CN101086021A - Primer and fluorescent probe for detecting sulfate reduction bacterium - Google Patents
Primer and fluorescent probe for detecting sulfate reduction bacterium Download PDFInfo
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- CN101086021A CN101086021A CN 200710072333 CN200710072333A CN101086021A CN 101086021 A CN101086021 A CN 101086021A CN 200710072333 CN200710072333 CN 200710072333 CN 200710072333 A CN200710072333 A CN 200710072333A CN 101086021 A CN101086021 A CN 101086021A
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Abstract
The invention discloses a kind of primer and fluorescent probe that are used for sulfate-reducing bacteria detection, relating to primer and fluorescent probe that are used for microbe detection. It overcomes shortcomings of long detection time, high non-specificity and smaller detection result compared to correct result which are existed in current quantitative determination method for SBR bacteria. The gene order of upstream primer is 5'-GTTCCCTGCTCGTGCCCT-3'and gene order of downstream primer is 5'-TTCCTTGAAGAAGATGTACGGGTT-3'. The fluorescent probe comprises report fluorophor, quenching fluorophor and probe gene order; the probe gene order is 5'-AATGGTGGATGGAAGAAGGC-3', the report fluorophor marked by 5'end in probe gene order is FAM, and the quenching fluoropho marked by 3' end in probe gene order is TAMRA. The invention is characterized by high specificity of primer and fluorescent probe, short SBR bacteria determination time, which needs only 6 hours for sample mould DNA extraction to fluorescence quantitative PCR detection.
Description
Technical field
The present invention relates to be used to detect primer and the probe of microorganism.
Background technology
(sulfate reducing bacteria SRB) is the general name of the class bacterium relevant with sulfate reduction to sulphate reducing bacteria.Constitute by multiple microorganism such as Gram-negative bacteria (as Desulfovibrio Desulfovibrio, Desulfobacter Desulfobacterium), gram-positive microorganism (Desulfotomaculum Desulfotomaculum), thermophilic bacterium (thermally desulfurizing Pseudomonas Thermodesulfobacterium) and thermophilic archeobacteria (ancient green-ball Pseudomonas Archaeoglobus).
The SRB bacterium becomes H by the biochemical reaction of a series of complexity with sulfate reduction
2S and a large amount of sulfide, sulfide and H
2S not only corrodes oilfield production equipment; And its corrosion product (metallic sulfide) is water insoluble, causes sewage blackout, suspended solids content to increase, make to handle that suspended solids content exceeds standard in the water of back, serious harm the ordinary production in oil field.Therefore, fast quantification detection sulphate reducing bacteria has important operation instruction meaning for the reasonable adjusting of oil field water quality detection and ground system concentration of sterilant.But at present the quantitative detecting method of SRB bacterium (as: disappearance dilution method etc.) exists length detection time, non-specific height, defective that detected result is on the low side.
Summary of the invention
The objective of the invention is for the quantitative detecting method that solves present SRB bacterium exists length detection time, non-specific height, defective that detected result is on the low side, and the primer and the fluorescent probe that are used to detect sulphate reducing bacteria that provide.
The upstream primer gene order that is used to detect sulphate reducing bacteria is 5 '-GTTCCCTGCTCGTGCCCT-3 '; The downstream primer gene order is 5 '-TTCCTTGAAGAAGATGTACGGGTT-3 '.
The fluorescent probe that is used to detect sulphate reducing bacteria is made up of report fluorophor, cancellation fluorophor and probe gene order; The probe gene order is 5 '-AATGGTGGATGGAAGAAGGC-3 ', and the report fluorophor of probe gene order 5 ' end mark is FAM, and the cancellation fluorophor of probe gene order 3 ' end mark is TAMRA.
