Development of duplex real time PCR for quick detection of Cryptosporidia in goats

Abstract

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. 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, 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 (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) 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 it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu 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 marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess 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.

A dual RPA-LFD assay for the simultaneous detection of Salmonella typhimurium and Salmonella enteritidis

“…Therefore, rapid, sensitive, and reliable detection of S. typhimurium and S. enteritidis was essential to reduce the risk factors caused by these two pathogens. There were many ways to detect pathogens in the stool, including real-time PCR (Sharma et al, 2023), enzyme-linked immunosorbent assay (Ghoshal et al, 2018), and electrochemical biosensors (Bacchu et al, 2022). However, these methods were not suitable for widespread application in field inspection due to the requirement of expensive instruments and time-consuming procedures.…”