| Listeria monocytogenes causes listeriosis in human and animal. The signs and symptoms of listeriosis include weakened immune systems, immunodeficiency, meningitisis, septicemia, miscarriage or premature labor, abscess to necrosis of visceralorgan. As a food-borne pathogen, the Listeria can cause as high as 30% to 50% death rate and has become one of the most deadly food-borne pathogens. Dengue virus and West Nile virus cause dengue fever and west nile fever, respectively. Symptoms may include fever, petechia or ecchymosis of skin and mucosa, damage and bleeding of organs, syndrome characterized by hypotension and shock. Both Dengue virus and West Nile virus are transmitted by mosquitoes, therefore called mosquito-borne virus. Infections caused by these viruses could be fatal.With the increase of fast food and raw food consumption, food safety incidents due to Listeria monocytogenes infection have happened more often in USA, Europe and other regions. The spread of these arboviruses, especially with Dengue virus and West Nile virus, have brought extensive attentions worldwide. Human and animal diseases caused by the three pathogens have become global public health issues; therefore strengthening rapid detection of these pathogens has become more important. Establishing early, rapid and accurate detection methods specific to Listeria monocytogenes, Dengue virus and West Nile virus not only plays a key role in controling diseases in a timely manner and raising the survival rate, but also is important for the port health quarantine organizations to monitor pollution sources, screen and quarantine of patients, preventing food contaminations or the epidemic situations from spreading into domestic.In recent years, with the rapid development of nanotechnology, immunolabelling technique and lateral flow technology, a comprehensive technology combined with nano-superparamagnetic technology, immuno-chromatographic technology and magnetic quantitative detection technique has appeared. This superparamagnetic chromatographic immunoassay technology is attracting wide attentions in the world with characteristics of high sensitivity, quantitativity, repeatability and wide linearity range. In this study we have developed a superparamagnetic chromatographic immunoassay for Listeria monocytogenes and a superparamagnetic chromatographic combined immunoassay for Dengue virus and West Nile virus simultaneously.In this study, we first established superparamagnetic chromatographic immunoassay for Listeria monocytogenes. After labeling and coating twelve Listeria monocytogenes antibodies, we screened out one pair, which can be used to detect Listeria monocytogenes. By comparing to the standard curve, we have determined the sensitivity of superparamagnetic chromatographic immunoassay for Listeria monocytogenes was about 104CFU/m L and the linearity range was from 104CFU/m L to 108CFU/m L. Using specificity detection we found that even though there were some weak cross-reaction effects for the detection of non-pathogenic Listeria spp.(L. ivanovii and L. seeligeri), superparamagnetic chromatographic immunoassay still possessed high specificity. Further evaluation with seventy three food samples indicated that the superparamagnetic chromatographic immunoassay showed 100% concordance with real-time PCR.We also developed a superparamagnetic chromatographic combined immunoassay for Dengue virus and West Nile virus. After labeling eight Dengue virus antibodies and four West Nile virus antibodies, we have selected one pair of antibodies, which can be used to detect Dengue virus and West Nile virus, respectively. We also determined the sensitivity of superparamagnetic chromatographic combined immunoassay for Dengue virus was 10ng/m L and the linearity range was from 10ng/m L to 10000ng/m L by comparing to the standard curve. The sensitivity of superparamagnetic chromatographic combined immunoassay for West Nile virus was 100ng/m L and the linearity range was from 100ng/m L to 10000ng/m L. Specificity detection of Dengue virus and West Nnile virus using combined detection card indicated that there was partial cross reaction between dengue virus and west nile virus, however, no cross reaction while detecting of yellow fever virus and Japanese encephalitis. In detection of 100 serum samples, including 25 Dengue virus positive samples and 75 negative samples, we found that the accuracy and false positive rate of superparamagnetic chromatographic combined immunoassay was 80.0% and 29.3% for Dengue virus, and false positive rate was 89.9% for West Nile virus.In the study, superparamagnetic chromatographic immunoassay for Listeria monocytogenes, Dengue virus and West Nile virus was successfully established. The sensitivity and linearity range of this method were more accurate than immunochromatography and the specificity for Listeria monocytogeneswas was equal to the PCR results. The Superparamagnetic chromatographic immunoassay is simple to operate, more reliable and fast to detect. In the study, one pair of antibodies for Listeria monocytogenes and dengue virus, respectively, were successfully screened and can be used as the reserve antibodies for diagnostic reagents research. Superparamagnetic chromatographic immunoassay for Listeria monocytogenes had been put into patent application, which will be beneficial to further popularization and application of this technology in the departments of disease prevention departments, port inspection and quarantine institutions, hospitals and other departments, improving the ability to detect in initial screening and on site testing of these pathogens. |
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