The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth ...factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories' expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required. Areas covered: The EGFR mutation testing landscape is manifold and includes both screening and targeted methods, each with their own pros and cons. Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its liquid-biopsy applications. Expert commentary: The Roche cobas® EGFR mutation test v2, based on real time RT-PCR, is a reliable option for testing EGFR mutations in clinical practice, either using tissue-derived DNA or plasma-derived cfDNA. This application will be valuable for laboratories with whose purpose is purely diagnostic and lacking high-throughput technologies.
OBJECTIVE: To determine whether microRNAs are differentially expressed in men with normal versus impaired spermatogenesis, and to find a biomarker for accurate diagnosis of male infertility. DESIGN: ...Microarray with real-time polymerase chain reaction (RT-PCR) validation. SETTING: University research and clinical institutes. PATIENT(S): Male partner of selected couples (n = 27) who were undergoing assisted reproduction techniques for infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Statistically significantly altered microRNA expression profiles in normozoospermic versus asthenozoospermic and oligoasthenozoospermic men. RESULT(S): There were 50 miRNAs up-regulated and 27 miRNAs down-regulated in asthenozoospermic males. In oligoasthenozoospermic males, 42 miRNAs were up-regulated and 44 miRNAs down-regulated when compared with normozoospermic males. The miRNAs that exhibited the highest fold changes and area under the receiver operating characteristic curve were miR-34b, miR-122, and miR-1973 in samples from asthenozoospermic men and miR-34b, miR-34b*, miR-15b, miR-34c-5p, miR-122, miR-449a, miR-1973, miR-16, and miR-19a in samples from oligoasthenozoospermic men. Furthermore, quantitative RT-PCR assays on specific miRNAs, including miR-141, miR-200a, miR-122, miR-34b, miR-34c-5p, and miR-16, yielded results that were largely consistent with the microarray data. CONCLUSION(S): Our results reveal an extended number of miRNAs that were differentially expressed in asthenozoospermic and oligoasthenozoospermic males compared with normozoospermic males. These data provide evidence for analysis of miRNA profiles as a future diagnosing tool for male infertility.
Trichuriasis is a serious threat to the economic development of animal husbandry. This research aimed to establish a droplet digital PCR (ddPCR) method to detect Trichuris spp. for the early ...diagnosis and prevention of trichuriasis in sheep. The real-time quantitative PCR (qPCR) and ddPCR methods were used for the detection of nematodes by targeted amplification of the ITS gene. Each means was evaluated to optimize the limit of detection and reproducibility. For a recombinant plasmid, the qPCR results showed that the detection limit was 31.7 copies per reaction. In contrast to qPCR, ddPCR was able to detect concentrations below 3.17 copies per reaction. Both assays exhibited good reproducibility. However, the ddPCR method was more stable for low-copy-number detection. This new assay was specific for Trichuris spp. and did not cross-react with other relevant gastrointestinal nematodes. A total of 98 clinical samples were tested with both assays. The results showed that the positive rate of ddPCR (80.6%) was higher than that of qPCR (72.4%). This method could be used as an efficient molecular biology tool to test for Trichuris spp. and could be a new valuable tool for the clinical diagnosis and prevention of trichuriasis.
Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted ...to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.
RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up ...experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 μg/μL - 0.24 ng/μL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 μL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.
•The variation pattern between different amounts of RNA template inputs and the RT yield was assessed using ddPCR.•125 ng of RNA template input had the highest RT yield in a 10 μL RT reaction system with the selected kit.•RNA extracted from stained samples was below 125 ng, so add as much RNA as possible in the RT step.•Systematic experiments should be performed to determine the amount of RNA input at the optimal RT yield for any selected kit.
We present a self-discretization and zero-water-loss microfluidic digital PCR (dPCR) device to enable low-cost and robust quantitative nucleic acid assays. In this device, a thin void is integrated ...beneath the reaction chamber array. By utilizing the permeability of polydimethylsiloxane (PDMS) film, the integrated void serves a dual function: vacuum “accumulator” and hydration “reservoir”. The combination of pre-stored pumping energy and water compensation for evaporation loss enables simple, robust and reliable single-DNA-molecule amplification and detection in 10,000 reactors of picoliter volume. Compared to the conventional degassing PDMS pumps, the vacuum accumulator exhibits superior performance due to more vacuum storage and shorter diffusion distance. We also evaluated the performance of the embedded hydration layer in suppressing evaporation loss at elevated temperatures, and verified that zero-water-loss could be achieved for all reaction chambers in our dPCR chip during thermal cycling. By performing quantitative detection of T790M DNA from 10 to 104 copies/μL, the proposed dPCR chip demonstrated high accuracy and excellent performance for the absolute quantification of the target gene with a dynamic range of 104. The simplicity and robustness of our dPCR chip make it well suited for low-cost molecular diagnostic assays under resource-limited settings.
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•A self-digitization and zero-water-loss microfluidic digital PCR (dPCR) chip is developed.•A dual function void is integrated in the dPCR chip.•The integrated dual function void serves as vacuum accumulator and hydration reservoir.•Our dPCR chip can provide low-cost and robust quantitative nucleic acid assays.
Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key ...experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.
The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of ...molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.
External quality assessment (EQA) is essential for ensuring reliable test results, especially when laboratories are using assays authorized for emergency use for newly emerging pathogens. We ...developed an EQA panel to assess the quality of real-time reverse transcription PCR assays being used in South Korea to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the participation of 23 public health organization laboratories and 95 nongovernmental laboratories involved in SARS-CoV-2 testing, we conducted qualitative and semiquantitative performance assessments by using pooled respiratory samples containing different viral loads of SARS-CoV-2 or human coronavirus OC43. A total of 110 (93.2%) laboratories reported correct results for all qualitative tests; 29 (24.6%) laboratories had >1 outliers according to cycle threshold values. Our EQA panel identified the potential weaknesses of currently available commercial reagent kits. The methodology we used can provide practical experience for those planning to conduct evaluations for testing of SARS-CoV-2 and other emerging pathogens in the future.
Abstract
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel β-coronavirus, causes severe pneumonia and has spread throughout the globe rapidly. The disease associated ...with SARS-CoV-2 infection is named coronavirus disease 2019 (COVID-19). To date, real-time reverse-transcription polymerase chain reaction (RT-PCR) is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection.
Methods
In this study, we developed a peptide-based luminescent immunoassay that detected immunoglobulin (Ig)G and IgM. The assay cutoff value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2.
Results
To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively.
Conclusions
Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19.
A peptide-based magnetic chemiluminescence enzyme immunoassay for the detection of SARS-CoV-2 antibodies was developed; 71.4% (197 of 276) and 57.2% (158 of 276) of the COVID-19 inpatients were positive for IgG and IgM against SARS-CoV-2.
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