key: cord-0253340-x6hm187g
authors: Sharma, Atul Kumar; Gururaj, K.; Sharma, Rama; Goel, Anjana; Paul, Souvik; Sharma, Dinesh Kumar
title: Development of duplex real time PCR for quick detection of Cryptosporidia in goats
date: 2022-02-25
journal: bioRxiv
DOI: 10.1101/2022.02.23.481731
sha: 99035e1bdc72d59950a66b52491ebed0cbf68f0b
doc_id: 253340
cord_uid: x6hm187g

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 7.08×1004 and 5.95×1005 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. Importance Cryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration and stunted growth. For diagnosing this condition, many diagnostic tests are available including microscopy, immunological tests, but none of the diagnostic tests available currently can differentiate between active and passive infection in the host. 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 the test was developed in a reverse transcriptional format in a duplex mode. The currently developed diagnostic assay for cryptosporidiosis was evaluated for sensitivity using Limit of detection (LOD). This diagnostic test will act as a quantitative marker to aid in detecting active stages of Cryptosporidium infection in neonatal goats and will eventually lead to better control strategies for managing cryptosporidial infections in the future.

of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x10 04 and 23 5.95x10 05 respectively. During active infections the oocyst wall protein will be active and so its 24 COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU 25 rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay 26 will be a quantitative marker that will help assess active stages of Cryptosporidium infection in 27 neonates. The disease dynamics will help better understand to formulate the control strategies 28 and contain infection among the healthy animals. 29 Importance: 30 Cryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration 31 and stunted growth. For diagnosing this condition, many diagnostic tests are available including 32 microscopy, immunological tests, but none of the diagnostic tests available currently can The already available diagnostic assays can only detect the presence or absence of 79 Cryptosporidium in a qualitative manner, and there is no scope to decipher the 80 metabolically/transcriptionally active oocysts from passive ones. Hence, a robust technique is 81 required that can able to differentiate the active oocysts from dead oocyst in passive infection. 82 In detail, the main aim of this study is to develop and standardize an mRNA based 83 Cryptosporidium spp.-specific real-time PCR assay using two important target genes viz. 18 84 small subunit ribosomal RNA (18SSU rRNA) and Cryptosporidium oocyst wall (COWP). The 85 18ssu rRNA gene is highly abundant in terms of genomic copies and it has high transcripts that 86 can increase the sensitivity of the assay, while the COWP is available only in transcriptionally 87 active live cryptosporidial oocysts. (13-23, 28-31). Hence, in the current study we report an mRNA based TaqMan® probe duplex real time PCR 89 that can able to detect the metabolically active Cryptosporidium oocysts from dead oocysts in 90 passively infected animals 91

Microscopic identification of Cryptosporidium 93 Cryptosporidium suspected faecal smear that were stained by the modified Ziehl-Neelsen The conventional PCR reactions for newly designed primers viz., COWP and 18ssu rRNA were 103 analysed by gel electrophoresis. The amplicon size for COWP and 18ssu rRNA is 157bp and 104 6 109bp respectively and the same was observed in the gel photo (Fig.2) . Whereas the negative 105 controls did not generate any visible amplicons in this technique. 112 After verifying the workability of the newly designed primers by conventional PCR, the same set 113 of primers, but this time along with their respective probes was assayed using a duplex format (Table 7) . While the no template 118 controls (NTC) produced mean Cq for COWP and 18ss rRNA of 33.32 and 35.88 respectively 119 which were above the threshold cut-off cq for their respective genes and hence are negative. After observing the workability of the duplex TaqMan® probe real time PCR using DNA 128 template, we further worked on the optimal concentrations of probe and primers by titration for The sensitivity of the assay was based on the copy numbers ranged from 5.37x10 11 to 5.37x10 06

for COWP (FAM) and 1.22x10 12 to 1.22x10 07 for 18ssu rRNA (HEX) separately in simplex 156 mode. The standard curve showed a linear curve over these dilutions to making the range of The sensitivity of duplex assay was based on the copy numbers ranged from 7.08x10 10 to 

In the present study, primer and probes were newly designed and checked for its working by To further understand the dynamics of these two target genes, a relative quantification based 261 dRT-qPCR assay was conducted. In this assay, the fold change was computed for COWP gene 262 which is taken as a gene of interest (GOI) due to its differential expression during infections, The mRNA based TaqMan probe real time PCR assay is simple, rapid and self-regulating Real time PCR primers and probes (Table 1) To check the workability each of the newly designed 18ssu rRNA and COWP primers, 364 conventional PCR was performed by following standard PCR reaction conditions. The (Table 7) and thermal conditions as mentioned below. to RNA extraction and cDNA synthesis as per the protocol described in the previous sections.

Reaction mix was prepared in duplicates for each of the sample using the master mix, probes, 458 primers, and nuclease free water as per the protocol described in previous sections ( also kept for interpretation of the results. The assay was performed as per the thermal conditions 461 described in previous sections (Fig.4) . The data analysis for gene expression studies was done 462 using the CFX-96 manager (CFX Real-time PCR system, Biorad ® , USA) using the ΔΔCq 463 algorithm. The ΔΔCq method was done using the formula described by Livak and Schmittgen 

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