Romero-Estévez, Gabriela C.

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    Detección e identificación de patógenos utilizando la reacción en cadena de la polimerasa (PCR) convencional y de alta fidelidad
    (2012) Romero-Estévez, Gabriela C.; Estévez-de Jensen, Consuelo; College of Agricultural Sciences; V. Rodrigues, José Carlos; Rivera Vargas, Lydia I.; Department of Crops and Agro-Environmental Sciences; Vega, Carmen A.
    The sensitivity of standard PCR and high fidelity PCR was compared to identify Candidatus Liberibacter asiaticus, causal agent of “citrus greening”, Ralstonia solanacearum, causal agent of “bacterial wilt in tomato” and Pythium dissotocum, causal agent of “root rot” in cilantro and pea. DNA was extracted using a “DNeasy Plant Mini Kit” of Qiagen. DNA amplification of Candidatus Liberibacter asiaticus was performed from 138 symptomatic and asymptomatic leaves of Citrus sinensis, Citrus latifolia, Citrus limon and Myrtus communis. The High Fidelity PCR was more sensitive to detect Ca. L. asiaticus with primers OI1 and OI2 from 138 samples, twelve samples, that were negative, amplified a 1160 bp band of the 16S rDNA with Hi-Fi PCR. Identification of 16S region of DNAr of Ralstonia solanacearum was carry out with primers 759 and 760. Standard PCR amplified bands of 280 bp corresponding to Ralstonia solanacearum in 18 stem sections of nine plants with bacterial wilt symptoms. High Fidelity PCR produced false negatives. Standard PCR was sensitive to identify R. solanacearum directly from tomato tissue. Identification of Pythium dissotocum was performed from pea and cilantro using primers ITS1 and ITS4. High Fidelity PCR was more sensible than standard PCR, amplified a band of 808 bp corresponding to Pythium dissotocum in all samples analized. High Fidelity PCR allowed increased sensitivity in detection of Candidatus Liberibacter asiaticus from citrus tissue and Pythium dissotocum from pea and cilantro tissue.