Environmental Water And Clinical Samples Testing For Legionella And Restriction Study

Legionella are facultative intracellular Gram-negative bacteria which are fastidious and ubiquitous worldwide in naturel water environment.It was the important pathogen that causes Legionnaires disease.More than 50 species and 70 sero-groups have been identified in the familly of Legionella, Nearly 20 legionella species have been docunmented as human illness agents.Legionella pneumophila is the most comman pathogenic species within the genus legionella and is the main cause of legionellosis.There have been many methods for Legionella detection on clinic. The traditional methods for detection of legionella are based on culture techniques.which is difficult and time-consuming.Serologic testing can only be used for the known species.Biochemical phenotypic identification has the low resolution and poor reproducibility. Other methods such as electrophoresis of soluble protein, fatty acid composition analysis, all have shortcomings. Therefore, a fast and effective detection and identification methods is meaningful.In recent years, the molecular biology methods for detecting legionella have been recognized by more and more laboratories. And digestion typing method combined molecular biology techniques has been widely used in microbial molecular typing test.The technology used restrictive enzymes to digest the specific gene fragment,It can not only combine the high sensitivity of PCR,but also provide a more objective, reliable and efficient results.To develop polymerase chain reaction (PCR) with enzymatic digestion analysis method for identification and differentiation of Legionella, and investigate distribution state of Legionella in environmental water systems in Guangzhou. One hundred-twelve strains of Legionella isolated from river and lake environment water system in Guangzhou were detected by PCR with enzymatic digestion and sequencing analysis method that was designed according to Legionella-specific 16S rRNA gene sequence. Sixty-six strains of Legionella pneumophila and 46 Non-L. pneumophila of 112 isolated Legionella were identified by PCR-enzymatic digestion and sequencing analysis method of 16S rRNA. Forty-six strains of Non-L. pneumophila were including 20 strains L. feeilei,17 strains L. gormanii,7 strains L. oakridgensis and 2 strains L. longbeachae. PCR with enzymatic digestion analysis is a simple, rapid, sensitive and specific method for the identification and differentiation of Legionella, while L. pneumophila were exists broadly, followed L. feeilei, L. gormanii, L. oakridgensis and L. longbeachae in water environment in Guangzhou.To establish the method of culture of sputum specimens, to improve the positive rate of cultured sputum specimens. Collecting 50 cases of fresh sputum samples and dividing them into two groups. One group directly was cultured after pre-treatment; the other group was cultured after adding Legionella reference strains and pre-treatment. Pretreatment was used by method of liquefying for sputum samples and was eradicated non-Legionella bacteria with acid and/or heat treatment. After pretreatment, culturing sputum specimens on Legionella selective BCYEa-DGVP medium, then checking the colony growth of legionella. It could be initially identified as Legionella according to the features of growth and morphology of colony. And then it was finally determined by biochemical reaction and PCR reaction. The 50 cases of sputum samples without adding standard strains were not found Legionella. The 50 cases of sputum samples added Legionella standard strains and directly cultured had an average positive rate of 58%; the samples of acid treatment had a positive rate of 70%, and the samples of heat treatment had a positive rate of 66%. Comparing three methods of pretreatment, the difference between heat treatment and no treatment was not significant, but the difference between acid treatment and no treatment was significant. In addition, the consequence between acid treatment and heat treatment was not consistent with each other. The two methods used acid and heat treatment can improve the detection rate of Legionella, while acid treatment can significantly improve the detection rate of Legionella.This study collected 100 sputum samples of patients with atypical pneumonia and investigated by 16S rRNA PCR, enzymatic digestion analysis method and PCR-sequencing of the 16S rRNA fragment of legionella. Result 13 cases were positive of polymerase chain reaction of 16S rRNA fragment, among 12 cases were identified as Legionella pneumophila by sequencing and enzymatic digestion analysis method.The result of PCR with enzymatic digestion analysis is agree with the sequencing result of 16S rRNA gene. PCR with enzymatic digestion analysis is a simple, rapid, sensitive and specific method for the identification and differentiation of Legionella.