scholarly journals KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

2020 ◽  
Author(s):  
Ana Alvarez-Fernandez ◽  
María J. Bernal ◽  
Isabel Fradejas Villajos ◽  
Alexandra Martín Ramírez ◽  
Noor Azian Md Yu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Control ◽  
Low Cost ◽  
Reference Method ◽  
Rapid Identification ◽  
Control Programs ◽  
Specific Pcr ◽  
Antimalarial Resistance ◽  
Allele Specific ◽  
Almost All

Abstract BackgroundThe emergence and spread of antimalarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known antimalarials. This antimalarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorfisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programs. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum.MethodsThree SNPs involved in most cases of resistance to the most widespread antimalarial treatments have been analyzed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analyzed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.ResultsThe KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analyzed.ConclusionsThe KASP assays developed in our study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

scholarly journals KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

2020 ◽  
Author(s):  
Ana Alvarez-Fernandez ◽  
María J. Bernal ◽  
Isabel Fradejas Villajos ◽  
Alexandra Martín Ramírez ◽  
Noor Azian Md Yu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Control ◽  
Low Cost ◽  
Reference Method ◽  
Rapid Identification ◽  
Control Programs ◽  
Specific Pcr ◽  
Antimalarial Resistance ◽  
Allele Specific ◽  
Almost All

Abstract Background The emergence and spread of antimalarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known antimalarials. This antimalarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorfisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programs. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Methods Three SNPs involved in most cases of resistance to the most widespread antimalarial treatments have been analyzed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analyzed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analyzed. Conclusions The KASP assays developed in our study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

scholarly journals KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ana Alvarez-Fernandez ◽  
María J. Bernal ◽  
Isabel Fradejas ◽  
Alexandra Martin Ramírez ◽  
Noor Azian Md Yusuf ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Control ◽  
Low Cost ◽  
Reference Method ◽  
Rapid Identification ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Control Programmes ◽  
Allele Specific ◽  
Almost All

Abstract Background The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Methods Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. Conclusions The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

scholarly journals Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 982
Author(s):  
Zhiliang Xiao ◽  
Congcong Kong ◽  
Fengqing Han ◽  
Limei Yang ◽  
Mu Zhuang ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Brassica Oleracea ◽  
Rapid Identification ◽  
Inbred Lines ◽  
Hybrid Lethality ◽  
Specific Pcr ◽  
Allele Specific ◽  
User Friendly ◽  
Allele Specific Pcr ◽  
Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

scholarly journals Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

PLoS ONE ◽  
2020 ◽  
Vol 15 (3) ◽  
pp. e0230445 ◽  
Author(s):  
Wei Suo ◽  
Xiujin Shi ◽  
Sha Xu ◽  
Xiao Li ◽  
Yang Lin
Keyword(s):  
Low Cost ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals A multiplex qPCR approach for detection of pfhrp2 and pfhrp3 gene deletions in multiple strain infections of Plasmodium falciparum

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tobias Schindler ◽  
Anna C. Deal ◽  
Martina Fink ◽  
Etienne Guirou ◽  
Kara A. Moser ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Control ◽  
High Throughput Screening ◽  
Malaria Diagnosis ◽  
The Novel ◽  
Essential Factor ◽  
Gene Deletions ◽  
Control Programs ◽  
Sub Saharan ◽  
Insight Into

Abstract The rapid and accurate diagnosis of Plasmodium falciparum malaria infection is an essential factor in malaria control. Currently, malaria diagnosis in the field depends heavily on using rapid diagnostic tests (RDTs) many of which detect circulating parasite-derived histidine-rich protein 2 antigen (PfHRP2) in capillary blood. P. falciparum strains lacking PfHRP2, due to pfhrp2 gene deletions, are an emerging threat to malaria control programs. The novel assay described here, named qHRP2/3-del, is well suited for high-throughput screening of P. falciparum isolates to identify these gene deletions. The qHRP2/3-del assay identified pfhrp2 and pfhrp3 deletion status correctly in 93.4% of samples with parasitemia levels higher than 5 parasites/µL when compared to nested PCR. The qHRP2/3-del assay can correctly identify pfhrp2 and pfhrp3 gene deletions in multiple strain co-infections, particularly prevalent in Sub-Saharan countries. Deployment of this qHRP2/3-del assay will provide rapid insight into the prevalence and potential spread of P. falciparum isolates that escape surveillance by RDTs.

scholarly journals Single tube allele specific PCR: a low cost technique for molecular screening of sickle cell anaemia in Nigeria

2018 ◽  
Vol 18 (4) ◽  
pp. 995
Author(s):  
Emuejevoke T Toye ◽  
Guido Van Marle ◽  
Wendy Hutchins ◽  
Olayinka Abgabiaje ◽  
Joy Okpuzor Okpuzor
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anaemia ◽  
Low Cost ◽  
Molecular Screening ◽  
Single Tube ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Rapid identification of capybara (Hydrochaeris hydrochaeris) using allele-specific PCR

2007 ◽  
Vol 67 (1) ◽  
pp. 189-190 ◽  
Author(s):  
F. Henrique-Silva ◽  
M. Cervini ◽  
WM. Rios ◽  
AL. Lusa ◽  
A. Lopes ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Hydrochaeris Hydrochaeris

scholarly journals Kompetitive Allele Specific PCR (KASP) Markers for Potato: An Effective Tool for Increased Genetic Gains

