Gene Probes Research Articles

SERS detection of DNA cancer with single-base mutation for KRAS-gene

We present a method based on surface enhanced Raman spectroscopy (SERS) to detect DNA KRAS-gene with single-base mutation. The KRAS gene belongs to the oncogenes class of genes. KRAS mutations cause normal cells to become cancerous. The detection utilizes DNA gene probes labeled by CY3 dye (as Raman marker) and plasmonic silver nanoparticles (Ag NPs) deposited on SERS substrates. SERS detect DNA via hybridization to the complementary DNA (target) sequences with single-base mutation. This SERS technique is a powerful tool and has a great potential gives both affectability and selectivity. Also, we utilized the Raman mapping to provide chemical information coupled with spatial information. The mapping image was obtained using scanning with three Raman bands over selected areas of the sample. The limit of detection (LOD) was accomplished down to a concentration 9 nM after the addition of magnesium sulfate (MgSO4) aggregated agent, causing hot spots of the aggregated Ag NPs.

Loss of iqgap1 (IQ Motif Containing GTPase Activating Protein 1) in Zebrafish Leads to Intracerebellar Hemorrhage, Morphological Abnormalities and Activates Hematopoietic Response

Defects in multiple cell signaling molecules lead to disruptions of vascular integrity given the need for fine-tuned regulation of the cell adhesion complexes. These genetic defects have been linked to the development of intracerebral hemorrhage (ICH). There is genetic evidence in humans for ICH due to some genetic variants, while other variants have been identified in preclinical animal models. Signaling adaptor proteins play a crucial role in cell signaling by promoting interactions between effector proteins and even enabling integration of different pathways. IQGAP1 is a conserved signaling adaptor known for its roles in cell adhesion, cancer and for other cell biological effects. We engineered a zebrafish null mutant in the zebrafish iqgap1 gene by introducing an 11-bp deletion using a CRISPR/Cas9 genome editing method and characterized its phenotype. Homozygous mutants exhibit severe brain hemorrhage and morphological abnormalities, which are ultimately lethal, in about 30-40% of cases, whereas the other embryos survive to adulthood. We visualized the expression pattern of iqgap1 relative to the established fli1a vascular marker and found that iqgap1 strongly overlapped with fli1a expression, but was expressed much more broadly in tissues, such as muscle, branchial arches, and the caudal hematopoietic tissue (equivalent to the mammalian fetal liver).Critically, iqgap1 exhibited co-localization with fli1a in the blood vessels of the central nervous system, whose disruption is likely responsible for the brain hemorrhage. Whole embryo RNA sequencing-based comparison of hemorrhage-positive iqgap1-/- embryo pools with wild-type embryos at 52 hours post-fertilization (hpf) shortly after the onset of hemorrhage identified approximately 800 differentially regulated genes. The most striking feature of this dataset was up-regulation of hematopoietic markers especially those of erythrocytes, neutrophils, mast cells and HSPCs (hematopoietic stem and progenitor cells), but not macrophages. We have confirmed by in situ hybridization with marker gene probes that erythrocyte and neutrophil production is up-regulated most strongly in iqgap1-/- embryos undergoing some level of hemorrhage. By contrast, fli1a endothelial and stem cell marker was downregulated. This animal model provides a compelling genotype-phenotype correlation, implicating IQGAP1 as a new player in vascular disorders such as ICH and identifying a previously unrecognized relationship between IQGAP and regulation of hematopoiesis. Furthermore, this model is now poised to identify ameliorating and exacerbating modifier lesions and potential therapeutic agents that restore normal vascular integrity and prevent ICH. DisclosuresRobitaille: Novartis: Consultancy. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

Genetic Variants in the Protein S ( PROS1 ) Gene and Protein S Deficiency in a Danish Population.

