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

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.

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

2006 ◽  
Vol 20 (1) ◽  
pp. 7-11 ◽  
Author(s):  
J. Tiwari ◽  
A. P. Kolte ◽  
S. Kumar ◽  
C. P. Swarnkar ◽  
D. Singh ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Specific Pcr ◽  
Benzimidazole Resistance ◽  
Allele Specific ◽  
Allele Specific Pcr

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

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
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.

scholarly journals Frequency of benzimidazole resistance inHaemonchus contortus populations isolated from buffalo, goat and sheep herds

2013 ◽  
Vol 22 (4) ◽  
pp. 548-553 ◽  
Author(s):  
Ronaldo Luiz Nunes ◽  
Livia Loiola dos Santos ◽  
Eduardo Bastianetto ◽  
Denise Aparecida Andrade de Oliveira ◽  
Bruno Santos Alves Figueiredo Brasil
Keyword(s):  
Haemonchus Contortus ◽  
Significant Variation ◽  
Genetic Basis ◽  
Anthelmintic Resistance ◽  
Allelic Frequency ◽  
Molecular Diagnostic ◽  
Economic Losses ◽  
Diagnostic Tools ◽  
Ruminant Species ◽  
Benzimidazole Resistance

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

scholarly journals Allele-Specific PCR for the Detection of Azoxystrobin Resistance in Didymella bryoniae

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1681-1684 ◽  
Author(s):  
Mavis J. Finger ◽  
Venkatesan Parkunan ◽  
Pingsheng Ji ◽  
Katherine L. Stevenson
Keyword(s):  
Dna Sequences ◽  
Cyt B ◽  
Didymella Bryoniae ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Sensitive Allele ◽  
Cyt B Gene ◽  
Allele Specific Pcr

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

Heterozygosity for the Non-Deletional O2 Allele Does Not Cause Discrepancies in Automated Blood Donor ABO Grouping.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 957-957 ◽  
Author(s):  
Mark H. Yazer ◽  
Jonathan McGuirt ◽  
Åsa Hellberg ◽  
Bahram Hosseini-Maaf ◽  
Annika Hult ◽  
...  
Keyword(s):  
B Cells ◽  
Consensus Sequence ◽  
Flow Cytometric Analysis ◽  
Antigen Expression ◽  
Blood Group System ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Digestion Pattern ◽  
Allele Specific ◽  
Group B

Abstract In the ABO blood group system any allele which does not give rise to A or B antigen expression is an O allele. The most common O alleles, O1 (O01) and O1v (O02) encode enzymatically inactive truncated proteins due to a single nucleotide polymorphism (SNP) compared to the consensus A1 allele, 261delG. Other O alleles containing the consensus nucleotide at residue 261 have been identified. The most common of these non-deletional O alleles is O2 (O03). It contains other SNPs which render the resulting protein non-functional. The most significant SNP is 802G>A (G268R); the arginine residue blocks the donor sugar’s access to the active site preventing its transfer. Recently O2 alleles were proposed to be the most common cause of ABO discrepancies (anti-A lacking or weakened in plasma despite apparent group O phenotype) in automated blood grouping (Transfusion2005;45:1331). Other clinical or biochemical reports have suggested that O2 may produce small amounts of A antigen on the RBC surface (Transfusion2005;45:70, 2005;45:359, J Biol Chem2005;280:525). To establish the effect of the O2 allele on automated ABO typing we extracted DNA from 562 randomly selected group O RBC units that were available for transfusion. Automated forward and reverse ABO grouping was performed on an Olympus PK7231 using A1 and B cells for reverse typing (Pittsburgh) and the IBG Multisampler Plato 3000 SI system using A1, A2 and B cells (Lund). A PCR-RFLP assay (Vox Sang 1995;69:242) involving amplification of exon 6 of the ABO gene followed by cleavage with KpnI to detect the presence of the 261delG SNP was performed and 34/562 (6%) of these units were heterozygous for a deletional and a non-deletional O allele. All 34 of these donors demonstrated an identical digestion pattern with BstEII which cleaves exon 6 in the presence of the consensus sequence at residue 261, confirming their heterozygosity for a non-deletional O allele. Allele-specific PCR to detect the O2-specific 802G>A polymorphism was performed on 32 of these donors for which additional DNA was available; the O2 allele was uniformly detected along with either an O1 or O1v allele. No homozygous O2O2 donors were identified. A sensitive flow cytometric analysis of the RBCs from 7 donors with the O2 allele was performed using monoclonal anti-A (ES-15) and a PE-labelled rat-anti-mouse kappa Ig secondary antibody. None of these RBCs demonstrated fluorescence levels in significant excess of the control O (O1O1) cells whilst A antigen on group B cells was demonstrable. Manual immediate spin and gel card forward typing was performed on these donors using a variety of murine monoclonal, polyclonal and experimental anti-A reagents; no A antigen was detected on their RBCs. Adsorption-elution studies were performed on the RBCs of 3 donors heterozygous for O2 using human-source polyclonal anti-A but no A antigen was detected. All 34 group O RBC units were transfused to group O recipients without reported hemolytic reactions. Our results suggest that in its heterozygous state the O2 allele does not necessarily induce ABO discrepancies in automated testing. Transfusing group O RBCs from donors with an O2 allele to recipients with anti-A did not result in reports of symptoms associated with hemolytic events in the recipients.

