CN104630371B - Molecular detection method for elm blight pathogen - Google Patents
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Abstract
The invention discloses a molecular detection method for elm blight pathogen. The molecular detection method for elm blight pathogen comprises the following steps: by using the DNA of the sample to be detected as a template, performing the first-round reaction of nest PCR via a universal primer ITS1-F/ITS4-B, by taking the product of the first-round reaction as a template and taking the primer OpF and primer OpR as the specific primers, performing the second-round reaction of nest PCR, performing the electrophoresis detection on the reaction product, if the electrophoretogram has 304bp strips, determining that the elm blight pathogen is detected, and otherwise, determining that the elm blight pathogen is not detected, wherein the sequence of the primer OpF is 5'-GCCCGCCACTGGTTTTGA-3'; and the sequence of the primer OpR is 5'-CCGCCCGAACCTTTTCCA-3'. An exact and rapid way is offered for detecting and evaluating the elm blight pathogen by the method, strong specificity is achieved by the primer OpF and the primer OpR, and the detection sensitivity of the method is 10 times of that of the general PCR.
Description
Technical field
The invention belongs to the field of molecular detection of Forest Protection is and in particular to a kind of Dutch elm disease bacterium
(ophiostoma ulmi(buisman) nannf.) molecular detecting method.
Background technology
Dutch elm disease is one of most destructive plant disease, is reported in Belgium and Switzerland earliest, about exists
1881 ~ nineteen twenty-one there is this disease in the state such as Belgian, French, Dutch in northwest Europe in succession, has spread all over meaning to nineteen thirty big
Sharp and Bulgarian.There occurs Outbreak big twice in the thirties in last century and this disease seventies, spread all over rapidly Europe
More than 30 countries and regions in continent, North America and the Central Asia, to these, regional elm causes destructive destruction, brings huge warp
Ji, Ecological Loss.China territory is vast, elm aboundresources, is distributed widely in China's north and south band, has according to incompletely statistics:
19 kinds such as American elm, black elm, Huang Yu, Chinese toon elm, Chinese elm, 2 mutation, 6 cultigens and some new varieties.Elm is because of it
Growth is rapid, reproductive capacity is strong, wide adaptability, is not only the fine tree species that China is township afforestation, or China shelter-forest, ecology
One of chief species of woods, Timber stands and scenic forest, to China's township afforestation, renewable resource using and improvement of the ecological environment rise
Very important effect.Dutch elm disease bacterium is mainly caused harm Elm seeds, once incoming China, it will to the elm kind of China
Cause very big loss.List China's quarantine harmful organisms register in.
At present, in the world for Dutch elm disease bacterium molecular detecting method report less, witthuhn etc. establishes
Pcr-rflp technology belongs to the Phylogenetic Relationships of some important kinds to distinguish long beak shell, but the method high cost, process complexity,
Detection sensitivity is low, and and the purity requirement for sample dna is higher it is difficult to quantitative, therefore is not suitable for promoting at each quarantine port
And application.
So, it is badly in need of it is set up a set of fast and accurately detection method, caused after the immigration of this pathogen to avoid
It is difficult to the economic loss making up.At present, can by tissue separate after morphological observation and molecular Biological Detection identification side
Method disease detection opportunistic pathogen.But the former exists, and separating difficulty is big, and the cycle is long, the shortcomings of inaccurate, in recent years, increasing research
Person to detect tree pathogen using the method for molecular biology.As being detection site using rdna in nucleus or in mitochondria
Pcr technology suffer from being widely applied in the different classifications level of Fungal identification.It is directed to Dutch elm disease bacterium urgently at this stage
A set of quick, accurate, reliable molecular detecting method must be set up, can quick detection enter a country entrained pathogen in timber,
In case causing domestic unnecessary loss.
Content of the invention
Goal of the invention: for the deficiencies in the prior art, it is an object of the invention to provide a kind of Dutch elm disease bacterium
Molecular detecting method, to providing one kind accurately and rapidly approach for the detection of Dutch elm disease bacterium.
