Target Copies Research Articles

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

BackgroundHuman coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. ObjectivesFor fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study designThe analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. ResultsOur HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. ConclusionsWe were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer’s disease and brain cancers

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer’s disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap® DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.

Droplet digital PCR for the identification of plant-derived adulterants in highly processed products

BackgroundThe high sensitivity of droplet digital PCR (ddPCR) contributes to its excellent performance in animal and microorganism identification, but the utilization of ddPCR is limited in plant adulterant identification of highly processed products for which effective methods are lacking. PurposeThis study investigated the feasibility of ddPCR in the identification of plant adulterants in Chinese patent medicine (CPM) as groundwork to develop ddPCR assays for other highly processed goods. MethodsThe original plant, processed and highly processed products of Mutong (Akebiae Caulis) and its two adulterants were used to analyze the specificity, sensitivity, and practical performance of the developed singleplex and triplex ddPCR assays. ResultsThe results revealed that the limit of detection (LOD) and limit of quantification (LOQ) for the selective ddPCR assays developed to identify Mutong and its adulterants were 0.00002 ng/μl and 0.00016 ng/μl, respectively, and that the regression equations representing the relationships between DNA concentration and target copy number all exhibited good linearity. Furthermore, the common adulterant of Mutong in three samples of Longdan Xiegan pills was successfully identified through ddPCR assays and confirmed by Sanger sequencing. ConclusionThis work comprehensively revealed the great ability of ddPCR technology in detecting plant adulterants in traditional Chinese medicine (TCM), providing a method for the quality control of highly processed plant products with complex components for commonly used goods.

Improving Environmental DNA Sensitivity for Dreissenid Mussels by Targeting Tandem Repeat Regions of the Mitochondrial Genome

The recent genetic revolution through the analysis of aquatic environmental DNA (eDNA) has become a powerful tool for improving the detection of rare and/or invasive species. For the majority of eDNA studies, genetic assays are designed to target mitochondrial genes commonly referred to as “barcode” regions. However, unlike the typical structure of an animal mitochondrial genome, those for the invasive zebra and quagga mussels are greatly expanded with large extended tandem repeat regions. These sections of repeated DNA can appear hundreds of times within the genome compared to a single copy for the mitochondrial barcode genes. This higher number of target copies per mitochondrial genome presents an opportunity to increase eDNA assay sensitivity for these species. Therefore, we designed and evaluated new eDNA assays to target the extended repeat sections for both zebra and quagga mussels. These assays lower the limit of detection of genomic DNA by 100-fold for zebra mussels and 10-fold for quagga mussels. Additionally, these newly developed assays provided longer durations of detection during degradation mesocosm experiments and greater sensitivity for eDNA detection from water samples collected across western Lake Erie compared to standard assays targeting mitochondrial genes. This work illustrates how understanding the complete genomic structure of an organism can improve eDNA analysis.

Evaluating Salmonella pullorum dissemination and shedding patterns and antibody production in infected chickens

BackgroundPullorum disease caused by Salmonella pullorum is one of the most important infectious diseases in the poultry industry, responsible for causing substantial economic losses globally. On farms, the traditional method to detect S. pullorum infection mainly involves the collection of feces and sera to test for antigens and antibodies, respectively, but the regularity of Salmonella pullorum dissemination in internal organs and shedding patterns and antibody production in infected chickens remains unclear. Herein we aimed to investigate the dissemination of S. pullorum to different organs and bacterial shedding patterns in the faeces as well as serum antibody production post-infection in chickens of different ages.ResultIn this study, the liver and heart of 2-day-old chickens showed the highest copy numbers of S. pullorum at 6.4 × 106 and 1.9 × 106 copies of DNA target sequences/30 mg, respectively. In case of 10-day-old chickens, the percentage of S. pullorum fecal shedding (0%–40%) and antibody production (0%–56.6%) markedly fluctuated during the entire experiment; furthermore, in case of 42-week-old chickens, the percentage of birds showing S. pullorum shedding in the faeces showed a downward trend (from 63.33% to 6.6% in the oral inoculation group and from 43.3% to 10% in the intraperitoneal injection group), while that of birds showing serum antibody production remained at a high level (38.3% and 80% in the oral inoculation and intraperitoneal injection groups, respectively). We also performed cohabitation experiments, showed that 15% 10-day-old and 3.33% 42-week-old chickens were infected via the horizontal transmission in cohabitation with S. pullorum infected chickens, and revealed a high risk of horizontal transmission of S. pullorum.ConclusionThis study systematically evaluated the dissemination of S. pullorum in internal organs and bacterial fecal shedding patterns, and antibody production in infected chickens. Collectively, our findings indicate how to effectively screen S. pullorum-negative chickens on livestock farms and should also help in the development of measures to control and eradicate S. pullorum.

