Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be ...relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
qPCR is more complex than perceived by many scientists.
The production of an amplification curve and an associated quantitative cycle value does not necessarily mean interpretable data.
The MIQE guidelines and associated methodology articles published thereafter, underline the ongoing drive to help scientists produce reproducible data from qPCR, culminating in a simple, stepwise methodology to ensure high-quality, reproducible data from qPCR experiments.
The concept of data normalization has led to the ongoing publication of articles solely focused on this subject for various sample types and experimental parameters.
The analysis of qPCR data can be challenging, especially as experiments grow in sample number and complexity of biological groups. A defined approach to qPCR data analysis is necessary to clarify gene expression analysis.
Introdução/objetivos: As gastroenterites agudas (GEA) são doenças que se constituem como a segunda maior causa de morte em crianças de todo mundo, especialmente em crianças menores de cinco anos, ...ocorrendo principalmente em países de baixa e média renda. Os agentes virais como rotavírus e norovírus têm sido demonstrados como as causas mais frequentes, no entanto alguns membros da família Picornaviridae também foram associados à diarreia em humanos. Diante disto, objetivou-se a realização da vigilância expandida dos picornavírus em amostras fecais de crianças com quadro de gastroenterite aguda em estados da região Norte e Nordeste do Brasil entre os anos de 2020 e 2021. Métodos: Para isso, foram utilizadas suspensões fecais provenientes de crianças com quadro de gastroenterite aguda e encaminhadas ao Laboratório de Enterovírus do Instituto Evandro Chagas através das redes de vigilância de rotavírus e póliovirus nos anos de 2020 e 2021. Para detecção foi realizada a extração do RNA viral utilizando o QIAmp Viral RNA Mini Kit (QIAGEN), posteriormente foi realizada a reação de RT-qPCR utilizando o kit comercial GoTaq Probe 1-Step RT-qPCR System (Promega) e oligonucleotídeos e sondas específicos para detecção de Parechovirus, Aichivirus e Cosavirus. Foram analisadas 419 amostras de suspensão fecal, nas quais 31,7% (133) foram positivas para parechovirus (HpeV), aichivírus (AiV) e cosavírus (CosV). Resultados: Com isso, foi observado à prevalência dos CosV, os quais estavam presentes em 57,1% (76/133) das amostras, seguido pelo HpeV com 37% (49/133) e por fim os AiV com 6% (8/133). Foram identificadas 18 codetecções entre os vírus investigados, sendo possível observar de HpeV com CosV em 13 amostras, duas codetecções entre AiV e CosV e três codetecções entre os três vírus investigados. Conclusão: Diante do exposto, conclui-se que é importante a vigilância desses vírus para fomentar estudos que visem comprovar sua atuação e comportamento como agentes causadores de gastroenterite, visto que sua prevalência em amostras vem se tornando cada vez mais prevalente e a escassez de estudos no mundo e no Brasil com esse objetivo acabam postergando esses resultados, por isso torna-se de extrema importância à continuidade dessa pesquisa nas regiões norte e nordeste, por conta de sua alta prevalência, para saber sua atuação e comportamento como agentes gastroentéricos.
•The amount of material collected by nasopharyngeal swabs is imprecise.•The determinations of SARS-CoV-2 viral loads from Cts ignore this error source.•SARS-CoV-2 Cts should be normalized with an ...internal marker to correct this errors.
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This study identified the occurrence of Holocene chronology hiatuses in the sedimentary record of the Patos-Mirim system caused by river avulsion processes, as well as evidence of sharp anthropogenic ...changes in the surrounding region of the lagoon water bodies. The presence of chronology hiatuses demonstrates the importance of considering the disturbance effect of paleo-drainage processes on the paleoenvironmental resolution and expression of the sedimentary record of such coastal plains. Anthropogenic activities especially those related to agriculture and forestry have increased significantly during the great acceleration, resulting in modifications of both the landscape and the environmental conditions of the lagoon bodies. Such impacts were clearly reflected in the sedimentary record where abrupt changes in palynological trends, sedimentary DNA, isotopic and granulometric analyses were inferred. The climatic conditions combined with regressive sea level can explain the chronology hiatuses. Pollen analyses demonstrated changes in the landscape, particularly indicated by the shift from Cyperaceae to Poaceae dominance after the 1960 CE. Therefore, all proxies together demonstrate the modification in the landscape and changes in the environment, clearly influenced by anthropogenic action from unsustainable agricultural practices.
