Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I‐based ...assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.—Ruijter, J. M., Ruiz‐Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis. FASEB J. 33, 14542‐14555 (2019). www.fasebj.org
To achieve rapid on-site nucleic acid detection of fish allergens in food, this study established a new direct and rapid qPCR detection technology for fish. The technology was able to complete ...nucleic acid extraction in 4 min using a food nucleic acid releaser, and the DNA samples were amplified by rapid qPCR within 25 min. The results showed that direct-rapid qPCR can specifically detect fish, and it can also detect 0.00001 % of fish components in artificially simulated samples, with high detection precision. This method can be used to effectively detect fish in various deep-processed foods. As a simple, rapid, and accurate molecular detection method for fish allergens, this method has broad application prospects for on-site food safety detection.
•A nucleic acid-release reagent for food was developed that could rapidly extract DNA from various food samples in 4 min.•A rapid qPCR technique adapted to nucleic acid release reagent was developed, and gene detection was completed within 25 min.•Direct rapid qPCR can be used for on-site detection of fish components in deep-processed food samples.
Virus inactivation validation studies have been widely applied in the risk assessment of biogenic material‐based medical products, such as biological products, animal tissue‐derived biomaterials, and ...allogeneic biomaterials, to decrease the risk of virus transmission. Traditional virus detection methods in an inactivation validation study utilize cell culture as a tool to quantify the infectious virus by observing cytopathic effects (CPEs) after virus inactivation. However, this is susceptible to subjective factors because CPEs must be observed by experts under a microscope during virus titration. In addition, this method is costly and time‐ and labor‐consuming. Molecular biological technologies such as quantitative polymerase chain reaction (qPCR) have been widely used for virus detection but cannot distinguish infectious and noninfectious viruses. Therefore, qPCR cannot be directly applied to virus inactivation validation studies. In this paper, methods to detect viruses and progress in the challenge of differentiating infectious and noninfectious viruses with the combination of pretreatment and qPCR techniques such as the integrated cell culture‐qPCR (ICC‐qPCR) method are reviewed. In addition, the advantages and disadvantages of each new method, as well as its prospect in virus inactivation validation studies, are discussed.
In this review, the basic concept about virus infectivity and mechanisms of virus inactivation, and the methods which currently were investigated for virus detection or virus inactivation validation studies were reviewed and their advantages and disadvantages, as well as the prospect to make it able to be applied in virus inactivation validation study of biogenic material‐based medical products, were discussed.
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how ...robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
Introdução: A identificação precisa e ágil das variantes circulantes do SARS-CoV-2 é fundamental para uma vigilância epidemiológica eficaz e para direcionar medidas de contenção da disseminação do ...vírus. Embora o Sequenciamento de Genoma Total (Whole Genome Sequencing - WGS) seja considerado o padrão ouro para a identificação de variantes, apresenta alto custo, tempo de entrega dos resultados prolongado e necessidade de equipe especializada. Por outro lado, técnicas baseadas na amplificação de ácidos nucleicos, como a transcrição reversa seguida de reação de polimerase em cadeia (RT-qPCR), têm a capacidade de identificar variantes por meio de mutações específicas de maneira mais rápida e econômica. Objetivo: Avaliar a concordância entre um kit comercial de genotipagem por RT-qPCR e o WGS na identificação de variantes do SARS-CoV-2. Métodos: Foram selecionadas 349 amostras positivas para SARS-CoV-2 de laboratórios públicos e privados da 4ª Coordenadoria Regional de Saúde do estado do RS, coletadas nas semanas epidemiológicas 13 a 27 de 2022. Essas amostras foram submetidas a RT-qPCR utilizando o kit 4Plex para a detecção de variantes de preocupação desenvolvido pelo Biomanguinhos. Além disso, as amostras foram sequenciadas nas plataformas MinION MK1C ou Illumina iSeq100. As sequências consenso foram geradas utilizando os protocolos de bioinformática ARTIC nCoV-2019 (MinION) ou Dragen COVID (Illumina). Os clados e linhagens foram atribuídos utilizando as ferramentas Nextclade e Pangolin, respectivamente. Resultados: No sequenciamento, 316 amostras foram classificadas como Ômicron, sendo a maioria pertencente à subvariante BA.2 (238 amostras). 33 amostras foram identificadas como variantes recombinantes, sendo a maioria da subvariante XAG (31 amostras). Na genotipagem por RT-qPCR, todas as variantes Ômicron foram identificadas corretamente, no entanto, não foi possível a identificação das variantes recombinantes. O Kappa de Cohen indicou 90,54% de concordância da RT-qPCR com o WGS. No entanto, não foi possível diferenciar as subvariantes utilizando a RT-qPCR. Conclusão: O RT-qPCR é uma metodologia rápida e econômica. No entanto, possui baixo poder discriminatório, sendo incapaz de identificar subvariantes e variantes recombinantes. Portanto, é necessário realizar o sequenciamento para obter essas informações. Assim, o RT-qPCR pode ser utilizado como uma metodologia complementar ao WGS para um rastreamento abrangente e mais rápido das variantes em circulação.
