CN101875981B - Primer group and kit for synchronously detecting multiple tobacco viruses - Google Patents
Primer group and kit for synchronously detecting multiple tobacco viruses Download PDFInfo
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Abstract
The invention relates to a primer group and a kit for synchronously detecting multiple tobacco viruses. The kit consists of a component I and a component II, wherein the all the reagents in the component I and the component II are separately packed; the component I comprises reverse transcriptase, an RNA enzyme inhibitor, a dNTP mixture, a reverse primer sequence group, and reverse transcription reaction buffer; and the component II comprises PCR reaction buffer, a dNTP mixture, MgCl2, Taq DNA polymerase and a PCR primer group. The primer group is characterized in that: the reverse primer sequence group comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10; and the PCR primer group comprises SEQ ID NO: 1 to 10.
Description
Technical field
The invention belongs to the plant virology technical field, particularly relate to primer sets and the test kit of the multiple tobacco virus of a kind of synchronous detection.
Background technology
Tobacco is one of topmost cash crop in the whole world, and the cultivated area of China occupies first place in the world.Yet, along with the development of industry, the expansion of cultivated area and industry restructuring, after 20 century 70s, tobacco virus harm increases the weight of year by year.Because infecting of virus, tobacco is chlorophyllous to be damaged, thereby has caused photosynthetic decline, has a strong impact on output and the quality of tobacco leaf, causes great financial loss.
The Major Diseases of harm China tobacco has tobacco mosaic virus (TMV) (Tobacco Mosaic Virus, TMV), cucumber mosaic virus (Cucumber Mosaic Virus, CMV), marmor upsilon (Potato Virus Y, PVY), marmor erodens (Tobacco Etch Virus, TEV) and tobacco vein banding mosaic virus (Tobaccovein banding mosaic Virus, TVBMV) etc.Wherein TMV, CMV, TEV, PVY and TVBMV are the main virus that causes tobacco virus.
The virus of harm tobacco is carried out accurately and the rapid detection diagnosis, be of great significance in prevention and control tobacco virus tool.Yet the main method that detects at present tobacco virus has plant indicator method, electron microscopy, serological method and regular-PCR technology.These detection methods mainly are to detect for single virus, waste time and energy.
Multiplex PCR (M-PCR) is improved on the basis of regular-PCR (polymerase chain reaction), in a PCR reaction system, add many to Auele Specific Primer, for the increase round pcr of a plurality of purpose fragments of the different zones of a plurality of dna profilings or same template.Compare with the single PCR of routine, multiplex PCR can detect multiple diseases simultaneously, greatly improves detection efficiency, reduces testing cost.The at present domestic report that only has substance PCR to detect TMV, CMV etc., and often show as the multiple diseases Combined Infection in the field, the workload of utilizing single PCR that multiple virus is carried out system's detection is larger, thereby the superiority of multiplex PCR in the detection of tobacco virus is more obvious, just lacking at present can the multiple tobacco virus of synchronous detection test kit.
Summary of the invention
For solving the problems of the technologies described above, the invention provides the primer sets of the multiple tobacco virus of a kind of synchronous detection, this primer sets comprises 5 couples of primer SEQ ID NO:1-10.
The invention provides the test kit of the multiple tobacco virus of a kind of synchronous detection, this test kit is comprised of component I and component I I; The independent packing of each reagent among component I and the component I I, component I comprises ThermoScript II, RNA enzyme inhibitors, dNTP mixture, reverse primer sequence set, reverse transcription reaction damping fluid; Component I I comprises the PCR reaction buffer, dNTP mixture, MgCl
2, the Taq archaeal dna polymerase, the PCR primer sets is characterized in that the reverse primer sequence set comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8 and SEQ ID NO:10; The PCR primer sets comprises SEQ ID NO:1-10.
