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.

Development of a sensitive and reliable droplet digital PCR assay for the detection of ‘Candidatus Liberibacter asiaticus'

Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

An Automatic Implementation of Oropharyngeal Swab Sampling for Diagnosing Respiratory Infectious Diseases via Soft Robotic End-Effectors

The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction(PCR)assays on patients’respiratory specimens,which are collected through either nasal or oropharyngeal swabs.The manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper sampling.In this paper,we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab sampling.The soft end-effector utilizes circumferential instability to ensure grasping stability,and exhibits several key properties,including high load-to-weight ratio,error tolerance,and variable swab-tip stiffness,leading to successful automatic robotic oropharyngeal swab sampling,from loosening and tightening the transport medium tube cap,holding the swab,and conducting sampling,to snapping off the swab tail and sterilizing itself.Using an industrial collaborative robotic arm,we integrated the soft end-effector,force sensor,camera,lights,and remote-control stick,and developed a robotic oropharyngeal swab sampling system.Using this swab sampling system,we conducted oropharyngeal swab-sampling tests on 20 volunteers.Our Digital PCR assay results(RNase P RNA gene absolute copy numbers for the samples)revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease diagnosis.In summary,we have developed a pharyngeal swab-sampling system based on an“enveloping”soft actuator,studied the sampling process,and imple-mented whole-process robotic oropharyngeal swab-sampling.

Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

Sphere-forming corneal cells repopulate dystrophic keratoconic stroma:Implications for potential therapy

BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.AIM To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.METHODS Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells.These spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in vitro.Tissue sections were used to maximise use of limited keratoconic tissue available for research.Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from implantation.Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.RESULTS Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue sections.No observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14.Spheres stained positively with Calcein-AM up to D14.Cell migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue edges.Cell proliferation marker,EdU,was detected at day 10.Implanted spheres stained positively for putative stem cell markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere detectable by droplet digital PCR up to D14.Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin(myofibroblast marker)in some migrated cells.Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers,indicating an appropriate response to repopulating diseased tissue.CONCLUSION Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.

The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation

Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain.The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses(herpes simplex virus 1,herpes simplex virus 2,varicella zoster virus,human cytomegalovirus,human herpesvirus 6,and human herpesvirus 7).From May 2019 to September 2020,64 serum samples of children following HSCT were collected.EBV infection and the viral load of serum samples were detected by cdPCR.The epidemiological characteristics of EBV infections were analyzed in HSCT patients.Results The limit of detection of EBV cdPCR was 110 copies/mL,and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid.The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain,and both were more sensitive than that of quantitative PCR.Using cdPCR,the incidence of EBV infection was 18.75%in 64 children after HSCT.The minimum EBV viral load was 140 copies/mL,and the maximum viral load was 3,209 copies/mL using cdPCR.The average hospital stay of children with EBV infection(184±91 days)was longer than that of children without EBV infection(125±79 days),P=0.026.Conclusion EBV cdPCR had good sensitivity and specificity.The incidence of EBV infection was 18.75%in 64 children after HSCT from May 2019 to September 2020.EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.

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.