Technical scheme: in order to realize foregoing invention purpose, the technical solution used in the present invention is:
A kind of molecular detecting method of Dutch elm disease bacterium: with testing sample dna as template, with universal primer its1-f/
Its4-b carries out the first round reaction of nido pcr, with first round product as template, is special with primer opf and primer opr
Property primer, carry out the second wheel reaction of nido pcr, product carries out electrophoresis detection, 304bp band and be in electrophoretogram
Detect Dutch elm disease bacterium, otherwise do not detect；Wherein, primer opf sequence is: 5'-gcccgccactggttttga-3'；Draw
Thing opr sequence is: 5'-ccgcccgaaccttttcca-3'.
The molecular detecting method of described Dutch elm disease bacterium, specifically comprises the following steps that
(1) mycelia taking 0.5g to freeze, is put in the flat centrifuge tube of 2ml, adds 2 a diameter of 3mm steel balls, shuts
Lid is placed on beveller, 30f/min, grinds 3min, takes out steel ball, adds 500 μ l ctab, freezes 2min in liquid nitrogen, then
It is put in 75 DEG C of water-baths and melts 2 minutes, be repeated twice, melt 30min in 75 DEG C of water-baths for the last time, be subsequently added into
The phenol of the 25:24:1 of 500 μ l: chloroform: isoamyl alcohol, mixing of turning upside down, 12000rpm is centrifuged 10min, takes supernatant, adds 2 times
The precooling absolute ethyl alcohol precipitation 1h of volume, 12000rpm are centrifuged 10min, abandon supernatant, and precipitation is washed with 70% ethanol, natural air drying,
50μl ddh2O dissolves dna, and -20 DEG C save backup；
(2) nido pcr reaction, the primer of nido pcr first round pcr reaction is universal primer its1-f/its4-b, pcr
Reaction cumulative volume is 25 l, comprises 1 l genome dna, each 1 l of upstream and downstream primer of 10 m, 2.5 l 10 × pcr buffer,
The mgcl of 2 l 25mm2, 2.5 l dntp, 1.25u taq dna polymerase, 14.75 l ddh2o；Pcr response procedures are: pre-
Amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C；Nest
Formula pcr second wheel reacts with specific primer opf/opr as primer, 50 times of nido pcr first round pcr product dilution, takes 2ul to make
For the second wheel reaction template, pcr reaction system is identical with the first round, and pcr reaction condition is: 95 DEG C of 180s of pre- amplification, and then 94
DEG C 30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C；
(3) take nido pcr product to carry out agargel electrophoresis detection, 304bp band occurs in electrophoretogram and as detects
Dutch elm disease bacterium.
In step (1), when carrying out mycelia dna extraction, after dna is dissolved in 100ul te solution, add 200ul again
Ctab repeats to extract once.
The present invention has been expanded in the ribosomes dna of Dutch elm disease bacterium using fungi universal primer its1-f/its4-b and has turned
Record spacer region has simultaneously carried out sequencing.Compared by sequence, devise a pair of Dutch elm disease bacterium primer opf/opr, by institute
The primer-blast that the specific primer of design is put back in genebank compares, and all strains testeds enter to primer pair to use this
Row nido pcr expands to verify the specificity of designed primer.Nido pcr is based on internal primer, carries out two-wheeled pcr amplification,
The concentration of dna amplified fragments can be increased, diluting effect is played to pcr inhibiting substances, therefore can avoid low dna concentration and pcr
The impact to detection for the inhibiting compound.
Beneficial effect: the present invention, with testing sample dna as template, with primer opf and primer opr as specific primer, enters
Row nido pcr reacts, and product i.e. can be with quick detection Dutch elm disease bacterium after carrying out electrophoresis detection.The method is Dutch elm disease
The detection of bacterium and assessment provide one kind accurately and rapidly approach, and primer opf and primer opr has very strong specificity, this method
Detection sensitivity can reach 10 times of conventional pcr.