Wherein tobacco virus is selected from tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), marmor upsilon (PVY), marmor erodens (TEV) and tobacco vein banding mosaic virus (TVBMV)
The sequence information of 5 couples of primer SEQ ID NO:1-10 is as follows:
The 1st pair of primer:
SEQ ID NO.1:5 '-TAGACCCGCTAGTCACAG-3 ' (forward sequence)
SEQ ID NO.2:5 '-CAGAGGTCCAAACCAAAC-3 ' (reverse sequence)
The 2nd pair of primer:
SEQ ID NO.3:5 '-GTGGGTGACAGTTCGTAAA-3 ' (forward sequence)
SEQ ID NO.4:5 '-GTGGGAATGCGTTGGT-3 ' (reverse sequence)
The 3rd pair of primer:
SEQ ID NO.5:5 '-T AG TGGATGGTGAGGAG-3 ' (forward sequence)
SEQ ID NO.6:5 '-GTGCCGTTCAGTGTCTT-3 ' (reverse sequence)
The 4th pair of primer:
SEQ ID NO.7:5 '-CCGAGAATCAAGGCTATC-3 ' (forward sequence)
SEQ ID NO.8:5 '-CGCTAAACCTACATCCC-3 ' (reverse sequence)
The 5th pair of primer:
SEQ ID NO.9:5 '-GAGGTCGTGAACTTACAGC-3 ' (forward sequence)
SEQ ID NO.10:5 '-GAGGTCGTGAACTTACAGC-3 ' (reverse sequence)
The mentioned reagent box, five pairs of primer concentration ratios are the 1st pair of primer in the PCR primer sets of component I I: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th couple of primer=1.2-1.6: 0.8-1.2: 0.8-1.2: 0.8-1.2: 1.3-1.7, preferably, 5 pairs of primer concentration ratios are the 1st pair of primer: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th pair of primer=1.4: 1: 1: 1: 1.5.
The mentioned reagent box, dNTP concentration is 0.12-0.32mmol/L among the component I I; Taq archaeal dna polymerase concentration is 0.02-0.12U/ μ L; MgCl
2Concentration is 1.2-3.2mmol/L, and preferably dNTPs concentration is 0.28mmol/L, and Taq archaeal dna polymerase concentration is 0.10U/ μ L, MgCl
2Concentration is 2.4mmol/L.
Preferably, in the component I, ThermoScript II is the M-MLV ThermoScript II.
Preferably, component I is composed as follows:
DNTP (each 2.5mmol/L) 5 μ L
Reverse primer sequence set (10 μ mol/L) 1 μ L
5×RT buffer 5μL
RNase inhibitor (40U/ μ L) 0.5 μ L
M-MLV ThermoScript II (200U/ μ L) 1 μ L
DEPC-H
2O 7μL
Preferably, the PCR reaction buffer is 750mmol/LTris-HCl (pH8.8) among the component I I, 200mmol/L (NH
4)
2SO
4, 0.1%Tween 20,
Preferably, component I I's is composed as follows:
DNTP mixture (each 2.5mmol/L) 2 μ L
MgCl
2(25mmol/L) 2μL
TaqDNA polysaccharase (5U/ μ L) 0.2 μ L
PCR primer sets 1 μ L
10 * PCR reaction buffer, 2.4 μ L
ddH
2O 14.4μL
5 pairs of primer concentration ratios are the 1st pair of primer in the PCR primer sets: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th pair of primer=1.4: 1: 1: 1: 1.5.
Tobacco virus often is Combined Infection, use detection kit of the present invention to carry out multiple RT-PCR, just can detect simultaneously five kinds of main tobacco viruses of China, greatly improve detection efficiency, reduce testing cost, this test kit successfully is applied to prove that test kit of the present invention is applied to multiple RT-PCR and has effectively detected plant virus in the synchronous detection of indoor and field tobacco virus disease Combined Infection.
