Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees
Trial chain tests of the three duplex transcription of the three duplex transcription (real-time RT-PCR) transcription based on Taqman chemistry, have been developed for simultaneous detection and the specific quantization of the virus Apple chlorotic leaves (ACLSV), plum pox virus (PPV), Prunus NecRotic Ringspot Virus (PNRSV), DNARE Virus (PDV), Latent Mosaic Virus (PLMVD) (PLMVD) and Fruit Phytoplasma European stone (Esfy), which are considered among the most important pathogens affecting stone fruit trees. The RT-PCR (RT-QPCR) quantitative analyzes have been optimized using RNA transcripts (plasmid linearized has been used for optimizing the determination of the esfy phytoplasm) of known concentrations. No difference in sensitivity was recorded between the duplex and RT-QPCR analyzes of RT-QPCR. The amplification efficiency of the recto-95.8% tests is 91.1 to 95.8%, while the linear quantization range was between 20 and 2 x 107 transcripts of linearized plasmid for PLMVD and phytoplasma esfy, 40 to 4 x 107 RNA transcripts for ACLSV, PPV and PDV and 102 to 108 RNA transcripts for PUNSV, respectively
Duplex RT-QPCR assays, which have been validated using all the two isolates characterized by all pathogens and field samples of Prunus species in northern Greece, have presented a wide range of detection . Overall, developed methods include useful tools that can be applied to simultaneous and reliable detection of transmissible transmissible pathogens in Prunus spp certification programs. Diarrheal diseases account for more than 50% of foodborne diseases around the world, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and takes a long time; Therefore, it is necessary to establish a fast and practical detection method that can detect multiple pathogens simultaneously.
In this study, we have developed a set of five PCR dosages SYBR GREEN I Time-time multiplex to simultaneously detect 15 common pathical agents based on the non-dimeric system assisted by homo-tag. These analyzes efficiently reduce the formation of dimeric primer and improve the stability, uniformity and efficiency of the amplification of multiplex PCR.
The role of polymorphisms Genes of the vitamin D receptor in the risk of colorectal cancer
Vitamin D deficiency has been associated with an increase in the risk of incidence and mortality of colorectal cancer (CRC). The median vitamin D its action through the binding of the vitamin D receiver (VDR) and the polymorphisms of the VDR could explain these reverse associations. The objective of the study was the Survey of the relevance of RS731236; Thermus Aquaticus I (Taqi), ; Acetobacter Pasteurianus Sub. Pasteurianus I, Bacillus Stearothermophilus I (BSMI) Polymorphisms of the VDR gene with colorectal carcinogenesis (CRC) and progression. The peripheral blood was obtained from 397 patients with staging II / III (n = 202) and step IV (n = 195) and IV (n = 195). In addition, samples of 100 healthy donors and 40 patients with adenomatic polyps were also included as control groups.
Genotyping in patient and control samples was carried out using polymorphisms of fragmentation length of the reaction of the polymerase reaction (PCR-RFLP). An important association has been revealed between the four polymorphisms and cancer. Individuals with homozygous mutant genotypes (TT, AA, FF or BB) were more sensitive to the disease (p <0.001). All detected mutant genotypes have also been significantly associated with Phase IV (p <0.001), resulting in a significant decrease in survival (p <0.001). In addition, the four polymorphisms have been significantly associated with Krasten mutations (Kirsten Ras oncogene) and genetic variants of the toll receptor (TLR2, TLR4 and TLR9).
Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees
Evaluation of seven African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples.
The African porcelain fever virus (ASFV) is a complex dual-stranded DNA virus, responsible for a highly infectious and mortal disease in pigs and shores and for a significant deterioration in animal welfare. Over the past decade, the disease has expanded to several European and Asian countries, causing unprecedented dramatic economic losses in the pork industry. In the absence of vaccine, affected countries are based on confidence diagnostic tests and test policies adapted to setting up control programs to combat disease.
In this study, we evaluated the sensitivity and specificity of seven real-time real-time PCR detection kits in the trade and three Taq polymerases on 300 samples of well-characterized boars collected in Belgium during the 2018 epidemic -2019. This study confirms that all commercial kits and two TAQ polymerases are suitable for ASFV detection in diagnostic laboratories. In addition, the use of endogenous controls is underlined when using field samples harvested on carcasses in an advanced decomposition step, in order to avoid falsely negative results. The functional efficacy of the expression cassettes integrated in a plasmid and a PCR-amplified fragment was relatively analyzed after the transitional transition transition in vitro or introduction into the development embryo of Danio Rerio.
The cassettes contained the journalist genes, the luciferase of photine pyral (LUC) or an improved green fluorescent protein, under the control of the human cytomegalovirus promoter, immediate and early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times greater than that of the PCR-amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR-amplified fragment was evaluated quantitatively. The mutations generated after 25 amplification cycles with the TAQ DNA the polymerase decreased the activity of the luciferase in cells transfected from 65 to 85%.
Jordan
