scholarly journals Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.)

2019 ◽  
Author(s):  
Karina Villanueva-Segura ◽  
Gustavo Ponce-Garcia ◽  
Beatriz Lopez-Monroy ◽  
Esteban de J. Mora-Jasso ◽  
Lucia Perales ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Multiplex Pcr ◽  
Management Strategies ◽  
Simultaneous Detection ◽  
Resistance Management ◽  
Single Amino Acid ◽  
Specific Pcr ◽  
Highly Effective ◽  
Allele Specific ◽  
Current Monitoring

AbstractBackgroundKnock down resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of nonsynonymous mutations in the he voltage-gated sodium channel (VGSC) gene, these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1,016I and F1,534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations.MethodsA multiplex PCR was developed to detect the V410L, V1,016I and F1,534C mutations in Ae. aegypti. The validation of the technique was carried out using wild populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies.ResultsThe standardized protocol for multiplex endpoint PCR was highly effective in detecting 12 genotypes in five wild Ae. aegypti populations from Mexico. A complete concordance with AS-PCR was found for the simultaneous detection of the three kdr mutations.ConclusionsOur diagnostic method is highly effective for the simultaneous detection of V410L, V1,016I and F1,534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

scholarly journals Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C in Aedes aegypti (L.)

2019 ◽  
Author(s):  
Karina Villanueva-Segura ◽  
Gustavo Ponce-Garcia ◽  
Beatriz Lopez-Monroy ◽  
Esteban de J. Mora-Jasso ◽  
Lucia Perales ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Multiplex Pcr ◽  
Management Strategies ◽  
Simultaneous Detection ◽  
Resistance Management ◽  
Single Amino Acid ◽  
Specific Pcr ◽  
Highly Effective ◽  
Allele Specific ◽  
Current Monitoring

Abstract BackgroundKnock down resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of nonsynonymous mutations in the he voltage-gated sodium channel (VGSC) gene, these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1,016I and F1,534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations.MethodsA multiplex PCR was developed to detect the V410L, V1,016I and F1,534C mutations in Ae. aegypti. The validation of the technique was carried out using wild populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies.ResultsThe standardized protocol for multiplex endpoint PCR was highly effective in detecting 12 genotypes in five wild Ae. aegypti populations from Mexico. A complete concordance with AS-PCR was found for the simultaneous detection of the three kdr mutations.ConclusionsOur diagnostic method is highly effective for the simultaneous detection of V410L, V1,016I and F1,534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

Distribution and Frequency of the kdr Mutation V410L in Natural Populations of Aedes aegypti (L.) (Diptera: Culicidae) From Eastern and Southern Mexico

2019 ◽  
Vol 57 (1) ◽  
pp. 218-223 ◽  
Author(s):  
Olga K Villanueva-Segura ◽  
Kevin A Ontiveros-Zapata ◽  
Beatriz Lopez-Monroy ◽  
Gustavo Ponce-Garcia ◽  
Selene M Gutierrez-Rodriguez ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Natural Populations ◽  
Resistance Management ◽  
Allelic Frequency ◽  
Quintana Roo ◽  
Southern Mexico ◽  
Specific Pcr ◽  
Allele Specific ◽  
Primary Vector ◽  
Allele Specific Pcr

Abstract Aedes aegypti (L.) is the primary vector of the viruses that cause dengue, Zika, and chikungunya, for which effective vaccines and drugs are still lacking. Current strategies for suppressing arbovirus outbreaks based on insecticide use pose a challenge because of the rapid increase in resistance. The widespread and excessive use of pyrethroid-based insecticides has created a large selection pressure for a kdr-type resistance, caused by mutations in the para gene of the voltage-gated sodium channel (vgsc). Our objective was to evaluate the allelic frequency of natural populations of Ae. aegypti of Mexico at codon 410 of the para gene. Twenty-six Ae. aegypti populations from east and southern Mexico were genotyped for the codon 410 using allele-specific PCR. The frequencies of the L410 allele in Ae. aegypti ranged from 0.10 to 0.99; however, most of the frequencies were in the range of 0.36 to 0.64. The highest frequencies were found in three populations from the state of Veracruz, namely, Minatitlan with 0.99, Poza Rica with 0.82, and Jose Cardel with 0.97, along with populations from Cancun in Quintana Roo with 0.93, Frontera in Tabasco with 0.91, and Ciudad del Carmen in Campeche with 0.86. The frequency of the L410 allele was high in all populations of Ae. aegypti with higher values in populations of the southeast of the country. The knowledge of specific substitutions in vgsc and their interaction to confer resistance is essential to predict and develop future strategies for resistance management in Ae. aegypti in Mexico.

