| In recent years,as people's living standards improved, the morbidity of Legionnaires disease is increasing year by year. Since its first outbreak in USA in 1976,many countries have reported about Legionnaires disease.In 1982, Nanjing confirmed the cases of Legionnaires disease in China firstly. Legionella mainly grows in water environments, especially in air conditioning systems and water supply systems.Many cities in China made Legionella surveys for air conditioning cooling towers, which suggested that the positive rate of Legionella was 50%～80%. There is a potential threat to human health because of air conditioning cooling tower water which contaminates of Legionella. At present,the detection of legionella mainly depends on isolation and cultivation of bacteria.However,the culture cycle is long,that affects the prevention and control of Legionnaires disease.This issue made surveys and analysis for the air-conditioning cooling tower Legionella contamination of a city in south China,and made genotypes for the isolated strains,and established molecular epidemiological data.In addition, the issue initially built a rapid method for detecting Lpl by fiber-optic evanescent wave biosensors,which provided a new direction for the on-site rapid screening of large size water samples that polluted seriously by Lpl.Objective1.Isolate and cultivate legionella from air conditioning cooling tower water in all kinds of sites,such as hotels,the market places,hospitals which understand the Legionella contamination to the air-conditioning cooling tower in a city of south China.Inspect the water temperature and the total of bacteria.And inquire into the air conditioning service life and the disinfection condition of air conditioning cooling tower water to know the relationships between the contamination conditions and these factors.2.Make genetyping to the isolated Lp strains by SBT technology, analyze the polymorphism of isolated strains, confirm the relation and variation among the genetic basis of strains, and understand the city epidemiological characteristics of the distribution of Legionella.3.Establishe rapid detection platform of Lpl.Choose the optimize detection conditions of the method,and confirme detection sensitivity and specificity under the optimum conditions,and at the same time to inspecte the artificial infected samples by the new method,and make count by traditional method to confirm the feasibility of the new method.Methods:1.Culture Legionella in the air-condition cooling towers with the couting those in plate and identify with the serum method.Inspect the water temperature and the total of bacteria of cooling tower water,survey the useful life and disinfection condition of air conditioning cooling tower,then analyze if there are relations between these factors and the contamination conditions of legionella.2.Use the SBT method to type the Legionella and measurate the OD260 and OD280 to calculate concentration of DNA.Amplify nucleic acid sequences within the fragment of 7 housekeeping genes (flaA, pile,asd, mip, momps, proA, neuA) separately, from 394bps to 710bps.Make nucleic acid purification to PCR amplification products obtained.Upload the sequence results of purified products to EWGLI database, to get allele of strains, and confirm the sequence types of the isolated strains.Draw phylogenetic trees to ST results using the method of UPGMA of the isolated strains by sofeware of MEGA4.Observe the similarity of the strains, and analyze the genetic relationships.3.Study the rapid detection method of fiber optic evanescent wave biosensor for Legionella pneumophila serotypel,and establish the platform for rapid detceion of Lpl according to the principle of double antibody sandwich method.Label the detection antibody by flourescent dye of Alexa fluor 647,and store the labeled antibody at-20℃by aliquots.The fibers are coated with streptavidin after washed by isopropanol, and the biotin-labeled capture antibodies are more effectively connected to the fibers by avidin-biotin reaction.Optimize for the conditions, including the best response times of capture antibody and target, reaction system and detection antibody, the optimal concentrations of capture antibody and detection antibody. Determine the detection sensitivity and specificity under the optimical condition, and conform sensitivity and specificity of the method in practical samples by detecting artificial infection samples, and preliminary establishe Lpl rapid detection method.Results:1.19 public places were test positive for Legionella,the positive rate was as high as 86.4%.There were31 isolates of Legionella pneumophila (Lp) from54 samples with the total positive rate of 57.4%. The positive rate of the samples from different kinds of places had no significant difference (P>0.05).The isolates were initially classified by serotyping as Lpland Lp2-14 with percent of 35.5%,64.5% respectively.The difference of water temperature,standard plate-count bacteria,service life of air conditioning or disinfection of the samples had little effect (P>0.05).2.There were a total of 19 kinds of STs for 31 strains of Separated Legionella pneumophila by using the SBT method.11 Strains of Lpl were divided into five kinds of STs, respectively, ST1,ST159, ST160, ST154 and ST52.Among them,5 strains of Lp1 were ST1 type, accounting for 45.5%,and 2 strains of Lpl were ST159.In addition,20 strains of Lp2-14 were divided into 14 kinds of STs, including ST242, ST 152,ST 8,ST 367, ST 560 that were known in the database.Among them,5 strains of Lp2-14 were ST1 type, accounting for 30.0%,and there were five new kinds of STs which named a～f,one strain for each type,and 0a,0b,0c types only got 6 genes amplified,the type of 0a had three strains, Ob and 0c each had one strain.3.We used the MEGA4 soft to draw the phylogenetic tree of the STs with the method of UPGMA.We divided the 26 strains of isolated Lp which all the seven genes were amplified into two homologous lineages. Among these, the strains of ST159, ST54, ST160 and the unknown type belonged to the same type isogenic lines, indicating a certain degree of genetic relationship;and the remaining STs belonged to the other homologous series.In addition, the results also revealed that for Legionella, the different serotype strains may also be derived from the same genetic ancestor, and the same ST type may have different origins.4.The Fiber optic evanescent wave biosensor automatically completed the detection within 40 minutes. The best response times of capture antibody and target, reaction system and detection antibody were both lOminutes, the optimal concentrations of capture antibody and detection antibody were respectively 100μg/ml and 20μg/ml.Under the best condition,the sensitivity of the method was 1.8×103cfu/ml.It had a better specificity to the Lpl,which the detection results were all negative for the othe 13 strains serotypes of legionella pneumophilia,2 strains of non-legionella pneumophilia and 4 strains of non-legionella.we used the method to detect the artificial infection samples made by 31 strains of isolated legionella pneumophilia to conform the sensitivity and specificity of the method. And we got the 100% positive rate for the samples that the concentrations of the Lp1 bacteria were 1.8 x 103cfu/ ml or more. And the three times decections for the samples that the concentrations of the Lp1 bacteria were below the specitivity but closely to 1.8×103 cfu/ml could be positive for 1 to 2 times.The samples that were infected by Lp2-14 were all negative.Conclusions:We got the data about epidemic in the public places of a city in south china,which could be the credibility data for the comparative studies.At the same time we used SBT method to get the genetypes of legionella pneumophilia,which could be more helpful to understand the strain polymorphism and to ensure the ralation and variation between heredity.The results provided the scientific basis for developing the monitoring measure of Legionella and controlling the outbreak of Legionnaires disease for the city.In addition,we initially developed the rapid detection method for Lpl using the fiber optic evanescent wave biosensor, which provided a new direction for the on-site rapid screening of large size water samples that polluted seriously by Lpl. |
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