Research Of A Rapid Detection Method For Legionella Pneumophila

Objective Legionella is widespread in the soil and water of nature.Legionnaires’ disease is an acute febrile respiratory disease caused by inhalation of aerosols contaminated with Legionella,with a mortality rate of 15% to 30%,and 90% of Legionella disease is caused by Legionella pneumophila.At present,the detection of Legionella mainly adopts the cultivation method,but due to the special nutritional requirements,the operation is cumbersome,takes a long time,and costs a lot,and the positive rate is not high.This paper studies three different detection methods of Legionella,and provides an important basis for the detection of Legionella pneumophila through the exploration of a new detection method for Legionella pneumophila.Methods 1.Legionella is isolated and cultivated using traditional culture methods.Samples were inoculated with GVPC plates and incubated at 37°C for 10 days.The growth was not Legionella the next day.Colonies were observed on the third day of incubation.Suspected colonies were inoculated with BCYE plates and BCYE-cys plates and incubated at 37°C for 2 days.Legionella colonies that grow on BCYE plates but do not grow on BCYE-cys plates.Legionella pneumophila was identified based on the biochemical reaction.Legionella diagnostic sera were used to classify Legionella pneumophila.2.PCR was used to detect the target gene of Legionella.According to the Legionella’s common gene 5Sr RNA fragment and the Legionella pneumophila’s mip gene fragment,primers were designed to pick colonies from BCYE plates and were suspended in EP tubes containing 100 μL of distilled water.The bacterial DNA was prepared by thermal lysis method and PCR amplification was performed.The gene of interest,agarose gel electrophoresis,was used to analyze whether the bacteria isolated were Legionella and Legionella pneumophila.3.Detection of Legionella pneumophila using nucleic acid isothermal amplification test strip rapid detection technology.A LAMP-LFD method for the Legionella pneumophila was established based on a primer probe designed for the Legionella pneumophila mip gene,and compared with LAMP-p H colorimetry,LAMP-real-time turbidimetry and fluorescence quantitative PCR.The sensitivity and specificity of the reaction system were tested and applied to the detection of environmental water samples.Results 1.The traditional culture method was used to isolate and culture Legionella in environmental water samples.A total of 44 samples were 34 pipe network biofilm samples and 10 air-conditioning condensate samples,of which 16 were detected Legionella,the positive rate was 36.4%.16 of the isolated Legionella biochemical reaction,biochemical identification results.According to the results,14 strains were Legionella pneumophila,which accounted for 87.5%.After diagnosis of serotyping by Legionella pneumophila,12 of the 14 Legionella pneumophila strains were LP1,1 strain was LP2,and 1 strain was LP5.2.The isolated strain preparation template was used for PCR detection of 5S r RNA and mip gene respectively.The sample was amplified by Legionella 5S r RNA gene fragment.The 16 isolates belonged to Legionella.The amplification of mip gene of Legionella pneumophila showed that 14 of them belonged to Legionella pneumophila,and consistent with biochemical identification results.3.In this study,primers were designed based on Legionella pneumophila-specific mip genes,and the LAMP-LFD detection reaction system and procedure were optimized.The samples were detected by LAMP-LFD,LAMP-PH discoloration,and LAMP-real-time turbidimetry respectively,and referenced by fluorescence quantitative PCR detection.The results showed that the minimum detection limit of LAMP-LFD method was 3 cfu/μL,and did not cross-react with other strains.The sensitivity and specificity were consistent with the results of real-time fluorescence quantitative PCR.Applied to environmental water samples to detect Legionella pneumophila,the test results are also consistent with real-time fluorescence quantitative PCR detection results.Conclusion The traditional method for the detection of Legionella in environmental waters is isolation and culture,combined with biochemical reactions,serotype diagnosis and PCR.In this study,primers were designed for Legionella pneumophila-specific mip genes,and LAMP-LFD detection reaction systems and procedures were determined through optimization experiments.The LAMP-LFD,LAMP-p H discoloration LAMP-real-time turbidity method was used to detect the samples,and the fluorescence quantitative PCR detection was used as a reference to determine the sensitivity and specificity of the method.The results showed that the minimum detection limit of LAMP-LFD was 3 cfu/μL and did not cross-react with other strains.The sensitivity and specificity were consistent with the results of real-time fluorescence quantitative PCR;applied to environmental water samples for detection of Legionella pneumophila,The test results are also consistent with the real-time fluorescence quantitative PCR test results.LAMP-LFD is simple in operation,does not require expensive instruments,and is stable in method and reliable in results.Compared with isolation and culture detection,the detection time is greatly shortened and the results are easy to observe.It is expected to be popularized and applied on the grass roots and on-site detection.