin and fluoroquinolones.[the Shapiro et al. such as Shapiro, J.Biotechnol., 2007,4 (132), pp487-493] compare the effect of two kinds of fusion cspA::lacZ and cspA::luc chlorine detection mycin, result display firefly luciferase is than beta galactosidase more rapid sensitive; SOS inducible promoter sulA and lucR1 fusion detect survey that Ofloxacin examines and limit and be equivalent to 15% of its MIC; CspA::lacZ and sulA::lucR1 is combined the biology sensor of chlorine detection mycin and Ofloxacin while of building a kind of by them.
Hua Zhong Agriculture University's national basic veterinary drug at the applicant place remains the application (Huang Mingwei of benchmarks room early-stage Study fluorescence labeling engineering bacteria technology in fluoroquinolones detects, Hua Zhong Agriculture University's master thesis, 2010, library of Hua Zhong Agriculture University, middle National IP Network), by plasmid pRecAlux3 (recA ':: luxCDABE) [this plasmid to be built by the people such as Vollmer and is used for detecting DNA damage agent (the Vollmer etal. such as mitomycin, Appl.Environ.Microbiol., 1997, 63 (7), pp2566-2571)] be converted in Escherichia coli ATCC128, construct a strain Sensitivity and Specificity good luciferase marker gene engineering bacteria Escherichia coli pR128, establish pig liver, kidney, the detection method of fluo quinolone drug residual in muscle and these four kinds, milk tissue, its lowest detectable limit (Limit of Detection, LOD) be 50 μ g/kg, lower than the MRL of European Union and China.But this research institute builds engineered strain existing defects in stability, tissue sample sensing range is wide not, need perfect further in actual applications.The object of the invention is to screen and build that a strain is responsive to fluoroquinolones, specificity is good and can the luciferase marker gene engineering bacteria of stable detection fluoroquinolones in succeeding generations, and setting up the multi-residue determination method that can be applicable to fluoroquinolones in multiple edibility animal product.
Summary of the invention
The object of the invention is to the defect overcoming prior art, screen and construct that a strain is responsive to fluoroquinolones, specificity is good and can the luciferase marker gene engineering bacteria of stable detection in succeeding generations.
Second object of the present invention utilizes this genetic engineering bacterium, sets up a kind of analytical approach that can be used for multiple fluo quinolone drug residual detection in tissue sample.The tissue sample scope wide (comprising milk, the flesh of fish, chicken, beef, pork, chicken liver, chicken kidney, cattle liver, Ren Bovis seu Bubali, pig liver and Ren sus domestica) that the tissue sample pretreatment process of the method is simple, contain, simple and efficient to handle.
3rd object of the present invention be the fluoroquinolones kind in tissue sample known time, semi-quantitative analysis can be carried out to it.
In order to realize task of the present invention, inventor constructs can be special, responsive, stable luciferase marker gene engineering bacteria Escherichia coli (Escherichia coli) pK12 identifying fluoroquinolones, its Classification And Nomenclature is Escherichia coli (Escherichia coli) pK12, this bacterial strain is recombination bacillus coli, containing plasmid pRecAlux3, this bacterial strain delivers China on August 28th, 2013. Wuhan. and Wuhan University's China typical culture collection center preservation, its deposit number be CCTCC NO:M2013385(it be by the plasmid pRecAlux3 transformation of E. coli K12 gained containing recA promoter and luciferase reporter gene, plasmid pRecAlux3 is given by Swarthmore College department of biology of U.S. Amy Cheng professor Vollmer and remains benchmarks room in Hua Zhong Agriculture University's national basic veterinary drug and preserve, e. coli k12 is purchased from China. Wuhan. and Wuhan University's China typical culture collection center, its deposit number is CCTCC NO:AB200068).
The invention provides a kind of detection method based on luciferase marker gene engineering bacteria simultaneously detecting 11 kinds of fluo quinolone drug residuals, the method comprises the steps such as the structure of luciferase marker gene engineering bacteria, the cultivation of engineering bacteria and sample pre-treatments, specific as follows:
(1) with plasmid pRecAlux3 transformation of E. coli K12, obtain Escherichia coli (Esherichia coli) pK12 recombinated, its preserving number is CCTCC NO:M2013385;
(2) tissue sample process: testing sample animal's liver, kidney and muscle phosphate buffer are extracted, milk sample can direct sample detection;
(3) Escherichia coli (Esherichia coli) pK12 of restructuring step (1) obtained in 26 DEG C, is cultured to OD in 250rpm constant-temperature table in the LB broth bouillon containing 25 μ g/mL kanamycins
600=1;
(4) testing sample that fresh bacterium liquid step (3) obtained and step (2) obtain at 26 DEG C of stationary incubation 60min, by multi-functional microplate reader amount of fluorescence detected;
(5) calculate derivative coefficient value IC, its formula is: IC=Li/Lb-1
IC in formula: derivative coefficient; Li: the fluorescence intensity of sample; Lb: the fluorescence intensity of blank tissue.