BioFire FilmArray Pneumonia Panel enhances detection of pathogens and antimicrobial resistance in lower respiratory tract specimens

BackgroundThis study investigated the diagnostic utility of the BioFire FilmArray Pneumonia Panel (PN panel), an automated and multiplexed nucleic acid detection system that rapidly detects 26 pathogens (18 bacteria and eight viruses) and seven antimicrobial resistance markers in a single assay.MethodsWe analyzed the targets in lower respiratory tract specimens using the PN panel and compared the detection results with those of bacterial culture methods and antimicrobial susceptibility testing.ResultsOf the 57 samples analyzed, the PN panel detected 97 targets (84 bacteria, four viruses, and nine antimicrobial resistance markers). Detection of bacteria and antimicrobial resistance was three times greater than that of the bacterial culture (25 bacteria and two resistant isolates) against the targets available in the panel. The overall positive and negative percent agreements between the PN panel and culture methods for bacterial detection were 100.0% and 92.9%, respectively. Multiple pathogens were detected by the PN panel in 24 samples (42.1%), ranging from two pathogens in 11 samples (19.3%) to six pathogens in one sample (1.8%). The PN panel semiquantitatively detected higher copies (≥ 106 copies/mL) of bacterial targets if the bacteria were positive by the culture method. In contrast, the semiquantitative values obtained by the panel varied (104 to 107 ≤ copies/mL) among bacteria that were negative by the culture method.ConclusionsThe PN panel enhanced the detection of pathogens and antimicrobial resistance markers in lower respiratory tract specimens.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

Recombinase Polymerase Amplification and Their Application in Phytopathogen Detection

DNA identification method is indispensable for the detection of a plant pathogen. However, established techniques, though reliable, requires advanced equipment, and their application outside specialized laboratories is limited. Along with the advancement of molecular techniques, several isothermal amplification methods, including Recombinase Polymerase Amplification (RPA), has been developed in this study. In fact, RPA is a rapid and sensitive amplification method, operating optimally at 37-42 degree celcius for 15 to 30 minutes with minimal sample preparation, and can amplify as low as 1-10 target copies. Furthermore, RPA has been a favourable method for the detection of plant pathogens due to its advantageous parameters. This review presents the current knowledge of RPA and its application in plant pathogen detection.

Recombinase Polymerase Amplification and Their Application in Phytopathogen Detection

DNA identification method is indispensable for the detection of a plant pathogen. However, established techniques, though reliable, requires advanced equipment, and their application outside specialized laboratories is limited. Along with the advancement of molecular techniques, several isothermal amplification methods, including Recombinase Polymerase Amplification (RPA), has been developed in this study. In fact, RPA is a rapid and sensitive amplification method, operating optimally at 37-42 degree celcius for 15 to 30 minutes with minimal sample preparation, and can amplify as low as 1-10 target copies. Furthermore, RPA has been a favourable method for the detection of plant pathogens due to its advantageous parameters. This review presents the current knowledge of RPA and its application in plant pathogen detection.

Digital PCR for Minimal Residual Disease Quantitation Using Immunoglobulin/T-Cell Receptor Gene Rearrangements in Acute Lymphoblastic Leukemia: A Proposed Analytic Algorithm

In minimal residual disease (MRD), where there are exceedingly low target copy numbers, digital PCR (dPCR) can improve MRD quantitation. However, standards for dPCR MRD interpretation in acute lymphoblastic leukemia are lacking. Here, for immunoglobulin/T-cell receptor-based MRD, we propose an objective, statistics-based analytic algorithm. In 161 postinduction samples from 79 children with acute lymphoblastic leukemia, MRD was performed by dPCR and real-time quantitative PCR (qPCR) using the same markers and primer-probe sets. The dPCR raw data were analyzed by using an automated algorithm. dPCR and qPCR results were highly concordant (P<0.0001): 98% (50 of 51) of qPCR positive were positive by dPCR, whereas 95% (61 of 64) of qPCR negative results were also negative by dPCR. For MRD quantitation, both qPCR and dPCR were tightly correlated (R2=0.94). Using more DNA (1 μg×7 versus 630 ng×3), dPCR improved sensitivity of MRD quantitation by one log10 (median MRD positive cutoff 1.6×10-5). With dPCR, 83% (29 of 35) of positive-not-quantifiable results by qPCR could be assigned positive/negative MRD status. Seven replicates of tested samples and negative controls were optimal. Compared with qPCR, dPCR could improve MRD sensitivity by one log10. We proposed an automatable, statistics-based algorithm that minimized interoperator variance for dPCR MRD.

