Development Of PCR-ELISA For The Detection Of Nosema Bombycis In Silkworm Eggs

Microsporidia are obligate intracellular eukaryotic parasites that are characterized as evolutionarily conserved opportunistic pathogens in hosts ranging from nearly all invertebrates to vertebrates.The microsporidian was firstly described in 1857 when pebrine,or“pepper disease,”was ravaging silkworms in southern Europe and threatening to destroy the European silk industry.The pathogen of pebrine is Nosema bombycis,which can not only infecte silkworm larva,pupa and moth,but also transmit to next generation via the silkworm eggs.Therefore,N.bombycis has been listed as the only statutory quarantine object in the production of silkworm eggs.In general,N.bombycis is commonly detected through microscopic examination of the female moth,which is the statutory detection method for pebrine in the sericulture industry^{[104][104][103][103]}.However,this method is an indirect detection,and the results can be easily influenced by the experience of inspectors,complicacy of sample and pathogen virulence.What's more not all the microsporidia that infect silkworms could be transmitted into the eggs^{[64][64][63][63]}.These human factors and objective reasons would cause that the final detection results could not represent the actual infection of silkworm eggs.At the same time,this method also has some problems such as time-consuming,laborious,subjective judgment.Thus,it is urgent to develop a direct,highly sensitive and labour-saving method for the detection of N.bombycis in silkworm eggs.This research aims to combine the PCR amplification with the enzyme linked immunosorbent assay?ELISA?to develop a fast and accurate,simple and high throughout method with high sensitivity and low cost.1.The development of PCR-ELISA for the detection of N.bombycisIn this study,we successfully established a detection method with the combination of PCR and ELISA,whose target was the nucleotide of pathogen and primers were digoxigenin-and biotin-labeled.PCR-ELISA assay was further set up for detecting N.bombycis in silkworm eggs.First,we used primer LSU 323 to establish the detection system of PCR-ELISA.The optimal concentration of streptavidin?coating buffer?and the optimal dilution of antidigoxigenin antibody were 1?g/mL and 1:1000 respectively.Besides,the optimize cycle numbers of PCR was 30cycels.Collectively,the sensitivity of PCR-ELISA could achieve the positive dectection with 20 fg of N.bombycis DNA,or 10³ spores in 50 silkworm eggs when using primer LUS323.Because primer LUS323 could detect other related species in previous experiments,then the we selected a specie-specific primer M247 based on the genome of the isolate,N.bombycis CQ1.When using M247 primers,the sensitivity was also 10³ spores in 50 silkworm eggs.In this follow-up study,primer LSU323 and M247 were simultaneously used in the application of PCR-ELISA.2.The application of PCR-ELISA for the detection of N.bombycisIn this part,the availability and reliability of PCR-ELISA was analyzed with different samples including artificially infected silkworm eggs,and the product eggs from silkworm eggs plant.We separately collected eggs from 29 female moths that were artificially feeding with N.bombycis,then light microscopy and PCR-ELISA were applied to detect the samples produced.The 29 female moths could be divided into four groups:serious infection,moderate infection,mild infection and no infection by microscopy examination.All silkworm egg samples?50 eggs?could be detected by PCR-ELISA as positive samples from seriously infected female moths and moderately infected female moths,conversely the silkworm egg samples from mildly infected female moths and no infected female mothswere detected as negative.Then we choose a silkworm egg sample from serious infected female moth,and mix these infected eggs with different number of healthy silkworm eggs to mimic the natural infected eggs.Then using PCR-ELISA to detect the mimics,1²infected eggs mixed with healthly eggs?total number is 50 eggs?could be detected as positive by PCR-ELISA and gel electrophoresis using primers M247 and LSU323,the results also indicate that PCR-ELISA is much more sentitive than gel electrophoresis.Then we use the PCR-ELISA to detect98 samples of foundation seed of silkworm eggs?Yuanyuan Zhong?,which all were positive by microscopic examination of female moth,the positive rate was 23.5%while the microscopy detection of newly hatched silkworms showed a positive rate of 18.3%.However in this experiment,PCR-ELISA did not get any positive results when using M247 primers.Thus in order to check the finess of M247 primers,we processed PCR using genomic DNA of isolate?-?of N.bombycis isolated from Guangxi Zhuang Automonous Region as template,then the PCR products were detected by gel electrophoresis.The results showed all isolates could be detected by LSU323primers,while only N.bombycis CQ1 and isolate?were detected with M247 primers.Based on these results,we know that PCR-ELISA can be applied to the detection of N.bombycis in in silkworm eggs.The primer LSU323 can be used in the detection of silkworm eggs in different regions because of its wide spectrum,while the primer M247 may only be used in detection of N.bombycis in silkworm eggs produced in Chongqing.In general,compared with the microscopic examination of female moth,the PCR-ELISA method can be directly used in the detection of silkworm eggs.The results are more objective,independent of the differences in the working environment,and the method can maintain the stability and authenticity of the test results.However,the improvement of sensibility,consistency between PCR-ELISA and corrective inspection of newly hatched larvae are needed in further study.