Detection Of Legionella Pneumophila And Influenza A Virus With Fluorescent Quantitation Methods

Objective To develop a rapid, sensitive and specific detecting methods for legionella pneumophila and influenza A virus respectively. To offer dependable approach for clinical diagnosis, environmental safety monitoring, import and export inspection and quarantine measures.Methods According to the unique patented technology named principle of composite probe quantitative assay, the specific genes were found and primers and quantitative probes were designed based on this gene as target sequence in pathogenic microorganisms, then various factors(the magnesium concentration, primer concentration, annealing temperature, quantity of probes and the proportionality of two probes) in the system of real-time fluorescence quantitative PCR were optimized to detect the correlative pathogenic microorganisms.Results The constructed plasmid, which included target fragment of mip gene at length of 108bp, was testified available to serve as Legionella pneumophila positive control in real-time PCR detection. Positive results and negative results were achieved in detecting Legionella pneumophila and non-Legionella pneumophila respectively. Optimized conditions with improved efficiency and sensitivity were confirmed as:3%formamide,5.0 mmol/L Mg2+, 0.5μmol/L primers,0.2μmol/L fluorescent probe,0.4μmol/L quench probe,53℃annealing 30s. The minimum detectable bacteria number was 102CFU/ml and the detection sensitivity was 102 CFU/ml in simulated wastewater by the protocol.The constructed plasmid, which included target fragment of HA gene at length of 540bp, was testified available to serve as influenza A virus positive control in real-time PCR detection. Optimized conditions were confirmed as:4.0 mmol/L Mg2+,50U/μl superscriptⅢand 2.0U/μl Taq enzyme blent as reverse transcriptase enzyme mixture,50℃reverse transcription 20 min, and 52℃annealing 20 s. Based on this protocol, negative results were achieved in detecting of 22 non-Influenza A virus strains, and posotive results were achieved in detecting of 4 samples of Influenza A virus. The value of CV% was under 5% in parallel experiments. The minimum detectable vitro transcribed RNA number was 10copies/μl. The coincidence rate was 100% in testing 38 clinical samples. Additional, an effective and economical RNA extraction kits was selected for influenza A viruse.Conclusion The Legionella pneumophila and influenza A viruse could be quantitatively detected by the technique of composite probe quantitative assay in characters of efficient, specific and sensitive. The protocol established in this study had significance in rapid diagnosis of disease, environmental monitoring, anti-terrorism and import and export inspection and quarantine.