Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Abstract

Sensitive and rapid detection of infection with Toxoplasma gondii in transplanted immunocompromised patients is crucial for a good prognosis. Two DNA fragments are used currently for detecting T. gondii infection by PCR, i.e., the B1 gene and a 529-bp repeat element that exists in 200-300 copies/genome. This study investigated whether targeting the 529-bp repeat element gives better sensitivity and accuracy than can be obtained when targeting the B1 gene (35 copies) when concentrations of T. gondii DNA are low. The results demonstrated that detection of the 529-bp repeat element increased diagnostic sensitivity and accuracy. Addition of an internal amplification control did not affect the PCR performance and was useful in order to monitor PCR inhibition by non-specific DNA in the LightCycler instrument. The real-time PCR was used successfully in a clinical context to monitor parasitaemia in the blood of a transplant recipient suffering from toxoplasmosis.

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens

“…This was true each time the two targets were tested within the same laboratory, when rep529-based assays systematically proved more efficient than B1-based ones, regardless of the primers used. This multicentric evaluation thus confirms previous findings by individual groups using a variety of primers (8,14,18,24,27). In contrast, interlaboratory comparisons show that B1-based assays in certain laboratories may be more efficient than rep529-based methods in others ( Fig.…”

Adapting a conventional pcr assay forToxoplasma gondiidetection to real-time quantitative pcr including a competitive internal control

“…Moreover, this control was included in the six points of the standard curve as in each sample. This type of IAC is used to detect false negative results due to PCR inhibitors and was reported in real-time PCR assay-SYBR Green to T. gondii quantification only by Edvisson et al (2006). Tachyzoites were spiked in 1 ml of amniotic fluid.…”

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

“…To increase the sensitivity of molecular diagnostics of toxoplasmosis nested PCR was introduced, although in recent years real-time PCR has shown a significantly higher sensitivity as well as specificity (Jauregui et al, 2001;Reischl et al, 2003;Contini et al, 2005;Calderaro et al, 2006;Edvinsson et al, 2006). Real-time PCR detection also has the capability of quantification of T. gondii in biological samples, which has found wide application in monitoring the kinetics and outcome of infection in patients undergoing therapy, as well as in experimental models (Lin et al, 2000;Jauregui et al, 2001;Contini et al, 2005;Djurković-Djaković et al, 2012).…”

“…However, it can be of great methodological significance to further clarify the specificity of using a multicopy target of unknown function before the introduction of such protocol into the laboratory diagnostics (Edvinsson at al., 2006) …”