Target-specific Primers Research Articles

Detection of the jellyfish Chrysaora pacifica by RPA-CRISPR-Cas12a environmental DNA (eDNA) assay and its evaluation through field validation and comparative eDNA analyses

Climate-driven environmental changes and anthropogenic activities can result in the proliferation of non-indigenous aquatic species such as jellyfish that may cause envenomation and various ecological disruptions. Here we developed a two-step RPA-CRISPR-Cas12a eDNA assay, consisting of target eDNA amplification followed by a CRISPR-Cas12 reaction, for the early detection of Chrysaora pacifica, a jellyfish species often considered non-indigenous to South Korea. The assay demonstrated high sensitivity, with a detection limit of two copies COI/μL for eDNA derived from C. pacifica, using target specific RPA primers and crRNA sequences. Field validation of the assay using eDNA samples from Jinhae Bay collected over eight months of time-series monitoring, revealed temporal distribution of the jellyfish which correlated with results of digital polymerase chain reaction (dPCR) and eDNA metabarcoding. The C. pacifica eDNA assays were also corroborated (R-square 0.7891) by reports from a citizen science–based jellyfish-monitoring program operated by the National Institute of Fisheries Science, South Korea. Our RPA-CRISPR-Cas eDNA assay can therefore, be an efficient alternative to traditional tools for the early detection of outbreaks of non-indigenous or harmful species in marine ecosystems.

Quantitative BCR::ABL1 Assay Using Digital Real-Time PCR System: Clinical Applications and Insights

Background: Accurate quantification of BCR::ABL1 transcripts is paramount for the management of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Despite advancements in real-time reverse transcription PCR (RT-qPCR), the need for more sensitive and precise assays remains. This study evaluated the diagnostic utility of digital reverse transcription PCR (dRT-PCR) for BCR::ABL1 quantification in assessing minimal residual disease (MRD). Material and methods. Twelve patients with CML or Ph+ ALL were included. Peripheral blood or bone marrow samples, collected in PaxGene RNA preservation tubes, were extracted with the automated QIASymphony SP instrument. dRT-PCR conducted on the LOAA analyzer, utilized the Dr. PCR BCR-ABL1 Detection Kit CE_IVD (OPTOLANE Technologies, South Korea). By employing a dual-labeled hydrolysis probe and target-specific primers, this protocol enabled the simultaneous amplification and detection of BCR::ABL1 transcripts e13a2 and e14a2 and ABL1. The resulting copy numbers/reactions were used to calculate the International Scale percentage (%IS) and the molecular reduction (MR) value and analyzed for clinical correlations. Results. Digital RT-PCR demonstrated high precision in quantifying BCR::ABL1 transcripts, the lowest %IS value being 0.02%. A consistent quality RNA sample totalizing 110 ng/reaction was crucial for successful reactions. This assay proved faster and easier than conventional RT-PCR and did not require standards or calibrators. Conclusion. This study supports the feasibility of digital RT-PCR for quantitative analysis. Future objectives for our laboratory include expanding our diagnostic repertoire using this platform, for enhancing patient care.

Effect of target gene sequence evenness and dominance on real-time PCR quantification of artificial sulfate-reducing microbial communities.

Quantitative real-time PCR of phylogenetic and functional marker genes is among the most commonly used techniques to quantify the abundance of microbial taxa in environmental samples. However, in most environmental applications, the approach is a rough assessment of population abundance rather than an exact absolute quantification method because of PCR-based estimation biases caused by multiple factors. Previous studies on these technical issues have focused on primer or template sequence features or PCR reaction conditions. However, how target gene sequence characteristics (e.g., evenness and dominance) in environmental samples affect qPCR quantifications has not been well studied. Here, we compared three primer sets targeting the beta subunit of the dissimilatory sulfite reductase (dsrB) to investigate qPCR quantification performance under different target gene sequence evenness and dominance conditions using artificial gBlock template mixtures designed accordingly. Our results suggested that the qPCR quantification performance of all tested primer sets was determined by the comprehensive effect of the target gene sequence evenness and dominance in environmental samples. Generally, highly degenerate primer sets have equivalent or better qPCR quantification results than a more target-specific primer set. Low template concentration in this study (~105 copies/L) will exaggerate the qPCR quantification results difference among tested primer sets. Improvements to the accuracy and reproducibility of qPCR assays for gene copy number quantification in environmental microbiology and microbial ecology studies should be based on prior knowledge of target gene sequence information acquired by metagenomic analysis or other approaches, careful selection of primer sets, and proper reaction conditions optimization.

