Development of a DNA Microarray for the Simultaneous Detection and Genotyping of Noroviruses

2008 ◽  
Vol 71 (7) ◽  
pp. 1434-1441 ◽  
Author(s):  
FRANCO PAGOTTO ◽  
NATHALIE CORNEAU ◽  
KIRSTEN MATTISON ◽  
SABAH BIDAWID
Keyword(s):  
Molecular Epidemiology ◽  
Molecular Characterization ◽  
Simultaneous Detection ◽  
Oligonucleotide Array ◽  
Rt Pcr ◽  
Major Drawback ◽  
Pcr Product ◽  
Reaction Specificity ◽  
Pcr Products

Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)–mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.

scholarly journals Identification and molecular characterization of onion yellow dwarf virus (OYDV) in Allium cepa crops in Poland

2021 ◽  
Vol 61 (3) ◽  
pp. 214-220
Keyword(s):  
Phylogenetic Analysis ◽  
Molecular Characterization ◽  
Coat Protein Sequence ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Onion Yellow Dwarf Virus ◽  
Rt Pcr ◽  
Pcr Products ◽  
Yellow Dwarf Virus

Onion yellow dwarf virus is distributed worldwide significantly reducing yield of crops from the Allium genus. The aim of the study was the detection and molecular characterization of newly identified OYDV isolates infecting onions in Poland. The virus was detected by transmission electron microscopy and RT-PCR techniques using two pairs of diagnostic primers: OYDV-NibCPF1/R1 and OYDV-CPF2/R2. The specificity of obtained RT-PCR products was confirmed by Sanger sequencing and received viral coat protein sequence was used for phylogenetic analysis. The phylogenetic analysis was carried out using CP sequences of the new Polish onion isolate obtained in this study and 37 other sequences of OYDV retrieved from GenBank. The analysis revealed that the Polish OYDV isolate is the most similar to the OYDV isolates derived from onions from Argentina and Germany, which may indicate their common origin. Moreover, it was observed that the Polish onion and garlic isolates are very diverse and belong to different phylogroups.

Molecular characterization of promoter of IGF-1 gene in chicken broiler line and its growth performance

2016 ◽  
Author(s):  
C. Paswan ◽  
T. K. Bhattacharya ◽  
R. N. Chatterjee ◽  
C. S. Nagaraja ◽  
K. Dushyanth
Keyword(s):  
Growth Performance ◽  
Molecular Characterization ◽  
Promoter Sequence ◽  
Growth Data ◽  
Male And Female ◽  
Nucleotide Variability ◽  
Pcr Product ◽  
Sscp Pattern ◽  
Pcr Products

A study was carried out to characterize the nucleotide variability in the promoter of the IGF-1 gene in broiler line of chicken. A PCR product of 375bp was amplified and nucleotide variability was studied using PCR-SSCP technique in chicken control broiler line. Selected sample PCR products were also sequenced to confirm the variability in promoter sequence. Present study revealed that the IGF-1 promoter was monomorphic having similar SSCP pattern in all individuals. Growth data was also analyzed to study the growth performance of the chicken broiler line at different age. Growth performance of male and female differed significantly at six week of age.

scholarly journals Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

2021 ◽  
Author(s):  
Katarzyna Trzmiel
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Brome Mosaic Virus ◽  
Mottle Virus ◽  
Grass Species ◽  
Replicase Gene ◽  
Rt Pcr ◽  
Pcr Products ◽  
Cereal Plants ◽  
Cocksfoot Mottle Virus

AbstractBrome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

scholarly journals Human Rhinovirus Molecular Epidemiology and Genotyping in Iranian Military Trainees with Acute Respiratory Symptoms

