scholarly journals Rapid, inexpensive methods for exploring SARS CoV-2 D614G mutation

2021 ◽  
Author(s):  
Sirwan M.A. Al-Jaf ◽  
Sherko Subhan Niranji ◽  
Zana Hameed Mahmood
Keyword(s):  
Fragment Length Polymorphism ◽  
Taqman Probe ◽  
Nasopharyngeal Swab ◽  
Amplification Refractory Mutation System ◽  
Common Mutation ◽  
Wild Type ◽  
Chain Reaction ◽  
Mutation System ◽  
Polymerase Chain ◽  
Restriction Fragment Length

A common mutation has occurred in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), known as D614G (A23403G). There are discrepancies in impacting of this mutation on the virus's infectivity, and the whole genome sequencings are expensive and time-consuming. This study aims to develop three fast economical assays for prompt identifications of the D614G mutation including Taqman probe-based real-time reverse transcriptase polymerase chain reaction (rRT PCR), an amplification refractory mutation system (ARMS) RT and restriction fragment length polymorphism (RFLP), in nasopharyngeal swab samples. Both rRT and ARMS data showed G614 mutant indicated by presence of HEX probe and 176bp, respectively. Additionally, the results of the RFLP data and DNA sequencings confirmed the prevalence of G614 mutant. These methods will be important, in epidemiological, reinfections and zoonotic aspects, through detecting the G614 mutant in retro-perspective samples to track its origins and future re-emergence of D614 wild type.

MUTATION IN THE FMO3 LOCUS AND ITS DISTRIBUTION IN A GROUP OF AYRSHIRE BREEDING BULLS USED IN FARMS OF THE KRASNODAR TERRITORY

2021 ◽  
pp. 10-15
Author(s):  
Н.В. КОВАЛЮК ◽  
Е.В. ШИРЯЕВА ◽  
Л.И. ЯКУШЕВА ◽  
Ю.Ю. ШАХНАЗАРОВА
Keyword(s):  
Polymerase Chain Reaction ◽  
Mutant Allele ◽  
Restriction Site ◽  
Fragment Length Polymorphism ◽  
Test System ◽  
Frequency Of Occurrence ◽  
Wild Type ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Рыбный привкус в коровьем молоке вызван наличием нонсенс-мутации (g.39523051C>T) в гене бычьего FMO3. Нами разработана тест-система для выявления FMO3- полиморфизма, основанная на полимеразной цепной реакции с последующим анализом полиморфизма длин фрагментов рестрикции с использованием эндонуклеазы TaqI. Фрагменты, которые амплифицировались с участка гена FMO3 «дикого» типа, расщеплялись эндонуклеазой TaqI  на 2 фрагмента: 136 и 99 пн. Фрагменты, амплифицированные с мутантного аллеля, сайта рестрикции не имели (их размер составлял  235 пн). Определена частота встречаемости носителей мутации в отечественной субпопуляции айрширского скота. Установлено, что среди айрширских быков-производителей (n=45), принадлежащих различным отечественным и зарубежным племорганизациям, частота встречаемости носителей мутации в гене FMO составила 9%. Учитывая, что выявленные носители мутации интенсивно используются и могут передавать эту аномалию значительному числу дочерей, генотипирование по локусу FMO3 должно стать обязательным для быков-производителей и групп быкопроизводящих коров племенных айрширских хозяйств. The fishy taste in cow's milk is caused by the presence of a nonsense mutation (g.39523051C>T) in the bovine FMO3 gene. We have developed a test system for detecting FMO3 polymorphism based on a polymerase chain reaction followed by analysis of restriction fragment length polymorphism using TaqI endonuclease. Fragments that were amplified from the wild-type FMO3 gene site were cleaved by TaqI endonuclease into 2 fragments: 136 and 99 bp. The fragments amplified from the mutant allele did not have a restriction site (their size was — 235 bp). The frequency of occurrence of mutation carriers in the domestic subpopulation of Ayrshire cattle was determined. It was found that among Ayrshire bulls (n=45) belonging to various domestic and foreign breeding organizations, the frequency of occurrence of carriers of the mutation in the FMO gene was 9%. Given that the identified carriers of the mutation are intensively used and can transmit this anomaly to a significant number of daughters, genotyping by the FMO3 locus should become mandatory for breeding bulls and groups of bull-producing cows of breeding Ayrshire farms.

scholarly journals The genotyping of Infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

2014 ◽  
Vol 26 (6) ◽  
pp. 721-733 ◽  
Author(s):  
Shr-Wei Huang ◽  
Chia-Fang Ho ◽  
Kun-Wei Chan ◽  
Min-Chung Cheng ◽  
Jui-Hung Shien ◽  
...  
Keyword(s):  
Amplification Refractory Mutation System ◽  
Wild Type ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Infectious Bronchitis ◽  
Melting Temperatures ◽  
Qrt Pcr ◽  
Multiplex Amplification ◽  
Mutation System ◽  
Polymerase Chain

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.

scholarly journals Greater frequency of K-ras Val-12 mutation in colorectal cancer as detected with sensitive methods

1996 ◽  
Vol 42 (6) ◽  
pp. 904-909 ◽  
Author(s):  
K M Carpenter ◽  
L G Durrant ◽  
K Morgan ◽  
D Bennett ◽  
J D Hardcastle ◽  
...  
Keyword(s):  
Amplification Refractory Mutation System ◽  
Colorectal Tumors ◽  
Wild Type ◽  
Chain Reaction ◽  
High Background ◽  
Mutation System ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Type Sequence ◽  
Bowel Mucosa

Abstract Assays for tumor mutations need to be sufficiently sensitive that a mutation can be readily detected in a high background of wild-type sequence. We have evaluated competitive allele-specific oligonucleotide (cASO) hybridization, the amplification refractory mutation system (ARMS), and a combination of cASO with mutant-enriched polymerase chain reaction (ME-PCR) for their respective sensitivities in detecting the K-ras Val-12 mutation. cASO hybridization detected 1 mutant allele in 100 wild-type alleles and identified 6 of 74 (8%) colorectal tumors as having the Val-12 mutation. ARMS detected 1 mutant in 1000 and 12 of 74 (16%) tumor samples, and the ME-PCR with cASO hybridization detected 1 in 5000 and 20 of 74 (27%) tumor samples. The mutation was not detected in normal bowel mucosa from selected Val-12 tumor-positive patients. ME-PCR combined with cASO resulted in a threefold higher detection rate than has previously been reported and suggests that other studies may have underestimated the frequency of K-ras mutations.

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