CN108192995A - For the probe set sequences of biochip test aspergillus flavus - Google Patents
For the probe set sequences of biochip test aspergillus flavus Download PDFInfo
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- CN108192995A CN108192995A CN201810228083.8A CN201810228083A CN108192995A CN 108192995 A CN108192995 A CN 108192995A CN 201810228083 A CN201810228083 A CN 201810228083A CN 108192995 A CN108192995 A CN 108192995A
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Abstract
The invention discloses a kind of probe set sequences for biochip test aspergillus flavus, belong to food-borne pathogenic microorganism detection technique field.The probe groups are：Capture probe CP (CP, Capture probe), detection probe (DP, detect probe), rolling ring probe (RCP, Rolling circle probe), the set probe set sequences are designed according to the whole genome sequence of Aspergillus flavus on GenBank, have the characteristics that high sensitivity, high specificity and accuracy are high in the biochip RCA detections aspergillus flavus method of above-mentioned probe groups exploitation, be widely portable to the detections such as food, cultivation and port.
Description
Technical field
The invention belongs to food-borne pathogenic microorganism detection technique fields, are related to detecting aspergillus flavus probe sequence, specifically relate to
And a kind of probe set sequences for biochip test aspergillus flavus.
Background technology
Aspergillus flavus (scientific name：Aspergillus flavus) or be yellow aspergillus, aspergillus flavus etc., it is a kind of fungi.
In natural environment, it is a kind of common mould, is widely present on soil, dust, plant and its fruit.Particularly in the torrid zone
With it is more common in semi-tropical kernel approaches and cereal.
Many Aspergillus flavus can generate enough compounds for having carcinogenicity and having acute toxicity.The toxicity of Aspergillus flavus is remote
Far above the toxicity of cyanide, arsenide and organic agricultural chemicals.When people's intake is big, acute poisoning can occur, Acute Hepatic occur
Inflammation, hemorrhagic necrosis, hepatic cell fattydegeneration and bile duct proliferation.Even if micro lasting intake, slow poisoning, growth barrier can be caused
Hinder, cause fibrous lesions, cause proliferation of fibrous tissue.The carcinogenicity of aflatoxin also ranks first, and is most strong in the world cause
One of cancer object.
Not only go mouldy food, and Huang may also be carried in those grains and peanut that pollute dust in transportational process and go mouldy
Aspertoxin and endanger the mankind.The carcinogenicity of aflatoxin and the effect of other carcinogenic substances such as N-nitrosodimethylamine have significantly
Relationship.
Aspergillus flavus mainly pollutes oil and foodstuffs, animals and plants food etc., such as peanut, corn, rice, wheat, beans, nut
Class, meat, milk and milk products, aquatic products etc. have aflatoxin contamination.Wherein with peanut and corn pollution most serious.Family
Self-control fermented food can also detect aflatoxin, and the especially grain and oil in high temperature and humidity area and product kind picks rate higher.Perhaps
More national regulations limiting value of the aflatoxin in food, the aflatoxin content in instant food do not allow more than this
Value.
In livestock and poultry cultivation production, the phenomenon that feedstuff and mixed feed by mould contamination are universal.Aspergillus flavus poison
Element is one of most common toxin in the mycotoxin found so far, when livestock and poultry eat the feed containing toxin or thalline, even
It can also cause whole acute and chronic poisoning after touching the substance containing toxin, seriously endanger livestock and poultry cultivation.
Aspergillus flavus is food-safe and mankind itself is healthy has constituted the threat that can not be ignored.According to world health group
The estimation knitted, the annual probability that aspergillus flavus cause disease number occurs in the whole world is also quite high, wherein food-borne micro- based on aspergillus flavus
Food origin disease accounts for 37.1% caused by biology.Traditional detection method includes Zengjing Granule screening and subsequent counter detection, life
Change the links such as reaction identification or serological Identification, these conventional methods are still the mainstream detection of current food hygiene regulatory agency
Method.The shortcomings that expansion of promotion and inspection scale however as examination requirements, conventional method, is also increasingly prominent, such as detects week
Phase is long, and it is low to prepare heavy with round-off work, specific deficiency, sensitivity, and needs professional operator etc..Along with reality
In detection, the result of most of Microbiological detection of foods is feminine gender, and biochip technology is provided for the detection of Aspergillus flavus
Good technology platform.Therefore great amount of samples is detected with biochip test method, is greatly improved the micro- life of food
The execution efficiency that quality testing is tested.But the detection that biochip is applied to carry out Aspergillus flavus must just obtain a large amount of specificity spy
Needle, a large amount of germplasm specific probe is the key that biological chip testing technology.