Early detection of oral bacteria causing gum infections and dental caries in children.

Periodontal diseases are resulted from gum infections and dental plaques, which are mainly caused by the bacterial agents. Since dental monitoring includes important prognostic roles, the aim of this study was to detect the most common periodontal pathogenic bacteria in children. A total of 200 clinical samples were collected from dental plaques and gingival grooves. Target-specific primers were designed for hbpA in Aggregatibacter actinomycetemcomitans, fimA in Porphyromonas gingivalis and 16S rRNA in Prevotella intermedia, Tannerella forsythia and Treponema denticola. Then, a multiplex polymerase chain reaction method was optimized for the highlighted bacterial agents. In general, the highest and the lowest bacterial prevalence rates belonged to Tannerella forsythia (88%) and Porphyromonas gingivalis (13%), respectively. Furthermore, prevalence rates of Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Treponema denticola were 25, 21 and 45% in samples, respectively. There were significant associations between dental/oral health and microbial community. Metabolism of the oral bacteria, including biofilm formation, can affect gums and develop dental plaques and hence dental caries, especially in children. Early diagnosis of dental caries in children via rapid, accurate molecular methods can increase the diagnostic capacity in clinical cases and therefore prevent periodontal infections in adulthood.

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyses the committed step in prostaglandin synthesis. Various invivo and invitro data suggest that COX-2 mediates the VEGF signalling pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co-expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC). Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT-PCR) using target-specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co-expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes. Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co-expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation. PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.

CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA)

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.

B-248 Rapid PCR Method for Sexually Transmitted Infection (STI) Multiplex Testing Based on Novel Microfluidic Chip

Abstract Background qPCR is being widely used for STI (Sexually Transmitted Infection) testing as the most sensitive and accurate diagnostic method. Genesystem developed a novel qPCR-based diagnostic platform, called Genoplex chip, which enables the simultaneous detection of multiple sexually transmitted pathogens with a single test. Genoplex chip realized high multiplex detection (up to 14 pathogens by a single pipetting), making it easy and fast to process more targets than conventional qPCR. In this study, Genoplex chip was used for diagnosing sexually transmitted infection(STI) to verify the usefulness in diagnostics. Methods Genoplex chip is a flat-shaped microfluidic chip where target-specific primers and probes are prelabeled free from contamination. This chip has 1 well for negative control and 9 connected wells for target detection. By a single pipetting into the inlet hole, samples are loaded and spread into every well of this microfluidic chip since all the wells are connected to each other. We completed performance validation of this new chip with STI diagnosis. Primers and probes for identifying STI pathogens are prelabeled on the inner surface of the Genoplex chip. We can detect and identify 12 major pathogens of STI, which are Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Neisseria gonorrhoeae (NG), Trichomanas vaginalis (TV), Ureaplasma parvum (UP), Gardnerella vaginalis (GV), Candida albicans (CA), Treponema pallidum (TP), Herpes simplex virus type 1 (HSV1) and Herpes simplex virus type 2 (HSV2) by using these probes. Amplification of the target genes was performed by Genechecker UF-300 PCR System. Results We estimated 50 copies of 12 STI pathogens by qPCR process in 40 min. We also confirmed that there was no contamination and no interference in 12 targets. Conclusion With Genoplex chip, various targets can be quantitatively detected in one sample and more accurate results can be provided in efficient way. This novel system gives an opportunity to overcome the limitations of current molecular diagnostics by innovative multiplex detection technology. Therefore, it is expected that the Genoplex chip system will attract attention in the molecular diagnostics market.

