CN1536090A - Food-originated pathogenic bactenium quick detection gene chip and its application - Google Patents
Food-originated pathogenic bactenium quick detection gene chip and its application Download PDFInfo
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- CN1536090A CN1536090A CNA031091490A CN03109149A CN1536090A CN 1536090 A CN1536090 A CN 1536090A CN A031091490 A CNA031091490 A CN A031091490A CN 03109149 A CN03109149 A CN 03109149A CN 1536090 A CN1536090 A CN 1536090A
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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
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Abstract
The present invention provides a gene chip for quickly detecting pathogens from food source, discloses the preparation method of said gene chip and provides 26 oligonucleotide probe sequences for detection. Said gene chip can quickly, accurately and high-effectively detect and identify the class of the pathogens in food, and its detection range includes staphylococcus aureus, Shiga's bacillus, salmonella, colibacillus 0157, bacillus proteus, mononuclear hyperplastic listerella, enterocolitis yersinia, aeruginous pseudomonads, vibrio parahaemolyticus, vibrio cholerae, bacillus cereus, beta hemolytic streptococcus, coconut fermentation pseudomonads, boticin, vibrio jejuni and bacillus perfringens, etc.
Description
Technical field
The invention belongs to the detection technique field, relate to a kind of gene chip (or claiming dna microarray), especially a kind of food-borne pathogens rapid detection gene chip.
Background technology
" bread is the staff of life ".Food and human existence and healthy closely related.Guarantee food safety and sanitation, prevent that hazardous and noxious substances from working the mischief to human body by food is vital.Food all might be subjected to the influence and the pollution of various factors in the environment in each links such as production, processing, storage, transportation and sale, and microbial contamination is particularly common.Food causes various food origin diseases owing to be subjected to the pollution of pathogenic bacterium, even dead.In order to judge whether safety of food, the propagation and the quick diagnosis bacterial food poisoning of prevention and control food source sexually transmitted disease, must have a kind of can be fast, accurately, in time, the methods of the detection pathogenic bacterium of efficient and energy automatization.
There is the deficiency of following two aspects in traditional detection method: the one, operate loaded down with trivial details, waste time and energy; The 2nd, on detected result, be prone to false positive or false negative result, be difficult for carrying out quality control.Along with the development of immunology and Protocols in Molecular Biology, technology such as immunofluorescence technique, monoclonal antibody technique, biosensor technology, gene probe technology and PCR have been used to the detection of pathogenic bacterium.Higher, the high specificity of these sensitivity has application promise in clinical practice aspect microorganism detection.Yet these detection techniques are once tested generally can only detect a kind of microorganism, and the pathogenic bacterium kind that exists in the food is a lot, so still can not satisfy the actual detected requirements of one's work.
Biochip technology is a kind of brand-new analysis and detection technology that the nineties is set up, and is progressively to grow up along with carrying out of the Human Genome Project.Protocols in Molecular Biology, ic manufacturing technology, computer technology, semiconductor technology, confocal laser scanning technique, fluorescent mark technology have been used in this technological synthesis, make the detection of sample have susceptibility height, high specificity, large scale integration, automatization, easy and simple to handle, quick, advantages such as high-level efficiency.At present, biochip technology is mainly used in aspects such as genetic expression correlative study and bio-pharmaceuticals research.Utilize biochip technology to realize the rapid detection of multiple common pathogen is identified still brand-new scientific research field.
Summary of the invention
One of content of the present invention provides a kind of gene chip that is used for the rapid detection food-borne pathogens, and this gene chip has overcome the deficiencies in the prior art, can detect and identify the kind of pathogenic bacterium in the food fast, accurately and efficiently.
Two of content of the present invention provides this gene chip preparation method and 26 sequence oligonucleotide probes that are used to detect is provided.
Three of content of the present invention provides the application of this gene chip in the food-borne pathogens rapid detection.
