Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine

Abstract

Diagnosis of Schistosoma haematobium relies primarily on microscopical analysis of urine. The method is time consuming and requires some expertise. Genus-specific real-time PCRs have been developed, but we still observed low sensitivity. In the present study, in order to achieve a more sensitive DNA detection of eggs of S. haematobium in urine samples, we wanted to develop a novel protocol of DNA extraction using mechanic disruption of eggs by bead beating as supplementary step. We tested Schistosoma spp. internal transcribed spacer 2 real-time PCR after both methods with and without bead beating. First, we preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL, and the Ct value analysis showed significant improved DNA detection per each point of egg concentration using the novel supplementary step. Twenty microscopy positive and five microscopy negative urine samples were used to validate the procedure. All urines came from imported cases and admitted at center for tropical medicine, and were examined by microscopy. PCR results after novel method with bead beating showed 100% to be positive for S. haematobium, compared with 85% positive by our standard extraction procedure. Results confirmed mechanic disruption of eggs by bead beating before DNA extraction to be highly effective method for the detection of S. haematobium DNA in urine.

Keywords: Bead beating; DNA; Real-time PCR; Schistosoma haematobium.

Fig. 1

Real-time PCR results and Ct values for evaluating the sensitivity with and without bead beating for DNA extraction from S. haematobium eggs in urine. Increasing number of eggs/10 mL was used: 2, 10, 50, and 90. Experiments were performed in triplicate and PCR results are expressed as mean of Ct values (GraphPad Prism v7.02). Each egg sample was processed by both procedures: A_PCR, directly frozen sample; B_PCR, bead beating supplemented on frozen sample

Fig. 2

DNA load distribution of n = 20 S. haematobium egg samples using two different preparation procedures A and B on urine samples. PCR results following the sample preparation procedure: A_PCR, directly frozen sample; B_PCR, bead beating supplemented on frozen sample