PlexProbes enhance qPCR multiplexing by discriminating multiple targets in each fluorescent channel.

The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched ‘on’ or ‘off’ in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.

Quantitative real-time PCR assays for the concurrent diagnosis of infectious laryngotracheitis virus, Newcastle disease virus and avian metapneumovirus in poultry.

Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log10 dynamic range, a reproducible (R2 > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%–98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.

Application of TaqMan Real-Time PCR for Detecting 'Candidatus Arsenophonus Phytopathogenicus' Infection in Sugar Beet.

The γ-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ is assigned as the major pathogen of “Syndrome des basses richesses”, a sugar beet disease characterised by a reduction in the sugar content of taproots and biomass yield. Despite the economic impact of this bacteriosis, diagnostics for this important pathogen currently rely on end-point PCR detection. Herein, we introduce a TaqMan qPCR for diagnostics of the agent targeting genes encoding a heat shock protein of the Hsp20 family and mannose-6-phosphate isomerase. Quantitation with synthetic oligonucleotides as standard showed that the developed TaqMan qPCR assays enable the detection of up to 100 target copies. A comparison between the TaqMan qPCR and end-point PCR for ‘Ca. A. phytopathogenicus’ detection was carried out on 78 sugar beet samples from different locations in southern Germany. The newly developed assays enable the fast, reliable and sensitive detection of ‘Ca. A. phytopathogenicus’ in sugar beet.

Calibration-free counting of low molecular copy numbers in single DNA-PAINT localization clusters

Single-molecule localization microscopy (SMLM) has revolutionized light microscopy by enabling optical resolution down to a few nanometer. Yet, localization precision commonly does not suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Because each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been persistently proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photophysics or on a calibration to a reference. Previously, we developed localization-based fluorescence correlation spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-PAINT (points accumulation for imaging in nanoscale topography), for the first time, to our knowledge, circumventing the necessity for reference calibrations. Here, we present an extended concept termed lbFCS+, which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting of fluorescent loci contained in individual nanoscopic volumes is achieved via precise measurement of the local hybridization rates of the fluorescently labeled oligonucleotides (“imagers”) employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to 10 docking strands. For N ≤ 4 target molecules, lbFCS+ is even able to resolve integers, providing the potential to study the composition of up to tetrameric molecular complexes. Furthermore, we show that lbFCS+ allows resolving heterogeneous binding dynamics, enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from biosensing to DNA computing.

CRISPR/Cas12a-assisted rapid identification of key beer spoilage bacteria

Beer spoilage bacteria present severe challenges for the beer fermentation industry. Here, we establish a rapid, specific and highly portable CRISPR/Cas12a-based key beer spoilage bacterial scanning method, dubbed CRISPR-Beer Scan. With the variable 16S rDNA segments of these major spoilage microbe species serving as targets, highly efficient and specific crRNAs were determined. By coupling with recombinase polymerase isothermal amplification, CRISPR-Beer Scan could detect as low as 10 copies of DNA targets. Indeed, trace amounts of target genome DNAs can be accurately identified within a mixture of genome DNAs conditioned in beer. Moreover, the extracted genome DNA samples can be conveniently distinguished through the visual fluorescent signals excited by blue light in 45 min with CRISPR-Beer Scan Method. Taken together, the CRISPR-Beer Scan represents a widely applicable microbe-monitoring platform for not only breweries, but also other industries.

Accessible detection of SARS-CoV-2 through molecular nanostructures and automated microfluidics

Accurate and accessible nucleic acid diagnostics is critical to reducing the spread of COVID-19 and resuming socioeconomic activities. Here, we present an integrated platform for the direct detection of SARS-CoV-2 RNA targets near patients. Termed electrochemical system integrating reconfigurable enzyme-DNA nanostructures (eSIREN), the technology leverages responsive molecular nanostructures and automated microfluidics to seamlessly transduce target-induced molecular activation into an enhanced electrochemical signal. Through responsive enzyme-DNA nanostructures, the technology establishes a molecular circuitry that directly recognizes specific RNA targets and catalytically enhances signaling; only upon target hybridization, the molecular nanostructures activate to liberate strong enzymatic activity and initiate cascading reactions. Through automated microfluidics, the system coordinates and interfaces the molecular circuitry with embedded electronics; its pressure actuation and liquid-guiding structures improve not only analytical performance but also automated implementation. The developed platform establishes a detection limit of 7 copies of RNA target per μl, operates against the complex biological background of native patient samples, and is completed in <20 min at room temperature. When clinically evaluated, the technology demonstrates accurate detection in extracted RNA samples and direct swab lysates to diagnose COVID-19 patients.