Agronomy ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2315
Author(s):  
Moctar Kante ◽  
Hannele Lindqvist-Kreuze ◽  
Leticia Portal ◽  
Maria David ◽  
Manuel Gastelo
Keyword(s):  
Quality Control ◽  
Low Cost ◽  
Yield Reduction ◽  
Genetic Gains ◽  
Specific Pcr ◽  
Breeding Populations ◽  
Allele Specific ◽  
Cultivated Potatoes ◽  
Allele Specific Pcr ◽  
Kasp Markers

Potato virus Y (PVY) and Phytophthora infestans (Mont.) de Bary that causes potato late blight (LB), pose serious constraints to cultivated potatoes due to significant yield reduction, and phenotyping for resistance remains challenging. Breeding operations for vegetatively propagated crops can lead to genotype mislabeling that, in turn, reduces genetic gains. Low-density and low-cost molecular marker assessment for phenotype prediction and quality control is a viable option for breeding programs. Here, we report on the development of kompetitive allele specific PCR (KASP) markers for LB and PVY resistance, and for routine quality control assessment of different breeding populations. Two KASP markers for LB resistance and two for PVY Ryadg were validated with an estimated assay power that ranged between 0.65 and 0.88. The developed QC KASP markers demonstrated the capability of discriminating tetraploid calls in breeding materials, including full-sibs and half-sibs. Routine implementation of the developed markers in a breeding program would assist with better allocation of resources and enable precise characterization of breeding material, thereby leading to increased genetic gains.

scholarly journals Evaluation of Etest for Susceptibility Testing of Multidrug-Resistant Isolates of Mycobacterium tuberculosis †

2000 ◽  
Vol 38 (12) ◽  
pp. 4599-4603 ◽  
Author(s):  
Manzour Hernando Hazbón ◽  
Maria del Socorro Orozco ◽  
Luz Angela Labrada ◽  
Rafael Tovar ◽  
Kristen A. Weigle ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistance ◽  
Susceptibility Testing ◽  
Low Cost ◽  
Reference Method ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Control Programs ◽  
Proportion Method ◽  
Testing Techniques

To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques forMycobacterium tuberculosis are needed, especially in regions with multidrug resistance. Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for M. tuberculosis in 5 to 10 days from a culture grown at low cost. The performance of Etest was compared to that of the reference proportion method, using 95 M. tuberculosisclinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method. Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin. This study supports the utility of Etest for timely detection of drug resistance in M. tuberculosis and for use in tuberculosis control programs.

scholarly journals Hybrid mosquitoes? Evidence from rural Tanzania on how local communities conceptualize and respond to modified mosquitoes as a tool for malaria control

2021 ◽  
Author(s):  
Marceline Francis Finda ◽  
Fredros Oketch Okumu ◽  
Elihaika Minja ◽  
Rukiyah Njalambaha ◽  
Winnfrida Mponzi ◽  
...  
Keyword(s):  
Malaria Control ◽  
Technology Development ◽  
Low Cost ◽  
Public Support ◽  
Community Leaders ◽  
Community Perceptions ◽  
Novel Technologies ◽  
Control Programs ◽  
High Level ◽  
Interpretive Frames

Abstract Background: Different forms of mosquito modification are being considered as potential high-impact and low-cost tools for future malaria control in Africa. Although still under evaluation, the eventual success of these technologies will require high-level public acceptance. Understanding prevailing community perceptions of mosquito modification is therefore crucial for effective design and implementation of these interventions. This study investigated community perceptions regarding genetically-modified mosquitoes (GMMs) and their potential for malaria control in Tanzanian villages where no research or campaign for such technologies has yet been undertaken.Methods: A mixed-methods design was used, involving: i) focus group discussions (FGD) with community leaders to get insights on how they frame and would respond to GMMs, and ii) structured questionnaires administered to 490 community members to assess awareness, perceptions and support for GMMs for malaria control. Descriptive statistics were used to summarize the findings and thematic content analysis was used to identify key concepts and interpret the findings. Results: Nearly all survey respondents were unaware of mosquito modification technologies for malaria control (94.3%), and reported no knowledge of their specific characteristics (97.3%). However, community leaders participating in FGDs offered a set of distinctive interpretive frames to conceptualize interventions relying on GMMs for malaria control. The participants commonly referenced their experiences of cross-breeding for selecting preferred traits in domestic plants and animals. Preferred GMMs attributes included the expected reductions in insecticide use and human labour. Population suppression approaches, requiring as few releases as possible, were favoured. Common concerns included whether the GMMs would look or behave differently than wild mosquitoes, and how the technology would be integrated into current malaria control policies. The participants emphasised the importance and the challenge of educating and engaging communities during the technology development. Conclusion: Understanding how communities perceive and interpret novel technologies is crucial to the design and effective implementation of new vector control programs. This study offers vital clues on how communities with no prior experience of modified mosquitoes might conceptualize or respond to such technologies when deployed in the context of malaria control programs. Drawing upon existing interpretive frames and locally-resonant analogies when deploying such technologies may provide a basis for more durable public support in the future.

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