Wherein:
Phosphate buffer in step (2) obtains as follows: take 0.523g KH
2pO
4with 16.73g K
2hPO
4be dissolved in 800mL distilled water, adjust ph to 8.0, is settled to 1L, sterilizing 20min under 121 DEG C of high pressure steam, for subsequent use.
The major advantage of this method is:
1, the genetic engineering bacterium Escherichia coli pK12 that the present invention builds can identify 11 kinds of fluoroquinolones simultaneously, and existing document and patent report still can not identify multiple fluoroquinolones like this;
2, the detection method that the present invention sets up can detect the residual condition of 11 kinds of fluoroquinolones, detectability is all at below the MRL that European Union specifies, and when fluoroquinolones kind is known, semi-quantitative analysis can be carried out to it, and existing micro-biological process detects limit for height, the medicament categories identified is limited, cannot be quantitative;
3, the tissue that the present invention is suitable for comprises pig, chicken, the liver of ox, 11 kinds of tissues such as kidney and muscle, the flesh of fish, milk, applied widely;
4, the sample treatment that the present invention relates to is easy, easy to operate, accuracy and precision good.
Accompanying drawing explanation
Fig. 1: be general technical route map of the present invention.
Fig. 2: be the collection of illustrative plates of the plasmid pRecAlux3 that the present invention builds.
Fig. 3: for Escherichia coli pK12 and Enrofloxacin hatch the susceptibility of different time, wherein X-axis is the logarithm value of Enrofloxacin standard solution concentration, and Y-axis is the derivative coefficient value to Enrofloxacin standard solution.
Fig. 4: for Enrofloxacin is to the induction typical curve of Escherichia coli pK12, wherein X-axis is the logarithm value of Enrofloxacin standard solution concentration, Y-axis is the derivative coefficient value to Enrofloxacin standard solution.
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
The structure of embodiment 1 luciferase marker gene engineering bacteria
The a small amount of e. coli k12 of picking (purchased from China. Wuhan. Wuhan University's China typical culture collection center, its deposit number is CCTCC NO:AB200068) freeze-dried powder, be seeded in the LB broth bouillon of 10mL, 37 DEG C, in 250rpm constant-temperature table, jolt overnight incubation.Choose the streak inoculation of a small amount of bacterium liquid in LB agar plate, 37 DEG C are spent the night.[method of bacterium competent cell is common method e. coli k12 to be prepared into competent cell, see: J. Pehanorm Brooker, EF is Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, Molecular Cloning: A Laboratory guide (third edition), Beijing, Science Press, 2002 editions] ,-70 DEG C are frozen.
By plasmid pRecAlux3 (given by Swarthmore College department of biology of U.S. Amy Cheng professor Vollmer and remain benchmarks room in Hua Zhong Agriculture University's national basic veterinary drug and preserve) transformation of E. coli K12 competent cell, obtain the luciferase marker gene engineering bacteria Escherichia coli pK12 recombinated.The mono-colony inoculation of picking Escherichia coli pK12, to (containing 25 μ g/mL kanamycins) in the LB broth bouillon of 10mL, 26 DEG C, jolts cultivation in 250rpm constant-temperature table, about 7 hours OD
600value can reach 1, gets 200-500 μ L bacterium liquid and goes down to posterity, about 5 ~ 6 hours, OD
600value can reach 1(and can preserve about one week in 4 DEG C for the bacterium liquid gone down to posterity).
The foundation of embodiment 2 fluoroquinolones detection method
The determination of 2.1 bacterium liquid best effort concentration
Escherichia coli pK12 is (containing 25 μ g/mL kanamycins) in LB fluid nutrient medium, 26 DEG C, cultivate, be cultured to OD under 250rpm condition
600when 0.7 and 1, detect Escherichia coli pK12 to the susceptibility of Enrofloxacin, incubation time is 45min; Result shows that in exponential phase, bacterial concentration is higher, and susceptibility is higher, therefore adopts OD
600bacterium liquid when=1 is work bacterium liquid.
