The use of RT-LAMP (reverse transcriptase-loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the ...RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.
Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection, which does great harm to the poultry industry all over the world. To diagnose the ...disease simply and quickly, 2 detection methods were established based on reverse transcription recombinase–aided amplification (RT-RAA) technology. One is reverse transcription recombinase–aided amplification-lateral flow dipstick (RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick; the other is real-time fluorescence-based reverse transcription recombinase–aided amplification (RF-RT-RAA) that is the combination of RT-RAA and exo probe. In this study, the reaction conditions such as reaction temperature and reaction time of the 2 methods were optimized, and their specificity and sensitivity were tested. The results showed that the RT-RAA-LFD method could be used to complete reaction within 23 min, and its lowest detectable limit was 102 copies/μL, 10 times higher than that of the conventional PCR method (103 copies/μL); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/μL, 100 times higher than that of conventional PCR method (103 copies/μL), and it was as sensitive as real-time fluorescence–based quantitative PCR (10 copies/μL). The 2 methods had no cross reaction to the nucleic acid of other avian pathogens and showed good specificity. A total of 86 clinical samples suspected of the Newcastle disease virus were tested by conventional PCR, real-time fluorescence–based quantitative PCR, RT-RAA-LFD, and RF-RT-RAA. Based on the commonly used conventional PCR method, the other 3 detection methods had a coincidence rate of higher than 93%. In summary, RT-RAA-LFD and RF-RT-RAA had high specificity, sensitivity, and efficiency, which were suitable for clinical and laboratory diagnosis, respectively, and provided technical support for the prevention and control of Newcastle disease.
Since the tumor is extremely heterogeneous, a single biomarker cannot reflect the exact symptoms of the disease or its stage. Exosomes are biomarker reservoirs that provide disease information with a ...high accuracy, especially when specific markers, including microRNAs (miRNAs) and proteins, are combined. However, currently available exosomal miRNA and protein detection methods are time consuming, expensive, and laborious. Meanwhile, simultaneous detection of an exosomal miRNA and protein in a single reaction is even more challenging. Thus, development of an efficient method for detecting multiple miRNAs and proteins in a single exosomal reaction is highly needed. Herein, to increase the value of using exosomes over other circulating biomarkers for prostate cancer (PCa) liquid biopsy, a method for simultaneous multiplexed in situ detection of exosomal miRNAs and proteins was developed. Exosomal miRNAs and surface proteins were simultaneously detected in captured exosomes with a high specificity, using nano-sized molecular beacons and fluorescent dye-conjugated antibodies. The method allowed the quantitative analysis of various disease-specific miRNAs and surface proteins in PCa cell-derived exosomes in a single exosomal reaction. Overall, simultaneous multiplexed in situ detection of exosomal miRNAs and surface proteins can be developed as a simple, cost-effective, non-invasive liquid biopsy method for diagnosing PCa.
•Simultaneous multiplexed in situ detection of exosomal miRNAs and proteins is achieved.•The method allowed the analysis of disease-specific miRNAs and surface proteins in exosomes in a single reaction.•The method offers a simple, cost-effective, and non-invasive liquid biopsy for diagnosing prostate cancer.
Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC ...cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Although preeclampsia is a common and serious complication of pregnancy, insight into its pathobiology and diagnosis is lacking. Circulating plasma exosomes, which contain RNA and other molecules and ...have recently become accessible for diagnostics, may be informative in this regard. We tested the hypothesis that preeclampsia may affect the miRNA cargo within circulating maternal blood exosomes. We collected plasma from 60 pregnant women at term, including 20 women with pregnancy complicated by preeclampsia, and 20 women with fetal growth restriction and 20 with healthy pregnancy, serving as controls. We isolated exosomes from the maternal plasma by continuous density gradient ultracentrifugation. Our main outcome variable was exosomal miRNA cargo, analyzed by quantitative polymerase chain reaction-based TaqMan advanced miRNA assay in a card format and the expression of differentially expressed exosomal miRNA in whole plasma from the same participants. We found that 7 miRNA species were differentially expressed in exosomes from women with preeclampsia and those from controls. In contrast, there was no significant difference in exosomal miRNA expression between women with fetal growth restriction and controls. The results were not affected by fetal sex. Only one of the preeclampsia-related, differentially expressed exosomal miRNAs was significantly different in whole plasma miRNA analysis. We concluded that unlike whole plasma miRNA, exosomes extracted from the plasma of women with preeclampsia exhibit a unique miRNA profile, suggesting that plasma exosomal miRNA could provide insight into the pathophysiology of preeclampsia, and may play a role in disease diagnostics.
Abstract Background Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective ...capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSCGATA-4 ) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells. Methods and results Exosomes were isolated and purified from MSCGATA-4 (ExoGATA-4 ) and control MSCs (ExoNull ). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in ExoGATA-4 . Rapid internalization of ExoGATA-4 by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with ExoGATA-4 compared to those treated with ExoNull . The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with ExoGATA-4 , which resulted in the activation of the Akt and ERK signaling pathways. Conclusions ExoGATA-4 upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.
