Separate PCR Research Articles

Diagnostic In Vivo Sensing of COVID-19 Antibody Detection Using DNA-Linking Graphene Oxide Synthetic Mimic Skin Tattoo Probes

COVID-19 antibody detection is dependent on highly specialized, time-consuming techniques, such as PCR separation, DNA amplification, and other methods such as spectrophotometric absorption. For these reasons, specialized technical training is necessary because individual diagnostic treatment is difficult. We have attempted to perform rapid sensing with a detection time of only 30 s. Additionally, we used a wearable multi-layer graphene oxide nanocolloid synthetic skin tattoo probe assay for influenza and COVID-19 virus detection with an electrochemical antigen–antibody redox ionic titration circuit. Cyclic voltametric−2 V~2.0 V potential windows were used. The diagnostic detection limit was determined using stripping anodic and cathodic amplifiers, and the working probe was fabricated with a graphene molecule structure with a virus antigen-immobilized amplifier. With redox potential strength obtained within −1.0 V~−1.3 V ionic activity, anodic and cathodic current linearly increased in the phosphate-buffered saline 5 mL electrolyte. The results indicate that instant detection was enabled via individual and wearable tattoo sensors.

Role of CRISPR-Cas System on Virulence traits and Carbapenem Resistance in Clinical Klebsiella pneumoniae Isolates

Background and Objective(s)The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence. Materials and MethodsA total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR. ResultsThe development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW. ConclusionCRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V H ) genes and the λ light chain variable region (V λ ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Genetic diversity and DNA fingerprinting of rice varieties of Manipur using microsatellite markers

Rice variety differentiation was based on morphological descriptors (DUS) before the advent of genomic and proteomic technology. DNA fingerprinting is becoming an important molecular marker approach because of its wide application in varietal protection, classification, and conflict settlement. In the present study, genetic diversity and variability among the 11 rice varieties of Manipur at the molecular level was studied using 42 SSR markers. The genotypic data of the SSR markers revealed four sub-populations. Cluster analysis indicated the close similarity between RC Maniphou 12 and RC Maniphou 15. To create a DNA fingerprinting database for 11 rice varieties, three separate multiplex PCR systems using different combinations of 11 polymorphic SSR markers based on amplicon size were successfully developed . These were able to distinguish the distinctness of all the eleven released rice varieties.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/μL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/μL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/μL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/μL (roughly 200 parasites/μL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/μL (<1 parasite/μL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Characterization of type-specific HPV prevalence in a population of persistent cutaneous warts in Flanders, Belgium

Cutaneous warts are benign skin lesions caused by the human papillomavirus (HPV). Even though they are considered benign, they can have a considerable impact on the quality of life and cause serious illness in certain immunocompromised populations. Studies have shown that the efficacy of wart treatment is dependent on the causative HPV type. Therefore, in this article, we aim to determine the HPV genotype-specific prevalence in cutaneous warts of a Flemish population as part of the Omnivirol-Salycilic acid randomized controlled trial. Swab samples of cutaneous warts (n = 269) were collected during enrollment. The DNA extraction was performed on the automated NucliSENS® easyMAG® system (bioMérieux). The samples were analyzed with two separate in-house PCR assays capable of detecting the most prevalent cutaneous HPV types (i.e. wart-associated HPV qPCR) as well as the most relevant mucosal types (i.e. RIATOL qPCR assay). In total, the type-specific prevalence of 30 distinct HPV genotypes was determined. The beta-globin gene was used as a cellularity control and for viral load quantification. Data concerning wart persistence, previous treatment, wart type, and other relevant wart and patient characteristics was collected through a baseline questionnaire. The study population consisted mostly of persistent warts considering that 98% (n = 263) of the sampled skin lesions were older than six months and 92% (n = 247) had undergone previous treatment. The most prominent wart type was the mosaic verruca plantaris (42%, n = 113). The most prevalent HPV types were cutaneous HPV types 27 (73%, n = 195), 57 (63%, n = 169), and 2 (42%, n = 113). Only 2% (n = 6) of the lesions was HPV negative. The highest median viral loads were observed with HPV27 and 57 (i.e. 6.29E+04 and 7.47E+01 viral copies per cell respectively). The multivariate analysis found significant associations between wart persistence and certain wart types, the number of warts, and HPV genotypes. Based on these findings, persistent warts are more likely to: (1) be verruca vulgaris, verruca plantaris simple or mosaic, (2) to manifest as multiple warts, (3) and to be negative for HPV type 2 or 4. These characteristics can be useful in the clinical setting for future risk stratification when considering treatment triage and management.Trial registration: NCT05862441, 17/05/2023 (retrospectively registered).