Description of drawings
The electrophoresis detection result of 5 kinds of single tobacco virus samples of detection of Fig. 1 substance RT-PCR
M: standard nucleic acid molecular weight Marker1; Swimming lane 1: health tobacco sample; Swimming lane 2-6: be followed successively by TMV, CMV, TEV, PVY and TVBMV viral sample
Fig. 2 multiple RT-PCR detects the 5 electrophoresis detection results that grow tobacco viral biased sample
M: standard nucleic acid molecular weight Marker1; Swimming lane A: health tobacco sample; Swimming lane B-F: be followed successively by TMV, TMV+CMV, TMV+CMV+TEV, TMV+CMV+TEV+PVY, TMV+CMV+TEV+PVY+TVBMV viral sample
Embodiment
In order to understand the present invention, the below further specifies the present invention with embodiment, but does not limit the present invention.
Used primer designs by the following method among the present invention:
According to TMV, CMV, TEV, the Coat protein gene sequence of PVY and TVBMV, use PrimerPrimier 5.0 primer-design softwares, every kind of virus is designed 1 pair of special primer, TMV, CMV, TEV, the special primer of PVY and TVBMV is followed successively by TMV cpF (SEQ ID NO.1)/TMV cpR (SEQID NO.2), CMV cpF (SEQ ID NO.3)/CMV cpR (SEQ ID NO.4), TEV cpF (SEQ ID NO.5)/TEV cpR (SEQ ID NO.6), PVY cpF (SEQ ID NO.7)/PVYcpR (SEQ ID NO.8) and TVBMV cpF (SEQ ID NO.9)/TBMV cpR (SEQ ID NO.10).The information of each primer is as shown in table 1 below:
The information of table 1 primer sequence SEQ ID NO.1-10
Above-mentioned primer is synthetic by match Parkson, Beijing company.
Used experiment material is as follows in the embodiments of the invention:
The tobacco virus sample picks up from respectively the Yangling Shaanxi Tobacco Farm take sick blade as material, packs with valve bag, and it is frozen for subsequent use in-80 ℃ of refrigerators to gather blade.The Taq box is excellent brilliant biotechnology company limited product, and cloning vector pMD18-T simple vector, molecular biology bacterial strain material are available from the biological company limited of TaKaRa (Dalian).
Embodiment 1: substance RT-PCR
The extraction of virus total RNA
Take by weighing respectively each 0.1g of sick leaf (being labeled as respectively sample 2, sample 3, sample 4, sample 5, sample 6) of the tobacco that infects TMV, CMV, TEV, PVY and five kinds of viruses of TVBMV in-80 ℃ of dark freezing in the mortar, adding liquid nitrogen fully grinds, be transferred to rapidly in the aseptic 1.5mL eppendof pipe without RNase, add 1mL BIOzol Extraction Reagent mixing, place 15min.Add 0.2mL chloroform and 0.3ml phenol solution turned upside down mixing, 4 ℃ of centrifugal 15min of 12000 * g.Get supernatant liquor and change in the new eppendof pipe, add and the isopyknic Virahol of supernatant liquor, the vibration mixing is placed 30min for-20 ℃.4 ℃ of centrifugal 15min of 12000 * g abandon supernatant liquor.With 1mL 75% ethanol (preparation of DEPC water) washing precipitation, 4 ℃ of centrifugal 5min of 12000 * g abandon supernatant liquor, seasoning.Add 30 μ L DEPC water dissolution precipitation.-80 ℃ save backup, and obtain the 5 total RNA that grow tobacco virus, and with the total negative contrast of RNA of health tobacco (being labeled as sample 1), extracting method is the same simultaneously.
Reverse transcription (RT) reaction
Respectively total RNA of every kind of virus carried out reverse transcription reaction.Synthesize each viral cDNA first chain with the reverse transcription of M-MLV ThermoScript II: 25 μ LRT reaction systems are as follows: the total RNA of 2 μ L, 1 μ L virus special downstream primer (10 μ mol/L), 7 μ LDEPC-H
2O, 70 ℃ of sex change 5min put rapidly 5min on ice; Add again 5 μ L, 5 * RT buffer, 5 μ LdNTP (each 2.5mmol/L), 0.5 μ LRNase inhibitor (40U/ μ L) and 1 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal after, 42 ℃ of water-bath 1h, 95 ℃ of deactivation 5min put stand-byly on ice, obtain the cDNA of 5 kinds of viruses.
