Fast pyrolysis method of bacteria.docVIP

 PAGE \* MERGEFORMAT 7 Fast pyrolysis method of bacteria 16S-23S rRNA gene is located on 16S rRNA gene and the interval between the 23S rRNA gene sequence, with a good conservative and relative variability, was considered a suitable location identification of bacteria [1,2]. We believe that the ability to quickly and efficiently amplified in the interval, polymerase chain reaction (PCR) amplification prior to sample preparation is the key. Therefore, lysis of bacteria compared three kinds of methods. One, materials and methods 1. Strains, the human genome DNA and virus strain Source: 6 Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, totaling 19; 21 gram-negative bacteria, including Escherichia coli, Klebsiella Reber bacteria, Pseudomonas aeruginosa, etc. A total of 40. More strains of clinical testing centers and by the Zhejiang Provincial Center for TB prevention and control, preservation of bacteria in my hospital room. The human genome DNA, Cryptococcus neoformans, cytomegalovirus provided by the hospital central laboratory. 2. Clinical specimens: June 1999 ~ March 2000 hospital newborns hospitalized children, some for the general ward hospitalized children. Sepsis, 42 blood samples, cerebrospinal fluid samples were 6. Control group, 15 were for the hospital neonatal wards recovered non-infected children look forward to and discharged from hospital blood samples. 3.16S-23S rRNA gene sequence of intervals primer design: In the bacterial 16S rRNA gene 3 ‘end and the 23S rRNA gene 5’ end of conserved sequence within a inspection by the computer software to design primers. 4. Clinical sample preparation: Blood samples: 0.5 ml of heparin anti-clotting Purchase at room temperature about 10 min to precipitate blood cells, in the serum and blood cell interfaces (ie, white blood cell layer) absorb 200 μ l, injection of 1.5 ml sterile Eppendorf tube. Adding 0.87% of the NH4Cl 1 ml, shaken, placed in 37 ℃ water bath