Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis

Abstract

Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

High‑resolution melting analysis for rapid and sensitive MYD88 screening in chronic lymphocytic leukemia

“…The dye fluoresces strongly when intercalated into the double-stranded DNA, but as the temperature increases during the HRM analysis, the DNA melts and the dye is release without fluorescence. The changes in fluorescence are sequence specific and can be recorded and analyzed by the designed program (16,17).…”

Molecular Techniques as Alternatives of Diagnostic Tools in China as Schistosomiasis Moving towards Elimination

“…The research conducted in the Philippines also showed similar trends, demonstrating that traditional copro-parasitological techniques underestimate the infection rate, signifying the advantages of qPCR for case finding and disease surveillance and monitoring [ 87 , 88 , 89 , 90 , 91 ]. Besides the gene of NADH I [ 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 ], several qPCR methods have been established targeting other genes, including COX I [ 96 ], 18S rRNA [ 97 , 98 , 99 , 100 , 101 ], ITS 2 [ 102 ], SjR2 [ 99 , 103 , 104 ], SjCHGCS20 [ 22 ], SjCHGC08270 [ 20 , 105 ], and Sjrh1.0 [ 99 , 106 ] ( Table 2 ). Those established qPCR methods have mostly completed the laboratory evaluation, but further validation should be conducted in field settings.…”

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain)

“…The experiment was performed in triplicate in a final volume of 15 µl by mixing 2 µl of first-strand cDNA with 1× iTaq Universal SYBR Green master mix (Bio-Rad Laboratories Inc., Hercules, CA, USA) and 100 nM each of Fw and Rv primers (Additional file 1: Table S1). 18S RNA was used as an internal control, as described previously [42, 43]. Amplification was performed using the MasterCycler Real-Time PCR system (Realplex 4 , Eppendorf, Hamburg, Germany) with pre-incubation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.…”