The PCR reaction
Respectively the cDNA of every kind of virus carried out the PCR reaction.25 μ LPCR standard reaction systems: 14.4 μ LddH2O, 2.4 μ L, 10 * PCR buffer[750mmol/LTris-HCl (pH8.8), 200mmol/L, (NH
4)
2SO
4, 0.1%Tween 20], 2 μ L25mmol/L, MgCl
2, 2 μ LdNTP (each 2.5m mol/L), 1 μ L upstream and downstream primer mixture (each 10 μ mol/L), 0.2 μ LTaqDNA polysaccharase (5U/ μ L) and 2 μ LcDNA templates.PCR reaction cycle parameter: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 48 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; 72 ℃ stop compensation and extend 10min.Get 5 μ LPCR products through 2% agarose gel electrophoresis analysis relatively.
Electrophoresis detection
Get 5 μ L PCR reaction product and carry out 2% agarose gel electrophoresis, in 0.5 * TAE buffered environment, 110V voltage stabilizing electrophoresis 40min, then observe with gel imaging system and the record result, obtain respectively 5 specific bands, obtain the amplimer pillar location of every kind of virus, referring to the Fig. 1 in the Figure of description.
Embodiment 2: multiple RT-PCR
The extraction of virus total RNA
Take respectively 6 samples: health tobacco, each 0.1 gram of the tobacco leaf that TMV, TMV+CMV, TMV+CMV+TEV, TMV+CMV+TEV+PVY, TMV+CMV+TEV+PVY+TVBMV infect, 6 sample labels are sample A, sample B, sample C, sample D, sample E, sample F.
Respectively sample is placed in-80 ℃ of dark mortars that freeze, add liquid nitrogen and fully grind, be transferred to rapidly in the aseptic 1.5mL eppendof pipe without RNase, add 1mL BIOzol Extraction Reagent mixing, place 15min.Add 0.2mL chloroform and 0.3ml phenol solution turned upside down mixing, 4 ℃ of centrifugal 15min of 12000 * g.Get supernatant liquor and change in the new eppendof pipe, add and the isopyknic Virahol of supernatant liquor, the vibration mixing is placed 30min for-20 ℃.4 ℃ of centrifugal 15min of 12000 * g abandon supernatant liquor.With 1mL 75% ethanol (preparation of DEPC water) washing precipitation, 4 ℃ of centrifugal 5min of 12000 * g abandon supernatant liquor, seasoning.Add 30 μ L DEPC water dissolution precipitation.-80 ℃ save backup, and obtain 6 total RNA of sample.
Reverse transcription (RT) reaction
Respectively total RNA of each sample carried out reverse transcription reaction.Synthesize each viral cDNA first chain with the reverse transcription of M-MLV ThermoScript II: 25 μ LRT reaction systems are as follows: the total RNA of 2 μ L, 1 μ L virus special downstream primer (10 μ mol/L), 7 μ LDEPC-H
2O, 70 ℃ of sex change 5min put rapidly 5min on ice; Add again 5 μ L, 5 * RT buffer, 5 μ LdNTP (each 2.5mmol/L), 0.5 μ LRNase inhibitor (40U/ μ L) and 1 μ L M-MLV ThermoScript II (200U/ μ L), of short duration centrifugal after, 42 ℃ of water-bath 1h, 95 ℃ of deactivation 5min put stand-byly on ice, obtain the cDNA of 5 samples.