The determination of 2.2 bacterium liquid and the best incubation time of medicine
Escherichia coli pK12 (containing 25 μ g/mL kanamycins) in 10mL LB fluid nutrient medium carries out being cultured to OD
600=1, add the Enrofloxacin (0.025 μ g/mL, 0.050 μ g/mL, 0.100 μ g/mL, 0.200 μ g/mL, 0.400 μ g/mL, 0.800 μ g/mL) of a series of concentration gradient respectively, medicine and each 50 μ L of bacterium liquid hatch certain hour altogether, carry out fluoroscopic examination at 30min, 45min, 60min respectively, calculate derivative coefficient according to formula 1.
Formula 1: the fluorescence intensity (Lb)-1 of fluorescence intensity (the Li)/blank tissue of derivative coefficient (IC)=sample
Although the derivative coefficient value of three time points (30min, 45min, 60min) and interpolation log concentration have good linear dependence, but the derivative coefficient value of 30min is too low, 60min derivative coefficient value is the highest, therefore selects the time that 60min is hatched as medicine and bacterium liquid.
The foundation of 2.3 typical curves
Enrofloxacin standard reserving solution LB broth dilution is mixed with these 8 concentration gradients of 0 μ g/L, 6.75 μ g/L, 12.5 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L, 200 μ g/L and 400 μ g/L, measure according to the condition after optimizing, with Enrofloxacin log concentration for horizontal ordinate, derivative coefficient value is ordinate, drawing standard curve.The regression equation of detection method and related coefficient are: y=1.4004x-0.6813, r=0.9983, and the range of linearity is 6.75-200 μ g/L.
2.4 cross reacting rate tests
The standard reserving solution LB broth dilution of 11 kinds of fluoroquinolones is become a series of concentration, detect according to the condition of above-mentioned optimization, the drug concentration that calculating derivative coefficient is corresponding when being 1, and comparing itself and Enrofloxacin, cross reacting rate is calculated according to formula 2, as shown in table 1.
Formula 2: cross reacting rate (%)=C
(IC=1)(Enrofloxacin)/C
(IC=1)(other drug) × 100%
Table 1 Escherichia coli pK12 is to the cross reacting rate of fluoroquinolones
Result shows, Escherichia coli pK12 all has higher cross reacting rate to 11 kinds of fluoroquinolones, can be used for the foundation of the Detection Methods of Fluoroquinolones Residue in animal tissue.
The implementation procedure of the method that embodiment 3 the present invention sets up
The preparation of 3.1 reagent
The compound method of phosphate buffer is: take 0.523g KH
2pO
4with 16.73g K
2hPO
4be dissolved in 800mL distilled water, adjust ph to 8.0, is settled to 1L, sterilizing 20min under 121 DEG C of high pressure steam, for subsequent use.
3.2 tissue sample pre-treatments
The pre-treating method of muscle and internal organ sample: get ox, chicken, the muscle of pig, liver, kidney and flesh of fish sample respectively, remove manadesma, shred, homogeneous 5min.Take the homogeneous 1.0g that organizes in centrifuge tube, add the phosphate buffer 2mL of aseptic pH8.0, vortex mixed 5min, room temperature leaves standstill 30min, and the centrifugal 10min of 12000g, gets supernatant to be measured.
The pre-treating method of milk sample: fresh milk sample can direct sample detection.
3.3 determination step
Sample and bacterium liquid are hatched: get the sample supernatant 50 μ L handled well and add 96 hole blanks, add bacterium liquid 50 μ L in 96 hole blanks with sample incubation, at 26 DEG C of stationary incubation temperature 60min; Then 96 hole blanks are placed in multi-functional microplate reader fluorescence intensity, calculate derivative coefficient according to formula 1.
3.4 results judge
With Enrofloxacin log concentration for horizontal ordinate, derivative coefficient value is ordinate, and drawing standard curve carries out linear regression, provides regression equation.
The calculating of fluoroquinolones concentration in tissue:
Calculation sample derivative coefficient is also brought in regression equation, is multiplied by dilution factor, calculates Enrofloxacin concentration (C in tissue
eNR), and calculate fluoroquinolones concentration (C in tissue according to formula 3
fQNs).
Formula 3:C
fQNs=C
eNR/ cross reacting rate
The sensitivity of the method that embodiment 4 the present invention sets up, precision, accuracy, replica test
The sensitivity test of 4.1 this method
The least concentration that the medicine that lowest detectable limit (LOD) obviously strengthens for fluorescence intensity adds.This test repeats five batches to often kind of medicine, and each concentration repeats 5 times, and namely 25 testing result derivative coefficient values are all greater than the Cmin of 0.5 is lowest detectable limit.The method good with above-mentioned optimization detects fluoroquinolones, and lowest detectable limit is as shown in table 2, and the lowest detectable limit of such medicine is all lower than the maximum residue limit(MRL) (MRL) that European Union specifies.
