Diagnosis of Benzimidazole Resistance in Haemonchus contortus of Sheep by Allele Specific PCR

The study investigated the presence of resistance to benzimidazoles in Haemonchus contortus helminths from ruminant species in Greece through the detection of the Phe/Tyr polymorphism in the amino acid at position 200 of the β-tubulin protein. In total, 288 adult female H. contortus helminths collected from the abomasum of various ruminant animals in Greece were tested. Of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and 48 from buffaloes. The frequencies of the homozygous and heterozygous resistant genotypes at the position 200 of the β-tubulin gene of helminths recovered from sheep were 96.9% and 3.1%, respectively. The frequencies of the homozygous and heterozygous resistant genotypes, respectively, were 100.0% and 0.0% in helminths from goats, 25.0% and 75.0% in helminths from cattle and 8.3% and 91.7% in helminths from buffaloes. In all parasitic populations, no homozygous susceptible genotypes were detected. The present study highlighted, for the first time, the emergence of benzimidazole-resistant H. contortus in goats, cattle, and buffaloes in Greece, using an allele-specific PCR. It is postulated that benzimidazole-resistant alleles were transferred from sheep or goats to cattle and buffaloes at the commonly grazing pastures in Greece.

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Molecular Detection of Benzimidazole Resistance in Haemonchus contortus Larvae of Goats in Chhattisgarh, India

Indian Journal of Animal Research ◽

10.18805/ijar.b-4532 ◽

2021 ◽

Author(s):

S. Nath ◽

S. Pal ◽

S. Mandal ◽

S. Jadhao ◽

M. Sankar ◽

...

Keyword(s):

Nested Pcr ◽

Haemonchus Contortus ◽

Central India ◽

Allelic Frequency ◽

Specific Pcr ◽

Benzimidazole Resistance ◽

Pcr Product ◽

Pcr Rflp ◽

Allele Specific ◽

Homozygous Resistant

Background: Benzimidazole resistance is one of the key problem in small ruminant production. A rapid, truthful and responsive system is required for detection benzimidazole resistance so that proper regulatory measure can be applied. Allele specific PCR is one of the tools to understand the mechanism and origin of benzimidazole resistance. Methods: A total 198 larvae of Haemonchus contortus were isolated from goats of Chhattisgarh region, central India were genotyped by allele specific polymerase chain reaction (AS-PCR). Faecal samples of goats were collected from three Government farms and adjoining field goats and were subjected for faecal culture, separately. DNA of third stage larva was used for nested PCR for amplification of β- tubulin gene. Restriction Fragment Length Polymorphism (RFLP) was applied on nested PCR product for species identification with RsaI enzyme. AS-PCR was applied on the nested-PCR product to know the genotypic and allelic frequency. Result: The nested PCR amplified product showed approximately 820 bp in all cases and PCR-RFLP revealed 462 bp, 211 bp and 147 bp fragments, which confirmed the species as H. Contortus. Frequency of resistant allele ('r') was 49.7% and 50.3% for susceptible allele ('S '). Frequency of homozygous resistant (rr), heterozygous susceptible (rS) and homozygous susceptible (SS) genotype were 33.83 per cent, 31.81 per cent and 34.34 per cent, respectively. The frequency of homozygous resistant (rr) genotype was low (19.61%) in field compare to farm (48.96%) indicating refugia in field region.

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Genotyping of benzimidazole resistant and susceptible isolates of Haemonchus contortus from sheep by allele specific PCR

Journal of Parasitic Diseases ◽

10.1007/s12639-016-0793-2 ◽

2016 ◽

Vol 41 (1) ◽

pp. 282-288 ◽

Cited By ~ 1

Author(s):

Karthik Mohanraj ◽

Subhra Subhadra ◽

Aravindan Kalyanasundaram ◽

Manikkavasagan Ilangopathy ◽

Muthusamy Raman

Keyword(s):

Haemonchus Contortus ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650362 ◽

1996 ◽

Vol 75 (05) ◽

pp. 757-759 ◽

Cited By ~ 11

Author(s):

Rainer Blasczyk ◽

Markus Ritter ◽

Christian Thiede ◽

Jenny Wehling ◽

Günter Hintz ◽

...

Keyword(s):

Direct Sequencing ◽

Activated Protein C ◽

Factor V Leiden ◽

Pcr Amplification ◽

Factor V ◽

Specific Pcr ◽

Pcr Rflp ◽

Allele Specific ◽

Specific Amplification ◽

Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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MUPrimer : a tool for finding allele specific PCR-primers for homologous gene sequences

10.32469/10355/6660 ◽

2009 ◽

Author(s):

M. Mursaleen Ahmad

Keyword(s):

Pcr Primers ◽

Homologous Gene ◽

Gene Sequences ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

BMC Plant Biology ◽

10.1186/s12870-021-02932-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhengjie Chen ◽

Dengguo Tang ◽

Jixing Ni ◽

Peng Li ◽

Le Wang ◽

...

Keyword(s):

Marker Assisted Selection ◽

Inbred Lines ◽

Average Density ◽

Snp Markers ◽

Data Sets ◽

Rna Seq ◽

Specific Pcr ◽

Maize Inbred Lines ◽

Allele Specific ◽

Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

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

Agronomy ◽

10.3390/agronomy11050982 ◽

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.

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