CN101086022A - Primer and fluorescent probe for quantitatively detecting sulfate functional bacterium - Google Patents
Primer and fluorescent probe for quantitatively detecting sulfate functional bacterium Download PDFInfo
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- CN101086022A CN101086022A CN 200710072334 CN200710072334A CN101086022A CN 101086022 A CN101086022 A CN 101086022A CN 200710072334 CN200710072334 CN 200710072334 CN 200710072334 A CN200710072334 A CN 200710072334A CN 101086022 A CN101086022 A CN 101086022A
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Abstract
The invention relates to a kind of primer and fluorescent probe that are used for sulfate functional bacteria detection. It overcomes shortcomings of long detection time, smaller detection result, disability of instant production direction and high detection price existed in current quantitative determination method for SBR bacteria in oil-field brine. The gene order of upstream primer is 5'-TCAGCTCGTGTCGTGAGATGTT-3'and gene order of downstream primer is 5'-ACGTCATCCCCACCTTCCTC-3'. The fluorescent probe used for sulfate functional bacteria detection is FAM: 5'-CAACGAGCGCAACC-3'MGB; the report fluorophor marked by 5'end in Tag Man-MGB probe is FAM, and the quenching fluoropho marked by 3' end is TAMR- MGB. The invention is characterized by accurate detection and result, short bacteria number count time, only 6 hours for sample mould DNA extraction to fluorescence quantitative PCR detection, reduced detection period and decreased detection cost.
Description
Technical field
The present invention relates to be used for the primer and the probe of sulfate functional bacterium detection by quantitative.
Background technology
The sulfate reduction function yeast is the general name of the class bacterium relevant with sulfate reduction, it is a classification to microorganism based on functional perspective, in the process of field produces, the breeding of sulfate reduction function yeast can cause a lot of harm, as: corrosion product in the ground system-water-fast metallic sulfide, cause sewage blackout, suspended solids content to increase, make and handle that suspended solids content exceeds standard in the water of back; The fast quantification of sulfate reduction function yeast detects, and for determining of reasonable bacteriocidal concentration in oil field water quality detection and the ground system, in time instructs production practice to have great importance.At present, the counting of sulfate functional bacterium bacterium is mainly carried out the China National Petroleum industry standard, " oilfield injection water bacteria analyzing method-disappearance dilution method " (ministerial standard), SY-T0532-93 in the oil field system.The problem that above-mentioned this method exists is: detecting needs 14 days time, because sense cycle is longer, can not be effective and instant instruct production practice, some strict anaerobism sulfate functional bacterium can't be survived in actual mechanical process, cause the numeration result of sulfate functional bacterium inaccurate, can not reflect the production practical situation really, detected result is meaningless, and testing cost is higher simultaneously.
Summary of the invention
The present invention be for solve existing oil field water quality SRB bacterium sense cycle long, can not be truly the comprehensively quantity, guidance production, testing cost problem of higher that can not be instant of the SRB bacterium in the reaction water, and provide a kind of primer and fluorescent probe that is used for the detection by quantitative sulfate functional bacterium.The primer that is used for the detection by quantitative sulfate functional bacterium: upstream primer: 5 '-TCAGCTCGTGTCGTGAGATGTT-3 ', downstream primer: 5 '-ACGTCATCCCCACCTTCCTC-3 '.The fluorescent probe that is used for the detection by quantitative sulfate functional bacterium is FAM:5 '-CAACGAGCGCAACC-3 ' MGB; The report fluorophor of 5 ' end mark of Tag Man-MGB probe is FAM, and the cancellation fluorophor of 3 ' end mark is TAMRA-MGB.The present invention adopts Tag Man-MGB probe, the 16SrDNA gene order that has sulphate salt restoring function bacterial strain by collection, design a pair of conservative specially property primer and a MGB probe specially, can diagnose accurately, the accurate and effective shortening gate time of numeration result, from sample DNA extract quantitative fluorescent PCR obtain at last detected result only need 6 hours, can reflect the production practical situation really, reduce to detect cost.