Protein S (PS) deficiency is a risk factor for venous thromboembolism (VTE) and can be caused by variants of the gene encoding PS ( PROS1 ). This study aimed to evaluate the clinical value of molecular analysis of the PROS1 gene in PS-deficient participants. We performed Sanger sequencing of the coding region of the PROS1 gene and multiplex ligation-dependent probe amplification to exclude large structural rearrangements. Free PS was measured by a particle-enhanced immunoassay, while PS activity was assessed by a clotting method. A total of 87 PS-deficient participants and family members were included. In 22 index participants, we identified 13 PROS1 coding variants. Five variants were novel. In 21 index participants, no coding sequence variants or structural rearrangements were identified. The free PS level was lower in index participants carrying a PROS1 variant compared with index participants with no variant (0.51 [0.32–0.61] vs. 0.62 [0.57–0.73] × 10 3 IU/L; p < 0.05). The p.(Thr78Met) variant was associated with only slightly decreased free PS levels (0.59 [0.53–0.66] × 10 3 IU/L) compared with the p.(Glu390Lys) variant (0.27 [0.24–0.37] × 10 3 IU/L, p < 0.01). The frequency of VTE in participants with a coding PROS1 variant was 43 and 17% in the group with normal PROS1 gene ( p = 0.05). In conclusion, we report 13 PROS1 coding variants including five novel variants. PS levels differ by PROS1 variant and the frequency of VTE was higher when a coding PROS1 variant was present. Hence, molecular analysis of the PROS1 gene may add clinical value in the diagnostic work-up of PS deficiency.

Alkaptonuria in Russia.

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

False-Negative Results in Taqman One-Step RT-PCR Test: Evaluation of Endogenous Internal Control Function Used in SARS-CoV-2 Detection Tests

Background: Taqman one-step real-time PCR (RT-PCR) has special importance due to its high sensitivity and specificity in the diagnosis of infectious diseases such as viral infections. In the recent pandemic of SARS-CoV-2, diagnostic kits based on this method are commonly used for molecular detection. One of the main systematic errors that misinterpret the results is using inaccurate internal control in RT-PCR diagnostic kits. Designing primers and probes that span exon-exon junction will avoid genomic DNA amplification and lead to obtaining high specific results. Objectives: This study aimed to evaluate the endogenous internal control of primers and probe for RNase P RNA to reduce false-negative results in respiratory samples. Methods: In this study, 30 samples of patients who were negative for SARS-CoV-2, influenza A, and influenza B were re-evaluated for SARS-CoV-2 using newly designed primers and probes for RNase P RNA (ultra-specific primers and probe). We also performed bioinformatics analysis on CDC-approved primers and probes of RNase P endogenous internal control. Results: In this analysis, we specified the location of these newly designed primers and probe on target mRNA and genomic DNA. Then, the Taqman one-step RT-PCR method was performed using both CDC-approved primers and probes along with our ultra-specific primers and probe for RNase P RNA. Based on bioinformatics analysis, the attachment sites of the CDC-approved primers and probe for endogenous internal control of RNase P are located on the first exon of this gene. In addition to identifying the target gene sequence, these primers and probe also non-specifically detect similar sequences on the genomic DNA. Conclusions: The present study showed that the use of specific primers and probes introduced by CDC for SARS-CoV-2 and influenza virus may cause false results due to non-specific binding to the genomic DNA. Therefore, choosing the right internal control for RNase P RNA can be useful in achieving very accurate results.

Cytogenetic findings of ectopic endometriotic tissue in women with endometriosis and review of the literature

ObjectiveTo determine chromosome and gene alterations in ectopic endometrial (EM) tissue which may be implicated in the clinical course or the progression of endometriosis and to review the literature concerning the cytogenetic findings of women with endometriosis. Study design15 women who underwent laparoscopic endometriosis surgery at the Athens Genesis Clinic were enrolled in the study. Ectopic endometrial tissue was surgically removed and further analyzed by conventional and molecular cytogenetic techniques. Fluoresent in situ hibridization (FISH) with probes for p53, ATM, MYC, MLL1 and IGH genes, the centromeres of chromosomes 7 and 8 and 7q22/7q31 chromosomal regions was carried out. ResultsKaryotypic analysis revealed no clonal chromosomal abnormalities. However, an increased frequency of polyploidy (55.6%) and sporadic chromosomal abnormalities (40.0%) concerning chromosomes 9, 11, 17 and X were noticed involving mainly deletions, trisomies or monosomies. FISH analysis showed IGH gene rearrangements in 54% of the EM cases and MLL gene rearrangements in 73% of the examined samples. Normal hybridization patterns were observed for p53, ATM and MYC. The increased frequency of polyploidy shown by conventional karyotyping was also confirmed by FISH. ConclusionPolyploidy, sporadic chromosomal abnormalities, as well as IGH and MLL gene rearrangements, may provoke genetic instability and play a potential role in the development of endometriosis. IGH and MLL gene rearrangements indicate a genetic relation between endometriosis and carcinogenesis. Confirmation of the above gene rearrangements in a large series of women may allow the determination of their possible involvement in the pathogenesis of this complex disease and their possible contribution in the early identification of women in danger for malignant transformation.

