scholarly journals Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern

Author(s):  
Dongyan Xiong ◽  
Xiaoxu Zhang ◽  
Mengjuan Shi ◽  
Nuo Wang ◽  
Ping He ◽  
...  

The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed.

2020 ◽  
Author(s):  
Md. Tanvir Islam ◽  
A. S. M. Rubayet Ul Alam ◽  
Najmuj Sakib ◽  
Md. Shazid Hasan ◽  
Tanay Chakrovarty ◽  
...  

SummaryTracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders the viral genotyping in low-income countries. Here, we propose a rapid, simple and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. Our multiplex PCR is designed in a mutually exclusive way to identify V-S and G-GH-GR clade variants separately. The pentaplex assay included all five variants and the quadruplex comprised of the triplex variants alongside either V or S clade mutations that created two separate subsets. The procedure was optimized in the primer concentration (0.2-0.6 µM) and annealing temperature (56-60°C) of PCR using 3-5 ng/µl cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentration for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is sample, cost-effective, and convenient that can successfully discriminate the circulating phylogenetic clades of SARS-CoV-2.


2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.


BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Raziyeh Khalesi ◽  
Ehsan Razmara ◽  
Golareh Asgaritarghi ◽  
Ali Reza Tavasoli ◽  
Yasser Riazalhosseini ◽  
...  

Abstract Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.


2007 ◽  
Vol 46 (2) ◽  
pp. 533-539 ◽  
Author(s):  
X. Lu ◽  
B. Holloway ◽  
R. K. Dare ◽  
J. Kuypers ◽  
S. Yagi ◽  
...  

Author(s):  
Eric M. Katz ◽  
Mathew D. Esona ◽  
Rashi Gautam ◽  
Michael D. Bowen

Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant ‘equine-like G3’ strains have emerged and spread across five continents among human populations in at least 14 countries. Here we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 2.3 × 10 9 – 227 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.


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