Development Of Quantum Dot Nuclear Acid Lateral Flow Strips For Detection Of Nosema Bombycis

Microsporidia are obligate intracellular and unicellular eukaryotic microorganism.More than 1400 species of microsporidia have been identified,and they could infect both vertebrates and invertebrates.Nosema bombycis was the first-identified microsporidia and the pathogen of silkworm pebrine disease.Because of vertical transmission and horizontal transmission of Nosema bombycis,pébrine lead to a huge economic loss in silkworm-feeding countries annually.Therefore,N.bombycis is the only inspection and quarantine object in sericulture.Silkworm industries still rely on the microscopic examination of mother moths to control and prevent the disease of pébrine.Yet it is a time-consuming,indirect and error-prone detection method.A rapid,direct,accurate detection method of Nosema bombycis is required in sericulture.To solve the problem,our group established a nucleic acid lateral flow strips(NALFS)detecting method of N.bombycis,which has a higher sensitivity and specificity.In addition,it is time-saving and has a good application prospect.However,some aspects like cost,qualitative analysis and detecting standard limit its application.Here,this study assembled a quantum dot nuclear acid lateral flow strip(QNALFS)based on the FAM monoclonal antibody(mAb)we produced,realized quantitative analysis of QNALFS estimated by qPCR detection,and the method was utilized to detect the egg samples from sericulture industry.Our study laid a foundation of QNALFS for further application in sericulture.1.Production of FAM monoclonal antibody and quantum dot strip9 FAM hybridoma cell lines were obtained,the subtypes of FAM mAb are IgG1-κ.We injected two cell lines into Balb/c mice and got high titers of FAM mAb(1:1600,000).The enriched FAM mAb were used for the assembly of quantum dot strips.After the optimization of various conditions,the prepared test strips had good detection sensitivity and specificity.The sensitivity was up to 102 copies/μL,when took LSU plasmid as templates.In addition,the sensitivity is up to 102 spores/mL when using gradient diluted Nosema bombycis genomic DNA as templates.The QNALFS Test value was 2.28.Therefore,we judge it as negative when the QNALFS Test value is below 2.28.Meanwhile,we detected the genomic DNA of different microsporidia,only Nosema bombycis could be detected.In order to realize large scale preparation of FAM monoclonal antibody and further cut the cost,we cloned the coding gene of FAM monoclonal antibody,introduced a flexible peptide between the heavy chain and light chain,and then constructed the stable expression vector of FAM single-chain fragment variable(scFv)which was transferred to sf9 cell line successfully.Western blot verified the function of the recombinant scFv-FAM that it could recognize FAM-tag.The production of scFv-FAM is benefit for large-scale preparation of FAM antibody,and therefore further decrease the cost of detection.2.Preliminary quantification of quantum dot nuclear acid lateral flow stripsFor quantitative analysis of QNALFS,this study established qPCR detection methods targeted on LSU and PTP2,respectively to calibrate the QNALFS method.NbPTP2 was a double-copy gene,while LSU was a multiple-copy gene.PTP2 qPCR detection method could reflect the quantity of N.bombycis in the samples.Both detection sensitivity was 102 copies/μL when detected the positive plasmid respectively.We used both two qPCR detection methods to detect the N.bombycis,the detection sensitivity of PTP2 qPCR detection system was 103 spores/mL,while the LSU qPCR was up to 101 spores/mL.We noticed that the copies of LSU were more than 10 times bigger than that of PTP2 which was basically consistent with whole genome analysis of N.bombycis,so that we could estimate the content of N.bombycis by copies of LSU.Therefore,gradient diluted plasmids of LSU were used as templates(l.0×102 copies/μL～1.0×109copies/μL)to perform QNALFS detection,a significant exponential relationship between QNALFS and LSU copies was found,the equation is y=0.3457e0.8531x,R2=0.9835.Therefore,the copies of LSU could be inferred from QNALFS detection value,and further estimate the quantity of N.bombycis to realize the preliminary quantification of QNALFS detection.Based on the knowledge we have,the samples with QNALFS detection value<2.28 were judged as negative samples,while it was positive when QNALFS value≥2.28.Samples with QNALFS detection value 2.28～12.84(Nb:102～105 spores/mL)were judged as light infected,samples with QNALFS detection value 12.84～404.67(Nb:105～108 spores/mL)were judged as medium infected,and those with QNALFS detection value>404.67(Nb:>108 spores/mL)were judged as heavy infected.3.Application of quantum dot nuclear acid lateral flow strips156 eggs samples were collected from Guangxi which were detected positive by microscopic examination of mother moths.They were then detected by QNALFS.And the results showed that 9.6%samples were detected negative(QNALFS value<2.28),25%samples were light infected(QNALFS value:2.28～12.84,pathogen load:102～105 spores/ml),43%were medium infected(QNALFS value:12.84～404.67,pathogen load:105～108 spores/ml)and 23.1%were heavy infected(QNALFS value>404.67,pathogen load>108 spores/ml).In addition,QNALFS and qPCR were used to detect the genomes of partial silkworm egg samples.It was found that the sensitivity of QNALFS was lower than that of qPCR,and the detection of low infected samples(Nb:102～103/mL)by QNALFS was not stable,while they were detected accurately and stably by LSU qPCR.In order to explore the qualification standard of infection-tolerance egg samples by QNALFS,we prepared the N.bombycis-infected eggs.These infected eggs with the QNALFS value 12.19 were mixed with normal eggs in incubation and culture process for monitoring the impact which infected eggs bring to the mixed silkworm group.Mixed groups were divided into 3%infected-eggs group and 10%infected-eggs group.The results showed that the survival rate of 3%mixed group and 10%mixed group was 68.75%and 54.25%,respectively,while normal group was over 90%.The average pupation rate of the normal group was over 90%,while that of the 3%group and 10%group was 46.67%and 32.02%,respectively,the pupation rate was significantly reduced.The infection rate of female moth and offspring in 3%mixed group were 55%and 27.27%respectively,while the rate of female moth and offspring in 10%mixed group were higher,reaching 100%and 35%respectively.The results show that when QNALFS value was 12.19,the survival rate of offspring would be seriously affected when 3%and 10%of the infectedeggs are mixed into the normal eggs.The development of QNALFS standard needs more comprehensive research on silkworm egg samples to determine the standard of QNALFS for N.bombycis-tolerant eggs.In summary,self-made quantum dot strip is sensitive,specific and low cost.Through combined analysis with quantitative PCR,the preliminary quantitative detection of QNALFS was realized,which could reflect the amount of N.bombycis infection in silkworm eggs.The self-assembled quantum dot strip has a good application prospect in the N.bombycis detection of eggs,but the corresponding sampling standards and detection standards need more experiments.