Brief description
Fig. 1 is the dna sensitivity technique that specific primer opf and primer opr routine pcr expands Dutch elm disease bacterium
1.2% agargel electrophoresis figure；Wherein, deposition condition, 100v, 57ma, electrophoresis 30min；In figure, swimming lane m is dl2000 dna
marker；Swimming lane 1 ~ 4 is Dutch elm disease bacteria strain dna；Swimming lane 5 ~ 8 be Ceratocystis fimbriata Strainses (ceratocystis sensu lato)dna；Swimming lane 9 ~ 10 be Ceratocystis fimbriata bacterium (ceratocystis sensu lato) dna；
Swimming lane 11 is distilled water；
Fig. 2 is 1.2% fine jade of the dna sensitivity technique that specific primer opf opr nido pcr expands Dutch elm disease bacterium
Fat gel electrophoresis figure；Wherein, deposition condition, 100v, 57ma, electrophoresis 30min；In figure, swimming lane m is dl2000 dna marker；
Swimming lane 1 ~ 10 is the 1pg of template containing dna respectively in 20ul pcr reaction system, and swimming lane 11 is distilled water；
Fig. 3 is will to separate the colony morphology characteristic the obtaining separator similar with Dutch elm disease bacterium cultural colony using special
1.2% agargel electrophoresis figure of specific primer opf and primer opr routine pcr detection；Wherein, deposition condition, 100v, 57ma,
Electrophoresis 30min；In figure, swimming lane m is dl2000 dna marker；Swimming lane 1 is Dutch elm disease bacterium positive control；Swimming lane 2 is to divide
From purifying Dutch elm disease bacterium dna；Swimming lane 3 is to separate pathogen mixing dna；Swimming lane 4 is distilled water；
Fig. 4 is Dutch elm disease bacterium specific primer opf and 1.2% agar gel of primer opr nido pcr response analysis
Electrophoretogram；Wherein, deposition condition, 100v, 57ma, electrophoresis 30min；In figure, swimming lane m is dl2000 dna marker；Swimming lane 1 ~
4 is Dutch elm disease bacteria strain dna；Swimming lane 5 ~ 8 be Ceratocystis fimbriata Strainses (ceratocystis sensu lato)dna；Swimming
Road 9 ~ 10 be Ceratocystis fimbriata bacterium (ceratocystis sensu lato) dna；Swimming lane 11 is distilled water；
Fig. 5 is that 1.2% agar of specific primer op1/op2 and specific primer op3/op4 nido pcr response analysis coagulates
Gel electrophoresis figure；Wherein, deposition condition, 100v, 57ma, electrophoresis 30min；In figure, swimming lane m is dl2000 dna marker；Swimming lane
1 ~ 4 is Dutch elm disease bacteria strain dna；Swimming lane 5 ~ 6 be Ceratocystis fimbriata Strainses (ceratocystis sensu lato)dna；
Swimming lane 7 ~ 8 be Ceratocystis fimbriata bacterium (ceratocystis sensu lato) dna.
Specific embodiment
With reference to specific embodiment, the present invention will be further explained.
Details as Follows for material that following examples are used and method:
For examination Dutch elm disease bacterium (ophiostoma ulmi(buisman) nannf.) 1 ~ 4 bacterial strain, by river
Entry-Exit Inspection and Quarantine Bureau of Shanxi Province of Soviet Union provides, and 5 ~ No. 10 6 bacterial strains are preserved by Nanjing Forestry University's forest Pathology Lab；Long beak
Shell bacterium (ceratocystis sensu lato) and Ceratocystis fimbriata bacterium (ceratocystis sensu lato) as reference strain, it is the strain excellent being provided by Jiangsu Entry-Exit Inspection and Quarantine Bureau of Shanxi Province for examination Dutch elm disease bacterium,
Therefore using Dutch elm disease bacterium as the positive strain of detection.
takara dna fragment purification kit、e.z.n.a.tmCycle-pure kit, dna are polymerized
Enzyme, dntp, pmd19-t carrier, dna marker etc. are purchased from Dalian treasured biotech firm, and primer is synthesized by Nanjing Jin Sirui company.
