Development Of Real-Time Fluorescent Quantitative PCR Assay For Detection Of Interferon Lambda1-3 MRNA And Study Of Relationship Between The Expression Of IFN-λ1 And Respiratory Tract Pathogen

Part 1 Development Of Real-Time Fluorescent Quantitative Pcr Assay For Detection Of Interferon Lambdal-3 mRNAObjectiveTo Establish a real-time fluorescent quantitative PCR assay method for detection of human interferon lambdal-3 mRNA.Method:According to the sequences of human interferon lambdal-3 mRNA, specific primers and fluorescent probes were designed. The standard products for real-time fluorescent quantitative PCR were prepared by the method of in vitro transcription.Results:The assay for the quantitation of human interferon lambdal-3 mRNA worked well with excellent linearity. Amplification efficiency were between 90%-100%, while the sensitivities of developed method for IFN-λ1-3 mRNA genes were100 copies/μL,101 copies/μL and 101 copies/μL. All the coefficient of variation of IFN-X1-3 mRNA genes were lower than 2%. The cross reaction between each type of interferon lambdal-3 mRNA is not obvious.Conclusion:Established real-time fluorescence quantitative reverse transcription PCR assays for IFN-X1-3 mRNA.Part 2 Study Of Relationship Between The Expression Of IFN-λ,1 And Respiratory Tract PathogenObjectiveTo explore the relationship between the expression of human IFN-λ1 and respiratory tract pathogen, and provide basic data and scientific basis for developing new treatments of children respiratory tract viral infection.Method:1.The children nasopharyngeal swab were collected in The First People Hospital of Chenzhou from July 2013 to June 2014. These children were all from Chenzhou area with acute respiratory tract infection and were hospitalized. The nasopharyngeal swabs samples were total 489. According to age and the date pairing of principles, health children nasopharyngeal swabs were 244 were collected in Child health clinic of the First People Hospital of Chenzhou as the control group. Meanwhile, the collected health children nasopharyngeal swabs mixed 2ml virus preservation solution were saved in-80℃ refrigerator.2.The IFN-λ1mRNA and GAPDH mRNA in samples were detected using Quantitative Fluorescence PCR (Real-time PCR) method. In the same time, these batch specimens were screened with common respiratory tract pathogen including 18 kinds of respiratory virus and 11 kinds of bacteria.ResμLts:1. The expression level of IFN-λ1 mRNA in respiratory tract infections group was significantly higher than those in the control group, (t=2.276 P=0.027).2. There were no difference of IFN-λ,1 expression level between viruses infection group and control group(t=0.57 P=0.57), but the IFN-λ, 1 mRNA of cases who with RSV infection was significantly higher than control group (t=2.25 P=0.O26).there were no relativity between IFN-λ1 expression level and RSV copies (r=0.12 P=0.41)3. There was significantly difference of IFN-λ1 mRNA level between the infection specimens with HRV infection than the control group with HRV Infection (t=2.8 P=0.015)4. There was significantly difference of IFN-λ1 expression level between RSV infection specimens and HRV infection specimens (t=2.19 P=0.045)5. Compared with the normal group, there was no difference of IFN-λ,1 expression level in bacterial infection cases (t=0.17 P=0.87) Conclusion:1. It was proofed that IFN-λ 1 was involved in the pathogenesis of respiratory infections.2. RSV can induce the mRNA expression of IFN-λ,1 and stronger than HRV.3. Respiratory tract bacterial infection may have no obvious effect on mRNA expression of IFN-λ-1.