scholarly journals Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis

2018 ◽  
Vol 3 (1) ◽  
pp. 10-17
Author(s):  
Heleen H Arts ◽  
Barry Eng ◽  
John S Waye
Keyword(s):  
Hereditary Hemochromatosis ◽  
Simultaneous Detection ◽  
Turnaround Time ◽  
Molecular Diagnostic ◽  
Hfe Gene ◽  
Specific Pcr ◽  
Liver And Pancreas ◽  
Allele Specific ◽  
Hfe Mutations ◽  
Allele Specific Pcr

Abstract Background Hereditary hemochromatosis (HH) is characterized by excessive iron absorption in the intestine, which can lead to failure of vital organs such as the heart, liver, and pancreas. Among northern Europeans, HH is most often associated with the C282Y and H63D mutations of the HFE gene. We developed a test that allows screening for both mutations in a single reaction. Methods A multiplex allele-specific PCR was developed for simultaneous genotyping of the H63D and C282Y HFE mutations. PCR fragments were designed such that the resulting PCR product can be analyzed in a single polyacrylamide gel lane. Results Test results from our multiplex assay were concordant with genotypes of 55 Canadian patients with suspected hemochromatosis, which had previously been established by allele-specific PCRs that targeted H63D and C282Y in separate reactions. Conclusions Molecular diagnostic detection of H63D and C282Y mutations can be achieved by a variety of methods, but these are not necessarily time-efficient or economical. Multiplex allele-specific PCR is an excellent tool for molecular diagnostic screening for H63D and C282Y mutations in patients with suspected hemochromatosis. This method is inexpensive, accurate, and highly efficient in terms of labor, throughput, and turnaround time.

Characterisation of DDT and Pyrethroid Resistance in Trinidad and Tobago populations of Aedes aegypti

2011 ◽  
Vol 101 (4) ◽  
pp. 435-441 ◽  
Author(s):  
K.A. Polson ◽  
S.C. Rawlins ◽  
W.G. Brogdon ◽  
D.D. Chadee
Keyword(s):  
Aedes Aegypti ◽  
Insecticide Resistance ◽  
Trinidad And Tobago ◽  
Pyrethroid Resistance ◽  
Management Strategies ◽  
Resistance Management ◽  
Activity Levels ◽  
Biochemical Assays ◽  
The Status ◽  
Control And Prevention

AbstractInsecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98–100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l−1, 0.2 mg l−1 and 1.0 mg l−1 for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80–98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.

scholarly journals Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261713
Author(s):  
Assalif Demissew ◽  
Abebe Animut ◽  
Solomon Kibret ◽  
Arega Tsegaye ◽  
Dawit Hawaria ◽  
...  
Keyword(s):  
Malaria Control ◽  
Pyrethroid Resistance ◽  
Management Strategies ◽  
Resistance Management ◽  
Indoor Residual Spraying ◽  
Resistance Mechanisms ◽  
Malaria Vectors ◽  
Insecticide Treated Nets ◽  
Irrigation Area ◽  
Specific Pcr

Background Indoor residual spraying and insecticide-treated nets are among the key malaria control intervention tools. However, their efficacy is declining due to the development and spread of insecticide resistant vectors. In Ethiopia, several studies reported resistance of An. arabiensis to multiple insecticide classes. However, such data is scarce in irrigated areas of the country where insecticides, pesticides and herbicides are intensively used. Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms were assessed in Arjo-Didessa sugarcane plantation area, southwestern Ethiopia. Methods Adult An. gambiae s.l. reared from larval/pupal collections of Arjo-Didessa sugarcane irrigation area and its surrounding were tested for their susceptibility to selected insecticides. Randomly selected An. gambiae s.l. (dead and survived) samples were identified to species using species-specific polymerase chain reaction (PCR) and were further analyzed for the presence of knockdown resistance (kdr) alleles using allele-specific PCR. Results Among the 214 An. gambiae s.l. samples analyzed by PCR, 89% (n = 190) were An. amharicus and 9% (n = 20) were An. arabiensis. Mortality rates of the An. gambiae s.l. exposed to deltamethrin and alphacypermethrin were 85% and 86.8%, respectively. On the other hand, mortalities against pirmiphos-methyl, bendiocarb, propoxur and clothianidin were 100%, 99%, 100% and 100%, respectively. Of those sub-samples (An. amharicus and An. arabiensis) examined for presence of kdr gene, none of them were found to carry the L1014F (West African) allelic mutation. Conclusion Anopheles amharicus and An. arabiensis from Arjo-Didessa sugarcane irrigation area were resistant to pyrethroids which might be synergized by extensive use of agricultural chemicals. Occurrence of pyrethroid resistant malaria vectors could challenge the ongoing malaria control and elimination program in the area unless resistance management strategies are implemented. Given the resistance of An. amharicus to pyrethroids, its behavior and vectorial capacity should be further investigated.