Comparisons of Gene Expression Indexes for Oligonucleotide Arrays

High density oligonucleotide arrays have become a standard research tool to monitor the expression of thousands of genes simultaneously. Affymetrix GeneChip arrays are the most popular. They use short oligonucleotides to probe for genes in an RNA sample. However, important challenges remain in estimating expression level from raw hybridization in tensities on the array. In this paper, we deal with the problem of estimating gene expression based on a statistical model. The present method is like Li and Wong model (2001a), but assumes more generality. More precisely, we show how the model introduced by Li and Wong can be generalized to provide new measure of gene expression. Moreover, we provide a comparison between these two models.

MOMC-3. Hypermethylation and overexpression of HOX genes are poor prognosticators in Lower-Grade Glioma

Abstract Diffuse gliomas represent over 80% of malignant brain tumors ranging from low-grade to aggressive high-grade lesions. Molecular characterization of these tumors led to the development of new classification system comprising specific glioma subtypes. While this provides novel molecular insight into gliomas it does not fully explain the variability in patient outcome. To identify and characterize a predictive signature of outcome in diffuse gliomas, we utilized an integrative molecular analysis (methylation, mRNA, copy number variation (CNV) and mutation data) using multiple molecular platforms, including a total of 310 IDH mutant glioma samples from University Health Network (UHN) and German Cancer Research Center (DKFZ) together with 419 samples from The Cancer Genome Atlas (TCGA). Cox regression analysis of methylation data from the UHN cohort identified CpG-based signatures that split the glioma cohort into two prognostic groups strongly predicting survival (p-value &amp;lt; 0.0001). The CpG-based signatures were reliably validated using two independent datasets from TCGA and DKFZ cohorts (both p-values &amp;lt; 0.0001). The results show that the methylation signatures that predict poor outcome also correlated with G-CIMP low status, elevated CNV instability and hypermethylation of a set of HOX gene probes. Further study in diffuse lower-grade glioma (LGG) using integrated mRNA and methylation (iRM) analyses showed that parallel HOX gene overexpression and hypermethylation in the same direction were significantly associated with increased mutational load, high aneuploidy and worse survival (p-value &amp;lt; 0.0001). Furthermore, this iRM high group was characterized by a 7-HOX gene signature showed significant survival differences not only in IDH mutant LGG but also in IDH wildtype LGG. These results demonstrate the importance of HOX genes in predicting the outcome of diffuse gliomas to identify relevant molecular subtyping independent of histology.

Abstract 327: High conversion library preparation with optimal hybridization capture panel design strategy in RNA-seq

