CN101580877A - Oligonucleotide microarray technique for detecting pathogen contamination in seawater - Google Patents
Oligonucleotide microarray technique for detecting pathogen contamination in seawater Download PDFInfo
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- CN101580877A CN101580877A CNA2009100694257A CN200910069425A CN101580877A CN 101580877 A CN101580877 A CN 101580877A CN A2009100694257 A CNA2009100694257 A CN A2009100694257A CN 200910069425 A CN200910069425 A CN 200910069425A CN 101580877 A CN101580877 A CN 101580877A
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Abstract
The invention relates to an oligonucleotide microarray technique for detecting pathogen contamination in seawater, belonging to the field of seawater contamination monitoring. The technique comprises the main technical schemes that a 16S-23S rRNA gene transcription interval sequence is used as a detection target and is amplified by a one-step polymerase chain reaction, a digoxin mark is obtained simultaneously, and then oligonucleotide hybridization is carried out; and the obtained monitoring result is interpreted in a manner that an enzyme-labeled antibody catalyzes the substrate colour development. Compared with the traditional product for detecting seawater contamination, the invention utilizes microarray detection to obtain the distribution situation of large numbers of pathogens and contamination index bacteria, and has the advantage of high flux; the invention can directly utilize seawater as a sample and truly obtain the contamination situation information of target bacteria under a condition of keeping the natural proportion of the flora number in the seawater; however, most existing detection techniques need the step of enrichment culture, destroy the original proportion of a flora composition, have lower reliability of the result and have longer detection procedure; and the oligonucleotide microarray detection operation has short procedure and is comparatively sensitive and fast.
Description
Technical field
The invention belongs to sea pollution monitoring field, to the effect that a kind of oligonucleotide microarray technique can be monitored pathogenetic bacteria pollution condition in the seawater in high-throughput ground.Can detect multiple malignant bacteria simultaneously, comprise vibrio fluvialis, Vibrio parahaemolyticus, vibrio alginolyticus, vibrio cholerae, Vibrio mimicus, Vibrio furnissii breathes out the arc maintenance bacterium, Vibrio vulnificus, enterococcus faecalis, the listeria bacteria, Pseudomonas aeruginosa, Salmonellas, streptococcus aureus, clostridium perfringens.And this microarray technology can not pass through enrichment culture, directly detects multiple index bacterium from seawater, and comprising can not be by the bacterium of artificial culture, and is not subjected to the influence in season.Except the pollution level of the pathogenic bacteria that can assess corresponding marine site, can also indicate this marine site whether to be subjected to sanitary wastewater pollution etc.
Background technology
The pathogen contamination of ocean is compared with other pollutions, and is more hidden, but but directly threatens littoral resident's health.And the characteristic distributions of the malignant bacteria kind of seawater pathogen contamination can reflect directly that seawater is subjected to the pollution condition of sanitary sewage, municipal effluent.The distribution situation that detects malignant bacteria in the seawater has multiple meaning.
It is various to pollute the pathogenic bacteria kind in the seawater, and some malignant bacterias of land finally import in the seawater through rainwashes, therefore variously can cause the pathogenic bacteria of human diseases all might exist in seawater.Itself surviving naturally in the seawater in addition has abundant pathogenic bacteria, and wherein many pathogenic bacterias can infect the mankind.These characteristics require the monitoring of ocean pathogenic bacteria that comprehensive as far as possible and representational detection information is provided.
The dna microarray detection technique can detect a plurality of pathogenic bacteria indexs simultaneously, and can carry out refinement to detection information according to demand.This advantage just in time satisfies the requirement of seawater pathogenic bacterium pollution monitoring.In addition, the dna microarray technology is in the dna molecular level detection, also has to be quick on the draw, and is simple to operate, weak point consuming time, and the accuracy height is easy to characteristics such as popularization.Increase bacterium owing to using the filtration enrichment, therefore need not cultivating.This technology can also detect can not artificial culture harshness bacterium and dead bacterium, improve positive rate greatly.In view of these advantages, the dna microarray technology can be as the good technique platform of exploitation seawater pathogenic bacteria detection.
