scholarly journals Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

Insects ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1034
Author(s):  
Darja Kušar ◽  
Bojan Papić ◽  
Urška Zajc ◽  
Irena Zdovc ◽  
Majda Golob ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Germination Rate ◽  
Single Copy ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Qpcr Assay ◽  
Paenibacillus Larvae ◽  
Reliable Quantification ◽  
Detection And Quantification

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

scholarly journals Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events

2020 ◽  
Author(s):  
Tigst Demeke ◽  
Monika Eng ◽  
Michelle Holigroski ◽  
Sung-Jong Lee
Keyword(s):  
Zero Tolerance ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Droplet Digital Pcr ◽  
Chain Reaction ◽  
Low Level ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Detection And Quantification

Abstract Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples.

Research Article One-Step RT-qPCR assay for detection and quantification of equine infectious anemia virus in-vitro

2018 ◽  
Vol 17 (3) ◽  
Author(s):  
B.L. Bueno ◽  
F.G. Oliveira ◽  
G.K. Lima ◽  
A.A. Fonseca Júnior ◽  
T.C. Kassar ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Qpcr Assay ◽  
Research Article ◽  
Equine Infectious Anemia ◽  
One Step ◽  
Anemia Virus ◽  
Detection And Quantification

scholarly journals Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of Mycobacterium tuberculosis

2020 ◽  
Vol 8 (5) ◽  
pp. 701 ◽  
Author(s):  
Raphael Nyaruaba ◽  
Jin Xiong ◽  
Caroline Mwaliko ◽  
Nuo Wang ◽  
Belindah J. Kibii ◽  
...  
Keyword(s):  
Target Genes ◽  
Annealing Temperature ◽  
Single Channel ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Sample Concentration ◽  
Specificity And Sensitivity ◽  
Probe Concentration ◽  
Detection And Quantification

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.

scholarly journals Short communication: Antimicrobial activity of indoleacetic, gibberellic and coumaric acids against Paenibacillus larvae and its toxicity against Apis mellifera

2020 ◽  
Vol 18 (1) ◽  
pp. e05SC01
Author(s):  
Nicolás Szawarski ◽  
Pablo Giménez-Martínez ◽  
Giulia Mitton ◽  
Pedro Negri ◽  
Facundo Meroi Arcerito ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Apis Mellifera ◽  
Inhibitory Concentration ◽  
Clinical Symptoms ◽  
Paenibacillus Larvae ◽  
National University ◽  
Entre Rios ◽  
Mic Minimum Inhibitory Concentration ◽  
Honey Bee Larvae

Aim of study: To explore three isolated phytomolecules: indoleacetic acid (IAA), gibberellic acid (GA), and the secondary metabolite p-coumaric acid (CUM): (1) evaluating their toxicity against Apis mellifera larvae and adults under controlled conditions in the laboratory; (2) searching for antimicrobial activity against Paenibacillus larvae.Area of study: Honey bee larvae and adults were collected from the experimental apiary of the “Centro de Investigación en Abejas Sociales (CIAS)” (-37.9348798, -57.682817), Institute of the National University of Mar del Plata (UNMdP), Argentina.Material and methods: Paenibacillus larvae strains were isolated from beehives from different provinces of Argentina (Buenos Aires, Córdoba and Entre Ríos) showing clinical symptoms of the American foulbrood. All strains (S1, S2, S3, S4) were genotypically identified using PL5 and PL4 primers and characterized as genotype ERIC1. Then standard essays were performed to determined toxicity of phytomolecules in honey bees and antimicrobial activity through the broth microdilution method.Main results:  The diet with GA, IAA and CUM did not present toxic effects in larvae or adult bees, and only CUM showed antimicrobial activity against P. larvae. In this study, we obtained in vitro values of MNIC (minimum non-inhibitory concentration) of 500 μg mL-1 and a MIC (minimum inhibitory concentration) of 650 μg mL-1 for CUM.Research highlights: The obtained results remark its potential as a natural alternative for the control of P. larvae, avoiding the problems generated by the use of synthetic antibiotics such as the resistance phenomena and the contamination of hive’s products.

scholarly journals Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR?

2019 ◽  
Vol 20 (9) ◽  
pp. 2249 ◽  
Author(s):  
Daniela Cilloni ◽  
Jessica Petiti ◽  
Valentina Rosso ◽  
Giacomo Andreani ◽  
Matteo Dragani ◽  
...  
Keyword(s):  
Residual Disease ◽  
Myeloproliferative Disorders ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Acute Leukemias ◽  
New Techniques ◽  
Standard Curve ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Digital Polymerase Chain Reaction

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.

scholarly journals Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

2021 ◽  
Author(s):  
Joan Xiaohui Yang ◽  
Xin Yuan Zhao ◽  
Dexi Bi ◽  
Citra Mattar ◽  
Joy Yan Ling Pang ◽  
...  
Keyword(s):  
New Technologies ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Variable Expression ◽  
Sample Extraction ◽  
Relative Standard ◽  
Extracellular Mirna ◽  
Digital Polymerase Chain Reaction

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72–16.95 copies/oocyte), Hprt1 (1.05–19.05 copies/oocyte) and ATPase 6, (5.55–32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

scholarly journals Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

2021 ◽  
Vol 22 (2) ◽  
pp. 687
Author(s):  
Tong Zhou ◽  
Bolan Zhou ◽  
Yasong Zhao ◽  
Qing Li ◽  
Guili Song ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Grass Carp ◽  
Single Copy ◽  
Type I ◽  
Western Blots ◽  
Paramisgurnus Dabryanus ◽  
Expression Activity ◽  
Transgenic Construct ◽  
Mucus Gland

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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