CN102199670A - Microcystin test chip, kit and detection method - Google Patents

Abstract

The invention discloses a microcystin test chip. The microcystin test chip comprises a substrate and probes combined on the substrate, wherein the probes are six specific probes related to six conserved region sequences in a microcystin synthetic gene cluster. The invention also discloses a kit provided with the test chip and a method for detecting microcystin by utilizing the kit. Compared with the prior art, the invention has the advantages that: a biochip and a molecular hybridization technology are combined, the fussy polymerase chain reaction (PCR) amplification is rejected, the detection time is shortened, and the high-throughput and high-sensitivity rapid detection can be performed.

Description

Microcystin detection chip, test kit and detection method

Technical field

The present invention relates to a kind of Microcystin detection chip, the invention still further relates to test kit and use the method that this test kit carries out the Microcystin detection with this detection chip.

Background technology

Blue-green algae has another name called " blue-green algae ", and in some nutritious water bodys, often a large amount of breedings form " wawter bloom " to some blue-green algae summer.When causing water quality deterioration, some " wawter bloom " kind also can produce the secondary metabolite with bio-toxicity, is called " cyanophycean toxin ".The release of these cyanophycean toxins directly produces harm to fish, people and animals.Cyanophycean toxin can be divided into four big classes according to the difference of its illness that finally causes: neurotoxin, hepatotoxin, cytotoxin and pungency toxin (irritant toxins).In this four toxoid, hepatotoxin is the Health hazard maximum of Microcystin (microcystin) to the public, and serious meeting causes its death.Therefore, around how detecting, controlling and eliminating cyanophycean toxin, the scientific worker of various countries has carried out number of research projects.

For the detection of Microcystin, carry out the bio-toxicity experiment whether to have cyanophycean toxin to exist in the detection water body by making up corresponding living model in early days.Yet a series of ethics problems that lower sensitivity, higher experiment expend and the bio-toxicity experiment brings make people have to select method for distinguishing to come contratoxin to diagnose.Along with the micro-example analytical instrument (as, HPLC, MALDI-TOF) development, rely on these instrument developments to go out the multiple method that is used for detecting the natural water cyanophycean toxin.These methods analyst process simple and fasts and have very high sensitivity.Yet, because analytical instrument has very high requirement to the pre-treatment of sample, therefore in practice, the processing of the sample not only time-consuming but also effort that becomes, and expensive analytical instrument and standard substance also make many laboratories can't carry out the diagnosis to cyanophycean toxin.Simultaneously, the diagnostic means that relies on immunology and biochemical method to develop also applies to the detection of cyanophycean toxin in laboratory or the physical environment water body gradually.As, ELISA method, colorimetry (protein phosphatase inhibitor) and cytotoxicity that hepatotoxin (microcystin and nodularin) detects are diagnosed.Yet aforementioned multiple diagnostic method is a detected object with the Microcystin that is released in the water body all, can not judge in advance it before toxin is discharged into water body in the frustule.Can't produce malicious Microcystis aeruginosa to potential by these methods and carry out preliminary examination.Thereby reduced the ageing of biological monitoring, influenced the monitoring/early warning of harmful algal bloom greatly.Similarly open source literature can referenced patent number be the Chinese invention patent " detection method of underwater trace Microcystin " (Granted publication CN100387985C) of ZL200310109105.2; Application number is 200710070274.8 Chinese invention patent application open " detection method of Microcystin in the water body " (publication number 101169414A); Can also be with reference to " hydrobiont journal " 04 phase in 2003, " suppress the method colorimetry with phosphoprotein phosphatase and detect Microcystin class material in the water " that the 27th volume the 4th interim Wang Xiao peak etc. is shown.