At present, the report of the pollution pathogenic bacteria in the complete detection seawater is few, and great majority are merely able to detect limited minority kind pathogenic bacteria.Such as multiple PCR technique.But detect the whole pollution situation that two or three kind of bacterium disease can not provide the seawater pathogenic bacteria effectively.And most seawater detection techniques of having reported are not to be purpose with assessment sea pollution situation.And the dna microarray technology can overcome these shortcomings well.
Summary of the invention
The oligonucleotide microarray technique that the purpose of this invention is to provide multiple pathogenic bacteria and contamination index bacterium in a kind of comprehensive monitoring seawater.This microarray technology as detecting target spot, by a polymerase chain reaction (PCR) amplification, and obtains digoxigenin labeled with region sequence between 16S-23S rRNA genetic transcription simultaneously, carries out oligonucleotide hybridization then; By the colour developing of enzyme labelled antibody catalytic substrate, interpretation obtains monitoring result.The detection abundant information is detailed, has high-throughout characteristics.And detecting does not need to cultivate enrichment by object bacteria, can obtain detected result in the short period of time fast.The present invention is achieved by the following technical solutions: a kind of 43 probe sequences of the oligonucleotide microarray technique that detects pathogen contamination in the seawater are as follows:
The oligonucleotide microarray technique using method is as follows among the present invention:
(1) oligonucleotide microarray is made:
1) preparation probe dilution liquid: 1 * salmon sperm dna solution (1g/L)
2) preparation probe solution: be configured to 0.1ug/ μ L probe solution with probe dilution liquid
3) probe solution is put on 5cm * 5cm nylon membrane matrix, 80 ℃ are incubated 2 hours.
The probe point sample is in proper order as following table:
| Vp710b | Keliarv | Vfu710b | Vvgpro | N1386 | Sal-1 | Cp-2 | Con | |
| Vpiapro | Val2 | Vm710b | Tetglavm | N1385 | PSE-3 | Cp-1 | ||
| Riverglavr | Val1 | Vm710e | Vviapro | N1384 | PSE-2 | Str-2 | ||
| Vf710e | Vp-n1 | Mchar | Vh710e | Lis-3-RV | PSE-1 | Str-3 | ||
| vfl-n2 | Hemglvm | Vc710e | Vh710b | Lis-2-RV | N1388 | Sal-3 | ||
| vfl-n1 | Vp710e | Vc710b | Vfu710e | Lis-1-RV | N1387 | Sal-2 | Cp-3 |
(2) seawater sample process and DNA extraction
1) sample process
1. get surface seawater 20L, be sub-packed in the plastic tank of sterilising treatment.The seawater of obtaining is handled in 3h.
2. earlier remove by filter suspended matter with 0.15 millimeter aperture sieve, use Mini-pellicon system (Millipore, the U.S.) to carry out pressure filtration then and collect thalline, the filter membrane aperture is 0.22 μ m (Milipo, the U.S.).The corresponding filter membrane of every filtration 2L seawater, promptly every 2L seawater be as a sample, corresponding 10 parallel sampleses of each sampling point.
3. with sterilized water the thalline on the filter membrane is washed, and collect thalline with the centrifugal 5min of 10000r/min immediately.The thalline and the contamination precipitation of results are used for the DNA extracting.
2) DNA extraction
1. the seawater thalline filters thalline and the centrifugal 2min of silt suspension liquid 12000r/min that obtains sample.
2. throw out adds TE damping fluid (10mM TrisHCl, the 1mM EDTA of 550 μ L; PH 8.0), piping and druming makes it to suspend again repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, mixing, 37 ℃ of incubation 1h.
