CN103184276A

CN103184276A - Water estrogen pollution detection method based on oryzias melastigma vitellogenin

Info

Publication number: CN103184276A
Application number: CN2011104521606A
Authority: CN
Inventors: 黄乾生; 方超; 伍辛泷; 董四君
Original assignee: Institute of Urban Environment of CAS
Current assignee: Institute of Urban Environment of CAS
Priority date: 2011-12-30
Filing date: 2011-12-30
Publication date: 2013-07-03

Abstract

The invention relates to a water estrogen pollution detection method based on oryzias melastigma vitellogenin, and belongs to the field of environmental pollution detection. Currently, no marine fish is used for environment estrogen biological detection, and biological detection technology of seawater estrogen pollution is insufficient. According to the invention, euryhalic male oryzias melastigma with strong adaptability is selected as an in-vivo model; a primer is designed; the expression of vitellogenin (VTG1) is detected by a real-time quantitative PCR method; and 18S ribosome RNA (18S) is used as reference genes to establish a detection system of the VTG1 transcriptional level. Male fish is exposed to 17-beta-estradiol and clean artificial seawater, which are used as a positive and a negative control of the system, and appropriate exposure time is explored. The detection method can be used for detection of environment estrogen pollution degrees of seawater samples, river estuary samples, and saltwater internal lake water samples.

Description

A kind of water body oestrogenic hormon contamination detection method based on Medaka vitellogenin

Technical field

The invention belongs to environmental pollution detection field, relate to a kind of water body environment oestrogenic hormon contamination detection method based on novel eurysalinity pattern fish vitellogenin, can be widely used in the coastal city inland lake, the mouth of the river, the environmental estrogens pollution level assessment in the zone such as seawater.

Background technology

At present, mainly adopt the method for chemical detection for the pollution problem of environmental estrogens, as liquid chromatography and MS (HPLC-MS) etc.The advantage of these methods is highly sensitive, but the required analytical instrument of these methods is expensive, and cost is very high, is difficult to be popularized.In addition, these methods having relatively high expectations to sample.Environmental sample is different from general sample, has that classes of compounds is various, content is low, sample composition is complicated, have the characteristics such as mobility and unstable.When Pollutant levels to be measured, lower than now methodical detection limit or sample complexity, in the situation of body serious interference, it is impossible directly measuring.With chemical process, compare, biological detection can be avoided composition detection, and the biological effect that the direct analysis contaminated samples causes can reflect the hazard rating of environmental pollution more intuitively.But current bioassay method is still in the initial development stage, and rarely seawater small scale mode animal is used Biological Detection.Thereby be necessary the detection technique of exploitation based on the seawater living living sample, from the comprehensive organism of seawater, learn and the toxicology activity is directly assessed the Health hazard degree of sea pollution.We have selected emerging model animals-ocean medaka.

The blue or green Medaka in ocean ( oryzias melastigmaor oryzias dancena), having another name called India's motor card (Indian medaka), the blue or green Medaka of stain, originate in the coastal and freshwater of Pakistan, India, Burma and Thailand.On taxonomy, the blue or green Medaka in ocean and Japanese blue or green Medaka belong to the blue or green Medaka of the strange jaw Medaka of Chordata spoke fin net-rope Beloniformes section together and belong to.The ocean medaka has following advantage as coastal ocean environmental estrogens live body detection model: 1. build is less, and tolerance is stronger, is easy to raise on a large scale in laboratory.2. gender difference can be distinguished fast by the difference of fin form.3. the raun egg laying amount is large, and the generation cycle is short, and reproductive order of generation is many.4. wide in range to the subject range of salinity, can be developed into the live body detection model that is different from the fresh water zebra fish model, under the envrionment conditions of various salinity, applied.

Fish vitellogenin specificity results from the jenny liver, under normal condition, in milter or juvenile fish body, is difficult to detect expression, but under oestrogenic hormon exposes, male also can produce stress and express vitellogenin.Thereby, the milter vitellogenin induce can indicative for environments in oestrogenic hormon pollute (Sumpter, J.P. and Jobling, S. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspect).For vitellogenin, develop the oestrogen-like hormone detection method both at home and abroad at present, but selected fish models is freshwater fish, comprises Cyprinidae and Acipenseridae fish (as patent publication No.: CN101519650 and CN1763090).By By consulting literatures (CNKI, the patent retrieval of Patent Office of the People's Republic of China (PRC), Science Direct, ISI Web of Science, Springer Journal, NCBI, Deng), have no the patent report of seawater fish for the context of detection of environmental estrogens.This research selects eurysalinity ocean medaka as living sample, has set up the live body environmental estrogens detection technique for vitellogenin, has higher novelty and practicality.