Invention content
Goal of the invention：In order to overcome the deficiencies in the prior art, the present invention provides a kind of for biochip test
The probe set sequences of aspergillus flavus.
Technical solution：To achieve the above object, the technical solution adopted by the present invention is：
A kind of probe set sequences for biochip test aspergillus flavus, each probe groups include capture probe CP, inspection
Probing needle DP, rolling ring probe RCP, wherein, the DP is sequentially connected by DP-A, DP-B and DP-C three parts and formed；The RCP is
Share probe.
Further, capture probe CP, detection probe DP share 5 groups, and sequence difference is as follows：
CP1 such as 5 '-NH2-SEQ ID NO：1-3 ' is shown, DP1 such as 5 '-SEQ ID NO：2~4-3 ' is shown, wherein,
DP1-A such as SEQ ID NO：Shown in 2, DP1-B such as SEQ ID NO：Shown in 3, DP1-C such as SEQ ID NO：Shown in 4；
CP2 such as 5 '-NH2-SEQ ID NO：5-3 ' is shown, DP2 such as 5 '-SEQ ID NO：6~8-3 ' is shown, wherein,
DP2-A such as SEQ ID NO：Shown in 6, DP2-B such as SEQ ID NO：Shown in 7, DP2-C such as SEQ ID NO：Shown in 8；
CP3 such as 5 '-NH2-SEQ ID NO：9-3 ' is shown, DP3 such as 5 '-SEQ ID NO：10~12-3 ' is shown, wherein,
DP3-A such as SEQ ID NO：Shown in 10, DP3-B such as SEQ ID NO：Shown in 11, DP3-C such as SEQ ID NO：Shown in 12；
CP4 such as 5 '-NH2-SEQ ID NO：13-3 ' is shown, DP4 such as 5 '-SEQ ID NO：14~16-3 ' is shown, wherein,
DP4-A such as SEQ ID NO：Shown in 14, DP4-B such as SEQ ID NO：Shown in 15, DP4-C such as SEQ ID NO：Shown in 16；
CP5 such as 5 '-NH2-SEQ ID NO：17-3 ' is shown, DP5 such as 5 '-SEQ ID NO：18~20-3 ' is shown, wherein,
DP5-A such as SEQ ID NO：Shown in 18, DP5-B such as SEQ ID NO：Shown in 19, DP5-C such as SEQ ID NO：Shown in 20.
Further, RCP sequences such as 5 '-SEQ ID NO：21-3 ' is shown.
Further, each CP and DP-A probe sequences and aspergillus flavus genome gDNA sequences are complementary；CP and DP-A probes
Sequence connects in the presence of aspergillus flavus genome gDNA sequences under T4 connection enzyme effects.
Further, each CP specific probes 5 ' are terminal modified amino, 5 ' 7 bases of end addition, as probe and substrate
Bonding pad.
Further, DP-A sequences and aspergillus flavus genome gDNA sequences are complementary, and DP-A and gDNA is combined during detection；DP-B
It is the bonding pad of DP-A and DP-C；DP-C sequences and the 5 ' ends of rolling ring probe RCP and the complementation of 3 ' terminal sequences and rolling ring probe
The primer of RCP and bonding pad.
Further, DP-B, DP-C and RCP are free of aspergillus flavus gDNA sequences.