Description of drawings
Fig. 1 is to be the amplification curve of substrate with pGEM-T-SRB in the embodiment three, wherein 1. is 1.496 * 10
7, 2. be 1.496 * 10
6, 3. be 1.496 * 10
5, 4. be 1.496 * 10
4, 5. be 1.496 * 10
3, 6. be 1.496 * 10
2, 7. be the water contrast; Fig. 2 is a sulphate reducing bacteria fluorescent quantitation examination criteria graphic representation in the embodiment three.
Embodiment
Embodiment one: the primer that is used for the detection by quantitative sulfate functional bacterium in the present embodiment: upstream primer: 5 '-TCAGCTCGTGTCGTGAGATGTT-3 ', downstream primer: 5 '-ACGTCATCCCCACCTTCCTC-3 '.
Embodiment two: the fluorescent probe that is used for the detection by quantitative sulfate functional bacterium in the present embodiment is FAM:5 '-CAACGAGCGCAACC-3 ' MGB; The report fluorophor of 5 ' end mark of Tag Man-MGB probe is FAM, and the cancellation fluorophor of 3 ' end mark is TAMRA-MGB.
Embodiment three: present embodiment specifies the present invention and how to implement, and present embodiment realizes by following steps:
One, design primer and probe:
Adopt Tag Man-MGB probe, have the 16SrDNA gene order of sulphate salt restoring function bacterial strain, design a pair of conservative specially property primer and a MGB probe specially by collection.
Upstream primer (Forward Primer) is: 5 '-TCAGCTCGTGTCGTGAGATGTT-3 ';
Downstream primer (Reverse Primer) is: 5 '-ACGTCATCCCCACCTTCCTC-3 '
Fluorescent probe is FAM5 '-CAACGAGCGCAACC-3 ' MGB; The report fluorophor FAM of 5 ' end mark of the probe (Tag Man-MGB probe) of fluorescent quantitation detection sulfate functional bacterium, the cancellation fluorophor TAMRA-MGB of 3 ' end mark.
Primer and probe are finished by bio-engineering corporation.
Two, working method:
The extraction of A, nucleic acid DNA: 1), 3000 leave the heart then, stay supernatant liquor with 1mL mud concussion 5min; 2) 10 * PBS that adds 2 times of volumes washs, and concussion 5min 12000 leaves heart 5min then, removes supernatant liquor; 3) add 10 * PBS damping fluid, 100 μ l, ultrasonic then 40s; 4) extract test kit with magnificent Shun DNA a small amount of bacterium and carry out the subsequent DNA extraction.
The preparation of B, real time PCR standard substance:
1, the DNA that extracts in the previous step is carried out pcr amplification: set up PCR reaction system (20 μ l): template (DNA that extracts in the previous step) 20ng, TagDNA polysaccharase (available from the precious biotechnology in Dalian company limited) 0.3U, 4 kinds of each 0.3mmol/L of dNTP, each 0.1 μ mol/L of upstream primer (Forward Primer) and downstream primer (Reverse Primer); The PCR reaction conditions: 94 ℃ of preheating 5min, 94 ℃ of sex change 30s, 58 ℃ of renaturation 45s, 72 ℃ are extended 90s, circulate 30 times, and last 72 ℃ are extended 10min.The PCR product is detected with 1.0% agarose electrophoresis.
2, connection, conversion, bacterium colony PCR detect
The PCR product reclaims test kit (precious Tyke) with glue and cuts the glue recovery, and the back is connected with pGEM-T (Promega) carrier, is transformed into intestinal bacteria TOP10 competent cell (sky is the epoch).Add penbritin Amp (5 μ g/mL) and X-gal in the LB solid medium, blue hickie screening transformant.Extract plasmid (Hua Shun), detect with carrier primer T7 and SP6, order-checking is finished by Shanghai biotechnology company limited, and the acquisition sequence length is 130bp.