3. add 100 μ L 5mol/L NaCl, mixing adds 80 μ L CTAB/NaCl solution (5g CTAB is dissolved in the 100mL0.5M NaCl solution), mixing, 65 ℃ of incubation 10min again.
4. add the abundant mixing of isopyknic phenol/chloroform/primary isoamyl alcohol, centrifugal 4-5min changes supernatant in the new pipe over to, adds the Virahol of 0.6-0.8 times of volume, softly puts upside down mixing, places 20min for-20 ℃.
5. precipitation is inverted drying with 70% washing with alcohol of 1mL, heavily is dissolved in 50 μ L TE damping fluids.
6. the DNA sample boils and was quenching on ice then in 5 minutes.-20 ℃ of preservations are standby.
(3) polymerase chain reaction and digoxigenin labeled
1) amplification and mark system:
Each component final concentration and volume (μ L) in the reagent system
10 * polymerase chain reaction Buffer, 5.0 μ L
F-E-T 10μmol/L(4.0μL)
B-E-T 10μmol/L(4.0μL)
dNTP 10mmol/L(0.3μL)
Digoxigenin-11-dUTP 25nmol/L(0.3μL)
Dna profiling 5.0 μ L
Taq DNA Polymerase 5U/μL(0.3μL)
Distilled water 31.1 μ L
Amount to 50.0 μ L
10 * polymerase chain reaction Buffer:Tris-HCl pH8.5,100mmol/L; KCl, 500mmol/L;
MgCl
2，15mmol/L。
2) polymerase chain reaction program
1. the pre-sex change stage: 95 ℃ of 5min
2. first cycle stage (5 circulations): 94 ℃ of 30sec
54℃30sec
72℃50sec
3. second cycle stage (5 circulations): 94 ℃ of 30sec
56℃30sec
72℃40sec
4. the 3rd cycle stage (10 circulations): 94 ℃ of 30sec
58℃30sec
72℃40sec
5. the 3rd cycle stage (15 circulations): 94 ℃ of 30sec
62℃40sec
6. ending phase: 72 ℃ of 5min
4 ℃ of insulations
(4) hybridization testing process
Hybridization uses U.S. Roch company digoxin detection kit.
1) each oligonucleotide microarray immerses 50 ℃ of prehybridization 45min in the 600 μ L hybridization solutions (Roch company).
2) the 95 ℃ of sex change 10min of pcr amplification product with digoxigenin labeled quench then.
3) the PCR product of 50 μ L sex change is joined in the hybridization solution (Roch company) that 600 μ L newly change, add the intact nylon membrane of prehybridization and rock hybridization 1 hour for 50 ℃.
4) after hybridization is finished with film cooling, use respectively under the room temperature 2 * SSC (+0.1%SDS), 0.5 * SSC (+0.1%SDS) and lavation buffer solution (Roch company) respectively wash 2min in order,
5) seal 30min with confining liquid, add enzyme and join anti--DigiTAb in conjunction with 30min.
6) wash 15min * 2 time with lavation buffer solution.With detecting damping fluid (Roch company) balance 5min, add the colour developing liquid lucifuge colour developing observations after 2 hours that contains the chromogen substrate.
20 * SSC:175.3g NaCl and 88.2g Trisodium Citrate are dissolved in 800mL water, add NaOH and regulate pH to 7.0, constant volume 1L.
2 * SSC: with 10 times of 20 * SSC dilute with waters.
0.5 * SSC: with 40 times of 20 * SSC dilute with waters.
A kind of oligonucleotide microarray technique that detects pathogen contamination in the seawater of the present invention can also be in the application in detecting other body of sea water pathogen contamination.
The present invention's advantage compared with prior art is: the utilization microarray technology detects the distribution situation that obtains numerous pathogenic bacterium and contamination index bacterium, promptly has high-throughout advantage; Can directly utilize seawater as sample, in keeping seawater, obtain the pollution situation information of object bacteria under the situation of flora number natural ratio truly, and existing detection technique great majority need the step of enrichment culture, not only destroyed the original scale that flora is formed, because the harshness bacterium is not by artificial culture, result's degree of reliability is also lower, and testing process is longer; Oligonucleotide microarray technique detecting operation flow process is short, the specificity height, relatively accurately, sensitivity, fast.