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed. It mainly includes a linear reverse transcription step using tailored-designed target specific capture primers, followed by an exponential amplification process using two universal primers to execute the multiplex assay. For proof of concept, four miRNAs were used as models to develop a multiplexed detection assay within one tube simultaneously and then evaluate the performance of the established STEM-Mi-PCR. The sensitivity of the 4-plexed assay was approximately 100 aM with an equivalent amplification efficiency (95.67±8.58%), and had no cross-reactivity each other with high specificity. Quantification of different miRNAs in twenty patients' tissues shown variation from approximately pM to fM concentration level, demonstrating the possibility of practical application of the established method. Moreover, this method was extraordinarily capable of single nucleotide mutation discrimination in different let-7 family members with no more than 0.7% nonspecific detection signal. Hence, the STEM-Mi-PCR we proposed here paves an easy and promising way for miRNA profiling in future clinical applications.

Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

Abstract 3339: Circulating long non-coding RNA HULC as an early indicator of hepatocellular carcinoma irrespective of etiology

Abstract Background: Hepatocellular carcinoma (HCC) is the 6th most common cancer worldwide and is highly prevalent in Asian countries. Long noncoding RNAs (lncRNAs) are non-coding transcripts with a size of more than 200 bases in length. Several studies reported that lncRNAs are highly deregulated in HCC and could be used as biomarkers for early diagnosis and prognostic purposes. In the present study, we measured the circulating lncRNA HULC as an early diagnostic marker for HCC with different etiology. Methods: Serum samples were collected from healthy controls, liver cirrhosis (LC), and HCC patients with different etiology and stored at -80°C until assay. Total RNA was extracted using Qiagen RNeasy kit and tested for RNA quantity and purity using NanoDrop spectrophotometer. The RNA extract that contains lncRNA HULC was transcribed into cDNA using a reverse transcription kit. Employing target specific primers for the lncRNA HULC, qRT-PCR was performed with the synthesized cDNA using Qiagen QuantiFast SYBR Green PCR kit on a Rotor-Gene qRT-PCR system. Results: Quantification of lncRNA HULC using qRT-PCR in the serum samples demonstrated significant increase in LC cases and in all HCC cases irrespective of etiology compared to the normal healthy controls. The highest concentration was observed in serum samples from HCC patients with hepatitis B virus (HBV) infection, which was about 8 fold higher compared to healthy controls. There was no significant difference between the mean HULC lncRNA levels in HCC patients with different etiology such as alcoholic cirrhosis, LC due to other reasons, hepatitis B and hepatitis C virus (HCV) infections, and HCC developed from other unknown causes. However, serum HULC lncRNA levels in LC cases without HCC was significantly lower compared to all HCC samples. Conclusions: The results of the present study demonstrated that there is a significant increase in HULC lncRNA levels in the serum samples of all HCC patients irrespective of etiology. Therefore, the circulating lncRNA HULC could be used as an early diagnostic biomarker for HCC before appearing other clinical symptoms. Citation Format: Joseph George, Mutsumi Tsuchishima, Mikihiro Tsutsumi. Circulating long non-coding RNA HULC as an early indicator of hepatocellular carcinoma irrespective of etiology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3339.

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

BackgroundIllumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing ‘N’ (0–10) spacers in target gene amplification primers that are pooled for simple handling.ResultWe evaluated the efficiency of ‘N’ (0–10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of ‘N’ (0–10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,“MetReTrim”, to trim the ‘N’ (0–10) spacers from the raw reads (https://github.com/Mohak91/MetReTrim). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.ConclusionThis method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using ‘N’ (0–10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

Evaluation of primers for the detection of deadwood-inhabiting archaea via amplicon sequencing.

Archaea have been reported from deadwood of a few different tree species in temperate and boreal forest ecosystems in the past. However, while one of their functions is well linked to methane production any additional contribution to wood decomposition is not understood and underexplored which may be also attributed to lacking investigations on their diversity in this substrate. With this current work, we aim at encouraging further investigations by providing aid in primer choice for DNA metabarcoding using Illumina amplicon sequencing. We tested 16S primer pairs on genomic DNA extracted from woody tissue of four temperate deciduous tree species. Three primer pairs were specific to archaea and one prokaryotic primer pair theoretically amplifies both, bacterial and archaeal DNA. Methanobacteriales and Methanomassiliicoccales have been consistently identified as dominant orders across all datasets but significant variability in ASV richness was observed using different primer combinations. Nitrososphaerales have only been identified when using archaea-specific primer sets. In addition, the most commonly applied primer combination targeting prokaryotes in general yielded the lowest relative proportion of archaeal sequences per sample, which underlines the fact, that using target specific primers unraveled a yet unknown diversity of archaea in deadwood. Hence, archaea seem to be an important group of the deadwood-inhabiting community and further research is needed to explore their role during the decomposition process.