The PCR reaction
Respectively the cDNA of each sample carried out the PCR reaction.25 μ LPCR standard reaction systems: 14.4 μ LddH2O, 2.3 μ L10 * PCR buffer[750mmol/LTris-HCl (pH8.8), 200mmol/L, (NH
4)
2SO
4, 0.1%Tween 20], 2.4 μ LMgCl
2(25mmol/L), 2.8 μ LdNTP (each 2.5mmol/L), (wherein the right ratio of each primer is the 1st pair of primer to 1 μ L primer: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th pair of primer=1.4: 1: 1: 1: 1.5) (concentration of forward and reverse sequence is 10 μ mol/L), 0.5 μ LTaq archaeal dna polymerase (5U/ μ L) and 4 μ LcDNA templates to mixture.PCR reaction cycle parameter: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 51 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ stop compensation and extend 10min.
Electrophoresis detection
Get 5 μ L PCR reaction product and carry out 2% agarose gel electrophoresis, in 0.5 * TAE buffered environment, 110V voltage stabilizing electrophoresis 40min, then observe with gel imaging system and the record result, obtain respectively the specific band of 5 samples, obtain the amplimer pillar location of virus in each sample, referring to the Fig. 2 in the Figure of description.
TMV, CMV, TEV, PVY and the TVBMV band through the multiple RT-PCR amplification reclaimed in rubber tapping, carries out external the connection with pMD18-T simple vector, and the picking positive colony extracts the plasmid order-checking.Sequencing result shows, the amplified production of TMV, CMV, TEV, PVY and TVBMV is comprised of 237,273,347,456 and 547 Nucleotide respectively, identical with the PCR product size of design, it all is the partial sequence of coat protein gene, the sequence homology analysis result shows that the homology of institute's calling sequence and reference sequences reaches respectively 98.60%, 99.20%, 100%, 99.00% and 100%, the conserved regions homology all reaches 100%, has proved the reliability of multiple RT-PCR detected result.
Method of the present invention is described by specific embodiment.Those skilled in the art can use for reference the links such as content appropriate change raw material of the present invention, processing condition and realize corresponding other purpose, its relevant change does not all break away from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and all be deemed to be included within the scope of the present invention.
Claims (9)
1. the test kit of the multiple tobacco virus of synchronous detection, this test kit is comprised of component I and component I I; The independent packing of each reagent among component I and the component I I, component I comprises ThermoScript II, RNA enzyme inhibitors, dNTP mixture, reverse primer sequence set, reverse transcription reaction damping fluid; Component I I comprises the PCR reaction buffer, dNTP mixture, MgCl
2, the Taq archaeal dna polymerase, the PCR primer sets is characterized in that the reverse primer sequence set comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10; The PCR primer sets comprises 5 couples of primer SEQ ID NO:1-10, and sequence information is as follows:
The 1st pair of primer:
Forward sequence: SEQ ID NO.1:5 '-TAGACCCGCTAGTCACAG-3 '
Reverse sequence: SEQ ID NO.2:5 '-CAGAGGTCCAAACCAAAC-3 '
The 2nd pair of primer:
Forward sequence: SEQ ID NO.3:5 '-GTGGGTGACAGTTCGTAAA-3 '
Reverse sequence: SEQ ID NO.4:5 '-GTGGGAATGCGTTGGT-3 '
The 3rd pair of primer:
Forward sequence: SEQ ID NO.5:5 '-TGATGGATGGTGAGGAG-3 '
Reverse sequence: SEQ ID NO.6:5 '-GTGCCGTTCAGTGTCTT-3 '
The 4th pair of primer:
Forward sequence: SEQ ID NO.7:5 '-CCGAGAATCAAGGCTATC-3 '
Reverse sequence: SEQ ID NO.8:5 '-CGCTAAACCTACATCCC-3 '
The 5th pair of primer:
Forward sequence: SEQ ID NO.9:5 '-GAGGTCGTGAACTTACAGC-3 '
Reverse sequence: SEQ ID NO.10:5 '-GAGGTCGTGAACTTACAGC-3 '
Wherein tobacco virus is tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), marmor upsilon (PVY), marmor erodens (TEV) and tobacco vein banding mosaic virus (TVBMV).
