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 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.dead and live oocysts, duplex real-time PCR assay, oocyst wall protein | INTRODUCTIONCryptosporidiosis disease is caused by the intracellular, extracytoplasmic protozoan parasite, which belongs to the phylum Apicomplexa. Cryptosporidiosis is the primary cause of chronic diarrhea among immunocompromised patients, especially in children suffering from HIV, AIDS-defining illness because of its high association with mortality. [1][2][3][4] Cryptosporidium is accountable for 8%-19% of diarrhea in less developed countries, 5 and till the year 2010, it already caused about 100,000 deaths globally. 6 More than 15% of the world's population is drinking contaminated water and suffering from waterborne parasitic protozoan diseases globally, 7 it is responsible for four billion cases of mild to severe diarrhea and 1.6 million deaths annually. 8,9 Cryptosporidium oocysts are

Standardization and evaluation of DNA extraction protocol for Cryptosporidium oocysts from faecal samples in goats

“…On the contrary, molecular techniques could aid species differentiation, possibly further characterization by sub-type grouping and phylogenetic analysis using sequencing methods. Similarly, Real-time PCR can also be an alternate for conventional PCR with its ability for quick detection and non-sequencing based characterization like 'endpoint genotyping' or multiplexing [14,31] . Hence, in the current study, we have used 18SSU rRNA gene-based TaqMan ® probe real-time PCR to assess the effectiveness of our extracted DNA for such protocols.…”

“…For 18SSU rRNA TaqMan ® probe Real-time PCR, we have used the oligonucleotide primers and probes designed in-house [14] in a previous study. The real-time PCR master mix was prepared in duplicates for each sample in a final reaction volume of 25μl in 8 strip PCR tubes with optically compatible caps assayed in the real-time thermal cycler (CFX96 Real-time PCR system ® , Bio-Rad) with the reagent preparation and thermal conditions as mentioned in the table (Table 2).…”

“…A wide range of polymerase chain reaction (PCR) assays are available with higher sensitivity and specificity including simplex PCR [11][12][13] , multiplex real-time PCR and duplex qRT-PCR for detecting Cryptosporidium spp. with the stage of infection and differentiating the live and dead oocysts [14] . Molecular assays are highly sensitive to the purity and quality of DNA material.…”