Controlled Rehydration of Dried Reagents for Robust Multiplex Digital PCR.

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).

The features of diagnostics of grain crop seeds intended for export

Russia occupies a leading position among the largest wheat producers and exporters. One of the key requirements for the biological safety of domestic grain products is the phytosanitary requirements of importing countries. The conformity of grain products with regard to phytopathogenic micromycetes is determined by their complete absence or the minimum content in the batch. When shipping exported products, it is necessary to carry out a phytosanitary estimation of a batch of plant products in a short time. In the current paper there has been estimated the effect of the proposed method of sample preparation of grain crop seeds on the quality of an isolated fungal pathogens’ DNA. The purpose of the study was to optimize the methods of sample preparation of grain products for the extraction of nucleic acids with further identification to reduce laboratory trials. The development of modern, reliable and low labor-consuming methods for diagnosing phytopathogens in plant products is of great urgency. In order to conduct the current research, wheat, barley and rye kernels were artificially inoculated with such fungal pathogens as Parastagonospora nodorum, the causative agent of Septoria leaf spot, and Alternaria infectoria, the causative agent of Alternaria leaf spot. The infection was carried out by soaking the kernels in a spore suspension followed by incubation. In order to assess infection, there were used the classical methods of mycology, as well as the patented E.Yu. Toropova’s method. The pathogens were identified by a classical PCR analysis using the target-specific primers. There has been given an assessment of the sensitivity of the proposed extraction method for each of the studied crops. The method allows identifying target objects even with a minimum number of infected kernels in the sample (1–5 infected seeds per 195–199 healthy ones). There were not identified any related pathogens, and there were no false-positive results during the conducted PCR analysis.

Establishment and Application of a Multiplex PCR Assay for the Rapid Detection of Rhizoctonia solani Anastomosis Group (AG)-3PT, the Pathogen Causing Potato Black Scurf and Stem Canker.

Rhizoctonia solani anastomosis group 3 (AG-3) is the main causative agent of the soil-borne disease known as potato black scurf, which poses a huge threat to potato production. Rapid and accurate identification of R. solani AG-3 isolates in soil and potato seed tubers prior to planting is essential for good production. In this study, a multiplex PCR assay was established for the detection of R. solani AG-3. Two pairs of target-specific primers were designed from sequences for endopolygalacturonase and pyridoxine biosynthesis genes downloaded from GenBank. The main factors influencing PCR amplification, such as annealing temperature and primer concentration, were optimized. Results show that the proposed multiplex PCR assay is highly sensitive and specific for the target genes in the pathogen even when the DNA concentration is reduced to 20 fg/μL. The resulting calibration plot shows a linear relationship between electrophoretic band peaks and genomic DNA concentration (R2 = 0.98). The primer specificity was confirmed by applying them to other R. solani AG groups and plant pathogen species on which no amplicons were produced. Using the primers, we successfully detected small amounts of R. solani AG-3 present in soil and potato tuber samples. Taken together, the detection assay developed in this study has high sensitivity, strong specificity, and accuracy and can be used to detect and identify soil and potato seed tubers infected with Rhizoctonia solani AG-3.

Development and application of DNA markers to detect adulteration with Scopolia japonica in the medicinal herb Atractylodes lancea.

The online version contains supplementary material available at 10.1007/s10068-021-01008-5.

Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools.

Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

Potential Clinical Utility of a Targeted Circulating Tumor DNA Assay in Esophageal Adenocarcinoma.

