Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Santos, Wagner Gouvêa dos; Metcheva, Ivelina S.; Buck, Gregory A.

doi:10.1590/s0074-02762000000100018

Cited by 8 publications

(3 citation statements)

References 15 publications

(10 reference statements)

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“…Indeed, reports in the literature show that engineering of T. cruzi strains to express beta-galactosidase or fluorescent proteins can alter parameters as infectivity to mice compared to their parental strains (Le-Senne et al, 2002, Guevara et al, 2005, Santos et al, 2000. We confirmed here that integration of the pTREXgfp vector does not affect the fitness of the parasite in vitro or in the mouse model of infection, maintaining throughout its life cycle the fluorescent signal without need of further re-selection.…”

Section: Myoblasts Loosely Adherent or Non-adherent Cells)supporting

confidence: 82%

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

et al. 2015

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Section: Myoblasts Loosely Adherent or Non-adherent Cells)supporting

confidence: 82%

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

et al. 2015

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“…Feline herpesvirus type 1, which is discharged from corneas of infected cats, can be detected by swabbing the conjunctiva, rinsing the swab in HBSS, and using the HBSS directly in the PCR (Burgesser et al 1999). In other PCR methods, cultures of microorganisms are boiled or microwaved for a few minutes and then added to the PCR mixture without further purification (Duenas et al 1999;dos Santos et al 2000;Menossi et al 2000).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Unpurified Cell Culture Supernatant as Template for the Polymerase Chain Reaction (PCR) with Largemouth Bass Virus

2005

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Polymerase chain reaction (PCR) has been used to identify largemouth bass virus (LMBV) in cell culture, and this method has included extraction of the sample DNA. To simplify this procedure, we centrifuged the cell culture fluid to remove cellular debris and then used the supernatant directly in the PCR without DNA extraction. For supernatants from cell cultures inoculated with 1:100 dilutions of homogenized swim bladder or pooled spleen and trunk kidney, only 70.0% of the LMBV-positive samples (based on all available evidence) tested positive with this new PCR method. After a 1:500 dilution of these cell culture supernatants, 85% of the failed samples became PCR positive. In contrast, 97.2% of LMBV-positive samples were PCR-positive when undiluted supernatants from subcultivations (cells inoculated with supernatant from cell cultures showing cytopathic effect [CPE]) were used directly in the PCR. Unpurified cell culture fluid supernatant from blind passage samples (cells inoculated with supernatant from CPE-negative cultures) was PCR-positive for 93.8% of the samples that were eventually shown to be LMBVpositive. The use of cell culture supernatant directly in the PCR reduced the cost and time required to identify LMBV isolated in cell culture. Extraction of DNA from cell cultures to obtain template for PCR identification of LMBV was required for only 2% of the LMBV-positive samples.

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“…As described for bacteria, yeasts and algae (Packeiser et al 2013), colony PCR could be the answer due to its quickness, simplicity and minimal amounts of required cells. Likely based on this idea, dos Santos et al (2000) proposed a colony PCR method to screen T. cruzi colonies that has been selected on solid medium (Mondragon et al 1999). However, although there are some advantages to growing transformants as colonies on agarose plates (Goldberg & Chiari 1980, Wittner et al 1982), liquid culture and limited dilution remain the preferred method.…”

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confidence: 99%

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

Alcantara

Fragoso

Picchi

2014

Mem. Inst. Oswaldo Cruz

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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.

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Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Cited by 8 publications

References 15 publications

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

Evaluation of Unpurified Cell Culture Supernatant as Template for the Polymerase Chain Reaction (PCR) with Largemouth Bass Virus

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

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