Summary of the invention

Target of the present invention is to provide the detection technique that a kind of environmental estrogens detected under each Variation of Salinity Condition pollutes, and applies to the environmental estrogens pollution detection in marine site.We first by novel pattern species-ocean medaka ( oryzias melastigma) as the fish models of this technology.Adopt real time fluorescence quantifying PCR method on method, detect the sexual maturity milter under environmental sample exposes, the changing conditions of vitellogenin (vitellogenin, VTG1) mrna expression level, and then the oestrogenic hormon of indication sample pollutes.

Adopt the SYBR real time quantitative PCR method to detect the expression of VTG1, and to take 18S ribosome-RNA(rRNA) (18S ribosomal RNA) be reference gene, set up the detection system of VTG1 transcriptional level, comprise the design of primer, mainly adopt primer3.0 primary design primer, with Oligo6.0, further screen primer, carry out the PCR experiment, specificity and amplification efficiency to primer are assessed, and each is a pair of with primer for the quantitative PCR that final optimization pass obtains VTG1 and reference gene 18S, and sequence is as follows:

The VTG1 fluorescence quantification PCR primer:

F:?5’-TTGGCAGAGATGCAGCAGCGGT-3’；

R:?5’-GGAAATGCAGGACACCCCAGTAGCC-3’；

The 18S fluorescence quantification PCR primer

5’-AACGCTGTGCTGCGTAGCCTCAATT-3’；

R:?5’-AGAAGAAGCCCCACTTTTCCTCGCA-3’。

Further SSR-PCR optimization, finally be defined as the two step method response procedures: 95 ℃ of 30 seconds denaturations, 95 ℃ of sex change in 5 seconds, 60 ℃ of 20 seconds renaturation, extension and collection fluorescent signals, 40 circulations.Data analysis adopts the maximum second derivative method, obtains Ct value (cycle threshold, fluorescent signal arrives the required reaction cycle number of setting threshold), adopts 2 ^{Δ Δ Ct}method is carried out relative quantitative assay.

Select sexually matured ocean medaka milter, the clean water sample of MilliQ water human configuration 35 ‰ salinity, exposed, as negative control, negative water sample adds 17-β-estradiol (E2,100 ng/L), expose as the positive, after 8 hours, put to death fish, extract liver rna, detect the expression amount of VTG1, respectively as feminine gender and positive control.

Gather water sample, carry out three parallel exposures, analyze the expression level of VTG1, compare with negative and positive controls, thus the environmental estrogens pollution level of analysis water-like.

The accompanying drawing explanation

Fig. 1 is 18S gene and VTG1 gene quantification PCR product melting curve figure.This figure shows that the PCR system has good specificity.

Fig. 2 is that environmental sample is induced ocean medaka VTG1 genetic expression relative level figure.That the ocean medaka of growing up carries out each water sample exposure, after 8 hours, stop exposing, obtain liver organization, detect 18S and VTG1 gene expression dose, the VTG1 expression level that the clean artificial seawater of take exposes fish is reference, the negative control sample is clean seawater, and three samples are respectively from San Ge marine site, Xiamen, and positive control sample is the clean artificial seawater that comprises 100 ng/L 17-β-estradiols (E2).Each sample is established three repetitions, and each duplicate packages contains three fishes,

p<0.05, significance rises,

p<0.01, utmost point significance rises.

Embodiment

1. sample collecting and pre-treatment

Gather seawater in required detection waters, gather the top layer water of plunging into the commercial sea.The seawater sample the gathered clarification of spending the night, adopt siphonage to obtain clarification seawater, solid deposits bottom discarding.

2. expose experiment

Select 3-4 month large male ocean medaka for exposing object, set up feminine gender and positive control.Adopt the negative contrast water of clean water sample of MilliQ water human configuration 35 ‰ salinity, add 100 ng/L 17-β-estradiols (E2), positive contrast water.Three Duplicate Samples are set, and each Duplicate Samples comprises 3 milters that obtain at random.Collected specimens exposes with contrasting simultaneously, and open-assembly time is 8 hours, between exposure period without feeding with change water.Fish is pulled small fish out after exposing 8 hours, with ice-water bath, puts to death fish, dissects, and obtains liver organization, in-80 ℃ of refrigerators of income, preserves rapidly.

