Reverse Transcription Loop-mediated Isothermal Amplification Detection And Epidemiological Analysis Of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is a non-segmented negative-sense RNA virus which belongs to the Family Paramyxoviridae, Order Mononegavirales. It replicated in cytoplasm of and buds at the plasma membrane. The RSV genome is15.2kb in length and has10genes encode11proteins. RSV can be divided into A and B two group based on the antigen diversity and each group can be subdivided into more genotypes.RSV is highly contagious and can re-infect a person throughout the life. Most children will be infected by RSV in their first year of life and virtually all are infected by the age of two. RSV is an important pathogen of respiratory tract infection in children, Hospitalization was observed in5percent of all RSV infection. Long term impact on respiratory function including asthma was associated with severe RSV infection and the highest mortality of RSV infection is found in the elder and population with comprised immunity or cardiopulmonary diseases. Therefore, the epidemiological analysis and the rapid diagnosis infection is very important for the prevention and the control of RSV.Several methods including cell culture, antigen based test, Polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA) was developed for the detection of RSV. They are not efficient for filed diagnosis of RSV infection. In the first section, a Reverse transcription loop-mediated isothermal amplification (RT-LAMP) based method for visual detection of RSV infection was developed. The RT-LAMP technology was firstly reported in2000which employed three pair of primer to form a stem-loop structure in the products of amplification, the single strand DNA in the loop part of amplification product can bind free primers and the strand replacement reaction can produce more single strand DNA as templates. This reaction can perform at an isothermal condition of60degree Celsius and the amplification products are100times higher than PCR. LAMP assay can achieve visual result when combined with fluorescent dye like Calcein.In order to design a common primer for all RSV, genomes available for A and B group RSV was retrieved from GenBank. The genomes was aligned and conserved region was searched for A and B group RSV respectively. Primers were designed in these conserved regions and orthogonal experiment was used to optimize the reaction system to get best reaction conditions. The reaction specificity of primers was assessed by the optimized reaction system and the detection limit of A and B group RSV was determined as5×10175TCID50/ml for group A and5×10-0.55TCID50/ml for group B. Finally, this assay was used to detect RSV infection in77clinical samples and the results were compared with RT-PCR. The positive rate of two methods were both47.9%and Of the RT-LAMP positive samples,36(95%) were also positive by RT-PCR, while two were negative by RT-PCR.The epidemiology data of RSV infection based on serology or DNA sequence had reported in many western countries. The epidemiology analysis of DNA sequence begin with the collection of clinical specimen, sequencing the genotyping region of viruses in the specimen, analyze the evolutionary status of sequences and then describe the epidemiological characteristic of the virus based on the space-time distribution of genotypes and infections. We can better understanding the pathogenesis based on the epidemiological statistics.In the second section of the research, an epidemiological survey of RSV infection in Shanghai was conducted. In the survey,2407nasopharyngeal swabs were collected between August2009and December2012from Nanxiang hospital.184RSV positive samples were sequenced and69sequences of C terminus of RSV G protein were obtained. These sequence were genotyped with genotyping reference sequences. Based on the genotyping results, firstly, there is a outbreak of RSV infection in the2009-2010epidemiological season; secondly, RSV infection outbrek was associated with the co-circulation of several genotype; thirdly, the evolutionary rates of RSV id very fast and RSV can transimit between continent within a epidemiological season.