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•The Patos-Mirim system is extensively dissected by Pleistocene/Holocene incised valleys.•The Holocene record is eroded by currents of meandering streams, which expression consisted of a Middle Holocene hiatus.•Novel palynological and 16S rRNA long-term data demonstrate changes in both landscape and lagoon anthropogenic transformation.
The prevalence of liver disease has led to its emergence as a major challenge. This challenge is also attributed to factors such as the challenging diagnosis, complexity of pathogenesis, and the ...absence of established therapies. When Hepatitis B virus (HBV) infections occurs in patients that do not have detectable hepatitis B surface antigen (HBsAg), it is referred to as occult infections. Despite the fact that these kinds of infections have been found in patients with chronic hepatitis C liver disease, there is still no evidence on their clinical implication and prevalence. HBV is a small partial deoxy ribonucleic acid (DNA) virus with like retroviruses. The virus falls under the group of Hepadnavirus infections and family of orthohepadna virus. About 66% of patients with acute HBV infection have an asymptomatic, mild, and sub-clinical illness that typically goes undetected. Thus, in this work, hematological parameters were used in detecting the virus, and the results of the hematological parameters revealed significant changes in white blood cell (WBC), lymphocyte and platelet area under curve (AUC) (0.95), (0.66), and (0.82). The mean values for ALT and ALP in HBV-positive patients increased significantly as compared to the control. Similarly, there was no significant difference between the AUC, CI for HBV-positivity for ALT 0.91, (0.8485-0.9854) and ALP 0.89 (0.8123-0.9633). This study revealed a significant positive correlation between ALP level and ALT and each lymphocyte, granulocyte and WBCs. Thus, each of them may be considered as major factors of development of HBV level.
Whole genome sequencing (WGS) is considered the best instrument to track both virus evolution and the spread of new, emerging variants. However, WGS still does not allow the analysis of as many ...samples as qPCR does. Epidemiological and clinical research needs to develop advanced qPCR methods to identify emerging variants of SARS-CoV-2 while collecting data on their spreading in a faster and cheaper way, which is critical for introducing public health measures. This study aimed at designing a one-step RT-qPCR assay for multiplex detection of the Omicron lineage and providing additional data on its subvariants in clinical samples. The RT-qPCR assay demonstrated high sensitivity and specificity on multiple SARS-CoV-2 variants and was cross-validated by WGS.
Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of ...sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly–CCC–1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly–CCC–1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly–CCC–1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.
•tsRNAs but not microRNAs are abundant in bovine preimplantation embryo conditioned medium (CM).•rsRNA-1044 is the most stable small non-coding RNA in the CM of the 2-cell embryo.•tDR-1:32-Gly–CCC–1 exhibited the best expression stability in the stages beyond the 2-cell stage.•bta-miR-155 and tDR-39:75-Arg-CCG-2 are significantly up-regulated in the CM of blastocysts compared to degenerates.
The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as ...the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.
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•Establishment of accurate and sensitive diagnostic detection of SARS-CoV-2 by reverse transcription digital PCR.•The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR.•The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively.•RT-dPCR is suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and close contacts.•The copy number of virus load was higher for aged patients, indicating a longer observation in the hospital may be needed.
The SARS‐CoV‐2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate ...the virus. Together, this calls for the development of safer methods for SARS‐CoV‐2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two‐step RT‐qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one‐step RT‐qPCR against SARS‐CoV‐2 using NP and OP samples. We furthermore tested a SARS‐CoV‐2 dilution series to determine the detection threshold. The method enables downstream detection of SARS‐CoV‐2 by RT‐qPCR with high sensitivity (~4 viral RNA copies per RT‐qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT‐qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR‐ready viral RNA and circumvents the need for commercial RNA purification kits.
Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic ...spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.
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•Optimized methods for SARS-CoV-2 detection in wastewater are highly needed.•This study compares different virus concentration method in municipal wastewater for subsequent qPCR detection of viral RNA•Evaluates recovery using spiked and internal reference viruses in municipal wastewater.•A modified ultrafiltration method is found to be the most sensitive for qPCR detection of SARS-CoV-2 in wastewater
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