scholarly journals Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring

2006 ◽  
Vol 72 (8) ◽  
pp. 5428-5435 ◽  
Author(s):  
Rosely A. B. Nichols ◽  
Brian M. Campbell ◽  
Huw V. Smith
Keyword(s):  
Environmental Water ◽  
18S Rrna Gene ◽  
Water Monitoring ◽  
Rrna Gene ◽  
Molecular Fingerprinting ◽  
Oocyst Wall ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Microscope Slides

ABSTRACT We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.

Comparison of PCR-RFLP with Allele-Specific PCR in Genetic Testing for Spinal Muscular Atrophy

2003 ◽  
Vol 7 (4) ◽  
pp. 277-281 ◽  
Author(s):  
Ruliang Xu ◽  
Shuji Ogino ◽  
Va Lip ◽  
Hong Fang ◽  
Bai-Lin Wu
Keyword(s):  
Genetic Testing ◽  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Molecular Study of Benzimidazole Resistance in Teladorsagia circumcincta Isolated from Sheep in North of Iran

2019 ◽  
Author(s):  
Rahim NEMATI ◽  
Aliasghar BAHARI ◽  
Pezhman MAHMOODI ◽  
Alireza SAZMAND
Keyword(s):  
Anthelmintic Resistance ◽  
Molecular Study ◽  
Nucleotide Polymorphisms ◽  
Northern Iran ◽  
The North ◽  
Specific Pcr ◽  
Teladorsagia Circumcincta ◽  
Pcr Rflp ◽  
North Of Iran ◽  
Allele Specific

Background: Resistance to benzimidazole (BZ) compounds is common in Teladorsagia circumcincta populations in sheep and goats worldwide. Given the importance of anthelmintic resistance and shortage of information on single nucleotide polymorphisms (SNPs) in this prevalent nematode in Iran, this study was conducted. Methods: From June to September 2016, abomasa of 139 sheep of different sexes and ages in Amol City slaughterhouse, northern Iran were examined for isolation of nematodes. Totally 45 male T. circumcincta confirmed by both microscopical and nested-PCR-RFLP methods were included in this study. Susceptibility or resistance of each single T. circumcincta worm to benzimidazoles was assessed using allele-specific PCR. Results: Frequency of genotypes in the present study were 33.33% heterozygote BZ and 66.67% BZ homozygote sensitive. No homozygote resistant worm was found. Conclusion: Resistance against BZs in T. circumcincta of sheep has occurred at a low prevalence in the north of Iran. However, mutated genes might get dominant under drug selection in future. Hence, periodic investigations for early detection of mutated alleles in nematode populations using accurate and sensitive molecular methods such as PCR-RFLP is recommended.

A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer

2021 ◽  
Author(s):  
Fatemeh Sheikhsofla ◽  
Behzad Poopak ◽  
Sajjad Firuzyar ◽  
Fatemeh Roudbari ◽  
Mojtaba Ghadiany
Keyword(s):  
Colorectal Cancer ◽  
Gene Mutations ◽  
Dot Blot ◽  
Braf Mutations ◽  
Kras Gene ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Reverse Dot Blot

Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods. Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively. Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D  and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.  Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

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