＞SRB16
5’-TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCTTTAGTTGCCATCAGTTCGGCTGGGCACTCTAAAGGGACTGCCGGTGTCAAACCGGAGGAAGGTGGGGATGACGT-3’。
3, the concentration determination of pGEM-T-SRB
The MJResearch Opticon TM 2 real-time fluorescence systems that adopt the U.S. to adopt MJ company to produce carry out quantitative fluorescent PCR.
Extract intestinal bacteria transformant plasmids (containing the pulsating plasmid of purpose) pGEM-T-SRB, measure its concentration and be scaled copy number.
Calculation result is 1.496 * 10
7Copies μ l
-1, do 10 * dilution then.Fig. 1 for-pGEM-T-SRB is the amplification curve of substrate, 7 curves from left to right are followed successively by 1.496 * 10
7, 1.496 * 10
6, 1.496 * 10
5, 1.496 * 10
4, 1.496 * 10
3, 1.496 * 10
2Copies μ l
-1, water contrast.After 40 loop ends of real-time fluorescence quantitative RT-PCR, computer utilizes analysis software Opticon Monitor2.02 according to the template amount of input and the fluorescent signal of generation, calculates a typical curve such as Fig. 2: y=-0.32x+10.67, and relation conefficient is r
2=1.000.The result shows, at least 1.496 * 10
0～1.496 * 10
7Within the copy scope, its corresponding CT value of sample copy number has good dependency.Therefore, this experiment can be to 1.496 * 10
0～1.496 * 10
7Template within the copy scope is carried out accurately quantitatively.
Three, detect:
1,1mL is treated test sample injects centrifuge tube after, in ultrasonic apparatus, shake 5min, centrifugal 2min in the 3000rpm/min whizzer gets supernatant liquor then; 10 * the PBS that adds 2 times of volumes in supernatant liquor washs, and shakes 5min in ultrasonic apparatus, and centrifugal 5min in the whizzer of 12000rpm/min removes supernatant liquor then; In centrifuge tube, add 10 * PBS damping fluid of 100 μ l, in ultrasonic apparatus, shake 5min; Extract test kit with magnificent Shun DNA a small amount of bacterium and extract DNA, obtain treating the DNA of test sample;
2, the DNA that treats test sample carries out fluorescence RT-PCR mensuration: set up RT-PCR reaction system (25 μ L): 5 * real time buffer, 5.0 μ L, dNTP (10mmol/L) 0.75 μ L, Mg
2+(250mmol/L) 0.5 μ L, upstream primer (Forward Primer) and downstream primer (Reverse Primer) be each 0.5 μ L (10mmol/L), fluorescent probe (5mmol/L) 0.6 μ L, Taq DNA enzyme (5U/ μ L) 0.3 μ L, treat the DNA 2 μ L of test sample, deionized water 15.4 μ L; Real time PCR reaction parameter: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 5s, 60 ℃ of annealing temperature 30s, 72 ℃ are extended 45s, 40 circulations, 72 ℃ are extended 2min.The temperature transition rate is 20 ℃/s, carries out fluorescent signal and detect when 60 ℃ of each round-robin.
3, the judgement of detected result: compare with standard, the CT value should be less than 28 in the positive control, and amplification curve is obvious in the positive; Feminine gender correlates this greater than 35 to the CT value, does not have amplification curve in the negative control.Different templates has the target segment of different quantity, curve at the fluorescent quantitation of detection by quantitative just has corresponding C T value, the typical curve that generates by system, system can provide the quantity of target fragment in the template, the i.e. quantity of sulfate reduction function yeast at last according to the CT value.