B-016 Automated Next Generation Sequencing Fluidic-Based Device for Sample to Library Preparation: A Flexible Solution for Rapid Decentralized Sequencing for Infectious Disease Diagnostics

Abstract Background In addition to the established use of Next Generation Sequencing (NGS), such as drug resistance monitoring in HIV, the recent COVID19 pandemic has shown the crucial need of NGS technologies as tools for identification of clinically relevant variants of the SARS-CoV-2 virus. Typically, in these settings, to be cost-effective most NGS analyses were conducted using larger batch sizes often resulting in long turn-around times of 2–3 weeks the need of trained personnel, and centralized testing facilities. Methods We have developed an easy to use automated fluidic-based benchtop device to bridge a perceived gap in NGS library preparation and provide a solution for decentralized testing that does not require specially trained personnel. With a batch size of 6, smaller hospitals and laboratories can perform NGS library preparation on fresh or archived samples with a fast turn-around-time of two days, facilitating earlier reporting to speed up diagnosis and treatment. The Helaxy NGS fluidic device performs sample nucleic acid extraction and a 2-step PCR in a plastic fluidic card, which is the sole consumable required for the extraction and library prep process.The sample is applied to the fluidic card by manual pipetting followed by loading of the card into the device. Nucleic acids are extracted with a combination of magnetic bead pull-down and centrifugal force in a purification chamber to give an eluate that is spun to a separate PCR chamber that uses the concept of thermal induction and cycling. Barcoding for library preparation follows within the same PCR chamber and the resulting product is transferred into another fluidic card for size selection purification, normalization and pooling to generate a library for input into compatible NGS sequencers, e.g., but not confined to, Illumina instruments. Results In order to demonstrate clinical utility we have shown that the Helaxy NGS fluidic device can successfully extract nucleic acid from at least three sample types: SARS-CoV-2 RNA from nasopharyngeal swabs, HIV RNA from plasma and 16S RNA from stools. Using in-house and commercial assays, we have generated libraries of target amplicons using the fluidic device workflow followed by sequencing with Illumina sequencers iSeq and MiSeq. Sequencing results analysed and reported by the Helaxy NGS Bioinformatics Reporting Pipeline revealed expected variants and mutations in the samples. We correctly identified all the unique mutations such as L211del and G446S in commercially available inactivated Omicron BA1 variant of the SARS-CoV-2 virus as well as characterised drug-resistant mutations such as L90M in the Protease gene and D67N and Y181C in the Reverse Transcriptase gene in commercially available inactivated HIV-1 virus. Conclusion The Helaxy NGS fluidic device can thus effectively provide an easy-to-use automated solution for sample library preparation in decentralized facilities to reduce the turn-around-time from sample collection to result reporting for clinical diagnosis and treatment.

Evaluation of Larger Test Portion Sizes for Escherichia coli Shiga Toxin Producer (STEC) on the Detection by Immunomagnetic Separation and Real-Time PCR in Meat and Vegetables

The aims of this study were (i) to evaluate the performance of the Assurance® GDS method combining immunomagnetic separation and real-time PCR for STEC detection in pooled samples (up to 375g) of vegetables and meat, and (ii) to compare its performances to that of the reference method ISO/TS-13136:2012 (25 g sample size) in artificially contaminated samples. The alternative Assurance® GDS method includes enrichment in proprietary broth at 41.5°C for 10 h followed by primary screening of TOP 7 STEC containing eae, stx genes and O157:H7 markers using MPX-Top 7 (IMS+PCR), secondary screening for serogroup identification using MPX-ID/EHEC-ID (IMS+PCR). And finally, cultural confirmation from same IMS beads on two selective agars is set up. For reference method, the enrichment was performed in BPW at 41.5°C for 18 h. A total of 120 samples of meat and vegetables, including 20 uninoculated and 100 samples spiked with stressed cells (<12 CFU/25 or 375g) of STEC, were analyzed using both methods. Our results showed that the Assurance® GDS method LOD50 ranged from 0.12 CFU/375 to 0.522 CFU/375 g and the LOD95 from 0.518 CFU/375 to 2.257 CFU/375 g. These data are similar to the LOD50 data of the reference method.

Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples following pre-enrichment culture

Molecular methods for diagnosing Lyme neuroborreliosis (LNB) have shown suboptimal diagnostic sensitivities. The objective of this study was to improve the clinical sensitivity of PCR detection of Borrelia burgdorferi sensu lato spirochetes by inoculating cerebrospinal fluid (CSF) from patients suspected of LNB directly into culture medium at the time of lumbar puncture, with this pursuing enrichment of Borrelia spirochetes before PCR analysis. Adult patients with symptoms suggestive of LNB were prospectively enrolled at two hospitals in the Region of Southern Denmark. The CSF-culture samples were incubated for at least eight weeks. During this period, culture sample aliquots were analysed for the presence of Borrelia DNA by separate PCR protocols in two independent clinical laboratories. The included patients were diagnosed with definite (n=12) or possible (n=2) LNB, and non-LNB (n=171) based on clinical and paraclinical findings. Patients in the LNB and the non-LNB group had a median duration from symptom onset to lumbar puncture of 40 days (IQR [23–90] days) and 120 days (IQR [32–365] days), respectively. Pre-enrichment growth of Borrelia spirochetes was accomplished from three patients (21 %) in the LNB group. The positive culture samples were confirmed by both the digital droplet PCR and the real-time PCR methods employed. All CSF samples were PCR negative in the non-LNB group. The results of this study do not support the use of Borrelia-specific PCR as a general routine diagnostic tool in adults. Still, they suggest it may prove of additional value in selected patients with a limited time from symptom onset to sample collection.

Detection of Clostridium perfringens types through genetic profiling and mouse neutralisation test.

The aim of this study was to detect the types of C. perfringens using PCR and mouse neutralisation test (MNT) and find the genetic profile of virulence factors tpeL and netB isolated from animal samples in Iran. For this purpose, 46 C. perfringens isolates were tested using MNT. Detection was also performed using 16S rRNA-PCR and multiplex PCR for alpha, beta, epsilon and iota toxins. For tpeL and netB gene profiling, separate PCR experiments were carried out. MNT detected that all isolates were C. perfringens type A. The same results were obtained in 16SrRNA-PCR and multiplex-PCR. Based on the obtained results, the molecular test was 100% consistent with MNT. Samples were positive for tpeL and netB genes in 52.1% and 60.8% isolates, respectively. Overall, four genetic virulence factor profiles were found in C. perfringens type A isolates.

Diversity and geographic distribution of rickettsial agents identified in brown dog ticks from across the United States

Rhipicephalus sanguineus sensu lato, or brown dog ticks, transmit a variety of pathogens of veterinary and public health importance globally. Pathogens vectored by brown dog ticks and identified in the United States include Babesia vogeli, Ehrlichia canis, and several spotted fever group Rickettsia spp. (SFGR). Due to the challenge of collecting canine blood samples nationwide to screen for exposure to these pathogens, we took an indirect approach and tested brown dog ticks for molecular evidence of infection. Brown dog ticks (616 adults and 65 nymphs) collected from dogs and cats across the nation were tested by separate PCR assays detecting Babesia spp., E. canis, and SFGR. While no Babesia sp. was found, we identified rickettsial agents in 3.5% (24/681; 95% CI 2.4–5.2%) of the ticks. Pathogens and related organisms detected in ticks included E. canis (n = 1), Rickettsia amblyommatis (n = 3), Rickettsia massiliae (n = 11), Rickettsia monacensis (n = 3), Rickettsia montanensis (n = 5), and an undefined Rickettsia species (n = 1). These data demonstrate a wider geographic distribution of R. massiliae than previously known, and to the authors’ knowledge, reports R. monacensis in brown dog ticks for the first time. Due to the close association that brown dog ticks have with domestic dogs and humans, more research is needed to understand the full array of organisms, some of which are zoonotic, potentially transmitted by this widespread tick complex.

Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Evaluation of Microbial Contamination in Cold Dishes and Prevalence of Foodborne Pathogens in Jilin Province

This study evaluated the microbial contamination status of cold dishes consumed by residents of Jilin Province and investigated to determine the incidence of four pathogenic bacteria in cold dishes. A total of 300 samples of cold dishes, including meat, vegetable, and mixed products, were collected from three purchasing places: supermarkets, farmers' markets, and mobile vendors. Viable bacteria were isolated using conventional culture methods. After separation, a quick and easy PCR was used to detect Listeria monocytogenes, Staphylococcus aureus, enterotoxigenic Escherichia coli, and Salmonella. The results showed that the total number of microbial colonies in the vegetable samples exceeded the standard rate of 8% and the total number of microbial colonies in the meat and mixed samples did not exceed the standard. The total microbial colony count exceeded the standard in all three procurement sites, with the highest exceedance of 7.4% in the mobile vendor sites. The detection rates of enterotoxigenic E. coli, S. aureus, L. monocytogenes, and Salmonella, among the four pathogenic bacteria detected in all samples, were 4.3, 3.3, 3.0, and 1.0%, respectively. This study can be used to qualitatively assess the microbiological quality associated with cold dishes. It provides data to support the detection of possible food safety problems.

Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses.

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI).

GJB2-RELATED NON-SYNDROMIC HEARING LOSS VARIANTS’ SPECTRUM AND THEIR FREQUENCY IN TURKISH POPULATION

Objective: Hearing loss (HL) is one of the most prevalent chronic conditions in children and has consequences in speech, language, education, and social functioning which impede the quality of life. Due to the major involvement of the genetic factors in HL, especially non-syndromic HL (NSHL), genetic diagnosis and genetic counseling have a major impact on early management of the affected individuals and their families. Herein, we report the GJB2 gene variants and their frequencies in NSHL cohort at a tertiary health center between 2002-2021 to contribute for the future genetic counseling of Turkish NSHL patients. Materials and Methods: Two exons of the GJB2 gene were amplified in 402 NSHL patients by two separate PCR reactions and sequenced using the Sanger technique. Results: We found 13 different GJB2 variants in 35% (141/402) of the patients with NSHL. 53.9% were homozygous and 33.3% were compound heterozygous for the most common (59.21%) variant, c.35delG. Approximately 13% of the patients were found to carry the variants in the heterozygous state. The most frequent GJB2 variant c.35delG was followed by c.71G&gt;A (6.38%), c.-23+1G&gt;A (3.54%) and c.233delG (2.48%). We found heterozygous p.Asp50Glu (c.150C&gt;A) alteration in four of eight patients with keratitis, ichthyosis, deafness (KID) and palmoplantar keratoderma (PPK) syndrome. Conclusion: Our results further emphasize the well-known prevalance of the GJB2 c.35delG alteration being the most predominant variant in the Turkish NSHL patients. The high rate of mono-allelic state could be considered as coincidental due to high allelic heterogeneity of NSHL, or possibly suggestive for digenic inheritance.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in meat using multiplex immunomagnetic separation and multiplex real-time PCR

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in meat using multiplex immunomagnetic separation and multiplex real-time PCR

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

High-accuracy quantitative principle of a new compact digital PCR equipment: Lab On An Array.

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

A one step nested PCR method for detection of Peronospora sparsa, the downy mildew pathogen, in boysenberry (Rubus ursinus)

Downy mildew is a major disease of boysenberries in New Zealand, caused by Peronospora sparsa. Most boysenberry plant material, including tissue culture propagated plants are systemically infected and this pathogen also presents as a latent infection. The current nested PCR method to detect latent infection of P. sparsa in asymptomatic boysenberry plants is time consuming as it employs two separate PCR, with potential contamination producing false positive and/or false negative results. To overcome these issues a one step nested PCR method was developed. The method was optimised for primer concentrations, PCR cycle number and DNA concentration. DNA was extracted using a CTAB method. The one step nested PCR method could detect latent infection of P. sparsa in both dormant and active plant growth at 0.4 pg genomic DNA. The most reliable detection was achieved from crown or root tissues. For surety of the infection status, replicate plant tissues should be assessed by PCR as inconsistency between the one step nested PCR and fluorescence microscopy indicated that P. sparsa colonisation is discontinuous through the plant. This method can be recommended for screening P. sparsa latent infection in boysenberry mother plants and daughter plants in nurseries, due to high sensitivity, improved throughput, low cost, and low contamination risk.

Characterization of the Immunoglobulin Heavy- and Light-Chain Repertoires in a Single Reaction

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a &amp;gt;90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.