2021 Volume 70 Issue 3 Pages 465-474
Entamoeba histolytica, which is the cause of a category 5 infectious disease, needs to be differentiated from the non-pathogenic species Entamoeba dispar and Entamoeba moshkovskii. PCR analysis is a specific and highly sensitive method of detecting E. histolytica, and the technology is shifting from conventional PCR to real-time PCR analysis. Therefore, we investigated a highly sensitive method to detect the Entamoeba complex by PCR-HRM analysis, which is a further evolution of real-time PCR analysis. Furthermore, we compared HRM analysis with multiplex PCR, allele-specific PCR, allele-specific real-time PCR, and nested PCR analyses. Results showed that the sensitivity of multiplex PCR analysis is inferior to that of other methods because the sensitivity decreased as the size of the PCR product increased. When the size of the PCR product is small, sufficient detection sensitivity can be obtained, so that it is not necessary to carry out nested PCR, which increases the risk of contamination. Since PCR-HRM analysis and allele-specific real-time PCR analysis use a real-time PCR thermal cycler, there is no need to open the lid of the PCR tube. Therefore, the risk of contamination is extremely low, and the turnaround time can be shortened. Moreover, since only one tube is used in PCR-HRM analysis, running costs are low, and there is a possibility that more Entamoeba species can be detected.
