ABSTRACT
Opisthorchiasis is one of the significant naturofocal diseases in Russia. The diagnosis of opisthorchiasis may mask some diseases, such as opisthorchiasis, metorchiasis, and clonorchiasis - biohelminthoses that are induced by various representatives of the family Opisthorchiidae-Opisthorchis felineus/O.viverrini, Metorchis bilis, and Clonorchis sinensis, respectively. Coproovoscopy and serologic methods fail to accurately define the species-specific affiliation; in this connection the identification of opisthorchids, by using DNA diagnostic techniques, becomes urgent. The present paper gives the results of development of DNA diagnosticum, which differentiates parasitic diseases induced by O. felineus and M. bills. The ribosomal RNA gene cluster fragment incorporating internal transcribed spacer 2 (ITS2) was used as a diagnostic marker. A system for diagnosing opisthorchiasis was developed as a multiplex polymerase chain analysis and tested on 37 patients infected with various species of opisthorchids.
Subject(s)
DNA, Helminth/analysis , Opisthorchiasis/diagnosis , Opisthorchis/isolation & purification , Polymerase Chain Reaction/methods , Trematoda/isolation & purification , Trematode Infections/diagnosis , Animals , Base Sequence , DNA, Helminth/genetics , DNA, Ribosomal Spacer/genetics , Diagnosis, Differential , Humans , Molecular Sequence Data , Opisthorchis/genetics , Sequence Alignment , Trematoda/geneticsABSTRACT
OBJECTIVE: To develop a test system for polymerase chain reaction (PCR) diagnosis of Opisthorchis felineus in human feces. Based on the Internet databases, the authors have developed oligonucleotide primers, as well as TaqMan tests (fluorescence label probes) specific to O. felineus genome to perform PCR with the real-time detection of a result. Conditions for PCR, including temperatures, the number of components of amplification, etc., were selected. Test systems on fecal samples from 254 subjects randomly selected and on those from 100 patients with verified opisthorchiasis, and on those from 100 healthy individuals were studies. The significance of the test systems was ascertained to be about 98%. Their sensitivity in determining O. felineus is about 10(3) copies of target DNA per ml. The efficiency of various techniques (coproovoscopy, enzyme immunoassay, PCR) was compared to detect Opisthorchis invasion.