Abstract RNA-seq is a powerful tool to detect tissue-specific gene expression, splicing, and genetic variations associated with disease states. However, current RNA-seq approaches have limitations due to poor signal from low-abundant transcripts. Furthermore, tissue-derived RNA samples are often highly degraded, thereby limiting gene detection and suffering from potential sequencing artifacts. Here we show that target capture of sequencing libraries tagged with unique molecular identifiers (UMIs) using the xGen™ Prism DNA library prep kit, optimized for low-input and degraded samples, can overcome these obstacles. Directional and UMI-tagged RNA-seq libraries were constructed with RNA extracted from a FFPE RNA Fusion Reference Standard and captured with different designs of the xGen Pan-Cancer Panel spiked with probes for fusion genes. The first design strategy used IDT's stocked Pan Cancer V1.5 panel targeted to gDNA coordinates. The second design involved extracting all associated RefSeq NM transcripts associated with the gene list. Probes were designed to each transcript and duplicate probes were removed based on exact sequence match. The third strategy leveraged a multi-strain design, which created probes from fasta inputs, but removed probes with 90% or greater homology. Normalized expression was highly correlated (&amp;gt; 85%) between captured and uncaptured samples regardless of rRNA depletion prior to library prep. Captured samples had a greater depth of coverage with over 90% on target bases. In addition, our panel design strategies identified low frequency fusions with deep sequencing regardless of rRNA depletion prior to library prep. The multi-strain design was more effective in reducing redundant capture probes compared the other design strategies. Enhanced coverage and PCR de-duplication with UMIs allowed us to reproducibly measure expression over a wide range of RNA inputs (5-500 ng). We show that target capture of RNA-seq libraries reliably maintains expression information present in uncaptured libraries while increasing coverage for poorly expressed genes and low frequency fusions. In addition, the target captured libraries without rRNA depletion prior to library prep have comparable on-target rate and target coverage with rRNA-depleted, target captured libraries. The addition of UMIs to differentiate between PCR duplicates and unique starting molecules also makes it possible to reliably analyze even highly amplified libraries. (For research use only). Citation Format: Tzu-Chun Chen, Katelyn Larkin, Shale Dames, Hsiao-Yun Huang, Kevin Lai, Jessica Sheu, Timothy Barnes, Katia Star, Manqing Hong, Bosun Min, Ryan Demeter, Ashley Dvorak, Ushati Das Chakravarty, Patrick Lau, Steven Henck. High conversion library preparation with optimal hybridization capture panel design strategy in RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 327.

A case of chlamydia psittaci caused severe pneumonia and meningitis diagnosed by metagenome next-generation sequencing and clinical analysis: a case report and literature review

BackgroundPsittacosis, which is also known as parrot fever, is Chlamydia psittaci (C. psittaci) caused infectious disease. The clinical manifestations vary from asymptomatic infection to severe atypical pneumonia or even fatal meningitis. Early recognition of psittacosis is difficult because of its nonspecific clinical manifestations. Culture and gene probe techniques for C. psittaci are not available for routine clinical use, which makes the diagnosis difficult too. Although psittacosis has increasingly been recognized and reported in recent years, cure of severe pneumonia complicated with meningitis, with etiologic diagnosis aided by the use of metagenomic next-generation sequencing (mNGS), is still uncommon. So, it is necessary to report and review such potentially fatal case.Case presentationThis report describes a 54-year-old woman with C. psittaci caused severe atypical pneumonia and meningitis. She presented with symptoms of fever, dry cough and dyspnea, accompanied by prominent headache. Her condition deteriorated rapidly to respiratory failure and lethargy under the treatment of empirical antibacterial agents, and was treated with invasive mechanical ventilation soon. She denied contact with birds, poultry or horses, but unbiased mNGS of both the bronchoalveolar lavage fluid (BALF) and the cerebrospinal fluid (CSF) identified sequence reads corresponding to C. psittaci infection, and there was no sequence read corresponding to other probable pathogens. Combined use of targeted antimicrobial agents of tetracyclines, macrolides and fluoroquinolones was carried out, and the patient’s condition improved and she was discharged home 28 days later. Her status returned close to premorbid condition on day 60 of follow-up.ConclusionsWhen clinicians come across a patient with atypical pneumonia accompanied by symptoms of meningitis, psittacosis should be taken into consideration. mNGS is a promising detection method in such condition and is recommended.