(dissimilatory sulfite reductase is that the sulphate reducing bacteria sulphate reducing generates H DSR) to alienation type sulfite reductase
2The key enzyme of S.The DSR gene has the conservative property structural framing in sulphate reducing bacteria, the six aggressiveness (α that DSR is made up of three different subunits
2β
2γ
2), include the conservative framework (C-X of typical vitriol-nitrite reductase in the alienation type sulfite reductase α subunit
5-C)-X
n-(C-X
3-C) and CP-X
n-C-X
2-C-X
2-C framework can with [Fe
4S
4]-sigh cough up iron iron porphyrin (siroheme) combination, be a total stable target site of SRB bacterium; So the present invention has designed according to the conservative region of alienation type sulfite reductase gene order has specific primer and fluorescent probe.Primer of the present invention and fluorescent probe specificity height, false positive significantly reduces under the dual function of primer and fluorescent probe.Use primer of the present invention and fluorescent probe to carry out the fluorescence quantitative PCR detection sulphate reducing bacteria, detection time is short, extract the fluorescence quantitative PCR detection whole process from the template DNA of sample only needs 6h.
Description of drawings
Fig. 1 is to be the pcr amplification graphic representation of substrate with pGEM-T-DSR in the embodiment four, among Fig. 1 curve 1. template concentrations be 2.31 * 10
7The pcr amplification curve of copies/ μ L, 2. template concentrations is 2.31 * 10
6The pcr amplification curve of copies/ μ L, 3. template concentrations is 2.31 * 10
5The pcr amplification curve of copies/ μ L, 4. template concentrations is 2.31 * 10
4The pcr amplification curve of copies/ μ L, 5. template concentrations is 2.31 * 10
3The pcr amplification curve of copies/ μ L, 6. template concentrations is 2.31 * 10
2The pcr amplification curve of copies/ μ L, 7. template concentrations is the pcr amplification curve of 0copies/ μ L; Fig. 2 is a sulphate reducing bacteria fluorescent quantitation examination criteria graphic representation in the embodiment four.
Embodiment
Embodiment one: the upstream primer gene order (FP) that present embodiment is used to detect sulphate reducing bacteria is 5 '-GTTCCCTGCTCGTGCCCT-3 '; Downstream primer gene order (RP) is 5 '-TTCCTTGAAGAAGATGTACGGGTT-3 '.
Present embodiment detects conservative property structural framing that primer has according to the DSR gene and specificity design, synthetic in sulphate reducing bacteria.
Embodiment two: present embodiment is used to detect the fluorescent probe of sulphate reducing bacteria and is made up of report fluorophor, cancellation fluorophor and probe gene order; The probe gene order is 5 '-AATGGTGGATGGAAGAAGGC-3 ', and the report fluorophor of probe gene order 5 ' end mark is FAM, and the cancellation fluorophor of probe gene order 3 ' end mark is TAMRA.
The conservative property structural framing that the gene order of present embodiment fluorescent probe has in sulphate reducing bacteria according to the DSR gene and specificity design, synthetic.
Embodiment three: present embodiment adopts fluorescence quantitative PCR method to detect sulphate reducing bacteria:
1, design, synthetic primer and the fluorescent probe of detecting
The conservative property structural framing that has in sulphate reducing bacteria according to the DSR gene designs, synthesizes Auele Specific Primer and fluorescent probe.Upstream primer gene order (FP) is 5 '-GTTCCCTGCTCGTGCCCT-3 '; Downstream primer gene order (RP) is 5 '-TTCCTTGAAGAAGATGTACGGGTT-3 '; The probe gene order is 5 '-AATGGTGGATGGAAGAAGGC-3 ', and the report fluorophor of probe gene order 5 ' end mark is FAM, and the cancellation fluorophor of probe gene order 3 ' end mark is TAMRA.The synthetic of primer and fluorescent probe finished by bio-engineering corporation.