Table 2 Escherichia coli pK12 is to the lowest detectable limit of fluoroquinolones in animal tissue
The precision test of 4.2 this method
Respectively derivative coefficient value corresponding to 12.5 μ g/L, 25 μ g/L, 50 μ g/L, 100 μ g/L and 200 μ g/L Enrofloxacin standard concentration is substituted into the measured value that its typical curve equation obtains detection, in the plate calculating typical curve with standard concentration measured value and the coefficient of variation between plate, result is as shown in table 3.The coefficient of variation is all less than 15% in the plate of typical curve and between plate, illustrates that the detection method that this research is set up has better precision.
The coefficient of variation in the plate of table 3 typical curve and between plate
The accuracy of 4.3 this method and replica test
Often kind of fluoroquinolones is added to respectively in blank milk, the flesh of fish (silver carp), the muscle of pig, kidney, liver, the muscle of chicken, kidney, the muscle of liver and ox, kidney, liver specimens, each concentration arranges 5 repetitions (1/2MRL, MRL, 2MRL) respectively, replication 3 times.The recovery is calculated, the accuracy of wire examination method according to formula 4; Calculate within-run and between-run analysis coefficient, the repeatability of wire examination method.Its TIANZHU XINGNAO Capsul and batch in interassay coefficient of variation measurement result in table 4 ~ table 14.The medicine recovery is between 51.01 ~ 124.65%, and most of medicine meets the Ministry of Agriculture to the recovery substantially in the requirement of 60 ~ 120%, illustrates that accuracy is good; Within-run and between-run analysis coefficient, respectively within 25% and 30%, illustrates that repeatability is good.
Formula 4: the recovery (%)=measured concentration/interpolation concentration × 100%
The TIANZHU XINGNAO Capsul of table 4 fluoroquinolones in milk and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 5 fluoroquinolones in the flesh of fish and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 6 fluoroquinolones in chicken and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 7 fluoroquinolones in beef and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 8 fluoroquinolones in pork and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 9 fluoroquinolones in pig liver and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 10 fluoroquinolones in Ren sus domestica and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 11 fluoroquinolones in chicken liver and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 12 fluoroquinolones in chicken kidney and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 13 fluoroquinolones in cattle liver and the coefficient of variation
The TIANZHU XINGNAO Capsul of table 14 fluoroquinolones in Ren Bovis seu Bubali and the coefficient of variation
Claims (3)
1. one kind can identify the genetic engineering bacterium Escherichia coli pK12 of the luciferase mark of fluoroquinolones, it is characterized in that: this bacterial strain is deposited in China typical culture collection center, preserving number is CCTCC NO:M2013385, and it is by the plasmid pRecAlux3 transformation of E. coli K12 gained containing recA promoter and luciferase reporter gene.
2. one kind based on the detection method to 11 kinds of fluo quinolone drug residuals while luciferase marker gene engineering bacteria Escherichia coli pK12, comprise structure and the sample pre-treatments step of luciferase marker gene engineering bacteria Escherichia coli pK12, characterized by further comprising following steps:
(1) with plasmid pRecAlux3 transformation of E. coli K12, obtain the Escherichia coli pK12 recombinated, its preserving number is CCTCC NO:M2013385;
(2) getting ox, chicken, the muscle of pig, liver, kidney and flesh of fish sample phosphate buffer respectively extracts supernatant is to be measured, the direct sample detection of fresh milk sample;
(3) the Escherichia coli pK12 of restructuring step (1) obtained in 26 DEG C, is cultured to OD in 250rpm constant-temperature table in the LB broth bouillon containing 25 μ g/mL kanamycins
600=1, obtain fresh bacterium liquid;
(4) testing sample that fresh bacterium liquid step (3) obtained and step (2) obtain at 26 DEG C of stationary incubation 60min, by multi-functional microplate reader amount of fluorescence detected;
(5) calculate derivative coefficient value IC, its formula is: IC=Li/Lb-1
IC in formula: derivative coefficient; Li: the fluorescence intensity of sample; Lb: the fluorescence intensity of blank tissue.
Wherein:
Phosphate buffer in step (2) is prepared as follows: take 0.523g KH
2pO
4with 16.73g K
2hPO
4be dissolved in 800mL distilled water; Adjust ph to 8.0; Be settled to 1L; Under 121 DEG C of high pressure steam, sterilizing 20min, for subsequent use.
3. the application of detection method according to claim 2 in edibility animal product in fluo quinolone drug residual detection.
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