Embodiment
One of content of the present invention is achieved in that
By the information biology means, design a series of detection probes at different pathogenic bacterium, probe according to microscope slide surface or other surface of solid phase carriers of certain arrangement mode point sample in aldehyde radicalization, is prepared into gene chip.Sample to be checked is by fluorescence on the PCR reaction marking, and the PCR product does not need purifying directly hybridize more than the 1h under proper temperature with gene chip, utilizes biochip scanner detection fluorescent signal, can judge the kind of pathogenic bacterium according to the position of fluorescent signal appearance.For isolating unknown bacterium colony, whole testing process can be finished within 3h.The bacterial species that this gene chip can detect has: streptococcus aureus, shigella, salmonella, Escherichia coli O 157, Bacillus proteus, monokaryon hyperplasia Lee Salmonella, enterocolitis yersinia enterocolitica, Pseudomonas aeruginosa, Vibrio parahaemolyticus, vibrio cholerae, bacillus cercus, beta hemolytic streptococcus, Paeudomonas Cocovenenans Subsp Farinofermentans, Clostridium botulinum, campylobacter jejuni, Vibrio flurialis, clostridium perfringens etc.
Two of content of the present invention is achieved in that
Utilize common sequence databases such as information biology software such as Oligo 6.0 and GenBank, design and screening are used for the oligonucleotide probe of bacterial detection 16S rRNA genovariation, and have designed the oligonucleotide probe of test section bacterium Disease-causing gene.Oligonucleotide has carried out 3 ' end amination modification when synthetic or 5 ' end amination is modified.
The carrier of gene chip is selected microslide for use.Used slide glass need carry out surface amination and aldehyde radical processing.
Utilize automatic point sample instrument or manual point sample instrument to finish whole point samples, make gene chip, the arrangement mode of dot matrix is set as required on the chip.Utilize on sealing such as the sodium borohydride chip not and oligonucleotide bonded aldehyde radical.
Gene chip is as a kind of technology platform, and its effect is that institute's fixed probe is realized on the dependence chip.Fixed probe and detection effect thereof are as shown in table 1 on the gene chip of the present invention.
Table 1: " food-borne pathogens rapid detection gene chip " goes up fixed probe and detection effect thereof
Sequence number sequence (5 '-3 ') detected object and purposes
1 gtacaaggcccgggaacgtattcaccg and the hybridization of all eubacteriums are used to detect all eubacteriums
2 gacataaggggcatgatgatttgacgtc and gram-positive bacterium hybridization are used to detect gram positive bacterium
3 gtcgtaagggccatgatgacttgacgtc and gram-negative bacterium hybridization are used to detect gram negative bacterium
4 gtcatgaatcacaaagtggtaagcgcc and intestinal bacteria hybridization are used to detect intestinal bacteria
5 acgacgcactttatgaggtccgcttg and intestinal bacteria, salmonella, shigella hybridization are used to detect this three genus bacterium
6 gctcctaaaaggttactccaccggctt are used to detect the probe of streptococcus aureus
7 cgacggctagctccaaatggttact are used for the negative staphylococcus probe of plasma-coagulase
8 cggctagctccaaaaggttactcta are used for the negative staphylococcus probe of plasma-coagulase
9 tcacggtcttgcgtcttattgtacctac are used to detect the probe of clostridium botulinum
10 tagataatcggcttcgggcgtctccaac are used to detect the probe of clostridium difficile
11 gaactgagactggtttttaagtttggct are used to detect the probe of clostridium perfringens
12 ccctagagcataaggggcatgatgattt are used to detect the probe of clostridium tetani
13 tcacggtcttgcgtcttattgtacctac detect the probe of shigella virA gene
14 tagataatcggcttcgggcgtctccaac detect the probe of salmonella invA gene
15 cgaactgggacatattttatagatttgc are used to detect the probe of campylobacter jejuni
16 ggtcgccccttcgccgccctctgtatca are used to detect the probe of Paeudomonas Cocovenenans Subsp Farinofermentans
