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A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

Stulberg MJ, Huang Q - PLoS ONE (2015)

Bottom Line: RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control.Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium.Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR.

View Article: PubMed Central - PubMed

Affiliation: Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, United States of America; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, United States of America.

ABSTRACT
Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

No MeSH data available.


Related in: MedlinePlus

Comparison of efficiency and sensitivity for RsSA2 (A, C, D, F) and RsII (B, C, E, F) sets under both uniplex (A, B, D, E) and multiplex qPCR (C, F) conditions with 10-fold serial dilutions of Ralstonia solanacearum strain UW551 cells diluted in either sterile water or Qiagen-purified healthy geranium extracts.Mean (M) Cq value and standard deviation (SD) for each bacterial colony forming unit (CFU) was calculated from 4 replicates in two separate experiments. NTC: no template water control.
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pone.0139637.g001: Comparison of efficiency and sensitivity for RsSA2 (A, C, D, F) and RsII (B, C, E, F) sets under both uniplex (A, B, D, E) and multiplex qPCR (C, F) conditions with 10-fold serial dilutions of Ralstonia solanacearum strain UW551 cells diluted in either sterile water or Qiagen-purified healthy geranium extracts.Mean (M) Cq value and standard deviation (SD) for each bacterial colony forming unit (CFU) was calculated from 4 replicates in two separate experiments. NTC: no template water control.

Mentions: The standard curve of each uniplex and multiplex qPCR assay was determined using 10-fold serial dilutions of bacterial cells of the IIB-1 strain UW551 at concentrations of 4 to 4 x 104 CFU per reaction in either sterile water (Fig 1A–1C) or in total plant DNA extracted from healthy geranium plants to mimic extracts of infected geranium plants (Fig 1D and 1E). When serial dilutions were made in water, the efficiencies of RsSA2 and RsII were 99% and 102%, respectively, under uniplex conditions (Fig 1A and 1B). When RsSA2, RsII and Cox1 were multiplexed together, the efficiencies were 101% and 99% for RsSA2 and RsII, respectively, similar to their efficiencies under uniplex conditions (Fig 1C). Similar efficiencies were obtained when purified healthy geranium DNA was used to dilute bacterial cell templates for qPCR (Fig 1D–1F). Although RsSA1 had an efficiency of 98% in uniplex assays, under multiplex conditions it increased to 155%, above the acceptable level of amplification efficiencies, and was thus not further studied in a multiplex qPCR assay. Cox1 efficiencies were confirmed to be acceptable under uniplex (101%) and multiplex (89%) conditions using serially diluted Qiagen-purified total DNA from geranium infected by UW551 (data not shown).


A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

Stulberg MJ, Huang Q - PLoS ONE (2015)

Comparison of efficiency and sensitivity for RsSA2 (A, C, D, F) and RsII (B, C, E, F) sets under both uniplex (A, B, D, E) and multiplex qPCR (C, F) conditions with 10-fold serial dilutions of Ralstonia solanacearum strain UW551 cells diluted in either sterile water or Qiagen-purified healthy geranium extracts.Mean (M) Cq value and standard deviation (SD) for each bacterial colony forming unit (CFU) was calculated from 4 replicates in two separate experiments. NTC: no template water control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591012&req=5

pone.0139637.g001: Comparison of efficiency and sensitivity for RsSA2 (A, C, D, F) and RsII (B, C, E, F) sets under both uniplex (A, B, D, E) and multiplex qPCR (C, F) conditions with 10-fold serial dilutions of Ralstonia solanacearum strain UW551 cells diluted in either sterile water or Qiagen-purified healthy geranium extracts.Mean (M) Cq value and standard deviation (SD) for each bacterial colony forming unit (CFU) was calculated from 4 replicates in two separate experiments. NTC: no template water control.
Mentions: The standard curve of each uniplex and multiplex qPCR assay was determined using 10-fold serial dilutions of bacterial cells of the IIB-1 strain UW551 at concentrations of 4 to 4 x 104 CFU per reaction in either sterile water (Fig 1A–1C) or in total plant DNA extracted from healthy geranium plants to mimic extracts of infected geranium plants (Fig 1D and 1E). When serial dilutions were made in water, the efficiencies of RsSA2 and RsII were 99% and 102%, respectively, under uniplex conditions (Fig 1A and 1B). When RsSA2, RsII and Cox1 were multiplexed together, the efficiencies were 101% and 99% for RsSA2 and RsII, respectively, similar to their efficiencies under uniplex conditions (Fig 1C). Similar efficiencies were obtained when purified healthy geranium DNA was used to dilute bacterial cell templates for qPCR (Fig 1D–1F). Although RsSA1 had an efficiency of 98% in uniplex assays, under multiplex conditions it increased to 155%, above the acceptable level of amplification efficiencies, and was thus not further studied in a multiplex qPCR assay. Cox1 efficiencies were confirmed to be acceptable under uniplex (101%) and multiplex (89%) conditions using serially diluted Qiagen-purified total DNA from geranium infected by UW551 (data not shown).

Bottom Line: RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control.Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium.Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR.

View Article: PubMed Central - PubMed

Affiliation: Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, United States of America; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, United States of America.

ABSTRACT
Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

No MeSH data available.


Related in: MedlinePlus