Bioinspired spiky-like double yolk-shell structured TiO2@ZnIn2S4 for efficient photocatalytic CO2 reduction

Photocatalytic carbon dioxide (CO2) reduction (PCR) into syngas is one of the sustainable approaches for recycling CO2 into value-added chemical feedstock. However, the PCR efficiency is often limited by the...

Use and Misuse of Cq in qPCR Data Analysis and Reporting

In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.

A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

Nano-Forensic: New Perspective and Extensive Applications in Solving Crimes

Nanotechnology has continued to prove its dominance with vast applications to traditional methods in medical, electronic, engineering, and other fields. In forensic science, nanotechnology research has provided a new perspective for real-time crime investigation and developed advanced nanosensors, nano-manipulators, and nano-imaging tools for visualization. Often, nanotechnology aids in enhancing and improving the efficiency of already existing and applied forensic techniques with high accuracy, sensitivity, and reducing the time required. So, this paper reviews the vital applications of classic tools of nanotechnology for examining questioned documents, age of bloodstains, and time since death, along with its application in improving PCR efficiency, DNA analysis, and explosive detection. It also attempts to highlight the use of advanced nanotechnology instruments for illicit drug screening. Nano-based techniques hold an immense future in fingerprinting and security features. Therefore, this paper also offers insights into the applications of nanoparticles in detecting latent fingerprints.

qPCR multiplex detection of microRNA and messenger RNA in a single reaction

Reverse Transcription-Quantitative PCR (RT-qPCR) is one of the standards for analytical measurement of different RNA species in biological models. However, current Reverse Transcription (RT) based priming strategies are unable to synthesize differing RNAs and ncRNAs especially miRNAs, within a single tube. We present a new methodology, referred to as RNAmp, that measures in parallel miRNA and mRNA expression. We demonstrate this in various cell lines, then evaluate clinical utility by quantifying several miRNAs and mRNA simultaneously in sera. PCR efficiency in RNAmp was estimated between 1.8 and 1.9 which is comparable to standard miRNA and random primer RT approaches. Furthermore, when using RNAmp to detect selected mRNA and miRNAs, the quantification cycle (Cq) was several cycles lower. This low volume single-tube duplex protocol reduces technical variation and reagent usage and is suitable for uniform analysis of single or multiple miRNAs and/or mRNAs within a single qPCR reaction.

SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

Variation in PCR Efficiencies between Quantification Standards and Clinical Specimens using Different Real-Time Quantitative PCR Interpretation Methods

The analysis of the Ct and standard curve produced by real-time polymerase chain reaction (PCR) is a well-established method for the quantification of nucleic acids. However, this method assumes that the PCR efficiency between the unknown specimen and standard is equal, resulting in the possibility of significant inaccuracies due to the presence of inhibitory agents in the unknown specimen. Although numerous methods have been proposed to correct this issue, the understanding of the differences in PCR efficiencies in clinical samples is limited. In this study, 1185 cytomegalovirus (CMV) DNA real-time PCR test results from 106 batches were analyzed. The PCR efficiencies were calculated using the cpD2, maxE, Cy0, maxRatio and window-of-linearity (WoL) methods. The concentrations were calculated using the cpD2, Cy0, maxRatio, WoL, and take off point (TOP) methods. The coefficient of variation (CV) in the efficiency of the quantification standards was less than 5% in all methods. Positive samples with high quantification values demonstrated lower PCR efficiency compared to the quantification standards. This suggests possible inaccuracies in quantification using quantification standards in clinical samples.

Comparison of commercial RT-PCR diagnostic kits for COVID-19

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.