Construction and validation of a polycompetitor construct (SWITCH) for usein competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii

Citation
Re. Lyons et al., Construction and validation of a polycompetitor construct (SWITCH) for usein competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii, PARASITOL, 123, 2001, pp. 433-439
Citations number
21
Categorie Soggetti
Microbiology
Journal title
PARASITOLOGY
ISSN journal
00311820 → ACNP
Volume
123
Year of publication
2001
Part
5
Pages
433 - 439
Database
ISI
SICI code
0031-1820(200111)123:<433:CAVOAP>2.0.ZU;2-U
Abstract
The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, g ive rise to slowly dividing bradyzoites that reside in tissue cysts. Reacti vation of disease is associated with conversion of bradyzoites to tachyzoit es. A sensitive method for detection and assessment of the number of each l ife-cycle stage would be useful for following these events. Herein we descr ibe the construction and validation of a plasmid (pSWITCH) containing a pol ycompetitor construct (SWITCH) for use in competitive reverse transcriptase -PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, whic h are exclusively expressed by tachyzoite and bradyzoite stages respectivel y, and for beta -tubulin, a gene expressed by both stages. Using cRT-PCR, s amples can first be accurately normalized for expression of the housekeepin g gene, beta -tubulin and then the relative levels of SAG2A and LDH2 expres sion compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonst rated here for the SAG2-related family of genes. This technique offers a po werful tool for studying the processes involved in tachyzoite and bradyzoit e interconversion.