NextGenPCR® Publications

NextGenPCR™ Publication Highlights

Last updated: 04 Feb 2025.

This document contains an overview of peer-reviewed publications, studies, and laboratory protocols that feature the use of NextGenPCR™ technology in various life science fields.

Original Articles:

Brons et al., 2020. Fast identification of Escherichia coli in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler.

Abstract

Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, but their applicability is complicated by high genomic variability within UPEC strains. Here, we describe a novel PCR-based method for the identification of E. coli in urine. Based on in silico screening of UPEC genomes, we selected three UPEC-specific genes predicted to be involved in pathogenesis (c3509, c3686 (yrbH) and chuA), and one E. coli-specific marker gene (uidA). We validated the method on 128 clinical (UTI) strains. Despite differential occurrences of these genes in uropathogenic E. coli, the method, when using multi-gene combinations, specifically detected the target organism across all samples. The lower detection limit, assessed with model UPEC strains, was approximately 10⁴ CFU/ml. Additionally, the use of this method in a novel ultrafast PCR thermal cycler (NextGenPCR) allowed a detection time from urine sampling to identification of only 52 min. This is the first study that uses such defined sets of marker genes for the detection of E. coli in UTIs. In addition, we are the first to demonstrate the potential of the NextGen thermal cycler. Our E. coli identification method has the potential to be a rapid, reliable and inexpensive alternative for traditional methods.

Source: PubMed: Read full article

Tutu-van Furth et al., 2022. Paediatric monkeypox patient with unknown source of infection, the Netherlands, June 2022. Euro Surveill 27(29):2200552.

Abstract

While repeated community outbreaks of monkeypox (MPX) have been reported in African countries and the United States, these were mainly caused by spillover events from animals to humans [1,2]. Many of these cases also involved children, with a case fatality rate (CFR) between 3.6% and 10.6% depending upon the MPX clade [3,4]. The current global outbreak of MPX does not appear to have a clear link to Africa and distinguishes itself from previous reported outbreaks in that there is more sustained transmission within the community of men who have sex with men (MSM) [5]. The infection of children is very rare and warrants further investigation.

Source: PubMed: Read full article

Struijk et al., 2023. Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach. Diagn Microbiol Infect Dis 107(1):115975.

Abstract

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/μL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

Source: PubMed: Read full article

Čurová et al., 2023. Detection of SARS-CoV-2 using a laboratory-developed ultra-fast NextGenPCR™ test versus a conventional RT-PCR test. Acta Virol 67

Abstract

The reverse transcription polymerase chain reaction (RT-PCR) is considered the gold standard method for the detection of viruses in a clinic. The aim of this study was to compare the ability of conventional RT-PCR test (FTDTM SARS-CoV-2 Test) and laboratory-developed ultra-fast PCR test (NextGenPCR™ TM SARS-CoV-2 RT-PCR Reagent Kit) to detect the coronavirus SARS-CoV-2 causing COVID-19. A total of 318 nasopharyngeal swab specimens were collected from people under investigation for COVID-19. Despite the collection of two swab specimens from each patient and their different processing, the analysis showed an overall agreement of 95.9% between the conventional and laboratory-developed tests. The positive percentage agreement was 90.5% (114/126) and the negative percentage agreement was 99.5% (191/192). The ultra-fast NextGenPCR™ method does not require the isolation of RNA, provides a result of 20–96 specimens within 57–82 min after sampling, and offers a simple procedure of sample processing, analysis, and evaluation. Our results indicate that this method can be considered a potential diagnostic method for the detection of SARS-CoV-2 virus in hospitals, healthcare facilities, and research laboratories.

Source: PubMed: Read full article

Gonzalez et al., 2024. Multiple introductions of monkeypox virus to Ireland during the international mpox outbreak, May 2022 to October 2023. Euro Surveill 29(16):2300505

Abstract

Monkeypox virus (MPXV) is a double-stranded DNA virus in the genus Orthopoxvirus within the Poxviridae family [1], causing the disease mpox. The MPXV genome is ca 197.2 kb encoding a predicted 191 proteins and is genetically closely related to the variola (smallpox) virus. Despite its name, the natural reservoir of the virus remains unknown, but it is transmitted among small mammals, such as rodents [2]. Two major clades of MPXV have been recognised as circulating endemically in Africa, one in Central Africa (Clade I) and the other one in West Africa (Clade II) with differing transmissibility and case fatality rates [3]. In May 2022, an increased number of mpox cases started appearing internationally [1,3], which were characterised as part of the West African Clade II. Based on viral phylogenetic properties and epidemiological evidence of increased human-to-human transmission outside of endemic geographic locations, MPXV causing the 2022 outbreak were classified within a distinct subclade, termed IIb [4]. The outbreak has been reported across multiple countries on different continents and differed from earlier outbreaks of Clade I and IIa in terms of patient age (54.3% of individuals in their 30s) and predominantly affecting men who have sex with men (MSM)

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Madadelahi et al., 2024. A roadmap to high-speed polymerase chain reaction (PCR): COVID-19 as a technology accelerator. Biosens Bioelectron 15:246:115830

