Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli

Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.

Multiple exosome RNA analysis methods for lung cancer diagnosis through integrated on-chip microfluidic system

Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos(e.g.,proteins,m RNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR(dd PCR).Gradient dilution experiments show great linearity over a large concentration range with R^(2)=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.

Performance of the point-of-care circulating cathodic antigen test in the diagnosis of schistosomiasis japonica in a human cohort from Northern Samar, the Philippines

Background:Zoonotic schistosomiasis,caused by Schistosoma japonicum,remains a major public health problem in the Philippines.This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen(POC-CCA)test in detecting individuals infected with S.japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines.Methods:Clinical samples were collectedin 18 barangays endemic for S.japonicum infection in Laoang and Palapag municipalities,Northern Samar,the Philippines,in 2015.The presence of CCA in flter-concentrated urine samples(n=412)was evaluated using the commercial kits and the results were converted to images,which were further analyzed by ImageJ software to calculate R values.The diagnostic performance of the immunochromatographic POCCCA test was compared using the Kato-Katz(KK)procedure,in-house enzyme-linked immunosorbent assays(ELISAs)and droplet digital(dd)PCR assays as reference.Results:The POC-CCA test was able to detect S.japonicum-infected individuals in the cohort with an eggs per gram of faeces(EPG)more than or equal to 10 with sensitivity/specifcity values of 63.3%/93.3%.However,the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals,of which the majority had an extremely low egg burden(EPG:1–9).The prevalence of S.japonicum infection in the total cohort determined by the POC-CCA test was 12.4%,only half of that determined by the KK method(26.2%).When compared with the ELISAs and ddPCR assays as a reference,the POC-CCA assay was further shown to be a test with low sensitivity.Nevertheless,the assay exhibited signifcant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort.Conclusions:By using in silico image analysis,the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability.Because of its low sensitivity,the commercially available POC-CCA assay had limited potential for determining the status of a S.japonicum infection in the target cohort.The assay should be applied with caution in populations where schistosome parasites(especially S.japonicum)are present at low infection intensity.