17 cgatccaccctgagaccggcttttaagg are used to detect the probe of bacillus cercus
18 tactcgtaagggccatgatacgacttaa are used to detect the probe of proteus vulgaris
19 cgcggcttggcaaccctttgtaccgacc are used to detect the probe of the false born of the same parents bacterium of verdigris
20 actgagaatagttttatgggattag are used to detect the probe of monokaryon hyperplasia listeria spp
21 gctccaccttcgcggtattcgctgccct are used to detect the probe of vibrio cholerae
22 tcactttcgcaagttggccgccctctgt are used to detect the probe of Vibrio flurialis
23 tggtaagcgtccccccgtagttgaaac are used to detect the probe of Vibrio parahemolyticus
24 tacgacagactttatgtggtccgcttgc are used to detect the probe of yersinia entero-colitica
25 agattggctttaagagattagcttgccg are used to detect the probe of beta hemolytic streptococcus
26 atccccaccttcctccagtt are as the probe of experiment positive control
Three of content of the present invention is achieved in that this gene chip can be used for the rapid detection evaluation of food common pathogen, cause that the food poisoning pathogenic bacterium detect aspects such as evaluation, food safety evaluation.During detection, bacterium to be checked is carried out pcr amplification, the sequence of amplification primers is as shown in table 2.For the PCR product is carried out fluorescent mark, a kind of way is that primer 1169U20 is carried out fluorescent mark, another kind of way is that utilization has fluorescently-labeled base such as Cy3-dCTP or Cy5-dUTP etc. mix the PCR reaction system by a certain percentage, and the mark effect of these two kinds of fluorescent methods is proximate.
Table 2 primer sequence
Primer title sequence (5 '-3 ')
1169U20????????AACTGGAGGAAGGTGGGGAT
1521L19????????AGGAGGTGATCCAACCGCA
Pcr amplification product mixes with hybridization solution through sex change, adds on the gene chip probes array, and remains on more than 45 ℃-60 ℃ hybridization 1h.The preparation washings treats that hybridization finishes, and washs gene chip according to ionic strength order from high to low.Read fluorescent signal on the chip with biochip dedicated scan instrument, and the scanning result that utilizes the analysis of corresponding biochip data analysis software to obtain, judge the kind of sample bacterium, reach pathogenic bacterium are detected the purpose of identifying.
By above-mentioned disclosed technical scheme as seen, this gene chip can detect various pathogens, has shortened detection time greatly, has improved the accuracy that detects and reduced cost again, will have profound significance.
Embodiment:
For further specifying food-borne pathogens rapid detection gene chip and uses thereof, describe with reference to the following example, these embodiment are in order to explain rather than limit by any way the present invention.
Embodiment oneThe preparation of food-borne pathogens rapid detection gene chip
1.1 the surperficial aldehyde radical processing of carrier sheet glass:
Microslide chromic acid lotion soaked overnight to remove impurity such as surface organic matter, is cleaned with distilled water then, and immerses in 25% ammoniacal liquor and spend the night.Afterwards, slide glass is immersed 20min in 95% ethanol (pH to 4.5) that contains aminopropyl trimethoxysilane, with 95% ethanol ultrasonic cleaning 30min, pure water ultrasonic cleaning 20min, 115 ℃ of oven dry 45min.At last slide glass is immersed in 5% the glutaraldehyde solution and soaks 50min, then ultrasonic cleaning 10min washes 2 times, places dry dust-separation place standby.
1.2 utilize automatic point sample instrument spot printing probe array on slide glass:
The model of the mechanical manipulator of biochip point sample instrument (Cartesian Technologies) is AxSys 5000, and the control software of using is PixSys 5500Workstation.Use a feather pen type point needle head to finish whole point samples.Spacing is 0.5mm between the point.Arranging of array middle probe (sequence number) is as shown in table 3:
On the transfer table of point sample instrument, place surface treated aldehyde radical slide glass.
