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A Highly Sensitive 18 S rRNA Gene Based Nested PCR Assay for Detection of Calodium Hepaticum in Human and Animals
Gokula Kannan Ragavan1, Azhahianambi Palavesam2,3, Purushothaman Selvaraj1
1Translational Research Platform for Veterinary Biologicals, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, 600 051, India.
Purpose:
Calodium hepaticum is a neglected zoonotic parasite of rodents, primarily affecting rats. Highly sensitive molecular assays such as nested PCR are not available to screen the parasite in rat, man and dog.
Methods:
In the present study, parasite specific nested PCR primers were designed to amplify 171 bp partial 18 S rRNA gene of C. hepaticum and compared with an existing semi nested PCR.
Results:
Both nested PCR and semi nested PCR assays were sensitive enough to detect at least 10 C. hepaticum eggs in rat liver samples. The limit of detection of nested PCR assay was 420 zM, and it was 35-fold more sensitive than that of semi nested PCR assay. The nested and semi nested PCR assays specifically amplified C. hepaticum egg DNA from human and dog DNA samples spiked with parasite DNA. The molecular prevalence of C. hepaticum in household rats in Chennai was 41.81% based on nested PCR assay.
Conclusion:
This study suggests that the 18 S rRNA gene based nested PCR assay could be a more sensitive detection system for molecular screening of C. hepaticum in rat liver samples and highly suitable for epidemiological studies. Further, both the 18 S rRNA gene based nested and semi nested PCR assays can potentially used for detection of C. hepaticum infection in man and dog.