2, primer and fluorescent probe check
(1) preparation standard product (sulphate reducing bacteria) gene fragment.Standard substance gene fragment PCR reaction system (20 μ L) is: sulphate reducing bacteria templet gene 20ng, TagDNA polysaccharase final concentration is 0.3U, 4 kinds of each 0.3mmol/L of dNTP, (gene order is: 0.1 μ mol/L 5 '-GTTCCCTGCTCGTGCCCT-3 '), (gene order is downstream primer RP upstream primer FP: 0.1 μ mol/L and balance of deionized water 5 '-TTCCTTGAAGAAGATGTACGGGTT-3 ').Standard substance PCR response procedures is: 94 ℃ of preheating 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 90s, circulate 30 times, and last 72 ℃ are extended 10min.
The TagDNA polysaccharase is available from precious biotechnology (Dalian) company limited.
(2) connect, transform.The PCR product (standard substance gene fragment) that previous step (step 2 (1)) is obtained reclaims test kit (precious Tyke) with gelatin and cuts glue and reclaim, and is connected with the pGEM-T carrier then, is transformed in the intestinal bacteria TOP10 competent cell again.
PGEM-T is available from U.S. Promega company, and intestinal bacteria TOP10 competent cell is available from being Time Technology company limited in sky, Beijing.
(3) blue hickie screening.At the enterprising row filter of LB solid medium that adds penbritin Amp (5 μ g/mL) and X-gal.
(4) detect the positive plasmid gene order.Extract white plasmid gene, again with pGEM-T carrier primer T7 and SP6 amplification, and order-checking.The DNA a small amount of bacterium that the plasmid gene extraction uses the Shanghai China biological company limited of Shun to produce is extracted test kit, and gene sequencing work is finished by bio-engineering corporation, and insertion sequence is 227p in the plasmid, shown in SEQ ID NO:4.
3, sulphate reducing bacteria fluorescent quantitation detection sensitivity detects and the drawing standard graphic representation
(1) concentration of detection pGEM-T-DSR.
(2) be scaled copy number.The copy number reduction formula is:
C is the concentration of pGEM-T-DSR in the copy number reduction formula, unit: μ g/ μ L; Carrier and purpose segmental unit: bp.
(3) plasmid solution (10 *) gradient dilution.With the plasmid solution after the dilution is template, is control group with water, is primer with FP, RP, carries out real-time fluorescence quantitative RT-PCR and detects.After 40 loop ends of real-time fluorescence quantitative RT-PCR, utilize analysis software Opticon Monitor2.02, according to the template amount of input and the fluorescent signal of generation, calculate sulphate reducing bacteria fluorescent quantitation examination criteria curve.
4, sulphate reducing bacteria detects in real time
(1) real-time PCR reactions system (25 μ L) is as shown in table 1.
Table 1
| 5 * real time buffer (damping fluid in real time) | 5.0μL |
| dNTP(10mmol/L) | 0.75μL |
| Mg 2+(250mmol/L) | 0.5μL |
| Sulphate reducing bacteria detects primers F P (10mmol/L) | 0.5μL |
| Sulphate reducing bacteria detects primer RP (10mmol/L) | 0.5μL |
| Fluorescent probe (5mmol/L) | 0.6μL |
| Taq DNA enzyme (5U/ μ L) | 0.3μL |
| Test sample | 2μL |
| Deionized water | 15.4μL |
And on orifice plate, establish negative control, be in the same old way with water.
(2) real-time PCR reactions program: 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.Carrying out fluorescent signal when each round-robin 60 ℃ (annealing) detects.
(3) detected result is judged: less than 28, amplification curve is obvious in the positive for CT value in the positive control (thresholding cycle number); Negative control CT value does not have amplification curve greater than 35 in the negative control.
Oil field sludge or sewage sludge sample obtain according to the following steps in the present embodiment step 4: (one) 1mL mud concussion 5min, and centrifugal 3～5min under the condition of 3000r/min stays supernatant liquor then; (2) 10 * PBS that adds 2 times of volumes of supernatant liquor washs, concussion 5min, and centrifugal 5min under the condition of 12000r/min removes supernatant liquor then; (3) add 10 * PBS damping fluid, 100 μ L, ultrasonic wave 40s then; (4) the DNA a small amount of bacterium extraction test kit of producing with the biological company limited of the magnificent Shun in Shanghai carries out the subsequent DNA extraction.
