Development of duplex Real-time PCR for quick detection of
Cryptosporidiosis in goats
Abstract
Cryptosporidium spp. is the most important foodborne and waterborne
pathogens and leading cause of mortality from foodborne and waterborne
gastrointestinal diseases. In neonates of domestic animals, it is
associated with consistent diarrhoea and dehydration. Cryptosporidium
infection begins with the ingestion of sporulated oocytes disseminated
by carrier animals that consistently contaminate the environment. Many
diagnostic tests are available including microscopy, and antigen
trap-ELISA, but none of the diagnostic tests available currently cannot
differentiate between active and passive infection in the host. In the
current study, to address this challenge an mRNA based duplex TaqMan®
probe PCR was developed to target the Cryptosporidium oocyst wall
protein gene and 18SSU rRNA gene in a single tube that can detect
metabolically active cryptosporidial oocysts. The mRNA transcripts are
the direct indicator of any actively replicating cell and they will help
decipher the active stages of its lifecycle in a host. This diagnostic
assay was standardized by computing transcript copy number-based limit
of detection. For COWP and 18SSU rRNA genes, the limit of detection was
7.08x1004 and 5.95x1005 respectively. During active infections, the
oocyst wall protein will be active and so its COWP gene transcripts will
act as a marker for active infection. While transcripts for 18SSU rRNA
are constitutively expressed in cryptosporidial life cycle. This current
diagnostic assay will be a quantitative marker that will help assess the
active stages of Cryptosporidium infection in neonates. The disease
dynamics will help better understand to formulate the control strategies
and contain infection among the healthy animals.