A Real-Time PCR-Based Method for the Sequence Determination of Cryptosporidium hominis and Cryptosporidium parvum gp60 Subtypes During Epidemiological Investigations.

The gp60 gene has been widely used to subtype Cryptosporidium for many years. Predominantly, this has involved nested polymerase chain reaction (PCR) amplification, gel electrophoresis, and Sanger sequencing of the amplicons. However, laboratories may no longer have gel electrophoresis capability, and during outbreak investigations a more timely approach may be required. Here, we describe a real-time PCR assay combined with high-resolution melt curve analysis that streamlines the PCR detection of gp60 amplicons of Cryptosporidium parvum and Cryptosporidium hominis for sequencing during public health investigations.