1. Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern.
- Author
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Wang D, Jiang P, Yang X, Zhang J, Chen T, Hu M, Cacciò SM, Yin J, and Zhu G
- Subjects
- Risk Factors, Public Health, Real-Time Polymerase Chain Reaction methods, Oocysts, Cryptosporidium parvum, Cryptosporidium genetics
- Abstract
While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guan Zhu reports financial support was provided by National Key Research and Development Program of China. Min Hu reports financial support was provided by National Key Research and Development Program of China. Jigang Yin reports financial support was provided by Jilin Provincial Science and Technology Department. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2024
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