| The outbreak and epidemic of natural focus infection diseases, such as human Streptococcus suis infectionsetc, anthrax, avian influenza H7N9, hemorrhagic fever, etc,have been a huge threat to human health for the past few years. The development tendency about rapid detection of pathogens of natural foci disease is to detect pathogenic nucleic acids in field as quick as possible.Currently, the sensitivity of the immunological colloidal gold test method is quite low.PCR and real-time PCR assays are highly sensitive and specific assays for pathogen detection, but they require expensive apparatuses, sophisticated techniques, and tedious operations. Further, their use is not suitable for on-site detection in the field or in primitive clinical laboratories, particularly in developing countries. Herefore, the development and evaluation of a new simple, rapid, and cost-effective assay for detecting pathogens are urgently needed. The characteristics of loop-mediated isothermal amplification(LAMP)assay are fast, sensitive, simple operation and with general equipment. Therefore, LAMP has great advantage in field detection for pathogens of natural focus diseases.The purposes of our research is to establish a LAMP platform for pathogen detection of natural focus infection disease and to develop appropriate reagents and assemble the portable box set which can complete the entire process of isothermal amplification and detection. We have been developed the isothermal amplification technique for H7N9 avian influenza, hemorrhagic fever, highly pathogenic streptococcus suis, bacillus anthraci by the LAMP platform. The research contents and results are as follows:1. Two loop-mediated isothermal amplification(LAMP) assays that can rapidly detect S. suis 2 and its high pathogenicity island(89K PAI), and can be performed simultaneously under the same conditions, have been established.2. The same set of serial diluted DNA as templates was used to compare the sensitivities of the different visual detection methods(SYBR Green I, gel electrophoresis,turbidimeter, calcein and HNB) for LAMP product detection.3. A double LAMP assay for rapid detection of bacillus anthracis PA gene(plasmid pX01) and capA gene(pX02 plasmid) on visualization methods, have been established,while also distinguishing virulent strain or attenuated strain of bacillus anthracis. Thelyophilized kit of LAMP assay for detecting bacillus anthracis, which can be stored at room temperature, has been assembled.4. A one-step reverse transcription loop-mediated isothermal amplification(RT-LAMP)method for rapid detection of the hemagglutinin(HA) and neuraminidase(NA) genes of H7N9 virus have been established, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method.5. Two assays for specifically identifying Hantaan virus(HTNV) and Seoul virus(SEOV) which belong to the genus Hantavirus, cause hemorrhagic fever with renal syndrome(HFRS), have been developed.6. A portable field detection box set(visual LAMP test box based on small metal bath temperature), using available vehicle power or battery supply, has been assembled. Even without AC power supply, the box set can complete the whole process of field detection for pathogens of natural focus diseases.The study shows that LAMP is a more suitable choice for pathogens of natural focus diseases in view of its easy sample handling, convenient operation and simple equipment.The LAMP platform established in this study can be added more species about the natural foci of disease pathogens(such as Ebola virus, Marburg virus, dengue virus, Yersinia pestis,Brucella bacteria). Combined with lyophilisation of LAMP recipe and portable detection box set, the LAMP platform can be progressed into a realistic point of care format for resource-poor endemic areas. Considering LAMP’s simplicity in operation and high sensitivity, there is potential use in clinical diagnosis and surveillance of natural focus diseases. |
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