Development of a sensitive detection system for Cryptosporidium in environmental samples
Norma E Ramirez1, Srinand Sreevatsan
1Food Animal Health Research Program and Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, 44691, USA.
Abstract:
The identification of Cryptosporidium species and genotypes is necessary to determine sources of infection in outbreaks and the risk factors associated with their transmission. Few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples, particularly in soil and manure. The objective of this study was to develop an easy to use method which can be applied to field samples to rapidly detect the presence of Cryptosporidium parasites and identify their species. The assay included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a real-time PCR-melting curve analysis for species assignment. An internal positive control (IPC) was developed to determine the presence of PCR inhibitory substances. Two oocyst recovery methods, sodium chloride and sucrose flotation techniques were compared. Two commercial DNA extraction kits were performed using feces, soil and water samples each inoculated with different concentration of Cryptosporidium oocysts. Subsequently, methods were used to test field samples. The sucrose flotation method provided the greatest analytical sensitivity detecting as few as 10 oocysts. The PCR-hybridization detection limit was 10 oocysts for feces and soil, and less than 10 oocysts for water samples. IPC was positive for all inoculated and field samples indicating 0% PCR inhibition. Cryptosporidium species DNA samples were detected with the real-time PCR and were differentiated by the melting curve analysis. The results of this study demonstrate the potential of the assay system for rapid detection of Cryptosporidium parasites in environmental samples.
Insights
A new method rapidly detects Cryptosporidium parasites in environmental samples like soil and water. This technique aids in identifying infection sources and transmission risks during outbreaks.
Area of Science:
- Environmental microbiology
- Parasitology
- Molecular diagnostics
Background:
- Accurate identification of Cryptosporidium species and genotypes is crucial for understanding infection sources and transmission risks.
- Detecting Cryptosporidium oocysts in environmental samples, especially soil and manure, presents significant challenges, limiting previous studies.
Purpose of the Study:
- To develop a user-friendly method for rapid detection and species identification of Cryptosporidium in field samples.
- To overcome limitations in detecting oocysts in challenging environmental matrices.
Main Methods:
- Combined oocyst recovery (sucrose flotation vs. sodium chloride flotation) with spin column DNA extraction.
- Utilized PCR-hybridization for parasite detection and real-time PCR-melting curve analysis for species assignment.
- Incorporated an internal positive control (IPC) to assess PCR inhibition in various sample types (feces, soil, water).
Main Results:
- Sucrose flotation demonstrated superior analytical sensitivity, detecting as few as 10 oocysts.
- PCR-hybridization achieved a detection limit of 10 oocysts for feces/soil and <10 for water.
- IPC confirmed no PCR inhibition in any tested samples, validating the assay's reliability.
Conclusions:
- The developed assay system shows significant potential for the rapid and accurate detection of Cryptosporidium in diverse environmental samples.
- This method facilitates improved source tracking and risk assessment for Cryptosporidium transmission.
- The ease of use and high sensitivity make it suitable for field applications.
