Development of duplex real‐time PCR for quick detection of cryptosporidiosis in goats. (18th October 2022)

• Record Type: Journal Article
• Authors: Sharma, Atul Kumar; Gururaj, K.; Sharma, Rama; Goel, Anjana; Paul, Souvik; Sharma, Dinesh Kumar
• Is Part Of: Cell biochemistry and function. Volume 41:Number 1(2023)
• Page Start: 45
• Page End: 57
• Publication Date: 2022-10-18
• Subjects: dead and live oocysts -- duplex real‐time PCR assay -- oocyst wall protein; Cytochemistry -- Periodicals; Cell metabolism -- Periodicals; Biochemistry -- Periodicals; Cytology -- Periodicals; 572
• DOI: 10.1002/cbf.3759
• Languages: English
• ISSNs: 0263-6484

Abstract

Abstract: Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea 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 (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 10 04 and 5.95 × 10 05, 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 healthy animals. Significance statement: Cryptosporidiosis is an important neonatal disease affecting goats causing diarrhea, dehydration, and stunted growth. The currently available diagnostic techniques are inadequate to identify the status of infection, whether "passive" or "active." Hence, we developed a novel molecular technique that is based on "RNA" with duplexing which is entirely different from conventional PCR that traditionally uses "DNA" as the template. The mRNA transcripts are the direct indicator of any actively replicating cell and especially in intracellular parasites, it will help decipher the infective stages of a lifecycle in the host, and hence this current diagnostic test was developed in the reverse transcriptional form to suit this aspect. The developed test in the current study is duplex reverse transcription TaqMan® probe quantitative real‐time PCR (dRT‐qPCR) assay that aids in the detection of Cryptosporidium infection and simultaneously differentiating the active from passive infection leading to better clarity on the infection status. This diagnostic test will act as a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonatal goats and will aid in understanding the disease dynamics that will eventually lead to better control strategies to contain and manage cryptosporidial infections in the future.