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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (2) : 140-145     DOI: 10.1007/s11703-009-0032-z
RESEARCH ARTICLE |
A multiplex PCR method for detection of Clavibacter michiganensis subsp. michiganensis with co-amplification of its host DNA
Yan ZHANG, Wenxiang YANG, Yaning LI, Daqun LIU(), Ting ZHANG
College of Plant Protection, Biological Control Center for Plant Disease and Plant Pests of Hebei Province, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum–specific primers NS-7-F/NS-8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×102 cfu·mL-1. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.

Keywords Clavibacter michiganensis subsp. michiganensis (Cmm)      molecular detection      multiplex PCR      internal PCR control      false negative     
Corresponding Authors: LIU Daqun,Email:ldq@hebau.edu.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0032-z     OR     http://academic.hep.com.cn/fag/EN/Y2009/V3/I2/140
item 123456789101112131415
PSA/(μmol·L-1) 0.750.720.700.670.660.640.60.530.400.340.20.130.100.080.05
NS/(μmol·L-1)0.050.080.100.110.130.160.20.270.400.670.60.670.700.720.75
ratio15.009.007.006.005.004.003.002.001.000.500.300.200.150.100.50
Tab.1  Concentration and ratio of the two specific primers used in the reaction system
item123456789
PSA/(μmol·L-1)1.381.030.830.690.550.400.280.150.07
NS/(μmol·L-1)0.230.150.140.140.090.070.050.020.01
Tab.2  Different concentrations of the primers used in the reaction system
Fig.1  Amplification results of uniplex PCR and multiplex PCR
Note: M represents DNA size marker 100+50 bp ladder. Lanes 1, 2, 3, and 4 represent amplification by primer PSA-4/PSA-R, amplification by primer NS-7-F/NS-8-R, amplification by primer PSA-4/PSA-R and NS-7-F/NS-8-R, and negative control, respectively.
Fig.2  The effect of different ratios of two pairs of primers
Note: M represents marker; Lanes 1-15 correspond to the series in Table 1.
Fig.3  The effect of different concentrations of two sets of the primers
Note: M represents marker; Lanes 1-15 correspond to the series in Table 2.
Fig.4  The effect of different annealing temperatures on amplification
Note: M represents marker; Lanes 1-12 represent 71.5°C, 70.6°C, 69.1°C, 67.1°C, 64.5°C, 61.8°C, 59.1°C, 56.5°C, 54.2°C, 52.5°C, 51.3°C, and 51.0°C, respectively.
Fig.5  Sensitivity of the multiplex PCR and the uniplex PCR for Cmm
Note: M represents marker; Lanes 1-7 represent the sensitivity of multiplex PCR with healthy tomato DNA mixed with dilutions of Cmm cells ranging from 50 to 5×10 cfu·mL; Lanes 8–14 represent the sensitivity of uniplex PCR with healthy tomato DNA mixed with dilutions of Cmm cells ranging from 50 to 5×10 cfu·mL.
Fig.6  Direct detection of inoculated tomato leaves with multiplex PCR
Note: M represents marker; Lanes 1-9 represent healthy plants, 2 days after inoculation (0 level), 4 days after inoculation (0 level), 6 days after inoculation (0 level), 8 days after inoculation (0 level), 10 days after inoculation (1 level), 15 days after inoculation (2 level), 20 days after inoculation (3 level), and 20 days after inoculation (4 level), respectively.
Fig.7  Detection of tomato seeds artificially mixed with Cmm
Note: M represents marker; Lanes 1-6 represent Cmm-free seeds, seeds soaked with 5×10 cfu·mL, seeds soaked with 5×10 cfu·mL, seeds soaked with 5×10 cfu·mL, seeds soaked with 5×10 cfu·mL, and seeds soaked with 5×10 cfu·mL, respectively.
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