Use Of qPCR Research Articles

Optimizing Palmer amaranth (Amaranthus palmeri) genetic testing of seeds using real-time (quantitative) PCR

Abstract The Amaranthus genus contains numerous agronomic weedy species that are widely distributed across the United States. The seeds of many Amaranthus species are morphologically indistinguishable. The Minnesota Department of Agriculture declared Palmer amaranth (Amaranthus palmeri S. Watson) a prohibited noxious weed seed in 2016. Any Amaranthus spp. seeds that are identified as contaminants during routine seed testing require genetic testing to determine whether A. palmeri is present. This research aimed to validate and optimize the molecular detection of A. palmeri in seed. We refined the DNA extraction from pools of Amaranthus spp. seeds ranging in size from 1 to 100 seeds to improve sample testing throughput. Real-time polymerase chain reaction (qPCR) using primers developed by Murphy and colleagues correctly identified the presence of A. palmeri genetics in 84 samples containing 0, 1, 2, 25, or 50 A. palmeri seeds in samples containing up to 100 seeds. The method specificity was examined using 17 Amaranthus species and 4 hybrids of unknown genetics. The Murphy regular qPCR cycle amplified Watson’s amaranth (Amaranthus watsonii Standl.), spleen amaranth (Amaranthus dubius Mart. ex Thell.), and spiny amaranth (Amaranthus spinosus L.), which would result in false-positive calls; however, the fast cycle only identified A. watsonii as a potential false positive. Examination of the Murphy primer binding site revealed an identical sequence for A. palmeri and A. watsonii. Additional markers were evaluated and optimized for use in qPCR to eliminate the risk of a false positive. The additional markers did eliminate the amplification of A. dubius and A. spinosus but did not eliminate the amplification for A. watsonii. Currently, A. watsonii is not known to be distributed outside of its limited native range and is not expected to be encountered in samples.

Peripartum depression symptom trajectories, telomere length and genotype, and adverse childhood experiences

BackgroundAs a biological marker for cellular senescence, telomere length (TL) has been linked to a variety of psychiatric disorders and adverse childhood experiences (ACE), though only preliminarily to peripartum depression (PPD). The present study sought to examine the association between TL and PPD, assessing the moderating role of ACE and genetic polymorphic variations related with the telomere machinery.MethodsAdversity was self-reported, likewise were depressive symptoms evaluated at pregnancy week 17 and 32, as well as six-weeks and six-months postpartum. TL was assessed by use of qPCR in blood samples collected during delivery from females with antenatal depression resolving postpartum, females with depression persisting to postpartum, and healthy controls. Twenty haplotype-tagging Single Nucleotide Polymorphisms in the Telomerase Reverse Transcriptase (TERT) and three in the Telomerase RNA Component (TERC) genes were genotyped.ResultsTL was negatively correlated with severity of PPD symptoms at pregnancy week 32 and postpartum week 6. PPD was associated with shorter TL. Lastly, ACE, but not the TERT/TERC genotype, moderated the TL-trajectory association; with increasing ACE, individuals with persistent PPD symptoms had shorter TL, whereas the opposite pattern (longer TL) was observed in the controls.ConclusionsThe findings contribute to further understanding of PPD underpinnings, suggesting a negative relationship with TL.

Clinical significance of Spinal Muscular Atrophy carrier detection in Guangdong Province, China: Insights from quantitative polymerase chain reaction and multiplex ligation-dependent probe amplification analysis

ObjectiveThis study aimed to identify carriers of Spinal Muscular Atrophy (SMA) among 10,630 pregnant women in Guangdong Province and provide prenatal diagnoses for high-risk fetuses from carrier couples. The goal was to prevent the birth of children affected by SMA. We evaluated the effectiveness of quantitative PCR (qPCR) and multiplex ligation-dependent probe amplification (MLPA) in detecting deletions in the SMN1 gene, with MLPA as the reference standard. MethodsFluorescent qPCR was used for initial SMA carrier screening, followed by confirmatory testing with MLPA for all detected carriers. ResultsOf the 10,630 women screened, 219 were identified as carriers (2.06 % detection rate). This included 17 cases of heterozygous deletion of exon 7 (E7), 145 cases with deletions of both E7 and exon 8 (E8), and 57 cases of E8 deletion alone. The carrier rate for E7 heterozygous deletion was established at 1.5 %. Prenatal diagnosis for seven carrier couples revealed five fetuses as carriers and one affected by SMA. The diagnostic concordance between qPCR and MLPA was 100 %. ConclusionThe combined use of qPCR and MLPA is vital in identifying SMA carriers, allowing for early diagnosis and informed reproductive decisions. The high sensitivity and specificity of qPCR, matching MLPA, demonstrate its value in clinical settings for SMA screening and prenatal diagnosis. Our findings emphasize the critical importance of selecting precise diagnostic methods to enhance clinical outcomes in genetic screening programs.

