Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

Abstract

-The objective of this work was to evaluate the survival of two Trichoderma harzianum cotransformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Reovirus as a successful ex vivo purging modality for multiple myeloma

“…Nested PCR against enhanced GFP was performed as previously reported. 24 Primers used were: 5 0 -CACCGGGGTGGTGCCCATCC-3 0 (sense) and 5 0 -GGCGGCGGTCA CGAACTCCAG-3 0 (antisense) for the first round and 5 0 -TGAACCGCATC GAGCTGAAG-3 0 (sense) and 5 0 -CAGCAGGACCATGTGATCG-3 0 (antisense) for the nested round. Sensitivity of RT-PCR was evaluated by admixing RPMI 8226-GFP þ cells with AP cells to result in tumor: AP cell ratios of 1:10 6 -10 6 :10 6 .…”