Simultaneous determination of Mycobacterium leprae drug resistance and single nucleotide polymorphism genotype using nested multiplex PCR with amplicon sequencing

2021 ◽  
Author(s):  
Yasuhisa Iwao ◽  
Shuichi Mori ◽  
Manabu Ato ◽  
Noboru Nakata
Keyword(s):  
Drug Resistance ◽  
Single Nucleotide Polymorphism ◽  
Multiplex Pcr ◽  
Simultaneous Determination ◽  
Mycobacterium Leprae ◽  
Amplicon Sequencing ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Mycobacterium leprae is the predominant cause of leprosy worldwide, and its genotypes can be classified into four single nucleotide polymorphism (SNP) types and 16 subtypes. Determining M. leprae drug resistance and genotype is typically done by PCR and Sanger DNA sequencing, which require substantial effort. Here we describe a rapid method involving multiplex PCR in combination with nested amplification and next generation sequence analysis that allows simultaneous determination of M. leprae drug resistance and SNP genotype directly from clinical specimens. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. Regions in folP1 , rpoB , gyrA , and gyrB that determine drug resistance and those for 84 SNP-InDels in the M. leprae genome were amplified from clinical samples and their sequences were determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. The method may allow more rapid genetic analyses of M. leprae in clinical samples.

scholarly journals P-0022 Association Between C3435T Single Nucleotide Polymorphism of Multi-Drug Resistance 1 Gene and Risk of Gastric Cancer

2012 ◽  
Vol 23 ◽  
pp. iv37-iv38
Author(s):  
Juliana Oliveira ◽  
Aledson Felipe ◽  
Paula Chang ◽  
Tiago da Silva ◽  
Célia Pimenta ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Single Nucleotide Polymorphism ◽  
Multi Drug Resistance ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Determination of Residual Feed Intake and Its Associations with Single Nucleotide Polymorphism in Chickens

2014 ◽  
Vol 13 (1) ◽  
pp. 148-157 ◽  
Author(s):  
Zhen-qiang XU ◽  
Jie CHEN ◽  
Yan ZHANG ◽  
Cong-liang JI ◽  
De-xiang ZHANG ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Feed Intake ◽  
Residual Feed Intake ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Single-Nucleotide-Polymorphism Genotyping ofCoxiella burnetiiduring a Q Fever Outbreak in The Netherlands

2011 ◽  
Vol 77 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
Cornelis J. J. Huijsmans ◽  
Jeroen J. A. Schellekens ◽  
Peter C. Wever ◽  
Rudolf Toman ◽  
Paul H. M. Savelkoul ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
The Netherlands ◽  
Q Fever ◽  
Snp Genotyping ◽  
Cow Milk ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Animal Reservoir ◽  
Single Nucleotide ◽  
Outbreak Area

ABSTRACTCoxiella burnetiiis the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of theC. burnetiistrain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination ofC. burnetiistrains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 differentC. burnetiigenotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 differentC. burnetiigenotypes are involved in the Dutch outbreak.

scholarly journals Heterozygous Single-Nucleotide Polymorphism Genotypes at Heat Shock Protein 70 Gene Potentially Influence Thermo-Tolerance Among Four Zebu Breeds of Nigeria

2021 ◽  
Vol 12 ◽  
Author(s):  
Gbolabo Olaitan Onasanya ◽  
George Mutani Msalya ◽  
Aranganoor Kannan Thiruvenkadan ◽  
Chirukandoth Sreekumar ◽  
Gopalan Krishnaswamy Tirumurugaan ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Genetic Variants ◽  
Heat Shock Protein 70 ◽  
Genotypic Frequency ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Unique Snps

Genetic variants at heat shock protein 70 gene and their influence on heat stress (HS) tolerance were studied among selected Nigeria zebu, namely, 25 White Fulani (WF), 21 Sokoto Gudali (SG), 21 Red Bororo (RB), and 23 Ambala (AM). Detection of single nucleotide polymorphism (SNP) followed by determination of genotype and genotypic frequency was made among the selected breeds. The heat tolerance coefficient (HTC) was determined from thermo-related parameters including body temperature, rectal temperature, and respiratory rate. Thermo-Tolerance was evaluated through the SNP–thermo-parameter relationship. Statistical analyses were done using the GLM procedure in SAS. A quantitative real-time/high-resolution melting-based assay detected twelve genetic variants. Five of these were common and shared across all breeds of cattle. Of the remaining seven variants, three were specifically identified in AM, two in SG, and two in RB. Also, SNPs were evaluated and four unique SNPs (C151T, C146T, G90A, and C219A) were identified. Heterozygous animals had lower HTC suggesting their potential to withstand HS than homozygous counterparts. The WF and RB animals had significantly lower values for all parameters (BT, RT, RR, and HTC) compared to AM and SG breeds. Thermo-related parameters were significantly different (P < 0.001), and it is recommended that screening of SNPs in zebu is needed to enable selection for improved thermo-tolerance.

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