Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides.
OBJECTIVE
A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides.


RESULTS
Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested.


CONCLUSIONS
This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides.


CONCLUSIONS
The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.
Amar
C F L
CF
Food Safety Microbiology Laboratory, PHLS Division of Gastrointestinal Infections, Central Public Health Laboratory, London.
Chalmers
R M
RM
Elwin
K
K
Tynan
P
P
McLauchlin
J
J
D000818:Animals; D003458:Cryptosporidium / Q000145:classification* / Q000235:genetics; D016785:Cryptosporidium parvum / Q000145:classification / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis* / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology; D005838:Genotype; D006801:Humans; D033661:Oocysts / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011196:Potassium Permanganate; D015800:Protozoan Proteins / Q000235:genetics; D012394:Rosaniline Dyes; D016037:Single-Blind Method; D013194:Staining and Labeling / Q000379:methods
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012394:Rosaniline Dyes; C106305:oocyst wall protein, Cryptosporidium; D011196:Potassium Permanganate; C006898:carbol-fuchsin solution