3. fish sample preparation

Adopt the Trizol method to extract liver rna, spectrophotometry is identified the RNA quality and is detected RNA concentration, gets 1 μ g RNA, adopts the reverse transcription test kit PrimeScript of Takara company ^?rT Master Mix(catalog number DRR036A) reverse transcription becomes cDNA, and 20 μ l reaction systems comprise 4 μ l 5 X PrimeScript ^?rT Master Mix, the total RNA of 10-100 ng.Response procedures is 37 ℃, 15 minutes; 85 ℃, 5 seconds.Reverse transcription obtains the first chain cDNA, for next step experiment.

4. carry out fluorescent quantitative PCR experiment

In the enterprising performing PCR reaction of real-time fluorescence quantitative PCR instrument LightCycler Roche480 instrument, adopt the fluorescence quantitative kit SYBR of Takara company ^?premix Ex Taq ^tMiI (catalog number DRR081A) is reacted, and 20 μ l PCR system components are as following table:

Reagent	Usage quantity (μ l)	Final concentration
			SYBR ^? Premix Ex Taq ^TM II	10	1X
Forward primer	0.4	0.2 μM
			Reverse primer	0.4	0.2 μM
The cDNA template	1	< 100 ng
			Sterilized water	8.2	/

The PCR program is two-step approach, is specially 95 ℃ of 30 seconds denaturations, 95 ℃ of sex change in 5 seconds, 60 ℃ of 20 seconds renaturation, extension and collection fluorescent signals, 40 circulations.Carry out melting curve analysis, guarantee to only have the target product amplification to produce.Adopt the analysis of maximum second derivative method, obtain the Ct value, adopt 2 ^{Δ Δ Ct}method is carried out relative quantitative assay, and the 18S gene is reference gene.The vitellogenin expression amount of negative control group fish of take is reference value, calculates the expression amount of the yellow proteinogen of the fish-egg that is exposed to sample with respect to negative control group.Positive controls is for confirming the normal operation of system, and provides qualitative cognition for the environmental estrogens pollution level of sample.

The present invention can effectively apply to the environmental estrogens pollution detection of the water sample of various salinity, for day by day serious ocean environment oestrogenic hormon pollutes, provides plant quarantine.

Example: gather surrounding sea areas, Xiamen and interior salina-Yuandang Ya lake seawater sample, carry out pollution detection.Seawater sample Bao Kuo Yuandang Ya lake, marine site, Baicheng and the marine site, ferryc terminal of adopting.Each basic parameter that gathers water sample is as shown in the table.Acquired results after the water sample in ， Yuandang Ya lake and Baicheng exposes the bull fish as shown in Figure 2, causes that the vitellogenin significance rises in (relatively clean seawater), and the water sample that this two place is described is subject to environmental estrogens and pollutes.

Place	Sampling depth (rice)	Temperature (℃)	PH value	Salinity (‰)
					Yuandang Ya lake	1	20	6.0	20.2
Baicheng	2	20	6.0	24.4
					Ferryc terminal	2	20	6.1	22.3

Claims

1. a new technology of polluting for detection of briny environment oestrogenic hormon, it is characterized in that it be with novel seawater pattern fingerling-ocean medaka milter ( oryzias melastigma) be study subject, carry out the estrogenic pollution level of Evaluation Environment with the biological effect after exposing.

2. detection technique according to claim 1, is characterized in that according to vitellogenin (VTG1) and 18S coding gene sequence, and (sequence is design VTG1 fluorescence quantification PCR primer: F:5 '-TTGGCAGAGATGCAGCAGCGGT-3 '; R:5 '-GGAAATGCAGGACACCCCAGTAGCC-3 ') and reference gene 18S fluorescence quantification PCR primer (sequence is: 5 '-AACGCTGTGCTGCGTAGCCTCAATT-3 '; R:5 '-AGAAGAAGCCCCACTTTTCCTCGCA-3 ').

3. detection technique according to claim 1, is characterized in that setting up relative quantification PCR method, and the analysis environments sample is induced the difference of fish VTG1 expression level.

4. detection technique according to claim 3, it is characterized in that the ocean medaka is carried out to the environmental sample exposure, take 8 hours as exposing duration, and set up feminine gender and positive control, negative control is that clean artificial seawater exposes, positive control is that 100ng/L 17-beta-estradiol (E2) exposes, and this system is for assessment of the environmental estrogens pollution level of sample.