Description of drawings
Fig. 1: seawater sample 1 detected result
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.
Embodiment 1: seawater sample 1
1 oligonucleotide microarray is made:
1) preparation probe dilution liquid: 1 * salmon sperm dna solution (1g/L)
2) preparation probe solution: be configured to 0.1ug/ μ L probe solution with probe dilution liquid
3) stationary probe is on the nylon membrane matrix: probe solution is put on 5cm * 5cm nylon membrane matrix, and 80 ℃ are incubated 2 hours.
2 seawater sample process and DNA extraction
1) sample process
1. get surface seawater 20L, be sub-packed in the plastic tank of sterilising treatment.
2. earlier remove by filter suspended matter with 0.15mm aperture sieve, use Mini-pellicon system (Millipore, the U.S.) to carry out pressure filtration then and collect thalline, the filter membrane aperture is 0.22 μ m (Milipo, the U.S.).The corresponding filter membrane of every filtration 2L seawater, promptly every 2L seawater be as a sample, corresponding 10 parallel sampleses of each sampling point.
3. with sterilized water the thalline on the filter membrane is washed, and collect thalline with the centrifugal 5min of 10000r/min immediately.The thalline and the contamination precipitation of results are used for the DNA extracting.
2) DNA extraction
1. the seawater thalline filters thalline and the centrifugal 2min of silt suspension liquid 12000r/min that obtains sample.
2. throw out adds TE damping fluid (10mM TrisHCl, the 1mM EDTA of 550 μ L; PH 8.0), piping and druming makes it to suspend again repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, mixing, 37 ℃ of incubation 1h.
3. add 100 μ L 5mol/L NaCl, mixing adds 80 μ L CTAB/NaCl solution (5g CTAB is dissolved in the 100mL0.5mol/L NaCl solution), mixing, 65 ℃ of incubation 10min again.
4. add the abundant mixing of isopyknic phenol/chloroform/primary isoamyl alcohol, centrifugal 4-5min changes supernatant in the new pipe over to, adds the Virahol of 0.6-0.8 times of volume, softly puts upside down mixing, places 20min for-20 ℃.
5. precipitation is inverted drying with 70% washing with alcohol of 1mL, heavily is dissolved in 50 μ L TE damping fluids.
6. the DNA sample boils and was quenching on ice then in 5 minutes.-20 ℃ of preservations are standby.
3 polymerase chain reactions and digoxigenin labeled
1) amplification and mark system:
Each component final concentration and volume (μ L) in the reagent system
10 * polymerase chain reaction Buffer, 5.0 μ L
F-E-T 10μmol/L(4.0μL)
B-E-T 10μmol/L(4.0μL)
dNTP 10mmol/L(0.3μL)
Digoxigenin-11-dUTP 25nmol/L(0.3μL)
Dna profiling 5.0 μ L
Taq DNA Polymerase 5U/μL(0.3μL)
Distilled water 31.1 μ L
Amount to 50.0 μ L
10 * polymerase chain reaction Buffer:Tris-HCl pH8.5,100mmol/L; KCl, 500mmol/L;
MgCl
2，15mmol/L。
2) polymerase chain reaction program
1. the pre-sex change stage: 95 ℃ of 5min
2. first cycle stage (5 circulations): 94 ℃ of 30sec
54℃30sec
72℃50sec
3. second cycle stage (5 circulations): 94 ℃ of 30sec
56℃30sec
72℃40sec
4. the 3rd cycle stage (10 circulations): 94 ℃ of 30sec
58℃30sec
72℃40sec
5. the 3rd cycle stage (15 circulations): 94 ℃ of 30sec
62℃40sec
6. ending phase: 72 ℃ of 5min
4 ℃ of insulations
4 hybridization testing processes
Hybridization uses U.S. Roch company digoxin detection kit.