Microdeletion in Chromosome 17 Cytoband q21.31 Identified As Located within the KANSL1 Gene Prevailed in Flt3-ITD Negative Normal Karyotype AML Patients As Opposed to Their AML Counterparts and Influenced the Phenotype of the Disease

Recent advances in genetic tools for leukemic diagnosis have facilitated studies on genetic traits which in concert with a driving mutation if present may influence the phenotype of the disease. Flt3-ITD is present in about 30% of AML patients and tends to be associated with a poor prognosis. Currently the use of inhibitors of mutated and in consequence activated kinases is undergoing clinical trials. The dim prognosis of Flt3-ITD positive AML and the hope associated with kinases inhibitors prompted us to evaluate in some depth the genetics of AML cases in relation to the presence of Flt3-ITD .Ninety-two AML patients (diagnosed according to the FAB/WHO criteria, F/M=49/43, age median: 57, range: 21-81 years) who were seen in our institution were recruited to the present study which was conducted as follows:The patients underwent GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). 52 lacking GTG and/or FISH abnormalities were called as normal karyotype (NK) patients, among those, 20 were Flt3-ITD positive. The other 40 patients had karyotype abnormalities or were FISH positive for AML-associated abnormalities among which 10 had Flt3-ITD .In all 92 patients microarray analysis was performed using Agilent - Catalog Agilent Cancer CGH+SNP 180K or Roche - WG Catalog NimbleGene 12x270K microarrays.We found (Partec Genomics Suite and Biodiscovery Nexus 8 software) that NK Flt3-ITD negative had, when the whole genome was analyzed, a higher number of total copy number variation (CNV) aberrations (amplifications and/or deletions, vs 32.21±8.70 vs 16.15±2.60, p=0.008) including one copy losses, (2.79±0.57 vs 1.20±0.35 p=0.019)as opposed to NK patients having Flt3-ITD mutation .During the study, an interim analysis was performed at the time we switched from Roche to Agilent microarrays which revealed the significant prevalence of CNVs in AML Flt3-ITD negative patients over Flt3-ITD positive ones within the region 17q21.31 specifically in the positions from 44281092 to 4476549, 44702953 to 44745272 and 44745272 to 44765496. The same region appeared again as significantly discriminating Flt3-ITD negative and positive patients at the final analysis. Therefore, this region was analyzed in some depth. Hg19 annotation was used and according to that the region encompasses the KANSL1 gene among two others (NSFP1 and NSF). The NSF gene encodes a protein involved in transport to the Golgi and NSFP1 is a pseudogene. KANSL1, a component of the MLL1 complex (thought to contribute to leukemogenesis), was the focus of our interest, and the gene was further investigated to validate the microarray-based platform results and to localize the CNV area more precisely. In the region 44281092 to 4476549 within the KANSL1 gene Flt3-ITD negative patients had more deletions and amplifications as compared to those Flt3-ITD positive (31/62 vs 7/30, p=0.012). The same was valid when only deletions were considered (19/62 vs 4/30, p=0.058).Then to validate microarray platform results the same 80 samples were examined using the TaqMan Copy Number Assay (ThermoFisher) for the KANSL1 gene (primers and probe designed for the region 44297111-44297201 lying in chr17), and as a reference the RNaseP gene was employed. The results obtained using the microarray platform correlated well (r=0.671, p<0.001) with those obtained using RT-PCR copy number assay for KANSL1 .61 DNA samples obtained from the patients' solid organ biopsies were examined using the Copy Number Assay for KANSL1 gene CNV. In all instances except one the same CNP was observed in the marrow as that in the solid organ biopsies. It lends credit to the technology employed in this study, but still we do not have enough data to exclude the somatic origin of the microdeletions.The microdeletions are of biologic significance, as suggested by the following findings: 1.All three patients with FISH-documented MLL amplification had deletions within the KANSL1 gene (chr:17 44281092 to 4476549).2.Microdeletions within the KANSL1 gene were more frequently observed in the group M0-M2 patients than in other subtypes of AML (18/51 vs 5/35, p=0.026).In conclusion, a lack of Flt3-ITD and normal karyotype was associated with the frequent presence of microdeletions within the chr17 cytoband q21.31. The latter was identified as (i) present within the KANSL1 gene and (ii) influencing the phenotype of AML.Supported by the INNOMED/I/1/NCBR/2014 DisclosuresNo relevant conflicts of interest to declare.

Presence of Immunoglobulin Heavy Chain Gene (IGH) Translocations in Chronic Lymphocytic Leukemia Is Related to Poor Prognosis.

