Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Integrated Cell Culture-PCR Detection of Enteroviruses and Reoviruses in Water Sources in Gyeonggi-do

The integrated cell culture-PCR (ICC-PCR) method has been suggested as an improved method for detection of viruses in water environments. We tested 57 source waters including finished water samples in Gyeonggi-do for enteric viral contamination using total culturable virus assay (TCVA) using BGMK cells and ICC-PCR. Nineteen of the 57 source water samples (33.3%) exhibited the cytopathic effect (CPE) on BGMK cells and no finished water did exhibited CPE. Nineteen samples (33.3%) of the 57 were positive for reoviruses. For the enteroviruses, only 3 samples (5.3%) of the 57 samples showed positive results. By using ICC-PCR method, 202 flasks from source water samples were positive for enteroviruses and reoviruses. Three samples from source water were positive for both viruses. However, any flasks tested was not co-infected with two types of viruses. While the enteric viral frequencies in TCVA and ICC-PCR were similar, the viral frequency for reoviruses at first passage in two type of method was higher in ICC-PCR (94.7%) than TCVA (56.9%).