Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues.
The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain).
Schneider
Renate
R
Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
Gollackner
Bernd
B
Edel
Bernhard
B
Schmid
Katharina
K
Wrba
Friedrich
F
Tucek
Georg
G
Walochnik
Julia
J
Auer
Herbert
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D002985:Clinical Protocols; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification; D004443:Echinococcosis / Q000175:diagnosis / Q000235:genetics / Q000469:parasitology*; D004446:Echinococcus / Q000235:genetics*; D005260:Female; D005557:Formaldehyde; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D012418:Ruminants / Q000469:parasitology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D018509:DNA, Helminth; D004272:DNA, Mitochondrial; D005557:Formaldehyde