Evaluation of a PlexZyme-Based PCR Assay and Assessment of COVID-19 Surge Testing Throughput Compared to Cobas SARS-CoV-2.

Reliable high-throughput methods are required for the detection of severe acute respiratory coronavirus 2 (SARS-CoV-2). We evaluated the new research use only (RUO) SpeeDx PlexZyme SARS-CoV-2 components (Plex) compared to the Roche cobas SARS-CoV-2 assay (cobas). A collection of positive (n = 214) and negative samples (n = 201) was tested in parallel comparing Plex with cobas. The overall agreement comparing the qualitative outcomes was 96.9%. Using an in-house quantitative PCR method, correlation comparing Plex ORF1ab to cobas ORF1a was r2 = 0.95. The median Plex ORF1ab change in target copy number compared to cobas ORF1a was +0.48 log10 copies/mL respectively. Inter- and intra-assay reproducibility of each assay was compared, including a limit-of-detection study. Reproducibility was comparable; however cobas was more sensitive than Plex by 1-log dilution. Throughput was evaluated during a COVID-19 testing surge of 4324 samples in a 30-h period. Plex demonstrated less hands-on time per reportable result (19% decrease) and increased throughput (155% increase of 102 results/hour) compared to cobas (40 results/hour). Our study demonstrates good qualitative and quantitative correlation of Plex compared to cobas and that Plex is well-suited for high throughput testing.

Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n = 238) and negative samples (n = 196) was tested in parallel comparing thermal treatment (75 °C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (P < 0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was ­0.07 (1.6%) and ­0.22 (4.2%) log10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r2 = 0.99). Good correlation was observed for ORF1a (r2 = 0.96) and E-gene (r2 = 0.98) comparing thermal treatment to room-temperature control.

Loop-mediated isothermal amplification (LAMP) assays targeting 18S ribosomal RNA genes for identifying P. vivax and P. ovale species and mitochondrial DNA for detecting the genus Plasmodium

BackgroundLoop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic blood-stage infections. LAMP has not previously been specifically evaluated for its diagnostic performance in detecting P. ovale in an epidemiological study, and no commercial LAMP or rapid diagnostic test (RDT) kits are available for specifically diagnosing infections with P. ovale.MethodsAn assay was designed to target a portion of mitochondrial DNA (mtDNA) among Plasmodium spp., the five human Plasmodium species and two other assays were designed to target the nuclear 18S ribosomal DNA gene (18S rDNA) of either P. vivax or P. ovale for differentiating the two species. The sensitivity of the assays was compared to that of nested PCR using defined concentrations of plasmids containing the target sequences and using limiting dilutions prepared from clinical isolates derived from Chinese workers who had become infected in Africa or near the Chinese border with Myanmar.ResultsThe results showed that 102 copies of the mitochondrial target or 102 and 103 copies of 18S rDNA could be detected from Plasmodium spp., P. vivax and P. ovale, respectively. In 279 clinical samples, the malaria Pan mtDNA LAMP test performed well when compared with a nested PCR assay (95% confidence interval [CI] sensitivity 98.48–100%; specificity 90.75–100%). When diagnosing clinical cases of infection with P. vivax, the 18S rDNA assay demonstrated an even great sensitivity (95.85–100%) and specificity (98.1–100%). The same was true for clinical infections with P. ovale (sensitivity 90.76–99.96%; specificity 98.34–100%). Using plasmid-positive controls, the limits of detection of Malaria Pan, 18S rDNA P. vivax and 18S rDNA P. ovale LAMP were 100-, 100- and tenfold lower than those of PCR, respectively.ConclusionThe novel LAMP assays can greatly aid the rapid, reliable and highly sensitive diagnosis of infections of Plasmodium spp. transmitted among people, including P. vivax and P. ovale, cases of which are most prone to clinical relapse.Graphical

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the β-tubulin gene

“Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.