Embodiment 1
Strains tested is carried out using cetyl trimethylammonium bromide (ctab) method and mycelia dna extracts, particularly as follows: taking
The mycelia that 0.5g freezed, is put in the flat centrifuge tube of 2ml, adds 2 a diameter of 3mm steel balls, closes upper tube cap and is placed in grinding
On instrument, 30f/min, grinds 3min, takes out steel ball, add 500 μ l ctab, freeze 2min, then be put in 75 DEG C of water-baths in liquid nitrogen
Melt 2 minutes in pot, be repeated twice, melt 30min in 75 DEG C of water-baths for the last time, be subsequently added into the 25:24:1 of 500 μ l
Phenol: chloroform: isoamyl alcohol, mixing of turning upside down, 12000rpm is centrifuged 10min, takes supernatant, adds the precooling of 2 times of volumes anhydrous
Ethanol precipitation 1h, 12000rpm is centrifuged 10min, abandons supernatant, and precipitation is washed with 70% ethanol, natural air drying, 50 μ l ddh2O dissolves
Dna, -20 DEG C save backup.Wherein, when carrying out mycelia dna extraction, after dna is dissolved in 100ul te solution, add again
200ul ctab repeats to extract once.Extracted dna accords with through ultraviolet specrophotometer and the detection of 1.2 % agarose gel electrophoresis
Close the requirement of pcr.
With the dna of Dutch elm disease bacterium as template, with fungi universal primer its1-f its4-b to Dutch elm disease bacterium
Carry out pcr amplification, reaction system is: each pcr reaction cumulative volume is 25 l, comprises 25 l genome dna, 10 m's is upper and lower
Trip each 1 l of primer, 2.5 l 10 × pcr buffer, the mgcl of 2 l 25mm2, 2.5 l dntp, 1.25 u taq dna gather
Synthase (takara), 14.75 l ddh2o.Pcr response procedures are: pre- amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C
30s, 72 DEG C of 60s, 35 circulations, finally extend 10min at 72 DEG C.Wherein, the sequence of its1-f is: 5'-
The sequence of cttggtcatttagaggaagtaa-3', its4-b is: 5'-caggagacttgtacacggtccag-3'.
Pcr product is reclaimed through e.z.n.a.tmcycle-pure kit, connects pmd19-t carrier, convert jm109, choose
Select positive colony to serve the sequencing of Hai Meiji company after Insert Fragment checking, the sequence of acquisition is carried out on genebank
Primer-blast compares, and result learns, therefore judgement be sequenced knot consistent with Dutch elm disease bacterium sequence existing on genebank
Fruit is Dutch elm disease bacterium.
Embodiment 2
Its sequence 9 obtaining Dutch elm disease bacterium on genebank and its belonging to remaining kind together, obtains together with embodiment 1
Obtained the sequence of Dutch elm disease bacterium, and carried out clustal w Multiple sequence alignments, devised using primer premier 5.0
The specific primer of a pair of Dutch elm disease bacterium: opf/opr, opf sequence is: 5'-gcccgccactggttttga-3', opr sequence
It is classified as: 5'-ccgcccgaaccttttcca-3', amplified production size is 304bp.
The primer of nido pcr first round pcr reaction is universal primer its1-f its4-b, according to the system in embodiment 1
Carry out pcr amplification with step.Conventional pcr and nido pcr second wheel reacts with specific primer opf/opr as primer, nido pcr
50 times of first round pcr product dilution, takes 2ul as the second wheel reaction template, pcr reaction system is identical with the first round, reaction interval
Sequence is: pre- amplification 95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 35 circulations, finally extends at 72 DEG C
10min.
The primer-blast designed specific primer opf/opr being put back in genebank compares, only elm
The rrna gene of wilt can produce the fragment of 304bp, illustrate this to primer the specificity in current genebank.This
Outward, using this to primer pair for 4 plants of Dutch elm disease bacterium of examination, 4 kinds of common Ceratocystis fimbriata Strainses, and the long beak of 2 kinds of common sweet potatoes
Shell bacterium carries out nido pcr, as shown in figure 1, all Dutch elm disease bacterium amplifications are all positive, obtain size is result
The amplified production of 304bp, other reference strain amplifications are feminine gender.Using fungi its universal primer to all for examination fungi
Dna carries out pcr, all obtain 750bp about size product.Result shows that this can only be withered to elm to nido pcr primer
Germ carries out specific amplification, and other fungi dna can not obtain corresponding amplified production.