‘A comparative study of traditional and molecular diagnostic methods for detection of gastrointestinal parasites in Nepalese migrants to the UK’

BackgroundWe evaluated the results of examining a single faecal sample for gastrointestinal parasites (GIP) using a combination of traditional methods with multiplex qPCR for helminths and protozoa, compared to a reference standard of examining three faecal samples from each person using traditional diagnostic methods alone. MethodsThree faecal samples were collected at weekly intervals from 596 healthy Nepalese men. Each sample underwent formalin-ethyl acetate (FEA) concentration and light microscopy, and charcoal culture. The combined results of these investigations for all three stool samples were designated the reference standard. The first sample was also analysed using a multiplex TaqManTM qPCR assay, screening for five helminths and three protozoa. We compared sensitivity and specificity of analysing the first faecal sample with qPCR alone, or a hybrid approach combining qPCR with traditional methods, to the reference standard. Additionally, a serum sample was taken from each participant for Strongyloides stercoralis IgG ELISA. ResultsThe reference standard identified 139 GIP infections in 133 (22.3%) participants. Use of qPCR alone in one stool identified 176 infections in 147 (24.8%) participants, rising to 187 infections in 156 (26.3%) when combined with FEA microscopy and charcoal culture. The sensitivity of this latter hybrid approach was 100% for Strongyloides spp., 90.9% for Trichuris trichiura, 86.8% for hookworm species and 75% for Giardia duodenalis compared to the reference standard. The hybrid approach increased the detected cases of G. duodenalis by 4.5% (46 cases) overall, T. trichiura by 2.9% (18 cases), Strongyloides spp. by 1% (6 cases), and hookworm by 0.5% (8 cases), compared to the reference standard. ConclusionExamination of a single faecal sample using qPCR alone showed superior or equivalent sensitivity to traditional methods for most GIP infections when both were compared to the reference standard. Combining molecular and traditional methods to analyse a single stool improved the detection rate for most studied parasites. This approach has value in settings where repeated sampling and/or faecal culture for helminths is impractical, but molecular diagnostics are available.

Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene blaCMY-2 in an activated sludge microcosm

ABSTRACT As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla CMY-2 was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla CMY-2 in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.

Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation

Nowadays, the use of qPCR for the diagnosis of intestinal microsporidiosis is increasing. There are several studies on the evaluation of qPCR performance but very few focus on the stool pretreatment step before DNA extraction, which is nevertheless a crucial step. This study focuses on the mechanical pretreatment of stools for Enterocytozoon bieneusi spores DNA extraction. Firstly, a multicenter comparative study was conducted evaluating seven extraction methods (manual or automated) including various mechanical pretreatment. Secondly, several durations and grinding speeds and types of beads were tested in order to optimize mechanical pretreatment. Extraction methods of the various centers had widely-varying performances especially for samples with low microsporidia loads. Nuclisens® easyMAG (BioMérieux) and Quick DNA Fecal/Soil Microbe Microprep kit (ZymoResearch) presented the best performances (highest frequencies of detection of low spore concentrations and lowest Ct values). Optimal performances of mechanical pretreatment were obtained by applying a speed of 30 Hz during 60 s with the TissueLyser II (Qiagen) using commercial beads of various materials and sizes (from ZymoResearch or MP Biomedicals). Overall, the optimal DNA extraction method for E. bieneusi spores contained in stool samples was obtained with a strong but short bead beating using small-sized beads from various materials.