Background
Detection of Helicobacter pylori in gastric biopsy is important for appropriate treatment and prevention of gastric carcinoma and lymphoma. A novel peptide nucleic acid probe (PNA)‐based ...real‐time polymerase chain reaction (PCR) method was developed for detection of H pylori and A2142G/A2143G mutation of the 23S rRNA gene, which is associated with clarithromycin resistance.
Methods
To evaluate the performance of the PNA probe‐based PCR method, a total of 409 gastric biopsy samples were analyzed by PNA probe‐based PCR and compared with other H pylori detection methods, including hematoxylin and eosin (HE) and Warthin‐Starry (WS) staining, immunohistochemistry (IHC). A2142G/A2143G mutation of the 23S rRNA gene was tested by dual priming oligonucleotide (DPO)‐based PCR and Sanger sequencing to evaluate PNA probe‐based PCR.
Results
Among 271 cases that were positive for H pylori on IHC which was considered as a standard method, 264 cases (97.4%) and 259 cases (95.6%) were positively detected by HE/WS and PNA probe‐based qPCR, respectively. Of 100 H pylori‐positive patients tested by IHC, H pylori was detected in 93 cases (93.0%) by PNA probe‐based PCR, 86 cases (86.0%) by DPO‐based PCR, and 93 cases (93.0%) by conventional PCR. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PNA probe‐based qPCR were 93.0%, 94.9%, 93.9%, 94.9%, and 93.0%, respectively, which were all higher than those of DPO‐based PCR. When Sanger sequencing was determined as a standard method to detect A2142G/A2143G mutations, the sensitivity of the PNA‐ and DPO‐based methods was 100% and 94.4%, respectively, and the specificity was 100% for both methods.
Conclusion
PNA probe‐based qPCR is an appropriate method for detecting H pylori and the clarithromycin resistance‐associated mutation type.
Objectives
This study aimed to evaluate the diagnostic usefulness of real‐time (RT) polymerase chain reaction (PCR) on blood samples for diagnosis of invasive aspergillosis and mucormycosis in ...patients with suspected invasive mould infection.
Methods
Adult patients with suspected invasive mould infection were prospectively enrolled at a tertiary referral hospital in Seoul, South Korea between 2017 and 2020. Standard tests for diagnosis of invasive mould infection and RT‐PCR for Aspergillus, Mucor and Rhizopus using blood samples were performed. We evaluated the diagnostic performance of RT‐PCR tests in patients diagnosed with proven and probable invasive aspergillosis or mucormycosis infection, according to the modified definitions of the EORTC/MSG 2019.
Results
A total of 102 patients with suspected invasive mould infection were enrolled. Of these patients, 46 (45%) were classified as having proven (n = 13) or probable (n = 33) invasive aspergillosis, 21 (21%) as proven (n = 17) or probable (n = 4) invasive mucormycosis and 18 (18%) as possible invasive mould infection. The remaining 13 (13%) were classified as not having invasive mould infection. Patients with possible invasive mould infection (n = 18) and coinfection of aspergillosis and mucormycosis (n = 4) were excluded from the final analysis. The sensitivity and specificity of the Aspergillus PCR were 54.3% (25/46, 95% confidence interval CI: 40.2–67.9%) and 94.1% (32/34, 95% CI: 80.9–98.4%), respectively. The sensitivity and specificity of the Mucor or Rhizopus PCR were 57.1% (12/21, 95% CI: 36.6–75.5%) and 76.3% (45/59, 95% CI: 64.0–85.3), respectively.
Conclusions
Our study suggests that blood PCR can be a useful adjunct test for diagnosing patients with suspected invasive mould infection.
The epithelial lining of the fallopian tube is of critical importance for human reproduction and has been implicated as a site of origin of high-grade serous ovarian cancer. Here we report on the ...establishment of long-term, stable 3D organoid cultures from human fallopian tubes, indicative of the presence of adult stem cells. We show that single epithelial stem cells in vitro can give rise to differentiated organoids containing ciliated and secretory cells. Continuous growth and differentiation of organoids depend on both Wnt and Notch paracrine signalling. Microarray analysis reveals that inhibition of Notch signalling causes downregulation of stem cell-associated genes in parallel with decreased proliferation and increased numbers of ciliated cells and that organoids also respond to oestradiol and progesterone treatment in a physiological manner. Thus, our organoid model provides a much-needed basis for future investigations of signalling routes involved in health and disease of the fallopian tube.
Gene Expression Analysis in Bacteria by RT-qPCR Rocha, Danilo J P G; Castro, Thiago L P; Aguiar, Eric R G R ...
Methods in molecular biology (Clifton, N.J.),
01/2020, Letnik:
2065
Journal Article
Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through ...relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.
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