In order to further confirm that the specificity of pcr product, with takara dna fragment purification kit
Nido pcr electrophoresis product is carried out cutting glue reclaim by (purchased from Dalian treasured biotech firm), connects pmd19-t carrier, converts jm109,
Select positive colony and serve the sequencing of Hai Meiji company after Insert Fragment checking, compare with aim sequence after sequencing to confirm to expand
The correctness of product.Nido pcr product gel reclaims sequencing result and shows, the amplified production of 304bp size and Dutch elm disease
One section of sequence in bacterium its area is mated completely.Result shows, primer opf/opr can specifically amplify Dutch elm disease bacterium
The fragment of its area 304bp, and there is no amplified production to other fungies.The sequence of the amplified production of 304bp size such as seq id
Shown in no.1.
Embodiment 3
In order to assess the sensitivity of nido pcr, Dutch elm disease bacterium dna is diluted to 10 gradients, 20ul pcr reacts
The 100ng of template containing dna, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag respectively in system, use respectively
Specific primer carries out conventional pcr and nido pcr, conventional pcr with specific primer opf/opr as primer, Dutch elm disease bacterium
Dna is template, and reaction system and reaction condition are with nido pcr second wheel reaction in embodiment 2.Result is as shown in figure 1, routine
Pcr is only able to detect the dna of 10pg, as shown in Fig. 2 nido pcr low energy detects the dna of 1pg, nido pcr detection is sensitive
Degree is 10 times of conventional pcr.
Embodiment 4
In order to study the Detection results of specific primer designed by this test further, to apply Dutch elm disease bacterium 2
Year, raw potted plant elm seedling carried out nido pcr detection, and nido pcr reaction system and reaction condition are with embodiment 3.Result such as Fig. 3 institute
Show, the elm seedling testing result applying Dutch elm disease bacterium is the positive, and the elm seedling not applying Dutch elm disease bacterium does not expand
Product.
Embodiment 5
In order to assess the specificity of nido pcr, respectively with design multipair specific primer carry out nido pcr, respectively with
The specific primer opf/opr of embodiment 2 is test primer, and two designing in conventional manner couple specific primer is comparison, with
Dutch elm disease bacterium dna be template, reaction system and reaction condition and method with embodiment 2, result as shown in Figure 4 and Figure 5,
Only specific primer opf/opr can amplify the clear band of specific 304bp, other primers in Dutch elm disease bacterium
All respective strap cannot be amplified in Dutch elm disease bacterium it is seen that the specificity to Dutch elm disease bacterium for the primer opf/opr.Its
In, the sequence of two couples of specific primers op1/op2, op3/op4 is respectively as follows: op1 sequence and is: 5'-atttcgctgcgttcttca-
3', op2 sequence is: 5'-ttactgctttggcgtggt-3', and amplified production size is 159bp；Specific primer op3/op4,
Op3 sequence is: 5'-tcctccgcttattgatat-3', op4 sequence is: 5'-ttactgctttggcgtggt-3'.
sequence listing
<110>Nanjing Forestry University
<120>a kind of molecular detecting method of Dutch elm disease bacterium
<130> 100
<160> 5
<170> patentin version 3.3
<210> 1
<211> 304
<212> dna
<213> ophiostoma ulmi (buisman) nannf.