Use of qPCR to Evaluate Efficiency of the Bulky DNA Damage Removal in Extracts of Mammalian Cells with Different Maximum Lifespan

Proteins of nucleotide excision repair system (NER) are responsible for detecting and removing a wide range of bulky DNA damages, thereby contributing significantly to the genome stability maintenance within mammalian cells. Evaluation of NER functional status in the cells is important for identifying pathological changes in the body and assessing effectiveness of chemotherapy. The following method, described herein, has been developed for better assessment of bulky DNA damages removal in vitro, based on qPCR. Using the developed method, NER activity was compared for the extracts of the cells from two mammals with different lifespans: a long-lived naked mole-rat (Heterocephalus glaber) and a short-lived mouse (Mus musculus). Proteins of the H. glaber cell extract have been shown to be 1.5 times more effective at removing bulky damage from the model DNA substrate than the proteins of the M. musculus cell extract. These results are consistent with the experimental data previously obtained. The presented method could be applied not only in fundamental studies of DNA repair in mammalian cells, but also in clinical practice.

Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.Graphical

Pediococcus acidilactici reduces tau pathology and ameliorates behavioral deficits in models of neurodegenerative disorders

BackgroundAlzheimer’s disease (AD), affecting many elders worldwide, is characterized by A-beta and tau-related cognitive decline. Accumulating evidence suggests that brain iron accumulation is an important characteristic of AD. However, the function and mechanism of the iron-mediated gut-brain axis on AD is still unclear.MethodsA Caenorhabditis elegans model with tau-overexpression and a high-Fe diet mouse model of cognitive impairment was used for probiotic function evaluation. With the use of qPCR, and immunoblotting, the probiotic regulated differential expression of AD markers and iron related transporting genes was determined. Colorimetric kits, IHC staining, and immunofluorescence have been performed to explore the probiotic mechanism on the development of gut-brain links and brain iron accumulation.ResultsIn the present study, a high-Fe diet mouse model was used for evaluation in which cognitive impairment, higher A-beta, tau and phosphorylated (p)-tau expression, and dysfunctional phosphate distribution were observed. Considering the close crosstalk between intestine and brain, probiotics were then employed to delay the process of cognitive impairment in the HFe mouse model. Pediococcus acidilactici (PA), but not Bacillus subtilis (BN) administration in HFe-fed mice reduced brain iron accumulation, enhanced global alkaline phosphatase (AP) activity, accelerated dephosphorylation, lowered phosphate levels and increased brain urate production. In addition, because PA regulated cognitive behavior in HFe fed mice, we used the transgenic Caenorhabditis elegans with over-expressed human p-tau for model, and then PA fed worms became more active and longer lived than E.coli fed worms, as well as p-tau was down-regulated. These results suggest that brain iron accumulation influences AD risk proteins and various metabolites. Furthermore, PA was shown to reverse tau-induced pathogenesis via iron transporters and AP-urate interaction.ConclusionsPA administration studies demonstrate that PA is an important mediator of tau protein reduction, p-tau expression and neurodegenerative behavior both in Caenorhabditis elegans and iron-overload mice. Finally, our results provide candidates for AP modulation strategies as preventive tools for promoting brain health.1zXKqRd2u82F96nqb8MmwuVideo

Evaluation of Various Diagnostic Strategies for Bacterial Vaginosis, Including a New Approach Based on MALDI-TOF Mass Spectrometry.

Bacterial vaginosis (BV) is a common dysbiosis of unclear etiology but with potential consequences representing a public health problem. The diagnostic strategies vary widely. The Amsel criteria and Nugent score have obvious limitations, while molecular biology techniques are expensive and not yet widespread. We set out to evaluate different diagnostic strategies from vaginal samples using (1) a combination of abnormal vaginal discharge and vaginal pH > 4.5; (2) the Amsel-like criteria (replacing the "whiff test" with "malodorous discharge"); (3) the Nugent score; (4) the molecular quantification of Fannyhessea vaginae and Gardnerella vaginalis (qPCR); (5) and MALDI-TOF mass spectrometry (we also refer to it as "VAGI-TOF"). Overall, 54/129 patients (42%) were diagnosed with BV using the combination of vaginal discharge and pH, 46/118 (39%) using the Amsel-like criteria, 31/130 (24%) using qPCR, 32/130 (25%) using "VAGI-TOF", and 23/84 (27%) using the Nugent score (not including the 26 (31%) with intermediate flora). Of the 84 women for whom the five diagnostic strategies were performed, the diagnosis of BV was considered for 38% using the combination of vaginal discharge and pH, 34.5% using the Amsel-like criteria, 27% using the Nugent score, 25% using qPCR, and 25% using "VAGI-TOF". When qPCR was considered as the reference, the sensitivity rate for BV was 76.2% for the combination of vaginal discharge and pH, 90.5% for the Amsel-like criteria, 95.2% for the Nugent score, and 90.5% for "VAGI-TOF", while the specificity rates were 74.6%, 84.1%, 95.3%, and 95.3%, respectively. When the Nugent score was considered as the reference, the sensitivity for BV was 69.6% for the combination of vaginal discharge and pH, 82.6% for the Amsel-like criteria, 87% for qPCR, and 78.7% for "VAGI-TOF", while the specificity rates were 80%, 94.3%, 100%, and 97.1%, respectively. Overall, the use of qPCR and "VAGI-TOF" provided a consistent diagnosis of BV, followed by the Nugent score. If qPCR seems tedious and for some costly, "VAGI-TOF" could be an inexpensive, practical, and less time-consuming alternative.

