Development Of A Novel Isothermal Multiple-self-matching-initiated Amplification (IMSA) And Its Application On Rapid Detection Of Infectious Pathogens Of EV71, CVA16, H7N9, And HIV-1

Infectious disease in China develops as one of rather serious diseases, such as hand, footand mouth disease (HFMD), acquired immune deficiency syndrome (AIDS), and the newlypopular human-infected influenza A (H7N9). Since no vaccine or antiviral drugs are currentlyavailable, the rapid nucleic acid-based detection of infectious pathogens is critical for preventionand control of infectious diseases. Presently, the recommended platform for nucleic acid-baseddetection in China is polymerase chain reaction (PCR) technology. However, PCR is either time-consuming or dependents on expensive and cumbersome equipment, which is not suitable forthe application to primary level quarantine station or medical institution. In comparison withPCR, the new detection like loop-mediated isothermal amplification (LAMP) has a strongpotential in the application of primary settings, because of simple and rapid detection with highspecificity and sensitivity. Whereas, LAMP method is well protected by patents, which causesthe actual price of existing detection kits relatively high and restricted its real application. In thisregard, a novel isothermal multiple-self-matching-initiated amplification (IMSA) wasdeveloped in the study.To rapidly detect human enterovirus71(EV71) and coxsackievirus A16(CVA16) ofHMFD causative agents, human immunodeficiency virus type1(HIV-1) of AIDS, and H7N9virus, both real-time and visual IMSA assays were established. Firstly, the highly conservedregions of VP1gene for EV71and CVA16, H7and N9gene for H7N9, and gag gene for HIV-1were selected to design the corresponding IMSA primers. After optimizing the reactionconditions, the specificities and sensitivities of IMSA assays were then evaluated with theartificial templates, and the reported LAMP methods were chose as parallel tests. Finally, all theestablished IMSA approaches were applied to detect clinical specimens for further evaluation.The test results indicated that the IMSA approaches possessed high specificities without cross-reactions observed. On detection sensitivity, IMSA methods were not only comparable to theexisting LAMP methods, but also even displayed higher detection limits than the latter, whichwas further confirmed in clinical assessment. Furthermore, in the study, the details of IMSAprinciple were introduced, and the reaction effect assays with different primer combinationswere conducted, indicating that the normal six-primers set was necessary to realize simple, fast,highly specific and sensitive detection. Additionally, the IMSA reaction was indirectly provedthrough enzyme digestion experiments.In conclusion, IMSA technology as a new type of isothermal amplification technology, onthe one hand, breaks application restrictions of the LAMP patent in China; on the other hand, it makes improvement and complementarity for nucleic acid-based amplification in vitro. Due tothe advantages of simplicity, rapidness, high sensitivity and specificity, IMSA approach can beas a new technical idea and choice for the grass-roots level settings, and its application can beenlarged to other fields such as food safety inspection, detection of genetically modifiedingredients and ecological conservation.