Objective:To explore the clinical utility of circulating tumor DNA (ctDNA) in esophageal adenocarcinoma (EAC) by developing a cost-effective and rapid technique utilising targeted amplicon sequencing.Summary of background data:Emerging evidence suggests that levels of ctDNA in the blood can be used to monitor treatment response and in the detection of disease recurrence in various cancer types. Current staging modalities for EAC such as computerised tomography of the chest/abdomen/pelvis (CT) and positron emission tomography (PET) do not reliably detect occult micro-metastatic disease, the presence of which signifies a poor prognosis. After curative-intent treatment, some patients are still at high risk of recurrent disease, and there is no widely accepted optimal surveillance tool for patients with EAC.Methods:Sixty-two patients with EAC were investigated for the presence of ctDNA using a tumor-informed approach. We designed a custom targeted amplicon sequencing panel of target specific primers covering mutational foci in 9 of the most commonly mutated genes in EAC. Serial blood samples were taken before and after neoadjuvant treatment (NAT), and during surveillance.Results:Somatic mutations were detected in pre-treatment biopsy samples of 55 out of 62 (89%) EAC patients. Mutations in TP53 (80%) were the most common. Out of these 55 patients, 20 (36%) had detectable ctDNA at baseline. The majority (90%) of patients with detectable ctDNA had either locally advanced tumors, nodal involvement or metastatic disease. In patients with locally advanced tumors, disease free survival (DFS) was more accurately stratified using pre-treatment ctDNA status [HR 4.34 (95% CI 0.93–20.21); P = 0.05] compared to nodal status on PET-CT. In an exploratory subgroup analysis, patients who are node negative but ctDNA positive have inferior DFS [HR 11.71 (95% CI 1.16-118.80) P = 0.04]. In blood samples taken before and following NAT, clearance of ctDNA after NAT was associated with a favourable response to treatment. Furthermore, patients who are ctDNA positive during post-treatment surveillance are at high risk of relapse.Conclusions:Our study shows that ctDNA has potential to provide additional prognostication over conventional staging investigation such as CT and PET. It may also have clinical utility in the assessment of response to NAT and as a biomarker for the surveillance of recurrent disease.

Molecular detection and identification of Culex flavivirus in mosquito species from Jeju, Republic of Korea

BackgroundMosquito-borne flaviviruses are prime pathogens and have been a major hazard to humans and animals. They comprise several arthropod-borne viruses, including dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. Culex flavivirus (CxFV) is a member of the insect-specific flavivirus (ISF) group belonging to the genus Flavivirus, which is widely distributed in a variety of mosquito populations.MethodsViral nucleic acid was extracted from adult mosquito pools and subjected to reverse transcriptase nested polymerase chain reaction (PCR) using target-specific primers for detecting CxFV nonstructural protein 5 (NS5). The PCR-positive samples were then sequenced, and a phylogenetic tree was constructed, including reference sequences obtained from GenBank.Results21 pools, belonging to Culex pipiens pallens (Cx. p. pallens) were found to be positive for the CxFV RNA sequence, with a minimum infection rate of 14.5/1000 mosquitoes. The phylogenetic analysis of the NS5 protein sequences indicated that the detected sequences were closely related to strains identified in China, with 95–98% sequence similarities.ConclusionOur findings highlight the presence of CxFV in Cx. p. pallens mosquito species in Jeju province, Republic of Korea. This is the first study reporting the prevalence of CxFV in Culex Pipiens (Cx. pipiens) host in the Jeju province, which can create possible interaction with other flaviviruses causing human and animal diseases. Although, mosquito-borne disease causing viruses were not identified properly, more detailed surveillance and investigation of both the host and viruses are essential to understand the prevalence, evolutionary relationship and genetic characteristic with other species.

Field-Deployable Recombinase Polymerase Amplification Assay for Specific, Sensitive and Rapid Detection of the US Select Agent and Toxigenic Bacterium, Rathayibacter toxicus

Simple SummaryEarly, accurate, and rapid detection of R. toxicus is extremely important to improve inspections of imported annual ryegrass hay and seed at ports of entry and enhance in-field detection. RPA is a comparatively new, easy to use, and robust technology that can be performed in the palm of the hand without losing specificity. The RPA assay was more sensitive than endpoint PCR and did not require lab equipment in the field. The developed assay has tremendous applications for in-field plant diagnostics and biosecurity surveillance.Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.