scholarly journals A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

2021 ◽  
Vol 62 (4) ◽  
pp. 371-377
Author(s):  
Rossana C. Jaspe ◽  
Yoneira Sulbaran ◽  
Mariana Hidalgo ◽  
Mariana Hidalgo ◽  
Carmen L. Loureiro ◽  
...  
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Restriction Enzyme ◽  
Restriction Analysis ◽  
Spike Protein ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Simple Method ◽  
Spike Gene ◽  
Detection Of Mutations

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

scholarly journals Importance of mutations in amino acid 484 of the Spike protein of SARS-CoV-2: rapid detection by restriction enzyme analysis

2021 ◽  
Vol 62 ◽  
pp. 18-26
Author(s):  
Rossana C Jaspe ◽  
Yoneira Sulbarn ◽  
Carmen L Loureiro ◽  
Pierina D´Angelo ◽  
Lieska Rodríguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Enzyme ◽  
Rapid Detection ◽  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Spike Gene ◽  
Detection Of Mutations ◽  
Viral Isolates

Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 484 of the Spike protein are particularly important and associated with some of these variants: E484K or E484Q. These mutations have been associated with evasion to neutralizing antibodies. Restriction enzyme analysis is proposed as a rapid method to detect these mutations. A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. E484K, present in some VOCs, has emerged in several other lineages and is frequently found in recent viral isolates. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, and MseI. The use of these two enzymes allows the detection of mutations at position 484, and to differentiate between these three conditions: non-mutated, and the presence of E484K or E484Q. A 100% correlation was observed with sequencing results. The proposed methodology, which allows for the screening of a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.

scholarly journals Importance of E484K and N501Y mutations in SARS-CoV-2 for genomic surveillance: rapid detection by restriction enzyme analysis

2021 ◽  
Author(s):  
Rossana C Jaspe ◽  
Yoneira Sulbaran ◽  
Carmen L Loureiro ◽  
Pierina D Angelo ◽  
Lieska Rodriguez ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Restriction Enzyme ◽  
Viral Genome ◽  
Rapid Detection ◽  
Spike Protein ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Whole Genome ◽  
Spike Gene ◽  
Gene Coding

Introduction: Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Two mutations in the gene coding for the Spike protein of the viral genome are particularly important and associated with some of these variants: E484K and N501Y. Restriction enzyme analysis is proposed as a rapid method to detect these two mutations. Methodology: A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, which allows detecting E484K mutation, and MseI, for detecting the N501Y one. Results: The mutations E484K and N501Y, associated with VOCs, have emerged in several other lineages, particularly E484K. A 100% correlation was observed with sequencing results. Conclusions: The proposed methodology, which allows screening a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.

scholarly journals Genotyping of the Pseudorabies Virus by Multiplex PCR Followed by Restriction Enzyme Analysis

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
Antônio Augusto Fonseca ◽  
Cristina Gonçalves Magalhães ◽  
Érica Bravo Sales ◽  
Régia Maria D'Ambros ◽  
Janice Ciacci-Zanella ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Restriction Enzyme ◽  
Pseudorabies Virus ◽  
Virus Isolation ◽  
Restriction Analysis ◽  
Infectious Agent ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Restriction Sites ◽  
Herpesvirus 1

Suid herpesvirus 1 (SuHV-1) is the causative agent of Aujeszky's disease. The infectious agent has only one serotype, but it was classified by restriction enzyme analysis of the whole genome into four genotypes, named I to IV. The aim of this study was to standardize a rapid method for genotyping SuHV-1 without virus isolation, using a multiplex-PCR followed by enzymatic restriction analysis. The complete genome of the virus was analyzed in silico to determine the restriction sites for the enzyme BamHI. Primers were designed to flank sites with emphasis on certain points of differentiation of genotypes. The standard PCRs were able to detect the SuHV-1 and also to differentiate genotypes from brain tissue of infected pigs. The BamHI-PCR is a rapid, practical, and sensitive way to genotype SuHV-1.

Varicella-Like Illness Caused by Live Varicella Vaccine in Children With Acute Lymphocytic Leukemia

PEDIATRICS ◽  
1987 ◽  
Vol 79 (6) ◽  
pp. 922-927
Author(s):  
Philip A. Brunell ◽  
Clementina F. Geiser ◽  
Valerio Novelli ◽  
Susan Lipton ◽  
Sarah Narkewicz
Keyword(s):  
Immune Response ◽  
Restriction Enzyme ◽  
Cellular Immune Response ◽  
Acute Lymphocytic Leukemia ◽  
Varicella Vaccine ◽  
Lymphocytic Leukemia ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Household Contacts ◽  
Cellular Immune

A varicella-like illness occurred in five of 52 children with acute lymphocytic leukemia following the administration of live varicella vaccine. Only one of the children required treatment with acyclovir. Virus isolated from two of the children was "vaccine-like" but differed slightly from the original vaccine strain when tested by restriction enzyme analysis. There did not appear to be a reversion to virulence because two of the household contacts who seroconverted had mild or subclinical infections. Vaccinees in whom this reaction developed tended to have a poor cellular immune response to varicellazoster virus.

scholarly journals Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

2006 ◽  
Vol 72 (2) ◽  
pp. 1072-1078 ◽  
Author(s):  
Isabelle Robène-Soustrade ◽  
Philippe Laurent ◽  
Lionel Gagnevin ◽  
Emmanuel Jouen ◽  
Olivier Pruvost
Keyword(s):  
Diagnostic Tool ◽  
Nested Pcr ◽  
Restriction Analysis ◽  
Detection Threshold ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Amplification Product ◽  
Xanthomonas Axonopodis ◽  
Sequence Characterized Amplified Region ◽  
Pcr Test

ABSTRACT Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (103 CFU ml−1) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.

Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus

1985 ◽  
Vol 5 (9) ◽  
pp. 2197-2203
Author(s):  
M S Lakshmikumaran ◽  
E D'Ambrosio ◽  
L A Laimins ◽  
D T Lin ◽  
A V Furano
Keyword(s):  
Dna Sequence ◽  
Restriction Enzyme ◽  
Allelic Variation ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repeated Dna ◽  
Coding Region ◽  
Sequence Determination ◽  
Polymorphic Region ◽  
Or Gene

The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7-kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this.

Restriction Enzyme Analysis (REA) of Group A Streptococcal (GAS) M-Serotypes 1, 3, and 28

1997 ◽  
pp. 221-223 ◽  
Author(s):  
Dwight R. Johnson ◽  
Cheryl L. Romana ◽  
Carey D. Rehder ◽  
Joanne Dehnbostel ◽  
Edward L. Kaplan
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Group A
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