2. test kit according to claim 1, wherein 5 pairs of primer concentration ratios are the 1st pair of primer in the PCR primer sets: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th couple of primer=1.2-1.6: 0.8-1.2: 0.8-1.2: 0.8-1.2: 1.3-1.7.
3. test kit according to claim 2, wherein 5 pairs of primer concentration ratios are the 1st pair of primer in the PCR primer sets: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th pair of primer=1.4: 1: 1: 1: 1.5.
4. test kit according to claim 1, wherein dNTP concentration is 0.12-0.32mmol/L among the component I I; The TaqDNA polymerase concentration is 0.02-0.12U/ μ L; MgCl
2Concentration is 1.2-3.2mmol/L.
5. test kit according to claim 4, wherein dNTPs concentration is 0.28mmol/L, Taq archaeal dna polymerase concentration is 0.10U/ μ L, MgCl
2Concentration is 2.4mmol/L.
6. test kit according to claim 1, wherein in the component I, ThermoScript II is the M-MLV ThermoScript II.
7. test kit according to claim 1, wherein component I is composed as follows:
The dNTP 5 μ L of each 2.5mmol/L
The reverse primer sequence set 1 μ L of 10 μ mol/L
5×RTbuffer 5μL
The RNase inhibitor 0.5 μ L of 40U/ μ L
The M-MLV ThermoScript II 1 μ L of 200U/ μ L
DEPC-H
2O 7μL。
8. test kit according to claim 1, wherein component I I's is composed as follows:
The dNTP mixture 2 μ L of each 2.5m mol/L
The MgCl of 25mmol/L
22 μ L
The TaqDNA polysaccharase 0.2 μ L of 5U/ μ L
PCR primer sets 1 μ L
10 * PCR reaction buffer, 2.4 μ L
ddH
2O 14.4μL
Wherein 5 pairs of primer concentration ratios are the 1st pair of primer in the PCR primer sets: the 2nd pair of primer: the 3rd pair of primer: the 4th pair of primer: the 5th pair of primer=1.4: 1: 1: 1: 1.5, the PCR reaction buffer is the 750mmol/L Tris-HCl of pH8.8,200mmol/L (NH
4)
2SO
4With 0.1%Tween 20.
9. the primer sets of the multiple tobacco virus of synchronous detection, this primer sets comprises 5 couples of primer SEQ IDNO:1-10, and wherein tobacco virus is tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), marmor upsilon (PVY), marmor erodens (TEV) and tobacco vein banding mosaic virus (TVBMV).
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| CN102242224B (en) * | 2011-07-15 | 2013-02-13 | 中国烟草总公司郑州烟草研究院 | Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction) |
| CN102286637B (en) * | 2011-07-21 | 2013-10-16 | 陕西省烟草研究所 | Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology |
| CN103898228B (en) * | 2014-04-15 | 2016-03-02 | 云南省烟草农业科学研究院 | A kind of molecule marker identifying tobacco PVY resistance |
| CN106834537A (en) * | 2016-12-30 | 2017-06-13 | 中国烟草总公司广东省公司 | A kind of TMV virus rapid identification methods and its application |
| CN110066888A (en) * | 2019-04-12 | 2019-07-30 | 华南农业大学 | A kind of triple real time fluorescent quantitative RT-qPCR methods of synchronous detection TMV, CMV and PVY |
| CN114075612A (en) * | 2020-08-20 | 2022-02-22 | 西北农林科技大学 | Primer group and kit for RT-PCR detection of 2 strawberry viruses |
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| 宋婷婷等.应用多重RT-PCR检测烟草上的TMV 和CMV.《辽宁农业科学》.2007,(第1期),53-54. * |
| 王威麟等.侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用.《植物病理学报》.2010,第40卷(第1期),27-32. * |
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