sybr-green-i and Rodent-Diseases

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.

Topics: Animals; Babesia microti; Babesiosis; Benzothiazoles; Coinfection; Diamines; DNA Fingerprinting; DNA Primers; DNA, Protozoan; DNA, Ribosomal; Fluorescent Dyes; Genotype; Humans; Limit of Detection; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Rodent Diseases; Rodentia; Sequence Analysis, DNA; Ticks

Dobrava hantavirus (DOBV) belongs to the genus Hantavirus of the family Bunyaviridae, and is carried by yellow necked and striped field mice (Apodemus flavicollis and Apodemus agrarius), respectively. The aim of this study was to detect and genetically characterize new DOBV strains in rodents captured in the Transdanubian region of Hungary. Rodent corpses were dissected and lung tissues were used for hantavirus detection by SYBR Green-based real-time RT-PCR using specific primers located in the S-segment of the virus genome. A total of 22 captured animals of the Apodemus species were tested for the presence of DOBV. Three out of the 22 mice were positive. Phylogenetic and molecular sequence analyses showed that Hungarian DOBVs were most closely related to those viruses detected from A. agrarius mice in Slovenia. Based on our new data from the region we concluded that extended reservoir studies would be necessary in the future.

Topics: Animals; Benzothiazoles; Diamines; DNA Primers; Hantavirus Infections; Hungary; Molecular Sequence Data; Murinae; Organic Chemicals; Orthohantavirus; Phylogeny; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Rodent Diseases; Sequence Analysis, DNA