RESUMEN
Two new species of Episymploce Bey-Bienko from China are described. Nine individuals of E. sichuanensis sp. nov. were collected from Sichuan Province and four individuals of E. maxima, sp. nov. were collected from Guangxi Province. Morphology, especially the wings, specialized abdominal tergum and genitalia of adults, are described and illustrated in detail. Episymploce sichuanensis sp. nov. is similar to E. kunmingi (Bey-Bienko, 1969), but can be easily distinguished by the reduced wings, bifurcated two processes at the hind margin of the supra-anal plate, and the unspecialized first abdominal tergum (T1). Episymploce maxima sp. nov. is similar to E. taiheizana Asahina, 1979 but is distinguished by its large size, the lateromedial margins of the subgenital plate without processes, and the unspecialized T1. A key to the recorded Episymploce species from China is provided in this paper.
RESUMEN
Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.
Asunto(s)
Clonorquiasis/parasitología , Clonorchis sinensis/aislamiento & purificación , Ciclooxigenasa 1/aislamiento & purificación , Opistorquiasis/parasitología , Opisthorchis/aislamiento & purificación , Animales , Asia , Clonorquiasis/transmisión , Clonorchis sinensis/genética , Clonorchis sinensis/patogenicidad , Ciclooxigenasa 1/genética , Peces/parasitología , Humanos , Opistorquiasis/transmisión , Opisthorchis/genética , Opisthorchis/patogenicidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 10(2) CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
Asunto(s)
Cronobacter/genética , Cronobacter/aislamiento & purificación , Contaminación de Alimentos/análisis , Técnicas de Genotipaje/métodos , Fórmulas Infantiles , Reacción en Cadena en Tiempo Real de la Polimerasa , Temperatura de Transición , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Lactante , Límite de Detección , Desnaturalización de Ácido Nucleico , Polvos , Factores de TiempoRESUMEN
Two bio-synthesizing chimeric peptide (CP) immunogens named CP1 and CP10 have been designed, which consist of three linear B cell epitopes (BCE) of human chorionic gonadotropin beta subunit (beta-hCG) and six foreign T cell epitopes including two "promiscuous" TCEs from hepatitis B surface antigen and tetanus toxoid. Two artificial genes encoding CP1 and its derivative CP10 were synthesized, which could be expressed in E. coli at the level of about 1% of the total cell proteins when inserted into the thermo-induction vector respectively. In Western blot tests, the expressed CP1 and CP10 proteins with about 16.5 kD shown on the SDS-polyacrylamide gel electrophoresis (PAGE) gel can be recognized by the monoclonal or polyclonal antibodies specific to each linear epitope of beta-hCG. Each of expressed proteins can be purified with 95% relative homogeneity using our improved method of preparative gel PAGE. Their yields were about 1-2 mg per 12 L culture. Also, the CPl and CP10 immunogens can induce antibodies in mice that recognize recombinant CP1 betar CP10 and natural beta-hCG, and there are three anti-beta5, beta9 and beta8 BCE antibodies in their antisera. The construction and expression of beta-hCG CP1 and CP10 will provide new immunogens for developing an ideal and superior hCG birth control and/or tumor therapeutic vaccine.
Asunto(s)
Gonadotropina Coriónica Humana de Subunidad beta/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Gonadotropina Coriónica Humana de Subunidad beta/genética , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Escherichia coli/genética , Humanos , Ratones , Modelos Genéticos , Péptidos/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1 alpha and P1 beta) and two P2 proteins (P2 alpha and P2 beta), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild-type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1 alpha/P2 beta and P1beta/P2 alpha pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function.