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Cryptosporidium detection by Fluorescence in situ hybridisation (FISH) (CAT#: STEM-MB-1230-WXH)

Introduction

Cryptosporidium, the most common non-viral cause of diarrhea worldwide, is a protozoan parasite belonging to the Phylum Apicomplexa. Species belonging to the genus Cryptosporidium are the causative agents of a gastrointestinal infection, termed cryptosporidiosis . This diarrhoeal illness is self-limiting in immunocompetent patients but can be life-threatening in immunocompromised hosts, particularly in persons receiving immunosuppressive drugs and AIDS patients. There is currently no effective therapy for the treatment or prevention of cryptosporidiosis in humans, despite intensive efforts over the past 20 years.




Principle

FISH uses fluorescent probes with complementary base sequences to locate the presence or absence of specific portions of DNA on chromosomes. The probe and target DNA must be denatured with heat or chemicals to break hydrogen bonds in the DNA and to allow hybridisation to occur once the two samples are mixed. The fluorescent probes form new hydrogen bonds with their complementary base pairs on the DNA, and these can then be detected via microscopy.

Applications

Detect and localize the presence or absence of specific DNA sequences on chromosomes.
Detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples.

Procedure

1. Sample preparation
2. Co-denaturation and hybridization
3. Probe detection
4. Wash off of unbound probe
5. Analysis by flow cytometer/fluorescence microscopy

Materials

• Flow cytometer
• Fluorescence microscopy