Get each probe solution 4 μ l, add successively in the respective aperture of brand-new 96 orifice plates.Add 4 μ l, 6 * SSC to every hole then, blow and beat mixing repeatedly.96 orifice plates are placed on the sample table of point sample instrument.Start PixSys 5500 Workstation, set each parameter, the beginning point sample.Robot arm control syringe needle, every some sample will pass through last sample, cleaning, dry plurality of processes.
After point sample finished, chip is seasoning 24h at room temperature, and the zone of containing dot matrix is with pencil mark overleaf.The chip that point is good is placed in the slide box of cleaning, preserves at shady and cool dry place.
Arranging of table 3 array middle probe
1????????????2?????????????3?????????????4?????????????5?????????????6
ABCDE
| ??26 | ?1 | ?2 | ?3 | ?-* | ?26 |
| ??4 | ?5 | ?6 | ?7 | ?8 | ?9 |
| ??10 | ?11 | ?12 | ?13 | ?14 | ?15 |
| ??16 | ?17 | ?18 | ?19 | ?20 | ?21 |
| ??26 | ?25 | ?22 | ?23 | ?24 | ?26 |
1.3 sealing: in order sealing on the slide not and oligonucleotide bonded aldehyde radical, to dispel salt and unconjugated oligonucleotide, slide to be handled according to following program:
0.2%SDS (25 ℃) washes 2 times, each 5min;
Pure water (25 ℃) is washed 2 times, each 5min;
Pure water (95 ℃) is washed 1 time, washes 2min, and is cool to room temperature;
Rapidly slide is put into confining liquid and wash 5min; (prescription of the confining liquid of use is: with 1.3g NaHB
4Be dissolved in the 375ml PBS solution, add the 125ml dehydrated alcohol to solution again, use immediately.)
0.2%SDS (25 ℃) washes 3 times, each 1min;
Pure water (25 ℃) is washed 2 times, each 1min;
The slide seasoning is used as early as possible.
Embodiment two: the present invention detects the applicating example of unknown bacterium
2.1 picking one ring is grown in the bacterium colony on the substratum, grinds mixing in 100 μ l pure water,, boil 10min, the centrifugal 1min of 10000rpm draws supernatant 2 μ l and adds in the PCR reaction mixture.PCR reaction mixture prescription is as table 4.
Table 4:PCR reaction mixture prescription
Components and concentration application of sample amount (μ l)
ddH
20???????????????????????????-??????????????37.3
10×PCR?Buffer(Mg2
+Plus)????????10×???????????????????????5.0
dNTP?Mixture?????????????????????2.5mmol/L??????4.0
1169U20 (Cy3 mark) 5 μ mol/L 1.0
1521L19??????????????????????????5μmol/L???????0.5
TaKaRa?Taq(5U/ml)????????????????5U/μl?????????0.2
2.2 reaction tubes is put into PCR instrument (PE 2400), sets the loop parameter loop parameter:
94℃，5min；
94 ℃, 25sec; 55 ℃, 25sec; 72 ℃, 25sec; , totally 40 circulations;
72℃，5min；
100℃，5min；
4℃，5min。
The PCR reaction finishes to take out reaction tubes immediately and carries out next step hybridization.
2.3 getting amplified production (4 ℃) 1 μ l mixes with 4 μ l hybridization solutions (Telechem company), point is in " food-borne pathogens rapid detection gene chip " (the preparation scheme is seen embodiment one) probe array zone, the covered film is noted can not leaving bubble between cover glass and the slide glass carefully.Put into hybridizing box, in box, add 10ml 3 * SSC to keep humidity.Tighten the screw of hybridizing box, put into 55 ℃ of water-bath hybridization 1h.Take out hybridizing box, take out slide, drop into washings A (55 ℃) immediately and wash 60sec, drop into washings B (25 ℃) again and wash 60sec, washings C (25 ℃) washes 60sec.Remove residual liquid and at air drying.