To be used to detect the upstream primer gene order of sulphate reducing bacteria consistent for 1bp～18bp and present embodiment in the gene order of inserting in the present embodiment positive plasmid; To be used to detect the downstream primer gene order of sulphate reducing bacteria consistent for 204bp～227bp and present embodiment in the gene order of inserting in the positive plasmid; To be used to detect the gene order of fluorescent probe of sulphate reducing bacteria consistent for 76bp～95bp and present embodiment in the gene order of inserting in the positive plasmid.
The MJResearch Opticon TM2 real-time fluorescence system that present embodiment adopts U.S. MJ company to produce carries out quantitative fluorescent PCR.
Data of measuring according to quantitative real time PCR Instrument and sulphate reducing bacteria fluorescent quantitation examination criteria curve can quantitative calculation go out the quantity of sulphate reducing bacteria in the sample.It is more accurate that present embodiment detects numeration, and false positive significantly reduces.The present embodiment detected result only needs 6 hours, has saved detection time greatly, has more actual directive significance in the actual production of oil field.
Embodiment four: present embodiment adopts fluorescence quantitative PCR method to detect sulphate reducing bacteria, with the difference of embodiment three be in the step 3 (1) after testing the concentration of pGEM-T-DSR be 2.31 * 10
7Copies/ μ L.
Step 3 (3) plasmid solution (10 *) gradient dilution.With the plasmid solution after the dilution is template, is control group with water, is primer with FP, RP, carry out fluorescence quantitative RT-RCR and detect, and to be gone out with pGEM-T-DSR by computer drawing is the pcr amplification graphic representation (as shown in Figure 1) of substrate.Among Fig. 1 from left to right 7 curves be that template concentrations is 2.31 * 10 successively
7Copies/ μ L, 2.31 * 10
6Copies/ μ L, 2.31 * 10
5Copies/ μ L, 2.31 * 10
4Copies/ μ L, 2.31 * 10
3Copies/ μ L, 2.31 * 10
2The pcr amplification curve of copies/ μ L and 0copies/ μ L (control group).After 40 loop ends of real-time fluorescence quantitative RT-PCR, utilize analysis software Opticon Monitor2.02, according to the template amount of input and the fluorescent signal of generation, calculate sulphate reducing bacteria fluorescent quantitation examination criteria curve (as shown in Figure 2).Other step and parameter are identical with embodiment three.
Present embodiment sulphate reducing bacteria fluorescent quantitation examination criteria curve equation: Y=-0.31X+11.13; Relation conefficient is r
2=0.999.The result shows that its corresponding CT value of sample copy number has good dependency, can be used for the detection by quantitative of sulphate reducing bacteria.
Present embodiment and conventional sense method disappearance dilution method (most probable number, MPN)-Griess method detected result compares, and the detected result of two kinds of detection methods is as shown in table 2.
Table 2
| 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 | 9.750 | 3452630 | 35000 |
| 2 | 17.979 | 11159 | 1200 |
| 3 | 18.233 | 9350.11 | 90 |
| 4 | 9.979 | 2943650 | 30000 |
| 5 | 17.785 | 12778.6 | 135 |
| 6 | 18.093 | 10311.9 | 100 |
| 7 | 11.736 | 864862 | 9000 |
| 8 | 17.850 | 12208 | 125 |
| 9 | 11.792 | 831698 | 8000 |
| 10 | 16.848 | 24542.4 | 250 |
| 11 | 11.404 | 1090150 | 12500 |
| 12 | 16.466 | 32030 | 350 |
| 13 | 16.728 | 26687.2 | 300 |
| 14 | 18.221 | 9427.75 | 90 |
| 15 | 17.221 | 18924.3 | 190 |
The accuracy of detection of detected result explanation present embodiment detection method improves two orders of magnitude than ordinary method (disappearance dilution method).
