Problem
The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the ...laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad‐range real‐time polymerase chain reaction with electrospray ionization time‐of‐flight mass spectrometry (PCR/ESI‐MS) to identify and quantify genomic material from bacteria and viruses.
Method of study
AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor and intact membranes (PTL) were analyzed using cultivation techniques (aerobic, anaerobic, and genital mycoplasmas) as well as PCR/ESI‐MS. The prevalence and relative magnitude of intra‐amniotic inflammation AF interleukin 6 (IL‐6) concentration ≥ 2.6 ng/mL, acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined.
Results
(i) The prevalence of MIAC in patients with PTL was 7% using standard cultivation techniques and 12% using PCR/ESI‐MS; (ii) seven of ten patients with positive AF culture also had positive PCR/ESI‐MS ≥17 genome equivalents per PCR reaction well (GE/well); (iii) patients with positive PCR/ESI‐MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra‐amniotic inflammation and acute histologic chorioamnionitis, a shorter interval to delivery median (interquartile range‐IQR), and offspring at higher risk of perinatal mortality, than women with both tests negative 90% (9/10) versus 32% (39/122) OR: 5.6; 95% CI: 1.4–22; (P < 0.001); 70% (7/10) versus 35% (39/112); (P = 0.04); 1 (IQR: <1–2) days versus 25 (IQR: 5–51) days; (P = 0.002), respectively; (iv) there were no significant differences in these outcomes between patients with positive PCR/ESI‐MS (≥17 GE/well) who had negative AF cultures and those with positive AF cultures; and (v) PCR/ESI‐MS detected genomic material from viruses in two patients (1.4%).
Conclusion
(i) Rapid diagnosis of intra‐amniotic infection is possible using PCR/ESI‐MS; (ii) the combined use of biomarkers of inflammation and PCR/ESI‐MS allows for the identification of specific bacteria and viruses in women with preterm labor and intra‐amniotic infection; and (iii) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection‐induced preterm birth.
The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based ...tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.
Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate ...verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.
We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute ...respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.
Purpose
The purpose of this study was to detect severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) ribonucleic acid (RNA) in urine and blood specimens, and anal and oropharyngeal swabs from ...patients with confirmed SARS‐CoV‐2 infection, and correlated positive results with clinical findings.
Methods
Patients with confirmed SARS‐CoV‐2 infections were included in this study. Patients' demographic and clinical data were recorded. Quantitative real‐time polymerase chain reaction was used to detect SARS‐CoV‐2 RNA in urine and blood specimens, and anal and oropharyngeal swabs. The study is registered at ClinicalTrials.gov (No. NCT04279782, 19 February, 2020).
Results
SARS‐CoV‐2 RNA was present in all four specimen types, though not all specimen types were positive simultaneously. The presence of viral RNA was not necessarily predictive of clinical symptoms, for example, the presence of viral RNA in the urine did not necessarily predict urinary tract symptoms.
Conclusions
SARS‐CoV‐2 can infect multiple systems, including the urinary tract. Testing different specimen types may be useful for monitoring disease changes and progression, and for establishing a prognosis.
Highlights
SARS‐CoV‐2 can be detected in multiple system specimens but without necessarily corresponding dysfunction. Further study is needed to determine the virus ability to destroy different organs.
Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts ...invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a ...diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10
50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and ...real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.
Abstract
Background
Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and ...management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.
Methods
We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT.
Results
Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.
Conclusion
Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.
Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data ...normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data.
In this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization.
A workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.
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