| Infectious disease is a great threat for human health and represent important killer factor of human being. Over 1/4 of the death each year resulted from infectious diseases. In recent years, the increasing emergence of new infectious diseases have brought great global disaster and panic. This makes it a great challenge for clinical diagnosis and treatment. Traditional diagnosis methods include etiological detection,immulogical assay and molecular detection. During outbreaks, due to the limitation of long processing time, requirement of skilled staff, low sensitivity and positivity, and high percent of false negatives, pathogen isolation is not appropriate for disease monitor and point-of-care diagnosis. Immunological methods are based on interaction between antigen and antibody, and the most popular assays include ELISA and immune-strip. However, these assays are restricted by the occurrence of antibody and cross-reactions, which results in false positives or false negatives. Molecular detection technologies, such as PCR, real time PCR, require denaturation, anneling and elongation, and are limited for their applications at remote area and on site detection.Therefore, development of new detection technology of rapid, convenient, sensitive is important for diagnosis, treatment and outbreak control of infectious diseases.Recombinase polymerase amplification(RPA) is a new technology based on recombinase and polymerase enzymes. With this technology, amplification of nucleic acid can be finished in 20 minutes at 25-42 centigrade with high sensitivity,specificity and convenience. In this study, by using Brucella, Adenovirus and Ebola virus as representatives of bacteria, DNA virus and RNA virus, we developed RPA assays for the 3 pathogens, making it possible for rapid point-of-care diagnosis of these diseases.1. Development and application of RPA for BrucellaIn this study, a new RPA assay was developed for Brucella and evaluated by detection of clinical samples. The procedures include: definition of target gene;designment and synthesis of primers and probe; screening of optimal primer combinations; construction of positive plasmid and sensitivity determination;specificity test; and evaluation with clinical samples.The results showed that the best primer combination is Bru-RPA-F1 and Bru-RPA-R3, with threshold time of 4 minutes and fluorescence intensity of 666.5Int.The sensitivity of Bru-RPA was 3 copies/reaction. Five samples positive by real time PCR were also positive by Bru-RPA. DNA sequencing of the products confirmed that the amplification is specific and correct.2. Development of RPA assay for adenovirusAfter systematic alignment of genome sequences, highly concerved region was selected as target sequence. Primers and probes were designed based the signature sequence, optimal primer combination was screened and RPA assay developed.Primer combination of Adv-RPA-F1 and Adv-RPA-R4 generated best performance in terms of threshold time of 2 minutes. The assay showed sensitivity of at least 10copies/reaction, and hight specificity.3. Development and application of RPA assay for Ebola virusBased on genome sequence of 2014 outbreak strain, N protein gene was selected as the signature sequence and primer and probe was designed. Positive plasmid was constructed from a synthesized fragment. Primer combination was screened and the assay was developed. A simplied sample treatment procedure was developed to reduce time for RPA assay. The results showed that primer combination of F2 and R2 generated the best performance, which gave a sensitivity of 10 copies/reaction and high specificity. In the evaluation, a total 375 samples were detected. With RT-PCR as the reference, the general sensitivity was 97%(95% CI: 95.5–99.3), and specificity of 97%(95% CI: 93.9-100). The positive predictive value(PPV), negative predictive value(NPV), positive likelihood ratio(PLR), and negative likelihood ratio(NLR)were 99%, 94%, 33.8, and 0.03, respectively. The sensitivity values for samples witha Ct value of <34, which accounted for 95.59% of the samples, was 100%. Discordant samples positive by RT-PCR but negative by EBOV-RPA had significantly high Ct values. Results of external quality assessment samples with EBOV-RPA were 100%,consistent with those of RT-PCR.In summary, we developed a RPA assay for simple, sensitive and reliable detection of Brucella, adenovirus, and ebola virus, representatives of bacteria, DNA virus, and RNA virus. The results showed that, key factors of RPA assays vary with the pathogen, which define the sensitivity of RPA. With the above studies, we identified important factors of RPA for different types of pathogens. This was helpful for future development of RPA assays for infectious diseases. The evaluation showed that, sensitivity and reliability of RPA is comparable to that of RT-PCR, but the time is greatly shortened. These characteristics of RPA assay make it appropriate for point-of-care diagnosis of infectious diseases. |
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