Development Of Nucleic Acid Lateral Flow Strips For The Detection Of Nosema Bombycis

Nosema bombycis is the pathogenic fungus of silkworm, causes serious pebrine disease and resulted in the big deaths of the silkworms, which often transmits through horizontal transmission via food and vertical transmission via eggs. As it brings huge economic losses to the sericulture, Nosema bombycis is considered as the biggest threat to silkworm feeding. Thus, Nosema bombycis was the only one pathogen for statutory quarantine in almost all silkworm feeding countries of the world. Currently, Nosema bombycis is inspected through microscopic examination of female moth, which was invented in 1875 by Louis Pasteur. This method is simple and efficient, however the results would be influencedby the experience of the inspector. What’s more, it is very hard to distinguish the Nosema bombycis from some shape-similar subjects, such as the pollen or the spores of Aspergillus spp. Thus, it is definitely an emergency to develop a new detection method for the diagnosis of Nosema bombycis. This research aims to combine the PCR amplification with the immunochromatographic lateral flow strips to develop a fast, accurate and simple method with high sensitivity and low cost. 1. The development of Nucleic acid lateral flow strips for the detection of Nosema bombycisA detection method which exhibited a high sensitivity and specificity was successfully established. The efficient modified primers and the suitable DNA polymerase were screened out while the PCR amplification conditions were optimized. Besides, a user-friendly and less time-consuming DNA extraction method for eggs samples was developed to provide the template for PCR process. Results showed that this nucleic acid lateral flow strips(NALFS) can distinguish N. bombycis from other silkworm pathogens, and the detection limitation is 10 pg genomic DNA of purified spores or 10 spores/100 eggs when use the DNA roughly extracted from the mixture of spores and silkworm eggs. 2. The application of Nucleic acid lateral flow strips for the detection of Nosema bombycisIn this part, the NALFS was used to detect the artificially infected female moths and the eggs they laid. The unqualified silkworm eggs are collected from the sericulture industry and 4 isolates of N. bombycis are isolated from the infected silkworms. First, the artificially infected female moths and relevant eggs were inspected under the microscopes to confirm the infection, then the results of NALFS also showed the positive results to both of them, which demonstrated that the NALFS can be used to judge whether the moths and eggs are infected or not. When the NALFS was used to detect 67 samples of hybrid silkworm eggs collected from the silkworm eggs factory, the positive rate was 77% while microscopy detection of hatched first hybrid silkworm eggs showed a positive rate of 49%. When the NALFS was used to detect 29 samples of foundation seed(Yuanzhong) of silkworm eggs, the positive rate was 31% while the microscopy detection of foundation seeds(Yuanzhong) showed a positive rate of 17%. These results suggested the NALFS is more sensitive than the microscopy detection. In addition, silkworm eggs were detected as positive results through microscopy were totally showed positive when using the strips. Besides, the NALFS was used to detect different isolates of Nosema bombycis to confirm its universality. The results illuminated that it can distinguish three of four isolates isolated from GuangXi province.In order to compare the corelationship between the NALFS and agarose gel electrophoresis(AGE), 18 samples that were proved to be positive by the NALFS were tested by agarose gel electrophoresis again. The results showed the AGE has a same positive rate with the NALFS. When detecting the silkworm eggs products, all results showed NALFS is a stable and more sensitive method compared to the microscopy examination of female moth. The stability and specificity as well as the sensitivity of NALFS are enough for meeting the requirements of N. bombycis inspection.