Establishment and Application of Semi-nested PCR for Detection of Legionella Species

ObjectiveTo establish a molecular biological method for detection of legionella species.MethodsRoutine Polymerase Chain Reaction(PCR)was used to amplify the specific fragment of legionella species 16SrRNA gene and lepionella pneumophila(Lp)mip gene while semi-nested PCR was used to validate the amplified product of routine PCR.ResultsThe 654bp specific fragment of 16SrRNA gene of 5 legionella species strains(including 3Lp strains)could be amplified,whereas non-legionella stains could not produce positive amplified fragments.The amplified product was validated by semi-nested PCR which yielded the 430bp specific fragment of 16SrRNA gene.The 649bp specific fragment of 3 Lp strains could be ampified by mip primers and non2Lp stains was negative.Semi-nested PCR was used to validate the amplified product,by which the 399bp specific fragment of 3 Lp stains mip gene could be amplified.The sensitivity was 10CFU/ml.The method was used successfully to detect a isolated suspected legionella strain in water sample.ConclusionSemi-nested PCR was economy,rapid,simple,sensitive,specific and a powerful tool in early detection,environmental monitoring and control infection outbreaks of legionella.