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

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.

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone

“…To determine the allelic frequency of V135A and P253S in the field, an allele-specific PCR method was developed and deployed. AS-PCR is a relatively simple, inexpensive method and has been widely used in drug resistance studies for several parasites such as Plasmodium falciparum [ 40 , 41 ] and Haemonchus contortus [ 42 ]. Results obtained in the present study demonstrated that overall frequency of alleles corresponding to V135A and P253S was less than 4% ( Table 5 ).…”

Analysis of SOD1 and C9orf72 mutations in patients with amyotrophic lateral sclerosis in Antioquia, Colombia

“…This, along with the complete absence of G93A and D90A mutations of SOD1 in the patients, is an unexpected and relevant finding about amyotrophic lateral sclerosis genetics, its physiopathology and other neurodegenerative conditions. It is therefore necessary to confirm whether this genotype of 8 repeats in C9orf72 is pathologic in our population and to determine its importance as a diagnostic biomarker or as a risk factor for amyotrophic lateral sclerosis, as it is currently being done in other populations (43)(44)(45).…”

A comprehensive assessment of insecticide resistance mutations in source and immigrant populations of the diamondback moth Plutella xylostella (L.)

“…We also examined temporal variation in resistance mutation frequencies across 9 years to understand the potential impact of migration on the evolution of resistance in DBM. In order to deal with a large number of samples and mutations, we used the Kompetitive Allele Specific PCR (KASP) assay to detect the mutations, which provides for high‐throughput and cost‐effective genotyping for bi‐allelic variation 44–49 . This method holds promise for the genomics‐based diagnosis of insecticide resistance loci in small invertebrate pests 50 and plants 51 .…”

“…In order to deal with a large number of samples and mutations, we used the Kompetitive Allele Specific PCR (KASP) assay to detect the mutations, which provides for highthroughput and cost-effective genotyping for bi-allelic variation. [44][45][46][47][48][49] This method holds promise for the genomicsbased diagnosis of insecticide resistance loci in small invertebrate pests 50 and plants. 51 Our results help understand the dynamics of insecticide resistance in DBM and the potential influence of migration on its incidence with implications for effective control of this insect pest.…”