Liang chaoqiong2013.9.29

Reporter: Liang Chaoqiong Supervisor: Li Jianqiang
Contents
1 Introduction Materials & Methods Results Discussion Acquirements
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3
4
5
1
Bean anthracnose
Introduction
Seed lesion area calculation
The area of each lesion was highlighted in green Photoshop CS2
Quantify the lesion area Assess2.0
4
Materials & Methods
TaqMan MGB assay
87 symptomless seeds 1.14 ～130.54 fg Mean=22.45 fg
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Discussion
This is the first report on the use of real-time PCR technology to
quantify the extent of bean anthracnose.
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Procedure
Hold
95℃ 10min 95℃ 3s
Cycle
60℃ 30s
Materials & Methods
Real-time PCR amplifications
TaqMan MGB probe assay
20 μL
1×TaqMan Fast Universal PCR Master Mix 500 nM of each primer ClF432/ClR533 100nM internal TaqMan MGB probe ClP442 1μL template DNA
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Results
3.1 Real-time PCR tests on bean seeds
13
Results
3.1 Real-time PCR tests on bean seeds
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Results
3.2 Real-time PCR tests on bean seeds
The symptomless seeds had a low amount of fungal DNA.
Real-time PCR is rapid, sensitive and accurate.
Explore the use of this real-time PCR assay in the diagnostics of
foliar diseases of dry beans
Study on the ecology and epidemiology of C. lindemuthianum
DNA extraction and ITS sequencing
DNA extraction tested bean seeds
tested isolates
PCR amplication and ITS sequencing ITS4/ITS5
Sequence alignment editor Clustal W and BioEdit
Initial Steps
Each of 40 cycles Melt Anneal Extend Cycle
Procedure
Hold
95℃ 20 s
95℃ 1s
60℃ 20s
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Materials & Methods
Real-time PCR amplifications
Three standard samples which contained 500, 50 and 5
ClF432(5′-GGAGCCTCCTTTGCGTAGTAAC-3′) ClR533(5′-ACCTGATCCGAGGTCAACCTTGTT-3′) Show high specificity for C. lindemuthianum, distinguish it from the other Colletotrichum spp. and the other bean pathogens.
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Results
1. Specificity of primer
ClF432/ClR533
Only the target amplicon was ampli
Figure S2. The melt curve of real-time PCR amplification with SYBR Green assay using the primer set ClF432/ClR533. The unique peak for each reaction showed the melting temperature of the target amplicon.
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Materials & Methods
Real-time PCR primers and probe design
Primers design PRIMERQUEST Forward primers and TaqMan MGB probe PRIMER EXPRESS ClP442 (6FAM-AGGGCACTCCTGCCGT-MGBNFQ) Evaluate the specificity of the primer set ①. homologous DNA sequences BLAST program
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Results
2. Standard curve and detect sensitivity
Figure S3. Standard curve derived from the real-time PCR amplification of Colletotrichum lindemuthianum with a)the TaqMan MGB Probe assay (using primer set ClF432/ClR533 and Probe ClP442) b)the SYBR Green assay (using primer set ClF432/ClR533). The templates were 10-fold serial dilutions of C. lindemuthianum genomic DNA from 5×100 to 5×106fg).
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Materials & Methods
Source of materials
Tested bean seeds Tested isolates (Table 1)
A quantitative real-time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds.pdf PDA, room temperature, 10～14 days PDB, shake culture 100 rpm, dark, 3～7 days
Table 3.2 SYBR Green vs. TaqMan MGB
SYBR Green assay
weak 89 symptomless seeds fluorescent signals amount of C. lindemuthianum DNA 4.33 ～ 438.83 fg Mean=79.61 fg
②. detect the fluorescent signal of the target DNA real-time PCR
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Real-time PCR amplifications
SYBR Green dye assay 20 μL
Materials & Methods
1×TaqMan Fast Universal PCR Master Mix 500 nM of each primer ClF432/ClR533 0.75×SYBR Green I 1μL template DNA Initial Steps Each of 40 cycles Melt Anneal Extend 1 cycle Dissociation 95℃, 15s 60℃, 1min 95℃, 15s
mean Ct value (n = 3) versus logarithmic concentrations
of C. lindemuthianum as measured with the primer set ClF432/ClR533.
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Results
1. Specificity of primer set ClF432/ClR533
Subgroup Seminar, 2013/9/29
A quantitative real-time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds
Y. Y. Chen R. L. Conner Morden Research Station, Agriculture and Agri-Food Canada (AAFC) Plant Pathology (2013) 62, 900–907
pg of genomic DNA from a pure culture of C.
lindemuthianum race 73 were used to check the efficiency of the PCR amplification and calculate the amount of pathogen DNA in the seed samples. Both standard curves were constructed by plotting the