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Fahimeh Safarnezhad Tameshkel ◽  
Ali Salimi Jeda ◽  
Ahmad Tavakoli ◽  
Mohammad Hadi Karbalaie Niya ◽  
Morteza Izadi ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Clinical Symptoms ◽  
Respiratory Infections ◽  
Direct Sequencing ◽  
Human Rhinovirus ◽  
P Value ◽  
Rt Pcr ◽  
Respiratory Problems ◽  
Pcr Products ◽  
Tract Infections

Background: Human rhinovirus (HRV) is still the most prevalent viral infection in humans and a significant cause of acute respiratory tract infections (ARTIs) in many communities, including military personnel undergoing basic training. Objectives: In this research, we assessed the molecular epidemiology, genotyping, and phylogenetic classification of HRVs in Iranian military trainees with respiratory infections (RI). Methods: For HRV identification and genotyping, respiratory specimens were obtained, and RT-PCR was conducted for genotyping and phylogenetic analysis of HRV utilizing primers for the 5-UTR region. Results: Among 400 Iranian military trainees (average age of 21 ± 4 years, the range of 18 - 57 years) with respiratory infections, HRV was detected in 29 patients (7%) using RT-PCR. The direct sequencing of PCR products from 10 specimens showed that the incidence of type A (n = 5, 50%) was higher than that of type B (n = 4, 40%) and type C (n = 1, 10%). There were no significant associations between HRV and respiratory and clinical symptoms, blood group, and indoor or outdoor conditions (P-value > 0.05). Conclusions: This research was the first to record HRV as a significant cause of respiratory problems among military trainees in Iran, with a frequency of 7%. The most prevalent genotype was HRV-A, which may be applicable in epidemiological and clinical studies, as well as vaccination plans.

scholarly journals AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

2005 ◽  
Vol 6 (5-6) ◽  
pp. 268-276
Author(s):  
Fabienne S. Giraudeau ◽  
Jean-Philippe Walhin ◽  
Paul R. Murdock ◽  
Nigel K. Spurr ◽  
Ian C. Gray
Keyword(s):  
Negative Impact ◽  
Cathepsin K ◽  
Chromosome 1 ◽  
Rt Pcr ◽  
Transcription Start ◽  
Aryl Hydrocarbon ◽  
Pcr Product ◽  
Close Proximity ◽  
Pcr Products ◽  
Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

Molecular characterization of Candidatus Phytoplasma aurantifolia isolate infecting chickpea (Cicer arietinum) in Dharwad, Karnataka

2019 ◽  
Author(s):  
Gurupada Balol ◽  
C Channakeshava ◽  
M S Patil
Keyword(s):  
16S Rdna ◽  
Molecular Characterization ◽  
Cicer Arietinum ◽  
Nested Pcr ◽  
Rdna Sequences ◽  
Pcr Product ◽  
16S Rdna Sequences ◽  
Axillary Proliferation ◽  
Pale Green

Chickpea plants showing phytoplasma symptoms were observed in the research plots at University of Agricultural Sciences, Dharwad, Karnataka, India. The symptoms included phyllody, pale green leaves, bushy appearance and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by nested PCR with primers P1/P7 and R16F2n/R16R2 and 1,800 bp and 1,200 bp size products were amplified in first round PCR and nested-PCR respectively. The PCR product was sequenced and compared with the reference phytoplasma sequences collected from the database (NCBI). 16S rDNA sequences of Dharwad chickpea phytoplasma shared the highest nucleotide identity of (>98%) with Periwinkle phyllody16SrII-E (EU096500). This study indicated the association of ‘Candidatus Phytoplasma aurantifolia’ the 16SrII-E group infecting chickpea from Northern Karnataka.

Validation of RT-PCR Assays for Molecular Characterization of Porcine Teschoviruses and Enteroviruses

2006 ◽  
Vol 53 (6) ◽  
pp. 257-265 ◽  
Author(s):  
G. La Rosa ◽  
M. Muscillo ◽  
A. Di Grazia ◽  
S. Fontana ◽  
M. Iaconelli ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Rt Pcr ◽  
Pcr Assays
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