Further, probe groups further include negative control probe NC and positive control probe PC, NC and PC sequence does not contain
With the sequence of aspergillus flavus gDNA sequence complementations；5 ' terminal modified the amino of NC and PC, the 3 ' terminal modified biotin of PC；The 5 ' of NC and PC
11 T bases of end addition；NC and PC sequences are respectively such as 5 '-SEQ ID NO：22-biotin-3 ' and 5 '-SEQ ID NO：23-3′
It is shown.
The above-mentioned probe set sequences for biochip test aspergillus flavus are being prepared for biochip test aspergillus flavus
Application in product.
Further, the probe set sequences are used for aspergillus flavus biochip test method, include the following steps：
[1], the slide of silanization treatment with 5% glutaraldehyde is handled, is then steamed successively with acetone, ethyl alcohol, deionization
Distilled water is thoroughly cleaned, and aldehyde group modified slide is obtained after dry；
[2], the surface of glass slide that will be prepared through 5 '-amido modified CP point samples in the 1st step, the microarray of point sample through aquation and
NaIO4After reduction treatment, it is prepared into CP microarrays；
[3], it will be added to after DP, RCP and aspergillus flavus genome gDNA combined degenerations on the microarray of preparation and anneal and connect
It connects；
[4], to the microarray of annealing after elution, rolling circle amplification RCA reactions are added in the gene structure of chip surface
System object carries out in piece rolling circle amplification RCA and introduces biotin-dUTP, after amplification and washing, adds in streptavidin and RCA
Amplified production combines；
[5], through elution, IR fluorescence imager scanning chip obtains testing result：" if target " microorganism exists, CP
It is connected with DP by T4DNA ligases, is carried out in piece rolling circle amplification RCA, then have fluorescence signal generation；" if target " microorganism is not
In the presence of then unstressed configuration signal generates.
Advantageous effect：Provided by the present invention for the probe set sequences of biochip test aspergillus flavus, advantageous effect is：
(1) Aspergillus flavus biochip test probe set sequences of the present invention are yellow bent for biochip method detection
Mould has many advantages, such as high specific, does not depend on antibody.
(2) present invention is combined using RCA technologies and chip technology, and linear RCA is up to the amplification ability of template
100000 times, index RCA has extraordinary sensitivity, can realize detection up to the amplification efficiency of 9 powers times higher than 10
To single molecules level.
(3) it is a kind of isothermal amplification based on RCA in the present invention, it is not necessary to reuse expensive thermal cycler
To complete amplification, vast market prospect is provided for commercial applications and popularization.
Description of the drawings
Fig. 1 is probe (group) sequence and feature of aspergillus flavus.
Fig. 2 is the probe set sequences biochip preparation method principle schematic of detection aspergillus flavus of the present invention；
Wherein, 1-coupled reaction, the 5 ' ends of 2-CP and the bonding pad of aldehyde group modified slide matrix, 3-aldehyde group modified
Slide matrix.
Fig. 3 structures the formation schematic diagram for PC, CP and NC microarray point sample.
Wherein, 1-PC, 2-NC, 3-CP1,4-CP2,5-CP3,6-CP4 and 7-CP5.
Fig. 4 detects aspergillus flavus design sketch for micro-array chip probe groups.
Fig. 5 detects aspergillus flavus specific effect figure for micro-array chip probe groups.
Wherein, A, B, C, D, E and F are respectively micro probe array group detection Escherichia coli, escherichia coli, golden yellow Portugal
Grape coccus, salmonella, aspergillus flavus and Listeria monocytogenes result figure.
Specific embodiment
The invention discloses a kind of probe set sequences for biochip test aspergillus flavus, belong to the micro- life of food-borne pathogenic
Analyte detection technical field.The probe groups are：Capture probe CP (CP, Capture probe), detection probe (DP, detect
Probe), rolling ring probe (RCP, Rolling circle probe), probe set sequences as described in Figure 1, the set probe set sequences
It is designed according to the whole genome sequence of Aspergillus flavus on GenBank, it is yellow bent with the biochip RCA detections of above-mentioned probe groups exploitation
Mould method has the characteristics that high sensitivity, high specificity and accuracy are high, is widely portable to the inspections such as food, cultivation and port
It surveys.