Integrating molecular microbial methods to improve faecal pollution management in rivers with designated bathing waters

Rivers are at risk from a variety of pollution sources. Faecal pollution is of particular concern since it disperses pathogenic microorganisms in the aquatic environment. Currently, faecal pollution levels in rivers is monitored using faecal indicator bacteria (FIB) that do not offer information about pollution sources and associated risks. This study used a combined molecular approach, along with measurements of water quality, to gain information on pollution sources, and risk levels, in a newly designated recreational bathing site in the River Wharfe (UK). Physico-chemical parameters were monitored in situ, with water quality multiparameter monitoring sondes installed during the 2021 bathing season. The molecular approach was based on quantitative PCR (qPCR)-aided Microbial Source Tracking (MST) and 16S rRNA gene metabarcoding to obtain a fingerprint of bacterial communities and identify potential bioindicators.The analysis from the water quality sondes showed that ammonium was the main parameter determining the distribution of FIB values. Lower faecal pollution levels were detected in the main river when compared to tributaries, except for samples in the river located downstream of a wastewater treatment plant. The faecal pollution type (anthropogenic vs. zoogenic) changed the diversity and the structure of bacterial communities, giving a distinctive fingerprint that can be used to inform source. DNA-based methods showed that the presence of human-derived bacteria was associated with Escherichia coli spikes, coinciding with higher bacterial diversity and the presence of potential pathogenic bacteria mainly of the genus Mycobacterium, Aeromonas and Clostridium. Samples collected after a heavy rainfall event were associated with an increase in Bacteroidales, which are markers of faecal pollution, including Bacteroides graminisolvens, a ruminant marker associated with surface run-off from agricultural sources. The combined use of qPCR and 16S rRNA sequencing was able to identify pollution sources, and novel bacterial indicators, thereby aiding decision-making and management strategies in recreational bathing rivers.

Quantitative PCR (qPCR) for estimating the presence of Fusarium and its mycotoxins in barley grains

Members within the Fusarium sambucinum species complex (FSAMSC) are able to produce mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN) and enniatins (ENNs) in food products. Consequently, alternative methods for assessing the levels of these mycotoxins are relevant for quick decision-making. In this context, qPCR based on key mycotoxin biosynthetic genes could aid in determining the toxigenic fungal biomass, and could therefore infer mycotoxin content. The aim of this study was to verify the use of qPCR as a technique for estimating DON, NIV, ENNs and ZEN, as well as Fusarium graminearum sensu lato (s.l.) and F. poae in barley grains. For this purpose, 53 barley samples were selected for mycobiota, mycotoxin and qPCR analyses. ENNs were the most frequent mycotoxins, followed by DON, ZEN and NIV. 83% of the samples were contaminated by F. graminearum s.l. and 51% by F. poae. Pearson correlation analysis showed significant correlations for TRI12/15-ADON with DON, ESYN1 with ENNs, TRI12/15-ADON and ZEB1 with F. graminearum s.l., as well as ESYN1 and TRI12/NIV with F. poae. Based on the results, qPCR could be useful for the assessment of Fusarium presence, and therefore, provide an estimation of its mycotoxins’ levels from the same sample.

Physiological fibrin hydrogel modulates immune cells and molecules and accelerates mouse skin wound healing.