The prescription of washings is:
Washings A:1 * SSC (0.2%SDS)
Washings B:0.1 * SSC 0.2%SDS)
Washings C:0.1 * SSC
Read to analyze 2.4 results of hybridization is swept: sweep with ScanArray 3000 biochip scanners and read gene chip, select green laser for use, setting laser intensity is 80%, and the multiplier gain value is 80%, and scanning resolution is 10 μ m.Scanning result saves as the file of tiff format.Starting Imagene4.0 software analyzes the result.
The kind that position that occurs according to fluorescent signal and intensity can be differentiated the sample bacterium.
Claims (10)
1, a kind of rapid detection gene chip of food-borne pathogens, this gene chip is made of carrier and many oligonucleotide probes that are fixed on carrier surface.Many oligonucleotide probes that it is characterized in that carrier surface are arranged according to certain way, by with the hybridization of sample to be checked, can detect the kind of identifying the sample bacterium.
2, gene chip according to claim 1 is characterized in that, oligonucleotide probe is fixed on the solid phase carriers such as sheet glass, silicon chip, polypropylene screen, nitrocellulose filter, nylon membrane, forms the array of n * n, and each array has the positive control probe.A plurality of such array distribution can be arranged on same solid phase carrier.The approach that probe is applied to solid phase carrier is mechanical manipulator printing, spray printing or manual point sample, and the probe array for preparing like this is exactly gene chip (or claiming dna microarray).
3, gene chip according to claim 1 is characterized in that, the design of oligonucleotide probe is a foundation with the 16S rRNA gene of bacterium, is intended to detect the variation on the different bacterium 16S rRNA gene order, thereby distinguishes the kind of bacterium.Probe is divided into five classes according to function:
A. all eubacteriums have probe;
B. distinguish the probe of gram-positive microorganism and Gram-negative bacteria;
C. section's specific probe;
D. genus/specific specificity probe
E. positive control probe and negative control probe
For nearly derived bacterium is distinguished better, the present invention provides the detection probes at part bacterium Disease-causing gene simultaneously.Comprise the probe that detects shigella virA gene, at the probe of salmonella invA gene etc.
4, gene chip according to claim 1 is characterized in that, the sequences Design of probe is in the variable region of 16s rRNA gene, and length is that 20-29 base do not wait.The base sequence of every probe can be consistent with the base sequence of given zone domain dna normal chain on the 16s rRNA gene, also can be consistent with the base sequence of this given zone domain dna minus strand on the 16s rRNA gene.Identical with the above-mentioned consistence of using in the hybridization of all probes.
5, gene chip according to claim 1, it is characterized in that, with consistent being as the criterion of base sequence of given zone domain dna minus strand on the base sequence of every probe and the 16s rRNA gene, sequence of a whole set of probe of the present invention (5 '-3 ') and the measuring ability that can reach thereof are:
1 gtacaaggcccgggaacgtattcaccg and the hybridization of all eubacteriums are used to detect all eubacteriums
2 gacataaggggcatgatgatttgacgtc and gram-positive bacterium hybridization are used to detect gram positive bacterium
3 gtcgtaagggccatgatgacttgacgtc and gram-negative bacterium hybridization are used to detect gram negative bacterium
4 gtcatgaatcacaaagtggtaagcgcc and intestinal bacteria hybridization are used to detect intestinal bacteria
5 acgacgcactttatgaggtccgcttg and intestinal bacteria, Salmonellas, Shigellae hybridization are used to detect this three genus bacterium
6 gctcctaaaaggttactccaccggctt are used to detect the probe of streptococcus aureus
7 cgacggctagctccaaatggttact are used for the negative staphylococcus probe of plasma-coagulase
