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