1) each oligonucleotide microarray immerses 50 ℃ of prehybridization 45min in the 600 μ L hybridization solutions (Roch company).
2) the 95 ℃ of sex change 10min of pcr amplification product with digoxigenin labeled quench then.
3) the PCR product of 50 μ L sex change is joined in the hybridization solution (Roch company) that 600 μ L newly change, add the intact nylon membrane of prehybridization and rock hybridization 1 hour for 50 ℃.
4) after hybridization is finished with film cooling, use respectively under the room temperature 2 * SSC (+0.1%SDS), 0.5 * SSC (+0.1%SDS) and lavation buffer solution (Roch company) respectively wash 2min in order,
5) seal 30min with confining liquid, add enzyme and join anti--DigiTAb in conjunction with 30min.
6) wash 15min * 2 time with lavation buffer solution.With detecting damping fluid (Roch company) balance 5min, add the colour developing liquid lucifuge colour developing observations after 2 hours that contains the chromogen substrate.The results are shown in accompanying drawing.
Accompanying drawing: seawater sample 1 detected result
Show the expression positive findings of round dot in the accompanying drawing medium square, not Xian Se expression negative findings.
Wherein 1 row 9 is listed as---positive controls,
The probe of 1 row, 3 row is Vfu710b, and expression has detected Vibrio furnissii;
The probe of 1 row, 6 row is Sal-1,
The probe of 6 row, 7 row is Sal-2,
The probe of 5 row, 7 row is Sal-3, and expression has detected Salmonellas;
The probe of 2 row, 1 row is Vpiapro,
The probe of 4 row, 2 row is Vp-n1, and expression has detected Vibrio parahaemolyticus;
The probe of 4 row, 6 row is PSE-1,
The probe of 2 row, 6 row is PSE-3, and expression has detected Pseudomonas aeruginosa;
The probe of 4 row, 3 row is Mchar, and expression has detected vibrio cholerae;
The probe of 5 row, 4 row is Vh710b, and expression has detected Kazakhstan arc maintenance bacterium.
Sequence list
SEQUENCE LISTING
＜110〉Nankai University
＜120〉a kind of oligonucleotide microarray technique that detects pathogen contamination in the seawater
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Claims (3)
1. an oligonucleotide microarray technique that detects pathogen contamination in the seawater is characterized in that, 43 probe sequences of microarray are as follows:
2. according to the described a kind of oligonucleotide microarray technique that detects pathogen contamination in the seawater of claim 1, it is characterized in that this oligonucleotide microarray technique using method is as follows:
(1) oligonucleotide microarray is made:
1) preparation probe dilution liquid: 1 * salmon sperm dna solution (1g/L),
2) preparation probe solution: be configured to 0.1ug/ μ L probe solution with probe dilution liquid,
3) probe solution is put on 5cm * 5cm nylon membrane matrix, 80 ℃ are incubated 2 hours;
The probe point sample is in proper order as following table:
(2) seawater sample process and DNA extraction
1) sample process
1. get surface seawater 20L, be sub-packed in the plastic tank of sterilising treatment, the seawater of obtaining is handled in 3h;
2. earlier remove by filter suspended matter with 0.15 millimeter aperture sieve, use Mini-pellicon system (Millipore, the U.S.) to carry out pressure filtration then and collect thalline, the filter membrane aperture is 0.22 μ m (Milipo, the U.S.).The corresponding filter membrane of every filtration 2L seawater, promptly every 2L seawater be as a sample, corresponding 10 parallel sampleses of each sampling point;