Multiple sclerosis (MS) is an autoimmune disease of the central
nervous system (CNS) caused by auto-reactive T cells against
myelin antigens. T-cell immunoglobulin mucin -3 (TIM-3) is a
negative ...regulator glycoprotein expressed by a range of immune
cells. A defect in TIM-3 regulation has been shown in multiple
sclerosis patients. This study was planned to investigate the
correlation between serum TIM-3 and TIM-3 gene expression in
a sample of Multiple Sclerosis Iraqi patients. Three ml of blood
samples were collected from fifty Iraqi patients who have
Multiple Sclerosis (men and women) with ages ranging between
20-57 years, and 50 healthy volunteers as a control group;
0.25ml of blood was put in Trizol tube for RNA extraction,
subsequently to estimate TIM-3 gene expression by one step
RT-qPCR, and 2.75 ml of blood placed into gel tube for
determination TIM-3 serum level by enzyme-linked
immunosorbent assay (ELISA), the Statistical analysis was done
by using program of Statistical Analysis System (SAS). There
was a significant increase (P ≤ 0.05) in TIM-3 gene expression
for patients (5.30-fold) when compared with control (7.86-fold).
Moreover, the result demonstrated a high significant elevated (P
≤ 0.01) in TIM-3 serum level of patients (0.398 pg/ml) as
compared to control (3.17 pg/ml. Furthermore, the findings
showed a strong positive association between TIM-3 serum
level and TIM-3 mRNA expression with significant differences.
The current study concluded that the TIM-3 gene expression and
TIM-3 serum level were high in MS patients, and there was a
direct positive relationship between TIM-3 gene expression and
TIM-3 serum level.
Keywords: MS, TIM-3, RT-qPCR.,ELISA
The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this ...proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96–7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08–6.46 log10 GC/L), RhV A (3.95–5.63 log10 GC/L), RhV B (3.74–5.61 log10 GC/L), and PeV (3.17–5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.
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•13 respiratory viruses were detected in four WWTPs in Queensland, Australia.•BoV, PeV, RhV A and RhV B were detected in all wastewater samples.•Wastewater IAV and RSV GC concentrations correlated with clinical cases.•12–17 days lead times were observed for IAV and RSV in wastewater samples.•The PPV for the presence of IAV and RSV in wastewater was 97 % for clinical cases.
To overcome drawbacks of M. ulcerans culture in terms of incubation time and low sensitivity for the detection of viable bacilli from clinical specimens, a highly sensitive and M. ulcerans-specific ...RNA-based viability assay was developed. The assay combines a 16S rRNA reverse transcriptase real-time PCR (16S rRNA RT qPCR) to determine bacterial viability with an IS2404 quantitative real-time PCR (IS2404 qPCR) to ensure specificity as well as simultaneous quantification of bacilli. This proved to be highly efficient in detecting viable bacilli in clinical samples when implemented in the field.
Food and feed are daily exposed to mycotoxin contamination which effects may be counteracted by functional compounds like carotenoids and fermented whey. Among mycotoxins, the most toxic and studied ...are aflatoxin B1 (AFB1) and ochratoxin A (OTA), which neurotoxicity is not well reported. Therefore, SH-SY5Y human neuroblastoma cells ongoing differentiation were exposed during 7 days to digested bread extracts contained pumpkin and fermented whey, individually and in combination, along with AFB1 and OTA and their combination, in order to evaluate their presumed effects on neuronal differentiation. The immunofluorescence analysis of βIII-tubulin and dopamine markers pointed to OTA as the most damaging treatment for cell differentiation. Cell cycle analysis reported the highest significant differences for OTA-contained bread compared to the control in phase G0/G1. Lastly, RNA extraction was performed and gene expression was analyzed by qPCR. The selected genes were related to neuronal differentiation and cell cycle. The addition of functional ingredients in breads not only enhancing the expression of neuronal markers, but also induced an overall improvement of gene expression compromised by mycotoxins activity. These data confirm that in vitro neuronal differentiation may be impaired by AFB1 and OTA-exposure, which could be modulated by bioactive compounds naturally found in diet.
•OTA was the most damaging treatment for TUBB3 and dopamine fluorescent expression.•Cell cycle alterations was mainly reported in OTA-contained bread in phase G0/G1.•The most altered genes were cyclins B, D and Wnt5a upon mycotoxins exposure.•Bioactive compounds improved neuronal markers expression and cell cycle disruption.•The alteration in gene expression was modulated by functional ingredients.
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