Background: Genetic analysis has an important role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IGH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency in CLL are less well defined and little is known about their prognostic significance. We studied a series of CLL patients using a FISH probe set which included an IGH probe and reviewed clinical data and disease course of CLL patients with IGH translocations.Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IGH. Patients with an IGH abnormality were further studied using FISH probes sets for IGH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. Between 5/15/03 and 5/15/07, samples from 4,976 patients with a presumed lymphoproliferative disorder were analyzed by FISH. Of these, 316 patients (6.3%) had translocations involving the IGH locus. Sixty-three (21%) of the 316 patients with IGH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review and analysis of associations to the FISH results.Results: We identified 39 patients with CLL and IGH translocations. Identified partner genes were: BCL-2 (n=22), BCL-3 (n=7), CMYC (n=2), MALT-1 (n=1). The partner gene was not identified despite using above mentioned panel of partner gene probes in 7 patients. Median age at time of diagnosis of patients with IGH translocations was 62 (45-82), 29 patients were males. The median time to initial treatment (TTT) of all 39 patients with IGH translocations was 3.1 years. The median TTT for patients with IGH/BCL-2 was 3.6 years; 70% of patients required treatment by 5 years. The median TTT for IGH/BCL-3 patients was significantly shorter, 1.5 years, p=0.017, with all patients requiring therapy 3 years from diagnosis (see Figure 1).Conclusion: The presence of IGH translocations in CLL appears to be associated with rapid disease progression and the need for an early initiation of therapy. The IGH partner gene seems to be important; IGH/BCL-3 fusion is associated with a worse prognosis than IGH/BCL-2. Further studies on the biological significance of IGH translocations in CLL are warranted and continue at our institution. [Display omitted]

ResCap: plant resistance gene prediction and probe generation pipeline for resistance gene sequence capture.

The discovery of novel resistance genes (R-genes) is an important component in disease resistance breeding. Nevertheless, R-gene identification from wild species and close relatives of plants is not only a difficult but also a cumbersome process. In this study, ResCap, a support vector machine-based high-throughput R-gene prediction and probe generation pipeline has been developed to generate probes from genomic datasets. ResCap contains two integral modules. The first module identifies the R-genes and R-gene like sequences under four categories containing different domains such as TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), Receptor-like kinase (RLK) and Receptor-like proteins (RLPs). The second module generates probes from extracted nucleotide sequences of resistance genes to conduct sequence capture (SeqCap) experiments. For the validation of ResCap pipeline, ResCap generated probes were synthesized and a sequence capture experiment was performed to capture expressed resistance genes among six spring barley genotypes. The developed ResCap pipeline in combination with the performed sequence capture experiment has shown to increase precision of R-gene identification while simultaneously allowing rapid gene validation including non-sequenced plants. The ResCap pipeline is available at http://rescap.ltj.slu.se/ResCap/. sandeep.kushwaha@slu.se or sandeep@niab.org.in. Supplementary materials are available at Bioinformatics Advances online.

A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

Since December 2019, SARS‐CoV‐2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS‐CoV‐2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre‐analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS‐CoV‐2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost‐effective multiplex reverse‐transcription real‐time PCR assay that can be used to detect SARS‐CoV‐2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS‐CoV‐2 by RT‐qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low‐throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1‐14 samples) Alternate Protocol 2: High‐throughput automated extraction on the Kingfisher Flex machine (1‐96 samples) Basic Protocol 2: Multiplex RT‐qPCR protocol to detect SARS‐CoV‐2 Alternate Protocol 3: Multiplex one‐step RT‐qPCR protocol to detect SARS‐CoV‐2 with S and E gene probes labeled with the same fluorochrome

Gene Expression Profiling and Biofunction Analysis of HepG2 Cells Targeted by Crocetin.