In recent years, along with reaching its maturity of biochip technology, efficient, fast, accurately, the sensitive biochip is applied to growing field such as gene sequencing, hybridization, detection in Gene Mutation and polymorphism analysis, gene type, disease detection, food microorganisms detection etc.; This technology can be simultaneously with a large amount of different probe stationary on same upholder, thereby can disposablely carry out a large amount of sequential detection and foranalysis of nucleic acids to same sample; The utilization biochip technology can overcome the problem that other conventional art commonly used exists, satisfy in actual detected work fast, the requirement that detects of high-throughput, automatization; In view of these advantages of biochip, successively developed multiple biochip both at home and abroad and be used for a plurality of fields such as transgenation and polymorphism analysis, gene insertion or disappearance, clinical diagnosis, microorganism discriminating.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of Microcystin detection chip with specific probe at the above-mentioned state of the art.

Another technical problem to be solved by this invention provides a kind of test kit with Microcystin detection chip of specific probe.

Another technical problem to be solved by this invention provides a kind of high-throughput, highly sensitive microcystic toxins checking method.

The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of Microcystin detection chip, and comprise substrate and be incorporated into on-chip probe, it is characterized in that described probe is six, comprises following gene order respectively:

g、5’-TTTTTTTTTTTTTTTGTTCCTGTGCCRTGAGCTTC-3’；

h、5’-TTTTTTTTTTTTTTTTGACCAAGTAATCCCAGAAC-3’；

i、5’-TTTTTTTTTTTTTTTCTTGAGTCATTTCGGGTTGG-3’；

j、5’-TTTTTTTTTTTTTTTATTCGGGACGATTGAGGTAT-3’；

k、5’-TTTTTTTTTTTTTTTTTACTAAACGGATTGGAGA-3’；

l、5’-TTTTTTTTTTTTTTTCATTCAAAGGATTAGGCACA-3’。

A kind of test kit with above-mentioned Microcystin detection chip.This test kit also has nucleic acid extraction agent, hybridization solution.

A kind of method of utilizing above-mentioned test kit to carry out the Microcystin detection is characterized in that comprising the steps:

1. sample DNA extracts: water sample is centrifugal, abandon supernatant, and extract grand genomic DNA in the centrifugal gained water sample with the nucleic acid extraction agent in conjunction with the adsorption column method;

2. hybridization: hybridization solution is dissolved back and macro genome DNA mixing, and after the sex change, ice bath is put into hybridizing box to chip, covers cover plate, and by the hybridization solution behind the well injection sex change ice bath, the tight hybridization of lid lid is hybridized in hybridization instrument;

3. clean: the chip of having hybridized cleans also centrifugal by washing dried instrument;

4. chip scanning and interpretation as a result: clean back centrifugal chip by micro-array chip scanner, and the result is carried out interpretation, provide detected result.

Compared with prior art, the invention has the advantages that: Microcystin detection chip and micro-array chip scanner and the supporting use of Microcystin detection chip detection system.When detecting, the sample macro genome DNA after the enrichment is placed on the chip, when target dna combines with specific probe, carry out qualitative analysis to whether there being the Microcystin gene in the target water body by the fluorescence situation that detects each detection site.Chip has the specific probes of many group Microcystis aeruginosa synthetic genes, the mutual rectification by many groups probe reach to Microcystin in the water body carry out fast, the accurate purpose of detection; Secondly, owing to can put 4 probe arrays on a chip, each array can detect a sample, therefore can detect 4 samples that come from different acquisition ground simultaneously, reach the target of a plurality of samples of rapid detection, simultaneously, also can disposablely carry out 4 times repetition, the accuracy of detected result is proofreaied and correct fast certain sample; In conjunction with biochip and molecular hybridization, rejected loaded down with trivial details pcr amplification, shortened detection time, can high-throughput, highly sensitively detect fast.

Description of drawings

Fig. 1 is an embodiment chips structural representation.

Embodiment

Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.