8 cggctagctccaaaaggttactcta are used for the negative staphylococcus probe of plasma-coagulase
9 tcacggtcttgcgtcttattgtacctac are used to detect the probe of clostridium botulinum
10 tagataatcggcttcgggcgtctccaac are used to detect the probe of clostridium difficile
11 gaactgagactggtttttaagtttggct are used to detect the probe of clostridium perfringens
12 ccctagagcataaggggcatgatgattt are used to detect the probe of clostridium tetani
13 tcacggtcttgcgtcttattgtacctac detect the probe of shigella virA gene
14 tagataatcggcttcgggcgtctccaac detect the probe of salmonella invA gene
15 cgaactgggacatattttatagatttgc are used to detect the probe of campylobacter jejuni
16 ggtcgccccttcgccgccctctgtatca are used to detect the probe of Paeudomonas Cocovenenans Subsp Farinofermentans
17 cgatccaccctgagaccggcttttaagg are used to detect the probe of bacillus cercus
18 tactcgtaagggccatgatacgacttaa are used to detect the probe of proteus vulgaris
19 cgcggcttggcaaccctttgtaccgacc are used to detect the probe of the false born of the same parents bacterium of verdigris
20 actgagaatagttttatgggattag are used to detect the probe of monokaryon hyperplasia listeria spp
21 gctccaccttcgcggtattcgctgccct are used to detect the probe of vibrio cholerae
22 tcactttcgcaagttggccgccctctgt are used to detect the probe of Vibrio flurialis
23 tggtaagcgtccccccgtagttgaaac are used to detect the probe of Vibrio parahemolyticus
24 tacgacagactttatgtggtccgcttgc are used to detect the probe of yersinia entero-colitica
25 agattggctttaagagattagcttgccg are used to detect the probe of beta hemolytic streptococcus
26 atccccaccttcctccagtt are as the probe of experiment positive control
6, gene chip according to claim 1 is characterized in that, probe is an oligonucleotide probe, and the 3 ' end or the 5 ' end of probe have passed through the amination modification.
7, gene chip according to claim 1, it is characterized in that, utilize two universal primers long fragment (at different bacterium, the product sheet segment length is different) of the about 370bp of the 16S rRNA gene of nearly all bacterium that increases, the target sequence that detects as next step gene chip.Article two, the sequence of primer (5 '-3 ') is as follows:
1169U20????AACTGGAGGAAGGTGGGGAT
1521L19????AGGAGGTGATCCAACCGCA
Wherein a primer is fluorescein-labelled through 5 ' end.Utilize above-mentioned primer to carry out pcr amplification, amplified production does not need purifying, is incubated 20min altogether to a few hours with gene chip under certain hybridization condition, utilizes the fluorescent scanning instrument can detect fluorescent signal.Can judge the kind of bacterium according to the position of signal appearance.
8, gene chip according to claim 1, it is characterized in that this gene chip can accurately detect streptococcus aureus, shigella, salmonella, Escherichia coli O 157, Bacillus proteus, monokaryon hyperplasia Lee Salmonella, enterocolitis yersinia enterocolitica, Pseudomonas aeruginosa, Vibrio parahaemolyticus, vibrio cholerae, bacillus cercus, beta hemolytic streptococcus, Paeudomonas Cocovenenans Subsp Farinofermentans, Clostridium botulinum, campylobacter jejuni, Vibrio flurialis, clostridium perfringens etc.
9, gene chip according to claim 1 is characterized in that, utilizes above-mentioned a whole set of probe or wherein a part of, can detect the corresponding bacterium in the laboratory sample by hybridization.Have higher sensitivity and specific degree, be particularly useful for detection based on the gene chip principle.
10, gene chip according to claim 1 is characterized in that, this gene chip has overcome the deficiencies in the prior art, can detect and identify the kind of pathogenic bacterium in the food fast, accurately and efficiently.The supervision and the fields such as detection, the detection of food poisoning pathogeny bacterium, bacteriology classification and epidemiology survey that can be used for food.
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