Development of a real‐time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection

Abstract

We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Utilization of a real-time PCR assay for diagnosis of Babesia microti infection in clinical practice

“…Recently, the use of real-time PCR has been described for detection of B. microti in ticks or clinical specimens from patients suspected of having babesiosis (Bloch et al, 2013;Chan et al, 2013;Hojgaard et al, 2014;Rollend et al, 2013;Teal et al, 2012). In these reports, the analytical sensitivity of real-time PCR assays was most often assessed by spiking plasmid DNA to negative blood samples, which may result in inaccurate results due to the potential for preferred amplification by PCR of small plasmid DNA fragments, compared with the same target sequence contained within the complete genome of a microorganism (Lin et al, 2011;Yun et al, 2006).…”

Molecular Diagnostics in the Diagnosis of Parasitic Infection

“…parasites is 10-50 parasites/mL or 0.0002-0.001% parasitemia. In comparison, a recently described real-time 18S rDNA PCR assay for B. microti reported an LOD of significantly less than 1 parasite/mL of whole blood (0.006 parasites/mL) (Bloch et al, 2013), while another 18S rDNA PCR reported an estimated LOD of 5-10 parasites/mL (Teal et al, 2012). The number of copies of 18S rDNA in B. microti is unknown, but is expected to be similar to that seen with Plasmodium (approximately 5 copies/parasite) (Garcia, 2007).…”

“…Published PCR assays target portions of the 18S ssu rRNA gene (Bloch et al, 2013;Teal et al, 2012), 16S-like small subunit rRNA gene (Persing et al, 1992), internal transcribed spacers (ITS) gene region of nuclear rRNA and the thiamine pyrophosphokinase gene (Chan et al, 2013). Assays may be species specific (Chan et al, 2013;Teal et al, 2012;Wilson et al, 2014) or use subsequent sequence analysis or electrospray ionisation MS for differentiation to the species level (Eshoo et al, 2014;Herwaldt et al, 2003;Liu, 2013;Persing et al, 1992).…”

Diagnostic approaches for tick-borne haemoparasitic diseases in livestock

“…Bishop et al (1993) were able to further increase the accuracy of the identification of isolates or strains by using random amplified polymorphic deoxyribonucleic acid 'DNA' (RAPD). Moreover, several real-time PCR assay has been developed for diagnosis and quantitation of many tickbone parasites (Dong et al, 2013;Schotthoefer et al, 2013;Bloch et al, 2013).…”