Embodiment four: present embodiment is got 15 samples to be tested and has been carried out the fluorescence RT-PCR detection, extract intestinal bacteria transformant plasmids (containing the pulsating plasmid of purpose)-pGEM-T-ADNB, be template with the plasmid solution after the dilution respectively, with FP, RP is a primer, carrying out real-time fluorescence quantitative RT-PCR simultaneously with detected sample detects, the initial copy number of each sample is calculated according to the CT value of typical curve and sample automatically by analysis software Opticon Monitor 2.02,15 samples that detect the results are shown in Table 1, water contrast and pure bacterium (intestinal bacteria) wherein are set, verify with the disappearance dilution method simultaneously, detect the sulfate functional bacterium in 14 samples to be tested as a result, detected result is with conventional sense method disappearance dilution method (mostprobable number, MPN)-detected result of Griess method fits like a glove, but on the precision that detects, than two orders of magnitude of disappearance dilution method raising of routine.
Table 1 test sample CT value and copy number (bacterial count) and disappearance dilution method
| Sample number | The CT value | The fluorescent quantitation bacterial count (individual/mL) | MPN counting bacterial count (individual/mL) |
| The water contrast | 0 | 0 | 0 |
| Intestinal bacteria | 0 | 0 | 0 |
| 1 | 10.895 | 13588 | 135 |
| 2 | 4.408 | 5389440 | 60000 |
| 3 | 5.534 | 1907940 | 20000 |
| 4 | 11.376 | 8724.92 | 90 |
| 5 | 37.399 | 3293630 | 35000 |
| 6 | 6.419 | 843278 | 8500 |
| 7 | 11.719 | 6354.62 | 70 |
| 8 | 7.199 | 410945 | 4000 |
| 9 | 13.091 | 1792.82 | 18 |
| 10 | 8.499 | 123911 | 1500 |
| 11 | 11.009 | 12236.5 | 150 |
| 12 | 6.776 | 606908 | 6500 |
| 13 | 14.633 | 432.515 | 10 |
| 14 | 13.096 | 1785.13 | 18 |
Sequence table
＜110〉Harbin Institute of Technology
＜120〉be used for the primer and the fluorescent probe of detection by quantitative sulfate functional bacterium
<160>4
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to detect the upstream primer of sulfate functional bacterium.
<400>1
tcagctcgtg tcgtgagatg tt 22
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to detect the downstream primer of sulfate functional bacterium.
<400>2
acgtcatccc caccttcctc 20
<210>3
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the probe gene order of detection sulfate functional bacterium.
<400>3
caacgagcgc aacc 14
<210>4
<211>130
<212>DNA
＜213〉artificial sequence
<220>
＜223〉gene order of inserting in the positive plasmid.
<400>4
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgcctttag 60
ttgccatcag ttcggctggg cactctaaag ggactgccgg tgtcaaaccg gaggaaggtg 120
gggatgacgt 130
Claims (2)
1, the primer that is used for the detection by quantitative sulfate functional bacterium is characterized in that being used for the primer of detection by quantitative sulfate functional bacterium: upstream primer: 5 '-TCAGCTCGTGTCGTGAGATGTT-3 ', downstream primer: 5 '-ACGTCATCCCCACCTTCCTC-3 '.
2, the fluorescent probe that is used for the detection by quantitative sulfate functional bacterium, the fluorescent probe that it is characterized in that being used for the detection by quantitative sulfate functional bacterium is FAM5 '-CAACGAGCGCAACC-3 ' MGB; The report fluorophor of 5 ' end mark of the probe of fluorescent quantitation detection sulfate functional bacterium is FAM, and the cancellation fluorophor of 3 ' end mark is TAMRA-MGB.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762916A (en) * | 2019-03-18 | 2019-05-17 | 中国海洋石油集团有限公司 | The method and primer special of quantitative detection sulfate reducing bacteria |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762916A (en) * | 2019-03-18 | 2019-05-17 | 中国海洋石油集团有限公司 | The method and primer special of quantitative detection sulfate reducing bacteria |
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Open date: 20071212 |