Specific probe (group) sequence the present invention is provided to biochip test aspergillus flavus is according to aspergillus flavus gene
Group sequence design.
Probe (group) sequence the present invention is provided to biochip test aspergillus flavus is：
The probe set sequences are used for aspergillus flavus biochip test method, including：
[1], the slide of silanization treatment with 5% glutaraldehyde is handled, is then steamed successively with acetone, ethyl alcohol, deionization
Distilled water is thoroughly cleaned, and aldehyde group modified slide is obtained after dry.
[2], the surface of glass slide that will be prepared through 5 '-amido modified CP point samples in the 1st step, the microarray of point sample through aquation and
After NaIO4 reduction treatments, it is prepared into CP microarrays.
[3], it will be added on the microarray of preparation and annealed simultaneously after DP, RCP and Aspergillus flavus genome gDNA combined degenerations
Connection.
[4], to the microarray of annealing after elution, rolling circle amplification RCA reactions are added in the gene structure of chip surface
System object carries out in piece rolling circle amplification RCA and introduces biotin-dUTP, after amplification and washing, adds in streptavidin and RCA
Amplified production combines.
[5], it elutes under certain conditions, scans chip with near-infrared fluorescence imaging instrument, obtain testing result.If
" target " microorganism exists, and CP can be connected by T4DNA ligases with DP, can be carried out in piece rolling circle amplification (RCA), then have fluorescence
Signal generates, if " target " microorganism is not present, unstressed configuration signal generates.
The present invention is further described with reference to the accompanying drawings and examples.
Standard Aspergillus flavus (Aspergillus flavus Link GIM3.475) bacterial strain used in the embodiment of the present invention,
Escherichia coli (Escherichia coli CMCC44102 freeze-dried powders), staphylococcus aureus (Staphylococcus
Aureus subsp.aureus CMCC26003, freeze-dried powder), salmonella (Salmonella sp.CICC21490) and single increase
Listeria (Listeria Monocytohenes GIM1.298) is purchased from Chinese medicine bacterium preservation administrative center
(CMCC)。
Embodiment 1：For detecting the biochip test method of the probe set sequences of aspergillus flavus
(1) slide of silanization treatment is placed in containing 5% glutaraldehyde (50% aqueous solution, Amresco) phosphate buffer
It is impregnated 2 hours in (0.1M PH7.4), with acetone, ethyl alcohol, deionized-distilled water, thoroughly cleaning is each twice, nitrogen drying, 4 DEG C of guarantors
It deposits spare；
(2) according to sequence described in claims and feature synthesising probing needle.
(3) CP, PC and NC using TE buffer solutions are configured to 100 μM of storage liquid, are stored in -20 DEG C.Before point sample, use
100 μM of CP, PC and NC are diluted to 20 μM by TE buffer solutions, then are diluted to 2 μM with 50% sampling liquid, brief centrifugation mixing, then
It takes at least 35 μ L to 384 microwell plates, the sample position that 384 microwell plates are placed in point sample instrument is prepared into point sample.
(4) slide that point sample finishes is placed in closed wet box, is covered with the filter paper that PBS solution is soaked in wet box, 37 DEG C
Water-bath overnight (be more than 12h), deionized water washing 2min, 0.2%SDS washing 5min, deionized water washing 2min, 0.15%~
0.3% sodium borohydride solution processing 5min, deionized water are washed 3 times, and 2min/ times, the drying of slide centrifuge are stored in 4
It is DEG C spare.
(5) aspergillus flavus gDNA, DP (0.05 μM of final concentration) and RCP (5 μ L) are added in EP pipe hybridization solutions, heat 95 DEG C,
5min is denaturalized, 25 μ L hybridization reaction systems in EP pipes are quickly transferred to be affixed in 25 μ L Gene Frame of aldehyde radical substrate,
Coverslip is covered, is placed in hybrid heater and starts hybridization reaction, 50 DEG C of hybridization 4h.