Sequence table
＜110〉Harbin Institute of Technology
＜120〉be used to detect the primer and the fluorescent probe of sulphate reducing bacteria
<160>4
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to detect the upstream primer of sulphate reducing bacteria, design according to the conservative property structural framing of DSR gene in sulphate reducing bacteria.
<400>1
gttccctgct cgtgccct 18
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to detect the downstream primer of sulphate reducing bacteria, design according to the conservative property structural framing of DSR gene in sulphate reducing bacteria.
<400>2
ttccttgaag aagatgtacg ggtt 24
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the probe gene order of detection sulphate reducing bacteria designs according to the conservative property structural framing of DSR gene in sulphate reducing bacteria.
<400>3
<210>4
<211>227
<212>DNA
＜213〉artificial sequence
<220>
＜223〉gene order of inserting in the positive plasmid.
<400>4
gttccctgct cgtgcccttc gtttcctgtg aagcccctta cgatgacgtg aaagaagtca 60
tcgaaaagat ttgggaatgg tggatggaag aaggcaagaa ccgcgagcgc gtgggtgaaa 120
ccatgaagcg cctgtcgttc cagaagctgc tggaagtcac cgacactccc gccgctgcct 180
accatgtgaa ggaaccgcgt tccaacccgt acatcttctt caaggaa 227
Claims (2)
1, be used to detect the primer of sulphate reducing bacteria, the upstream primer gene order that it is characterized in that being used to detecting sulphate reducing bacteria is 5 '-GTTCCCTGCTCGTGCCCT-3 '; The downstream primer gene order is 5 '-TTCCTTGAAGAAGATGTACGGGTT-3 '.
2, be used to detect the fluorescent probe of sulphate reducing bacteria, the fluorescent probe that it is characterized in that being used to detecting sulphate reducing bacteria is made up of report fluorophor, cancellation fluorophor and probe gene order; The probe gene order is 5 '-AATGGTGGATGGAAGAAGGC-3 ', and the report fluorophor of probe gene order 5 ' end mark is FAM, and the cancellation fluorophor of probe gene order 3 ' end mark is TAMRA.
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| CN200710072333A CN101086021B (en) | 2007-06-08 | 2007-06-08 | Primer for detecting sulfate reduction bacterium |
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| CN101086021B CN101086021B (en) | 2010-05-26 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088133A (en) * | 2013-01-22 | 2013-05-08 | 华东理工大学 | Detection method for sulfate reducing bacteria |
| 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 |
| CN110062881A (en) * | 2016-12-09 | 2019-07-26 | 3M创新有限公司 | System and method for quickly detecting analytes of interest analytes |
| CN110241240A (en) * | 2019-06-25 | 2019-09-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Detection primer, kit and the quantitative approach of cable bacterium Candidatus Electronema |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITMI20022479A1 (en) * | 2002-11-22 | 2004-05-23 | Enitecnologie Spa | METHOD FOR IDENTIFICATION OF SULFUR-REDUCING BACTERIA. |
| CN1570139A (en) * | 2003-07-25 | 2005-01-26 | 上海复旦悦达生物技术有限公司 | Atypical pneumonia coronary virus gene detecting method and kit |
-
2007
- 2007-06-08 CN CN200710072333A patent/CN101086021B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088133A (en) * | 2013-01-22 | 2013-05-08 | 华东理工大学 | Detection method for sulfate reducing bacteria |
| CN103088133B (en) * | 2013-01-22 | 2015-05-20 | 华东理工大学 | Detection method for sulfate reducing bacteria |
| CN110062881A (en) * | 2016-12-09 | 2019-07-26 | 3M创新有限公司 | System and method for quickly detecting analytes of interest analytes |
| 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 |
| CN110241240A (en) * | 2019-06-25 | 2019-09-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Detection primer, kit and the quantitative approach of cable bacterium Candidatus Electronema |
| CN110241240B (en) * | 2019-06-25 | 2020-11-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Detection primer, kit and quantitative method for Candidatus Electron |
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| CN101086021B (en) | 2010-05-26 |
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