Wound healing is a complex process to restore homeostasis after injury and insufficient skin wound healing is a considerable problem in medicine. Whereas many attempts of regenerative medicine have been made for wound healing with growth factors and cell therapies, simple pharmacological and immunological studies are lagging behind. We investigated how fibrin hydrogels modulate immune cells and molecules in skin wound healing in mice. Physiological fibrin hydrogels (3.5 mg/mL fibrinogen) were generated, biophysically analyzed for stiffness and protein contents and were structurally studied by scanning electron microscopy. Physiological fibrin hydrogels were applied to full thickness skin wounds and, after 3 days, cells and molecules in wound tissues were analyzed. Leukocytes, endothelial cells, fibroblasts and keratinocytes were explored with the use of Flow Cytometry, whereas cytokines and matrix metalloproteinases were analyzed with the use of qPCR, ELISAs and zymography. Skin wound healing was analyzed microscopically at day 3, macroscopically followed daily during repair in mice and compared with commercially available fibrin sealant Tisseel. Exogenous fibrin at physiological concentrations decreased neutrophil and increased non-classical Ly6Clow monocyte and resolutive macrophage (CD206+ and CX3CR1+) populations, at day 3 after injury. Fibrin hydrogel reduced the expression of pro-inflammatory cytokines and increased IL-10 levels. In line with these findings, gelatinase B/MMP-9 was decreased, whereas gelatinase A/MMP-2 levels remained unaltered. Frequencies of dermal endothelial cells, fibroblasts and keratinocytes were increased and keratinocyte migration was enhanced by fibrin hydrogel. Importantly, physiological fibrin accelerated the healing of skin wounds in contrast to the highly concentrated fibrin sealant Tisseel, which delayed wound repair and possessed a higher fiber density. Collectively, we show that adding a tailored fibrin hydrogel scaffold to a wound bed positively influences the healing process, modulating leukocyte populations and inflammatory responses towards a faster wound repair.

Review: JAK2V617F Allele Burden in Diagnosis and Therapeutic Monitoring of Myeloproliferative Neoplasms

Characterized by overproduction of differentiated cells of myeloid lineage, polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are Philadelphia chromosome negative myeloproliferative neoplasms (MPNs). Found in 95% of PV patients and 50-60% of ET and PMF patients, the JAK2V617F mutation is the most common molecular abnormality shared by the three MPN phenotypes. Although the JAK2 mutation is recommended for diagnosis of MPNs by the World Health Organization (WHO), its presence alone is insufficient to discriminate among the 3 subtypes. This implication of single mutation (JAK2V617F) in all three MPN phenotypes has long been an objective under question and several studies investigating on the gene dosage hypothesis have discovered the promising role of the JAK2V617F allele burden in MPN phenotype. The significant differences of the JAK2V617F allele burden in PV, ET and PMF patients as well its associations with specific clinical and haematological characteristics bear high utility in diagnosis, prognosis, and therapeutic monitoring. Although great strides have been achieved with the use of qPCR and new molecular biology techniques in allele burden quantification, addressing the deficits in the current understandings and further improvement of technology will be highly beneficial. Therefore, we have reviewed PubMed database from 2005 to 2022. Using keywords such as JAK2V617F mutation, Allele burden, Myeloproliferative neoplasms etc. and the present review discusses the significance of JAK2V617F allele burden in diagnosis and therapeutic monitoring of myeloproliferative neoplasms.

BDNF Spinal Overexpression after Spinal Cord Injury Partially Protects Soleus Neuromuscular Junction from Disintegration, Increasing VAChT and AChE Transcripts in Soleus but Not Tibialis Anterior Motoneurons.

After spinal cord transection (SCT) the interaction between motoneurons (MNs) and muscle is impaired, due to reorganization of the spinal network after a loss of supraspinal inputs. Rats subjected to SCT, treated with intraspinal injection of a AAV-BDNF (brain-derived neurotrophic factor) construct, partially regained the ability to walk. The central effects of this treatment have been identified, but its impact at the neuromuscular junction (NMJ) has not been characterized. Here, we compared the ability of NMJ pre- and postsynaptic machinery in the ankle extensor (Sol) and flexor (TA) muscles to respond to intraspinal AAV-BDNF after SCT. The gene expression of cholinergic molecules (VAChT, ChAT, AChE, nAChR, mAChR) was investigated in tracer-identified, microdissected MN perikarya, and in muscle fibers with the use of qPCR. In the NMJs, a distribution of VAChT, nAChR and Schwann cells was studied by immunofluorescence, and of synaptic vesicles and membrane active zones by electron microscopy. We showed partial protection of the Sol NMJs from disintegration, and upregulation of the VAChT and AChE transcripts in the Sol, but not the TA MNs after spinal enrichment with BDNF. We propose that the observed discrepancy in response to BDNF treatment is an effect of difference in the TrkB expression setting BDNF responsiveness, and of BDNF demands in Sol and TA muscles.