scholarly journals Allele-specific PCR detection of sweet cherry self-incompatibility alleles S3, S4 and S9 using consensus and allele-specific primers in the Czech Republic

2014 ◽  
Vol 41 (No. 4) ◽  
pp. 153-159 ◽  
Author(s):  
K. Sharma ◽  
P. Sedlák ◽  
D. Zeka ◽  
P. Vejl ◽  
J. Soukup
Keyword(s):  
Sweet Cherry ◽  
Prunus Avium ◽  
Management Strategies ◽  
Self Incompatibility ◽  
Specific Primers ◽  
Specific Pcr ◽  
Allele Specific ◽  
Considerable Length ◽  
S Allele ◽  
Consensus Primers

&nbsp; Prunus avium species of the Rosaceae family exhibit gametophytic self-incompatibility. Determination of the self-incompatibility genotype of individuals is essential for genetic studies and the development of informed management strategies. The PCR-based detection of S-allele helps to promote and speed up traditional breeding activity and hence molecular analysis of the perspective genotypes has become more intensive in all cherry growing countries. The alleles S<sub>3</sub>, S<sub>4</sub> and S<sub>9</sub> from 34 accessions of Czech collections were determined using the polymerase chain reaction (PCR) method. Initially, DNA extracts were amplified with consensus primers that amplify across the first, second, or both introns of the S-ribonuclease gene which shows a considerable length polymorphism. The new allele specific primers were designed with the goal to overcome some occurring difficulties in the detection of expected alleles by previously published allele specific primers. S-alleles fragments of standard cultivars used in this study were PCR amplified, sequenced to validate the designed primers. The study demonstrates the advantage of newly designed primers application in testing of sweet cherry genotypes. &nbsp; &nbsp;

scholarly journals Identification of CYP2B6 Sequence Variants by Use of Multiplex PCR with Allele-Specific Genotyping

2004 ◽  
Vol 50 (8) ◽  
pp. 1372-1377 ◽  
Author(s):  
Robyn M Jacob ◽  
Elaine C Johnstone ◽  
Matt J Neville ◽  
Robert T Walton
Keyword(s):  
Multiplex Pcr ◽  
Large Scale ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Cytochrome P450 2B6 ◽  
Specific Pcr ◽  
Genotypic Analysis ◽  
Cyp2b6 Gene ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: Cytochrome P450 2B6 (CYP2B6) has a role in the metabolism of many clinically important substances, but the variation within the CYP2B6 gene has not been fully characterized. The aim of the present study was to develop a reliable and robust assay for determining genotypic variants. Methods: We used a two-stage procedure. An initial multiplex PCR reaction amplified the relevant gene fragments in exonic and regulatory regions to ensure isolation of CYP2B6 from its similar pseudogene (CYP2B7). This product was then genotyped by allele-specific PCR. Results: The assay detected the following published single-nucleotide polymorphisms: C64T (Arg22Cys), C78T, G216C, G516T (Gln172His), C777A (Ser259Arg), A785G (Lys262Arg), and C1459T (Arg487Cys), as well as additional loci found within the single-nucleotide polymorphism (SNP) databases: A1190G, C1268A, C1330T, A1382G, A1402T, and an A/T SNP in intron 2 (A12917T). This approach detected all common, previously reported alleles and identified a new allele (CYP2B6*4C) present in 2.2% of a Caucasian population. Genotypic frequencies obtained were consistent with previously published results. Conclusions: This method is simple, reliable, rapid, and amenable to automation and could facilitate the large-scale genotypic analysis of CYP2B6.

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