Sensitive Detection of Babesia microti by mPCR-HRM

The detection limit of mPCR-HRM performance and detection limit studies were performed on serial dilutions of B. microti 18S rRNA fragment, amplified with the primers CRYPTOF (5′-AACCTGGTTGATCCTGCCAGTAGTCAT-3′) and CRYPTOR (5′-AATGATCCTTCCGCAGGTTCACCTAC-3′). The primer pair CRYPTOF and CRYPTOR (Herwaldt et al. 2003 (link)) spans 1770 bp DNA fragment of B. microti 18S rRNA. The region amplified by mPCR-HRM is located within the 1770 bp fragment. The amplified 1770 bp 18S rRNA fragment was subjected to agarose gel electrophoresis and isolated from the agarose gel using the Gel-Out® kit (A&A Biotechnology). The concentration of the purified DNA fragment was measured by UV spectroscopy and 7 ten-fold; serial dilutions of the PCR product in water were prepared. Seven dilutions from 5 × 10⁶ copies/μl to 5 copies/μl were used as a template for mPCR-HRM. The mPCR-HRM reactions were performed in triplicate for each of the dilutions, and such independent reactions were performed in three independent PCR runs.