Understanding quantitative polymerase chain reaction bioanalysis issues before validation planning: Japan Bioanalysis Forum discussion group.

Investigating the biodistribution of cell and gene therapy products may play an important role in evaluating their safety and pharmacology. As quantitative polymerase chain reaction (qPCR) is often used for these analyses, it is essential to improve the reliability of bioanalysis performed using qPCR. In this report, the authors discuss the use of qPCR in nonclinical studies, as it can be used to detect target DNA/RNA and it is quantitative and applicable for long-term analysis. The authors also discuss points to consider during bioanalysis using qPCR and present appropriate validation items and their criteria. The authors anticipate the discussion provided herein to contribute to the development of validation and sample analysis for pharmaceuticals analyzed using qPCR.

Tidyqpcr

Software is intended to be a tool used to complete tasks efficiently. However, the intent is not always matched by the execution as users may become frustrated with design idiosyncrasies or suboptimal implementations. In order to support reproducibility and the user experience, research software needs to be rewarded on its usability and documentation as well as its functionality. Quality documentation needs to cover design decisions, functionality, and how to contribute to the software. Here we describe our work to tackle the reproducibility crisis in qPCR analysis by creating the open-source software package tidyqpcr. We also introduced the extensive infrastructure available for creating software documentation in the R programming language. 
 Quantitative polymerase chain reaction (qPCR) is a fundamental technique in molecular biology to detect and quantify DNA and RNA. However, the ubiquitous use of qPCR across research disciplines has led to inconsistencies in implementation and reporting, leading to a reproducibility crisis and the publication of the Minimum Information in a Quantitative PCR experiment (MIQE) guidelines. In addition, each stage of a qPCR experiment can be customised to extract a wide variety of information from numerous biological processes. Developing versatile and reliable software built with best-practices and thorough documentation would promote reproducible qPCR analysis across diverse disciplines. tidyqpcr is an open source software package for user-friendly qPCR analysis using the tidyverse suite of R packages. tidyqpcr offers a consistent user interface and structure for qPCR analysis, within the tidyverse paradigm of spreadsheet-like rectangular data frames and generic functions that build up complex analyses in a series of simple steps. tidyqpcr focuses on experimental design in microwell plates, and relative quantification using changes in quantification cycle (∆Cq). tidyqpcr has been improved in response to software review from the rOpenSci non-profit initiative, which co-ordinates with the Journal of Open Source Software. Overall, tidyqpcr empowers scientists to conduct reproducible, flexible, and best-practice compliant quantitative PCR analysis.

Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

An undergraduate laboratory to detect viruses in human DNA samples using qPCR

The COVID-19 pandemic has necessitated the need to reliably detect the presence of viral genomes in human clinical samples. The most accurate viral tests involve the use of qPCR. Thus, it is important for students to understand the mechanism to detect viral genomes by qPCR including critical qPCR controls and how to properly interpret qPCR data. Herein, we describe a 2-week undergraduate laboratory to detect a viral genome in a human DNA sample. The strategy follows a SARS-CoV-2 qPCR test in numerous ways. Students are provided isolated DNA representing a mock human patient sample, and determine if a viral genome (bacteriophage lambda) is present using qPCR. A battery of control samples and patient pooling strategies are utilized. The laboratory exercise is successful based on high rates of student success in detecting viral genomes, pre-quiz and post-quiz assessments focusing on viral qPCR testing, and positive student comments.

The Use of qPCR to Detect Cryphonectria parasitica in Plants.

Cryphonectria parasitica is a fungal pathogen that causes lethal bark necrosis in chestnut. A duplex qPCR allowing detection of the pathogen and its host, Castanea sativa, is described. The method can be used for early detection of the pathogen in chestnut bark tissues with an internal control of false-negative results caused by PCR inhibitors and/or DNA extraction failure. A positive amplification control of qPCR that allows detection of any deviation from a normal qPCR run based on a control chart is also described. As C. parasitica is a regulated pathogen in Europe, the protocol also provides information on the way to collect and handle bark samples to fulfil biosecurity rules.