<400> 1
gcccgccact ggttttgagg gccctgccgc cacagagggc ggaggagccc caacgccagc 60
gcacccaaaa gggcatgctg aggggggaaa tgacgctcgg acaggcatgc ccgccagaat 120
gctggcgggc gcaatgtgcg ttcaaagatt cgatgactcg ctgaattctg caattcgcat 180
tacgtatcgc atttcgctgc gttcttcatc gatgccagag ccaagagatc cgttgttgaa 240
agttttggct gtttttgttt gtttctcaga cgtttcgtta ctggtttgga aaaggttcgg 300
gcgg 304
<210> 2
<211> 22
<212> dna
<213> artificial
<220>
<223>sequence of its1-f
<400> 2
cttggtcatt tagaggaagt aa 22
<210> 3
<211> 23
<212> dna
<213> artificial
<220>
<223>sequence of its4-b
<400> 3
caggagactt gtacacggtc cag 23
<210> 4
<211> 18
<212> dna
<213> artificial
<220>
<223>opf sequence
<400> 4
gcccgccact ggttttga 18
<210> 5
<211> 18
<212> dna
<213> artificial
<220>
<223>opr sequence
<400> 5
ccgcccgaac cttttcca 18
Claims (2)
1. a kind of molecular detecting method of Dutch elm disease bacterium it is characterised in that: with testing sample dna as template, drawn with general
Thing its1-f/its4-b carries out the first round reaction of nido pcr, with first round product as template, with primer opf and primer
Opr is specific primer, carries out the second wheel reaction of nido pcr, and product carries out electrophoresis detection, occurs in electrophoretogram
304bp band as detects Dutch elm disease bacterium, does not otherwise detect；Wherein, primer opf sequence is: 5'-
gcccgccactggttttga-3'；Primer opr sequence is: 5'-ccgcccgaaccttttcca-3'；
The sequence of its1-f is: 5'-cttggtcatttagaggaagtaa-3',
The sequence of its4-b is: 5'-caggagacttgtacacggtccag-3'.
2. the molecular detecting method of Dutch elm disease bacterium according to claim 1 is it is characterised in that specifically comprise the following steps that
(1) mycelia taking 0.5g to freeze, is put in the flat centrifuge tube of 2ml, adds 2 a diameter of 3mm steel balls, closes upper tube cap
It is placed on beveller, 30f/min, grinds 3min, take out steel ball, add 500 μ l ctab, freeze 2min in liquid nitrogen, then be put in
Melt 2 minutes in 75 DEG C of water-baths, be repeated twice, melt 30min in 75 DEG C of water-baths for the last time, be subsequently added into 500 μ l
25:24:1 phenol: chloroform: isoamyl alcohol, mixing of turning upside down, 12000rpm is centrifuged 10min, takes supernatant, adds 2 times of volumes
Precooling absolute ethyl alcohol precipitates 1h, and 12000rpm is centrifuged 10min, abandons supernatant, and precipitation is washed with 70% ethanol, natural air drying, 50 μ l
ddh2O dissolves dna, and -20 DEG C save backup；
(2) nido pcr reaction, the primer of nido pcr first round pcr reaction is universal primer its1-f/its4-b, and pcr reacts
Cumulative volume is 25 l, comprises 1 l genome dna, each 1 l of upstream and downstream primer of 10 m, 2.5 l 10 × pcr buffer, 2 l
The mgcl of 25mm2, 2.5 l dntp, 1.25u taq dna polymerase, 14.75 l ddh2o；Pcr response procedures are: pre- amplification
95 DEG C of 180s, then 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C；Nido pcr
Second wheel reaction with specific primer opf/opr as primer, 50 times of nido pcr first round pcr product dilution, take 2 l as the
Two wheel reaction template, pcr reaction system is identical with the first round, and pcr reaction condition is: 95 DEG C of 180s of amplification in advance, then 94 DEG C
30s, 58 DEG C of 30s, 72 DEG C of 60s, 30 circulations, finally extend 10min at 72 DEG C；
(3) take nido pcr product to carry out agargel electrophoresis detection, 304bp band occurs in electrophoretogram and as detects elm
Wilt.
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| CN108315392A (en) * | 2018-05-15 | 2018-07-24 | 北京市园林科学研究院 | A kind of nest type PCR detection reagent and its application in the detection of smoke tree wilt |
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Application publication date: 20150520 Assignee: Nanjing Forest Protection Bird Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000399 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 Application publication date: 20150520 Assignee: Nanjing biological pest control technology, Limited by Share Ltd Assignor: Nanjing Forestry University Contract record no.: 2018320000400 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 Application publication date: 20150520 Assignee: Guizhou Linxin Ecological Agriculture Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2018320000401 Denomination of invention: Molecular detection method for elm blight pathogen Granted publication date: 20170118 License type: Common License Record date: 20181218 |
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