Crocetin is a carotenoid extracted from Gardenia jasminoides, one of the most popular traditional Chinese medicines, which has been used in the prevention and treatment of various diseases. The present study is aimed at clarifying the effect of crocetin on gene expression profiling of HepG2 cells by RNA-sequence assay and further investigating the molecular mechanism underlying the multiple biofunctions of crocetin based on bioinformatics analysis and molecular evidence. Among a total 23K differential genes identified, crocetin treatment upregulated the signals of 491 genes (2.14% of total gene probes) and downregulated the signals of 283 genes (1.24% of total gene probes) by ≥2-fold. The Gene Ontology analysis enriched these genes mainly on cell proliferation and apoptosis (BRD4 and DAXX); lipid formation (EHMT2); cell response to growth factor stimulation (CYP24A1 and GCNT2); and growth factor binding (ABCB1 and ABCG1), metabolism, and signal transduction processes. The KEGG pathway analysis revealed that crocetin has the potential to regulate transcriptional misregulation, ABC transporters, bile secretion, alcoholism, systemic lupus erythematosus (SLE), and other pathways, of which SLE was the most significantly disturbed pathway. The PPI network was constructed by using the STRING online protein interaction database and Cytoscape software, and 21 core proteins were obtained. RT-qPCR datasets serve as the solid evidence that verified the accuracy of transcriptome sequencing results with the same change trend. This study provides first-hand data for comprehensively understanding crocetin targeting on hepatic metabolism and its multiple biofunctions.

Conditionally Activated ("Caged") Oligonucleotides.

Conditionally activated (“caged”) oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical.

Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction.

Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.

Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs)

Multiple nucleic acid amplification tests (NATs) are available for the detection of SARS-CoV-2 in clinical specimens, including Laboratory Developed Tests (LDT), commercial high-throughput assays and point-of-care tests. Some assays were just recently released and there is limited data on their clinical performance. We compared the Xpert® Xpress SARS-CoV-2 (Cepheid) and Vivalytic VRI Panel (Schnelltest COVID-19) (Bosch) point-of-care tests with four high-throughput assays and one LDT, the cobas® SARS-CoV-2 test (Roche), the Allplex™ 2019-nCoV Assay (Seegene), the SARS-CoV-2 AMP (Abbott) Kit, the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona) as well as an assay using a SARS-CoV-2 RdRP gene specific primer and probe set. Samples from patients with confirmed SARS-CoV-2 infection, samples from the first and second SARS-CoV-2-PCR External Quality Assessment (EQA) (INSTAND e.V.) and a 10-fold serial dilution of a SARS-CoV-2 cell culture (SARS-CoV-2 Frankfurt 1) supernatant were examined. We determined that the NAT assays examined had a high specificity. Assays using the N gene as target demonstrated the highest sensitivity in the serial dilution panel, while all examined NAT assays showed a comparable sensitivity when testing clinical and EQA samples.

Transcriptomic Analysis of Laser Capture Microdissected Tumors Reveals Cancer- and Stromal-Specific Molecular Subtypes of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival. LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples. Gene expression profiling identified four subtypes of cancer cells (C1-C4) and three subtypes of cancer-adjacent stroma (S1-S3). These stroma-specific subtypes were associated with DFS (P = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; P < 0.02). Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.

Development of an electrochemical genosensor for detection of viral hemorrhagic septicemia virus (VHSV) using glycoprotein (G) gene probe

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of a highly contagious disease of both saltwater and freshwater fish around the world. VHSV, a member of viral Rhabdoviridae family, listed as a notifiable pathogen by the World Organization for Animal Health (OIE). This study was aimed to fabricate an electrochemical DNA genosensor for detection of the VHSV Glycoprotein gene. Immobilization of the DNA probe was carried out through self-assembly by thiol binding on RGO/Au-nanocomposite-modified pencil graphite electrode (PGE). The modified PGE was characterized by field emission scanning electron microscope (FESEM). The hybridization between the target sequences and the probe was analyzed by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods. Detection in these methods was performed by a decrease in the current and an increase in the charge transfer resistance (Rct) followed by target nucleic acids hybridization with biosensor probe. Genosensor sensitivity was tested by some mismatched sequences by 1, 2, and 3 bases in the methylene blue solution. The limit of detection (LOD) was at 125 pM of DNA target within the linear range of 10−4 to 10−10 M. The selectivity, stability, regeneration, and reproducibility of the fabricated electrochemical DNA biosensor were acceptable. A pilot analysis on real samples confirmed its applicability. Altogether, the genosensor can be suggested for direct detection of negative sense RNA genome of VHSV because of its fast assay, and high sensitivity.