(6) after hybridization reaction, deionized water washing 1min, drying.According to following coupled reaction system, connected
It connects, 25 μ L of coupled reaction system is affixed in gene structure (Gene Frame).
| Component | Reaction 1 |
| T4 DNA ligases (14 U/ μ L) | 1μL |
| 10 × T4 buffer solutions | 2.5μL |
| Aqua sterilisa | 21.5μL |
| Total volume | 25μL |
(7) 37 DEG C of 30 min of coupled reaction condition, the 1min of deionized water washing later, drying.
(8) the solid phase RCA systems of aldehyde radical substrate, reaction system are as follows：
25 μ L RCA amplification reaction systems in EP pipes are transferred in above-mentioned Gene Frame, by slide be placed in 30 DEG C it is miscellaneous
In the closed wet box for handing over stove, 2h is expanded.
(9) RCA after reaction, takes Gene Frame, 2 min of deionized water washing slide off.
(10) brief centrifugation slide adds in 1 × confining liquid submergence slide, room temperature, and shaking table shakes at a slow speed, closes 30 min.
(11) according to streptavidin (streptavidin-IRDye800):PBS=1:15000 ratio, configuration
The PBS solution of streptavidin-IRDye800 submerges slide, and room temperature, shaking table shakes at a slow speed, with reference to 30 min.
(12) deionized water washing 10 min of slide, brief centrifugation slide are swept using 800 nm laser channelings of Odyssey
Retouch signal.
Embodiment 2：The specific assay of Aspergillus flavus detection is carried out using the probe groups of the present invention
(1) PC, NC and CP are used on point sample instrument point sample to aldehyde radical substrate, 2 μM of concentration and probe concentration.
(2) probe fixation procedure is identical with (1) in embodiment 1-(4).
(3) Escherichia coli, escherichia coli, staphylococcus aureus, salmonella and single increasing Liszt are cultivated respectively
Bacterium, and its gDNA is extracted respectively.
(4) gDNA, DP of said extracted (0.05 μM of final concentration) and RCP (5 μ L) are separately added into EP pipe hybridization solutions, added
95 DEG C of heat is denaturalized 5min, and 25 μ L hybridization reaction systems in EP pipes are quickly transferred to be affixed on to 25 μ L of aldehyde radical substrate respectively
In Gene Frame, coverslip is covered, is placed in hybrid heater and starts hybridization reaction, 50 DEG C of hybridization 4h.
(5) connection, RCA, washing and scanning, step are identical with (6)-(12) of example 1.
Testing result is as shown in Figure 5.Except positive control probe (PC) fluorescence signal normally in addition to, only aspergillus flavus successfully realizes rolling ring
, there is fluorescence signal, detects target in amplification.And fluorescence signal is not detected in negative probes (NC) and other bacterial strains,
Illustrate that reaction cannot be attached, it is impossible to carry out rolling circle amplification, illustrate that the probe groups of the present invention carry out Aspergillus flavus detection and have
Higher specificity.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
<110>Xuzhou Engineering Institute
<120>For the probe set sequences of biochip test aspergillus flavus
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Claims (10)
1. a kind of probe set sequences for biochip test aspergillus flavus, it is characterised in that：Each probe groups include capture
Probe CP, detection probe DP, rolling ring probe RCP, wherein, the DP is sequentially connected by DP-A, DP-B and DP-C three parts and formed；
The RCP is to share probe.
2. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：Capture is visited
Needle CP, detection probe DP share 5 groups, and sequence difference is as follows：
CP1 such as 5 '-NH2-SEQ ID NO：1-3 ' is shown, DP1 such as 5 '-SEQ ID NO：2~4-3 ' is shown, wherein, DP1-A is such as
SEQ ID NO：Shown in 2, DP1-B such as SEQ ID NO：Shown in 3, DP1-C such as SEQ ID NO：Shown in 4；
CP2 such as 5 '-NH2-SEQ ID NO：5-3 ' is shown, DP2 such as 5 '-SEQ ID NO：6~8-3 ' is shown, wherein, DP2-A is such as
SEQ ID NO：Shown in 6, DP2-B such as SEQ ID NO：Shown in 7, DP2-C such as SEQ ID NO：Shown in 8；
CP3 such as 5 '-NH2-SEQ ID NO：9-3 ' is shown, DP3 such as 5 '-SEQ ID NO：10~12-3 ' is shown, wherein, DP3-A
Such as SEQ ID NO：Shown in 10, DP3-B such as SEQ ID NO：Shown in 11, DP3-C such as SEQ ID NO：Shown in 12；
CP4 such as 5 '-NH2-SEQ ID NO：13-3 ' is shown, DP4 such as 5 '-SEQ ID NO：14~16-3 ' is shown, wherein, DP4-A
Such as SEQ ID NO：Shown in 14, DP4-B such as SEQ ID NO：Shown in 15, DP4-C such as SEQ ID NO：Shown in 16；
CP5 such as 5 '-NH2-SEQ ID NO：17-3 ' is shown, DP5 such as 5 '-SEQ ID NO：18~20-3 ' is shown, wherein, DP5-A
Such as SEQ ID NO：Shown in 18, DP5-B such as SEQ ID NO：Shown in 19, DP5-C such as SEQ ID NO：Shown in 20.
3. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：RCP sequences
Row such as 5 '-SEQ ID NO：21-3 ' is shown.
4. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：Each CP
It is complementary with DP-A probe sequences and aspergillus flavus genome gDNA sequences；CP and DP-A probe sequences are in aspergillus flavus genome gDNA sequences
In the presence of row, connected under T4 connection enzyme effects.
5. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：Each CP
Specific probe 5 ' is terminal modified amino, 5 ' 7 bases of end addition, the bonding pad as probe and substrate.
6. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：DP-A sequences
Row and aspergillus flavus genome gDNA sequences are complementary, and DP-A and gDNA is combined during detection；DP-B is the bonding pad of DP-A and DP-C；
DP-C sequences and the 5 ' ends of rolling ring probe RCP and primer and the bonding pad of the complementation of 3 ' terminal sequences and rolling ring probe RCP.
7. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：DP-B、
DP-C and RCP is free of aspergillus flavus gDNA sequences.
8. the probe set sequences according to claim 1 for biochip test aspergillus flavus, it is characterised in that：Probe groups
It further includes negative control probe NC and positive control probe PC, NC and PC sequence does not contain sequence with aspergillus flavus gDNA sequence complementations
Row；5 ' terminal modified the amino of NC and PC, the 3 ' terminal modified biotin of PC；5 ' 11 T bases of end addition of NC and PC；NC and PC sequences
Row are respectively such as 5 '-SEQ ID NO：22-biotin-3 ' and 5 '-SEQ ID NO：23-3 ' is shown.
9. the probe set sequences according to any one of claims 1 to 8 for biochip test aspergillus flavus, feature exists
In：Application in preparing for biochip test aspergillus flavus product.
10. the probe set sequences according to claim 9 for biochip test aspergillus flavus, it is characterised in that：It is described
Probe set sequences are used for aspergillus flavus biochip test method, include the following steps：
[1], the slide of silanization treatment is handled with 5% glutaraldehyde, then successively with acetone, ethyl alcohol, deionized-distilled water
Thoroughly cleaning obtains aldehyde group modified slide after dry；
[2], the surface of glass slide that will be prepared through 5 '-amido modified CP point samples in the 1st step, the microarray of point sample is through aquation and NaIO4
After reduction treatment, it is prepared into CP microarrays；
[3], it will be added on the microarray of preparation after DP, RCP and aspergillus flavus genome gDNA combined degenerations and anneal and connect；
[4], to the microarray of annealing after elution, rolling circle amplification RCA reaction systems are added in the gene structure of chip surface
Object carries out in piece rolling circle amplification RCA and introduces biotin-dUTP, after amplification and washing, adds in streptavidin and is expanded with RCA
Product combines；
[5], through elution, IR fluorescence imager scanning chip obtains testing result：If " target " microorganism exists, CP and DP
It is connected by T4DNA ligases, is carried out in piece rolling circle amplification RCA, then have fluorescence signal generation；" if target " microorganism is not deposited
In then unstressed configuration signal generation.
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