Direct detection of Plasmodium female gametocyte-specific transcript s25 mRNA in blood by CLIP-PCR

Abstract

Objective To establish a molecular detection method for the Plasmodium female gametocyte. Methods Probes for capture and ligation probe-PCR (CLIP-PCR) were specific designed based on Plasmodium female gametocyte special RNA targets, which were the transcripts of the ookinetes surface protein (s25). After the whole blood cells were lysed, the RNA targets were directly released and captured on the 96-well plate by “sandwich” hybridization without extraction. Washing away the unbound probes, the ligation probes which also hybridized to the RNA target were ligated to form a single-strand amplification template with specific-designed sequence at both ends. The SYBR Green qPCR was carried out with universal primers. Or with TaqMan probe binding sequence was designed at the end of the templates, the TaqMan qPCR was carried out with universal primers and universal fluorescent probes. The sensitivity, specificity and repeatability of CLIP-PCR were evaluated and compared with the RT-qPCR. And it was also used to detect a small number of clinical samples. Results This method was successfully applied to detect s25 mRNA with high sensitivity and specificity. Both RT-qPCR and CLIP-PCR could detect s25 mRNA targets as low as 11 copies. However, CLIP-PCR was simpler compared to RT-qPCR. And it could accurately detect gametocytes in blood of malaria patients. The detection of 96 samples could be completed within 3 h. Conclusions SYBR Green CLIP-PCR and TaqMan CLIP-PCR for the female gametocyte of Plasmodium detection provides a sensitive and efficient method for large-scale screening of gametocytes and control of malaria transmission.

Keywords

• plasmodium|gametocyte|detection method|capture|ligation