scholarly journals Validation of PfSNP-LAMP-Lateral Flow Dipstick for Detection of Single Nucleotide Polymorphism Associated with Pyrimethamine Resistance in Plasmodium falciparum

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 948
Author(s):  
Suganya Yongkiettrakul ◽  
Fassou René Kolié ◽  
Darin Kongkasuriyachai ◽  
Jetsumon Sattabongkot ◽  
Wang Nguitragool ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Amino Acid Position ◽  
Lateral Flow ◽  
Management Policy ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Simple Method ◽  
Single Nucleotide ◽  
Lateral Flow Dipstick ◽  
Antifolate Resistance

The loop-mediated isothermal amplification coupled with lateral flow dipstick (PfSNP-LAMP-LFD) was recently developed to detect single nucleotide polymorphism (AAT → ATT), corresponding to substitution of asparagine to isoleucine at amino acid position 51 in the P. falciparumdhfr-ts gene associated with antifolate resistance. In this present study, the PfSNP-LAMP-LFD was validated on 128 clinical malaria samples of broad ranged parasite densities (10 to 87,634 parasites per microliter of blood). The results showed 100% accuracy for the detection of single nucleotide polymorphism for N51I mutation. Indeed, the high prevalence of N51I in the Pfdhfr-ts gene detected in the clinical samples is in line with reports of widespread antifolate resistant P. falciparum in Thailand. The relationship between enzyme choice and reaction time was observed to have an effect on PfSNP-LAMP-LFD specificity; however, the method yielded consistent results once the conditions have been optimized. The results demonstrate that PfSNP-LAMP-LFD is a simple method with sufficient sensitivity and specificity to be deployed in routine surveillance of antifolate resistance molecular marker and inform antimalarial management policy.

Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick

2017 ◽  
Vol 66 (1) ◽  
pp. 964-971 ◽  
Author(s):  
Suganya Yongkiettrakul ◽  
Jantana Kampeera ◽  
Wanwisa Chareanchim ◽  
Roonglawan Rattanajak ◽  
Wichai Pornthanakasem ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Plasmodium Falciparum ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Lateral Flow Dipstick ◽  
Simple Detection

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 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 Single-Nucleotide Polymorphism Leading to False Allelic Fraction by Droplet Digital PCR

2017 ◽  
Vol 63 (8) ◽  
pp. 1370-1376 ◽  
Author(s):  
Eric S Christenson ◽  
W Brian Dalton ◽  
David Chu ◽  
Ian Waters ◽  
Karen Cravero ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Careful Analysis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Unexpected Finding ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Patients With Cancer

Abstract BACKGROUND Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA. RESULTS Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele. CONCLUSIONS These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed.

scholarly journals A Lactotransferrin Single Nucleotide Polymorphism Demonstrates Biological Activity That Can Reduce Susceptibility to Caries

2013 ◽  
Vol 81 (5) ◽  
pp. 1596-1605 ◽  
Author(s):  
Daniel H. Fine ◽  
Gokce A. Toruner ◽  
Kabilan Velliyagounder ◽  
Vandana Sampathkumar ◽  
Dipti Godboley ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Amino Acid Position ◽  
Salivary Protein ◽  
Affinity Column ◽  
Dependent Manner ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
K Variant

ABSTRACTStreptococcus mutansis prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutansactivity, characterize its genotype, and determine genotypic variants associated withS. mutansactivity and reduced caries. AnS. mutansaffinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killedS. mutansin vitro. Saliva from a subject with moderate caries and with the LTF “wild-type” R form at position 47 had no such activity. A pilot genetic study (n= 30) showed that KK subjects were more likely to have anti-S. mutansactivity than RR subjects (P= 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reducedS. mutanskilling in a dose-dependent manner (P= 0.02). KK subjects were less likely to have caries (P= 0.02). A synthetic 11-mer LTF/K peptide killedS. mutansand other caries-related bacteria, while the LTF/R peptide had no effect (P= 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutansactivity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.

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