3. with sterilized water the thalline on the filter membrane is washed, and collect thalline with the centrifugal 5min of 10000r/min immediately.The thalline and the contamination precipitation of results are used for the DNA extracting;
2) DNA extraction
1. the seawater thalline filters thalline and the centrifugal 2min of silt suspension liquid 12000r/min that obtains sample;
2. throw out adds TE damping fluid (10mM TrisHCl, the 1mM EDTA of 550 μ L; PH 8.0), piping and druming makes it to suspend again repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, mixing, 37 ℃ of incubation 1h;
3. add 100 μ L 5mol/L NaCl, mixing adds 80 μ L CTAB/NaCl solution (5g CTAB is dissolved in the 100mL0.5M NaCl solution), mixing, 65 ℃ of incubation 10min again;
4. add the abundant mixing of isopyknic phenol/chloroform/primary isoamyl alcohol, centrifugal 4-5min changes supernatant in the new pipe over to, adds the Virahol of 0.6-0.8 times of volume, softly puts upside down mixing, places 20min for-20 ℃;
5. precipitation is inverted drying with 70% washing with alcohol of 1mL, heavily is dissolved in 50 μ L TE damping fluids;
6. the DNA sample boils and was quenching on ice then in 5 minutes, and-20 ℃ of preservations are standby;
(3) polymerase chain reaction and digoxigenin labeled
1) amplification and mark system:
Each component final concentration and volume (μ L) in the reagent system
10 * polymerase chain reaction Buffer, 5.0 μ L
F-E-T 10μmol/L(4.0μL)
B-E-T 10μmol/L(4.0μL)
dNTP 10mmol/L(0.3μL)
Digoxigenin-11-dUTP 25nmol/L(0.3μL)
Dna profiling 5.0 μ L
Taq DNA Polymerase 5U/μL(0.3μL)
Distilled water 31.1 μ L
Amount to 50.0 μ L
10 * polymerase chain reaction Buffer:Tris-HCl pH8.5,100mmol/L; KCl, 500mmol/L;
MgCl
2，15mmol/L；
2) polymerase chain reaction program
1. the pre-sex change stage: 95 ℃ of 5min
2. first cycle stage (5 circulations): 94 ℃ of 30sec
54℃30sec
72℃50sec
3. second cycle stage (5 circulations): 94 ℃ of 30sec
56℃30sec
72℃40sec
4. the 3rd cycle stage (10 circulations): 94 ℃ of 30sec
58℃30sec
72℃40sec
5. the 3rd cycle stage (15 circulations): 94 ℃ of 30sec
62℃40sec
6. ending phase: 72 ℃ of 5min
4 ℃ of insulations
(4) hybridization testing process
Hybridization uses U.S. Roch company digoxin detection kit;
1) each oligonucleotide microarray immerses 50 ℃ of prehybridization 45min in the 600 μ L hybridization solutions (Roch company);
2) the 95 ℃ of sex change 10min of pcr amplification product with digoxigenin labeled quench then;
3) the PCR product of 50 μ L sex change is joined in the hybridization solution (Roch company) that 600 μ L newly change, add the intact nylon membrane of prehybridization and rock hybridization 1 hour for 50 ℃;
4) after hybridization is finished with film cooling, use respectively under the room temperature 2 * SSC (+0.1%SDS), 0.5 * SSC (+0.1%SDS) and lavation buffer solution (Roch company) respectively wash 2min in order;
5) seal 30min with confining liquid, add enzyme and join anti--DigiTAb in conjunction with 30min;
6) wash 15min * 2 time with lavation buffer solution, with detecting damping fluid (Roch company) balance 5min, add the colour developing liquid lucifuge colour developing observations after 2 hours that contains the chromogen substrate;
20 * SSC:175.3g NaCl and 88.2g Trisodium Citrate are dissolved in 800mL water, add NaOH and regulate pH to 7.0, constant volume 1L;
2 * SSC: with 10 times of 20 * SSC dilute with waters;
0.5 * SSC: with 40 times of 20 * SSC dilute with waters.
3. application that detects oligonucleotide microarray technique pathogen contamination in detecting other body of sea water of pathogen contamination in the seawater.
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