Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar.
A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.
Liang
Shih-Yu
SY
Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.
Hsia
Kan-Tai
KT
Chan
Yun-Hsien
YH
Fan
Chia-Kwung
CK
Jiang
Donald Dah-Shyong
DD
Landt
Olfert
O
Ji
Dar-Der
DD
D017931:DNA Primers / Q000592:standards; D015342:DNA Probes / Q000592:standards; D016054:DNA, Protozoan / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D004747:Entamoeba / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004748:Entamoeba histolytica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D005243:Feces / Q000469:parasitology; D006801:Humans; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D015342:DNA Probes; D016054:DNA, Protozoan