肺炎支原体巢式PCR的优化.ppt

Comparison of P1 and 16SrRNA genes for detection of Mycoplasma pneumoniae by optimized nested PCR in adult patients in Zhejiang, China 汇报人：周子博 Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia for 10-40% in children and adults. Because of the treatment of M. pneumonia infection with β-lactam antibiotics is ineffective and the clinical manifestations of M. pneumoniae infection are complicated and nonspecific, so a rapid, sensitive and specific laboratory test is vital for early diagnosis of M. pneumoniae infection. Conventional tests for detecting M. pneumoniae have their limitations. Introduction Introduction Several PCR-related methods provide enhanced sensitivity and have been successfully applied for research purposes such as nested PCR. The P1 adhesion gene and the 16SrRNA gene have been utilized widely in PCR techniques as the targets for detection of M. pneumoniae. In this study, we sought to identify the more sensitive and specific target (P1 or 16SrRNA) in M. pneumoniae detection and to evaluate the use of nested PCR for the diagnosis of MP infection from patients in whom M. pneumoniae was suspected. Materials and methods Strains and clinical samples DNA preparation Orthogonal array design Optimization of single factor conditions Nested PCR sensitivity test Detection of clinical samples Orthogonal array design ? Factors Levels 1 2 3 Primer (μΜ) 0.1 0.3 0.5 Mg2+ (mM) 1.5 2.5 4 Annealing temperature(℃) 58(54) 60(56) 62(58) Dilution multiple NO 50 100 Table 1 Nested PCR factors and their levels for orthogonal projects (The annealing temperature of 16SrRNA gene is expressed in the brackets) Orthogonal array design Factors Reaction