Abstract

The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reactions (PCR), have all improved because of the financial and governmental support for the epidemic. The most notable improvement in overall efficiency among them has been seen with PCR. Its significance for human health increased during the COVID-19 pandemic, when it emerged as the commonly used approach for identifying the virus. However, because of problems with speed, complexity, and expense, PCR deployment in point-of-care settings continues to be difficult. Microfluidic platforms offer a promising solution by enabling the development of smaller, more affordable, and faster PCR systems. In this review, we delve into the engineering challenges associated with the advancement of high-speed microfluidic PCR equipment. We introduce criteria that facilitate the evaluation and comparison of factors such as speed, LOD, cycling efficiency, and multiplexing capacity, considering sample volume, fluidics, PCR reactor geometry and materials, as well as heating/cooling methods. We also provide a comprehensive list of commercially available PCR devices and conclude with projections and a discussion regarding the current obstacles that need to be addressed in order to progress further in this field.

Source: PubMed: Read full article

Schuele et al., 2024. Circulation, viral diversity and genomic rearrangement in mpox virus in the Netherlands during the 2022 outbreak and beyond. J Med Virol 96(1):e29397

Abstract

Mpox is an emerging zoonotic disease which has now spread to over 113 countries as of August 2023, with over 89,500 confirmed human cases. The Netherlands had one of the highest incidence rates in Europe during the peak of the outbreak. In this study, we generated 158 near-complete mpox virus (MPXV) genomes (12.4% of nationwide cases) that were collected throughout the Netherlands from the start of the outbreak in May 2022 to August 2023 to track viral evolution and investigate outbreak dynamics. We detected 14 different viral lineages, suggesting multiple introductions followed by rapid initial spread within the country. The estimated evolutionary rate was relatively high compared to previously described in orthopoxvirus literature, with an estimated 11.58 mutations per year. Genomic rearrangement events occurred at a rate of 0.63% and featured a large deletion event. In addition, based on phylogenetics, we identified multiple potential transmission clusters which could be supported by direct source- and contact tracing data. This led to the identification of at least two main transmission locations at the beginning of the outbreak. We conclude that whole genome sequencing of MPXV is essential to enhance our understanding of outbreak dynamics and evolution of a relatively understudied and emerging zoonotic pathogen.

Source: PubMed: Read full article

Vasilita et al., 2024. Express barcoding with NextGenPCR™ and MinION for species-level sorting of ecological samples. Mol Ecol Resour 24(3):e13922

Abstract

The use of DNA barcoding is well established for specimen identification and large-scale biodiversity discovery, but remains underutilized for time-sensitive applications such as rapid species discovery in field stations, identifying pests, citizen science projects, and authenticating food. The main reason is that existing express barcoding workflows are either too expensive or can only be used in very well-equipped laboratories by highly-trained staff. We here show an alternative workflow combining rapid DNA extraction with HotSHOT, amplicon production with NextGenPCR™ thermocyclers, and sequencing with low-cost MinION sequencers. We demonstrate the power of the approach by generating 250 barcodes for 285 specimens within 6 h including specimen identification through BLAST. The workflow required only the following major equipment that easily fits onto a lab bench: Thermocycler, NextGenPCR™, microplate sealer, Qubit, and MinION. Based on our results, we argue that simplified barcoding workflows for species-level sorting are now faster, more accurate, and sufficiently cost-effective to replace traditional morpho-species sorting in many projects.

Source: PubMed: Read full article

Treutiger et al., 2024. First case of mpox with monkeypox virus clade Ib outside Africa in a returning traveller, Sweden, August 2024: public health measures. Euro Surveill 29(48):2400740

Abstract

On 14 August 2024, the World Health Organization (WHO) declared a public health emergency of international concern (PHEIC) in response to the current monkeypox virus (MPXV) clade I outbreak in Africa [1]. Here, we report the first mpox case of clade Ib outside of Africa in a Swedish traveller returning in August 2024 from an area in Central Africa affected by the current clade I outbreak.

Source: PubMed: Read full article

Laboratory Protocols:

van den Ouden et al., 2023. 30 or 50 minutes NextGenPCR™ tiling for SARS-CoV-2 Nanopore sequencing (“Midnight primer set”)

Abstract

Source: Protocols.io: Read full article

Welkers et al., 2022. Monkeypox virus whole genome sequencing using combination of NextGenPCR™ and Oxford Nanopore

Abstract

Rapid genomic surveillance of monkeypox virus (MPXV) can provide valuable insights in order to guide public health interventions. Current sequencing protocols make use of direct Oxford Nanopore Sequencing. However, the obtained depth is a limiting factor which prevents multiplexing samples on a flowcell making sequencing very costly. Here, we provide the protocol for a PCR-based amplicon tiling approach (inspired by SARS-CoV-2 Midnight Protocol by Nikki Freed et. al. and the ARTIC network) for MPXV consisting of a total of 88 primer sets divided over 2 amplicon pools. The amplicon size is ~2,5kB. Our approach will increase the coverage (depth) significantly and allow for multiplexing up to 20 samples on a single Nanopore flowcell. In our experience clinical samples can be successfully sequenced with CT-values <25. Homopolymer regions will remain an issue in our approach, requiring manual curation of obtained consensus sequence genomes. Source: Protocols.io: Read full article