| Background:Acute lower respiratory infection(ALRI)is the leading cause of global child mortality as well as the frequent cause of hospitalization in pediatric care.Respiratory syncytial virus(RSV)is believed to be the most common viral pathogen causing ALRI in young children.While children with RSV infection tend to experience mild cold-like symptoms,less robust infants-preterm or those with congenital heart disease-may face severe illness and need to be hospitalized,the outcomes of illness can be fatal.In adults,RSV generally causes mild upper respiratory tract symptoms.However,RSV infection in susceptible adult populations including the elderly and the immunocompromised will induce severe respiratory tract illness such as asthma,bronchiolitis and pneumonia.Therefore,RSV infection will result in a substantial medical and economic burden on society.An accurate and timely identification of etiology is of great importance for early diagnosis and treatment and the detection of pathogen is a prerequisite for the exact diagnosis of the disease.Recombinase aided amplification(RAA)is a novel isothermal amplification and detection assay with China owned intellectual property rights,which utilizes specific enzymes and protein to rapidly amplify target sequences at 37-42℃ in less than 30 min,acquires the advantages of more rapidity,easier operation and cost-effectiveness and overcomes the drawbacks of existing molecular detection assays.RAA has been successfully integrated with different detection strategies,from real-time fluorescent detection to end-point lateral flow dipstick(LFD)amongst others,which will have the great potential to be used in local health care centers and in field or resource-limited clinical settings.Objective:In the present study,we aim to develop two sensitive and specific methods based on RAA technique for rapid detection of RSV,of which one is a real-time RT-RAA assay and the other is a RT-RAA-LFD assay.The sensitivity and the specificity of the two novel assays were examined respectively.The clinical performances of these assays were evaluated and compared with a published RT-qPCR assay using clinical samples.Methods:1.Development of real time RT-RAA for RSV detection:(1)Definition of target gene of RSVA and RSVB,respectively.(2)Design and synthesis of specific primers and probes according to the most conserved genes in the RSVA and RSVB genomes.(3)Screening of optimal primer and probe combinations.(4)Construction of standard recombinant plasmids and evaluation of analytical sensitivity and specificity of real time RT-RAA assay for the detection of RSVA and RSVB,respectively.(5)The clinical performance of the real time RT-RAA assay for the detection of RSVA and RSVB was evaluated using a total of 306 clinical specimens and a published RT-qPCR assay for the detection of RSVA and RSVB was chosen as a parallel test as well as the reference method.2.Development of RT-RAA-LFD for RSV detection:(1)Design and synthesis of specific primers and probe according to the design principles of RAA-LFD assay.(2)Construction of standard recombinant plasmids and evaluation of analytical sensitivity and specificity of RT-RAA-LFD assay for the detection of RSVA and RSVB,respectively.(3)The clinical performance of the RT-RAA-LFD assay for the detection of RSVA and RSVB was evaluated using a total of 306 clinical specimens and the test results were compared with those obtained by RT-qPCR.Results:In this study,a rapid real time RT-RAA assay was developed to detect RSVA and RSVB,respectively.The reaction was performed at 39℃ in less than 30 min.The analytical sensitivities of RSVA and RSVB RT-RAA assay at 95%probability by probit regression analysis were 38 copies per reaction and 35 copies per reaction,respectively,and no cross reaction with other related respiratory viruses were observed.The RT-RAA assay was further utilized to detect 306 clinical specimens and the results showed that 79(25.82%,79/306)samples were positive for RSV,of those 16(20.25%,16/79)were identified as RSVA and 63(79.75%,63/79)were RSVB,which is completely consistent with the results obtained by RSV RT-qPCR assay.In conclusion,the developed RAA assay will be of benefit as a faster,sensitive and specific alternative tool for detection of RSV.A rapid RT-RAA-LFD assay was developed to detect RSVA and RSVB,respectively.The whole time of the detection was 35 min including 30 min for amplification and 5 min for LFD detection.The analytical sensitivities of RSVA and RSVB RT-RAA-LFD assay were 10 copies per reaction respectively,and no cross reaction with other related respiratory viruses were observed.The RT-RAA-LFD assay was further utilized to detect 306 clinical specimens and the result is completely consistent with the results obtained by real time RT-RAA assay and RT-qPCR assay.In conclusion,the developed RT-RAA-LFD assay has the characteristics of high sensitivity,specificity,which provided a new tool for rapid detection of human respiratory syncytial virus.Conclusion:In the present study,we successfully established real time RT-RAA assay and RT-RAA-LFD assay based on the RAA technique for rapid detection of RSVA and RSVB,respectively.The two developed methods have the characteristics of time-saving,accuracy,convenience,high sensitivity and specificity,which provided two novel tools for rapid nucleic acid detection of RSV.In addition,both new assays will have the great potential to be used in field or resource-limited clinical settings,and the improvement and application of RAA technique in this study will overcome the barriers in molecular isothermal diagnostic techniques for the detection of pneumonia pathogens.And more importantly than all of that,rapid and accurate diagnosis of the etiology of ALRI will facilitate early intervention and improve outcome of treatment. |
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