RESUMO
Simultaneous multiple gene detection is indispensable for the detection of various genes in a small sample obtained by an invasive method. A typical detection method is probe-based fluorescence melting curve analysis by means of real-time PCR. It is very limited because, for each target, a probe sequence with at least a different Tm must be designed. To overcome this limitation, we developed a simultaneous multiple gene detection method based on a giant amplicon molecular beacon. PCR was performed by attaching stem sequences with different Tm values to each primer set, and the melting Tm was measured by hybridizing the stem sequences at both ends of the amplified amplicon; this generated well-separated Tm signals. The important point here is that the stem sequence that produces the Tm signal is an arbitrarily selectable sequence unrelated to the target gene. Because it is arbitrarily selectable, the desired Tm can be freely adjusted. As a result, we succeeded in the simultaneous detection of four samples with the use of only one fluorophore. Theoretically, a combination of five fluorophores could detect more than 20 multiple genes simultaneously.
Assuntos
Chlamydia trachomatis/genética , Primers do DNA/química , DNA Bacteriano/análise , DNA Viral/análise , Papillomavirus Humano 16/genética , Mycoplasma hominis/genética , Neisseria gonorrhoeae/genética , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/genética , DNA Viral/genética , Fluorescência , Papillomavirus Humano 16/isolamento & purificação , Humanos , Mycoplasma hominis/isolamento & purificação , Neisseria gonorrhoeae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sitios de Sequências RotuladasRESUMO
BACKGROUND: Vivax malaria occurring in the Republic of Korea is occasionally characterized by a long latent infection induced by hypnozoites in the liver. So far, the mechanisms responsible for short and long latent infections of vivax malaria are not known. Therefore, the present study classified the parasite isolates according to the long and short latent periods and then analysed the genetic diversity of the Plasmodium vivax merozoite surface protein 1 (PvMSP-1). METHODS: Blood samples containing P. vivax isolates were collected from 465 patients from 2011 to 2013 at health centers in the Republic of Korea. PvMSP-1 gene sequences were analysed in groups classified by the collection year, and short or long latent periods. The samples in short and long latent periods were selected by the timing of vivax malaria occurrence, July-August and January-May, respectively. RESULTS: Three PvMSP-1 types (Sal-1, Belem, and recombinant) were observed in P. vivax isolates collected from 2011 to 2013. Interestingly, the recombinant and Sal-1 types were dominant in vivax malaria of the long and short latent periods, respectively. In addition, the S-b like subtype of the PvMSP-1 Sal-1 type was first identified in 2013. CONCLUSION: This study revealed that the genetic type of PvMSP-1 is likely related to the duration of its latent period. Moreover, trends of the genetic types of PvMSP-1 seem to be stable in recent years compared with those of previous years in which various new types were observed.
Assuntos
Malária Vivax/parasitologia , Proteína 1 de Superfície de Merozoito/genética , Plasmodium vivax/genética , Estudos de Coortes , DNA de Protozoário/análise , DNA de Protozoário/genética , DNA Recombinante/genética , Humanos , Malária Vivax/epidemiologia , Polimorfismo de Nucleotídeo Único/genética , República da Coreia/epidemiologiaRESUMO
BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK). METHODS: Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK. RESULTS: Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants. CONCLUSIONS: No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.