Bacterial DNA decontamination for reverse transcription polymerase chain reaction (RT-PCR)

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 microg of nucleic acid using 10 U of RNase-free DNase I in 10-microl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.     

单细胞RT-PCR研究方法及其应用

在单细胞水平对基因表达进行研究,可有效地避免多细胞水平研究的诸多局限。可借助膜片钳电极所形成的吸收通道,或借助其它方法,获取单个细胞,对其进行单细胞水平的分子生物学研究。采用嵌套PCR方法,或三元尾端PCR扩增方法,对单细胞mRNA进行RT－PCR扩增,可收获足量,可靠和稳定的产物。单细胞RT－PCR可应用于受体研究,离子通道研究等方面。     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.