1. sample DNA extracts: water sample is centrifugal, abandon supernatant, and extract grand genomic DNA in the centrifugal gained water sample with the nucleic acid extraction agent in conjunction with the adsorption column method

2. hybridization: hybridization solution is dissolved back and macro genome DNA mixing, and after the sex change, ice bath is put into hybridizing box to chip, covers cover plate, and by the hybridization solution behind the well injection sex change ice bath, the tight hybridization of lid lid is hybridized in hybridization instrument;

3. clean: the chip of having hybridized cleans also centrifugal by washing dried instrument;

4. chip scanning and interpretation as a result: clean back centrifugal chip by micro-array chip scanner, and the result is carried out interpretation, provide detected result.

Wherein, nucleic acid extraction agent Qbiogene, the FastDNA spin kit that U.S.A company produces.Hybridization instrument, wash dried instrument and scanner three parts constitute microcystis waterbloom detection chip check and analysis system jointly, hybridization instrument, wash dried instrument and scanner and be Boao Biological Co., Ltd and produce, product type is respectively: brilliant core II chip hybridization instrument, brilliant core

Washer 8 chips are washed dried instrument and brilliant core

The 10K-A micro-array chip scanner.

Hybridization solution is the hybridization solution that does not comprise denatured products (macro genome DNA), by the mixing solutions of plurality of reagents preparation to be used for the crossover process of chip.Concrete proportion of composing following (is example with 8 μ L):

As shown in Figure 1, the specific probe on the chip is attached on the aldehyde group modified substrate by schiff base reaction; Various probes are formed array, its whole formation Microcystin detection chip.Wherein the gene order at improvement back probe is as follows according to Microcystin synthetic gene bunch 6 improvement probes of 6 conservative region designs:

The probe label	Sequence
		1	TTTTTTTTTTTTTTTGTTCCTGTGCCRTGAGCTTC
2	TTTTTTTTTTTTTTTTGACCAAGTAATCCCAGAAC
		3	TTTTTTTTTTTTTTTCTTGAGTCATTTCGGGTTGG
4	TTTTTTTTTTTTTTTATTCGGGACGATTGAGGTAT
		5	TTTTTTTTTTTTTTTTTACTAAACGGATTGGAGA
6	TTTTTTTTTTTTTTTCATTCAAAGGATTAGGCACA

Annotate: modify the mark kind, 5 ' Aminolinker C6.

Claims (4)

1. a Microcystin detection chip comprises substrate and is incorporated into on-chip probe, it is characterized in that described probe is six, comprises following gene order respectively:

a、5’-TTTTTTTTTTTTTTTGTTCCTGTGCCRTGAGCTTC-3’；

b、5’-TTTTTTTTTTTTTTTTGACCAAGTAATCCCAGAAC-3’；

c、5’-TTTTTTTTTTTTTTTCTTGAGTCATTTCGGGTTGG-3’；

d、5’-TTTTTTTTTTTTTTTATTCGGGACGATTGAGGTAT-3’；

e、5’-TTTTTTTTTTTTTTTTTACTAAACGGATTGGAGA-3’；

f、5’-TTTTTTTTTTTTTTTCATTCAAAGGATTAGGCACA-3’。

2. test kit with the described Microcystin detection chip of claim 1.

3. test kit according to claim 2 is characterized in that this test kit also has nucleic acid extraction agent, hybridization solution.

4. one kind is utilized the described test kit of claim 2 to carry out the method that Microcystin detects, and it is characterized in that comprising the steps:

1. sample DNA extracts: water sample is centrifugal, abandon supernatant, and extract grand genomic DNA in the centrifugal gained water sample with the nucleic acid extraction agent in conjunction with the adsorption column method;

2. hybridization: hybridization solution is dissolved back and macro genome DNA mixing, and after the sex change, ice bath is put into hybridizing box to chip, covers cover plate, and by the hybridization solution behind the well injection sex change ice bath, the tight hybridization of lid lid is hybridized in hybridization instrument;

3. clean: the chip of having hybridized cleans also centrifugal by washing dried instrument;

4. chip scanning and interpretation as a result: clean back centrifugal chip by micro-array chip scanner, and the result is carried out interpretation, provide detected result.

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