Allele-specific PCR Amplification Research Articles

Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 104 copies/μL and 103 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2–3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.

An Integrated Strategy for Analyzing the Complete Complex Integrated Structure of Maize MON810 and Identification of an SNP in External Insertion Sequences.

Genetically modified maize (Zea mays L.) MON810 was approved for importation into China for feed use in 2004; however, the localization data concerning exogenous insertion sequences, which confer insect resistance, have been questionable. MON810 maize plants discovered in northeastern China were used to analyze the molecular characteristics of the exogenous insertion. Using PacBio-HiFi sequencing and PCR assays, we found the insertion was located in chromosome 8, and there was a CaMV35S promoter, hsp70 intron, and insecticide gene cry1Ab, except for genome sequence insertion in the MON810 event. Importantly, the 5' and 3' flanking sequences were located in the region of 55869747-55879326 and 68416240-68419152 on chromosome 5, respectively. The results of this study correct previous results on the genomic localization of the insertion structure for the MON810 event. We also found a single-nucleotide polymorphism (SNP) in the hsp70 intron, which is most likely the first SNP found in a transgenic insertion sequence. PCR amplification in conjunction with Sanger sequencing, allele-specific PCR (AS-PCR), and blocker displacement amplification (BDA) assays were all effective at detecting the base variance. The integrated strategy used in this study can serve as a model for other cases when facing similar challenges involving partially characterized genetic modification events or SNPs.

Association of SERPING1 gene single nucleotide polymorphism with resistance to natural strongyle infection among native goats in humid tropical Kerala

Strongylosis poses a significant threat to goat populations, particularly in humid tropical regions like Kerala, a southern peninsular state in India. This study aimed to compare breed differences in host resistance and resilience to natural strongyle infection between Attappady Black and Malabari goats reared under semi-intensive systems. Additionally, we analyzed the association of a potential single nucleotide polymorphism (SNP) in the Serine Proteinase Inhibitor Clade G member 1 (SERPING1) gene with these traits. Faecal egg count (FEC) and volume of packed red cells (VPRC) were employed as indicators of host resistance and resilience, respectively. Over the course of one year, fortnightly phenotypic data on FEC and VPRC were collected from 109 native goats, including 58 Malabari and 51 Attappady Black goats. The Malabari goats exhibited greater resistance and resilience to strongylosis, evidenced by significantly lower FEC (p≤0.01) and higher VPRC (p≤0.05) compared to Attappady Black goats. Pearson correlation analysis revealed a highly significant negative correlation between FEC and VPRC (r= −0.41, p<0.01). Genotyping of the −312-C>T SNP in SERPING1 gene was conducted using modified bidirectional PCR amplification of specific alleles (Bi-PASA). While the association between SERPING1 genotypes and FEC was not significant, a significant association was observed between SERPING1 genotypes and VPRC (p<0.05). These findings underscore the importance of breed-specific differences in goat resilience to strongyle infection and highlight the potential role of SERPING1 gene polymorphism in modulating host resilience traits.

Global DNA Methylation Levels Viz-a-Viz Genetic and Biochemical Variations in One Carbon Metabolic Pathway: An Exploratory Study from North India.

Despite the importance of one carbon metabolic pathway (OCMP) in modulating the DNA methylation process, only a few population-based studies have explored their relationship among healthy individuals. This study aimed to understand the variations in global DNA methylation levels with respect to selected genetic (CBS 844ins68, MTRR A66G, MTR A2756G, and MTHFR C677T polymorphisms) and biochemical (folate, vitamin B12, and homocysteine) markers associated with OCMP among healthy North Indian adults. The study has been conducted among 1095 individuals of either sex (69.5% females), aged 30-75 years. A sample of 5 mL of blood was collected from each participant. Homocysteine, folate, and vitamin B12 levels were determined using the chemiluminescence technique. Restriction digestion was performed for genotyping MTRR A66G, MTR A2756G, and MTHFR C677T polymorphisms and allele-specific PCR amplification for CBS 844ins68 polymorphism. Global DNA methylation levels were analyzed using ELISA-based colorimetric technique. Of the selected genetic and biochemical markers, the mutant MTRR A66G allele was positively associated with global DNA methylation levels. Further, advanced age was inversely associated with methylation levels. MTRR 66GG genotype group was hypermethylated than other genotypes in folate replete and vitamin B12 deficient group (a condition prevalent among vegetarians), suggesting that the G allele may be more efficient than the wild-type allele in such conditions. Global DNA methylation levels appeared to be more influenced by genetic than biochemical factors. MTRR 66G allele may have a selective advantage in vitamin B12 deficient conditions. Further research should be undertaken to understand how genetics affects epigenetic processes.

Kdr mutations and deltamethrin resistance in house flies in Abu Dhabi, UAE

BackgroundThe house fly, Musca domestica, is a significant carrier of diseases that can impact public health. Repeated use of pyrethroid insecticides may act as a selection pressure for mutations and amino acid substitutions in the house fly voltage-sensitive sodium channel (VSSC), which ultimately confers resistance. The objectives of this study were to determine the presence of knockdown resistance (kdr) mutations using molecular tools and to set up a CDC bottle bioassay specific for house flies in the United Arab Emirates (UAE) to screen for deltamethrin resistance.MethodsAdult flies were collected from 19 locations in Abu Dhabi, UAE, and DNA was extracted, followed by PCR amplification of specific alleles (PASA) and conventional PCR using several primers to amplify regions of the VSSC gene. Sanger sequencing was performed on PCR products. We also designed primers that detect four kdr mutations using complementary DNA (cDNA) in reverse transcriptase (RT)-PCR followed by Sanger sequencing. Additionally, a CDC bottle bioassay was set up for detecting deltamethrin resistance in adult house flies.ResultsIn PASA, the primers successfully amplified the target bands (480, 280 and 200 bp). The kdr allele was found in flies collected from 18 of the 19 locations, at the highest and lowest prevalence of 46.9% and 9.4%, respectively. Resistant homozygous (RR) insects constituted 5.0% of the tested populations, and heterozygous (RS) insects accounted for 36.5%. The RR genotype was prevalent in house flies collected at 10 of 19 sampling locations. House fly populations were mostly in Hardy–Weinberg equilibrium, except in three locations. In addition to verifying the presence of the previously identified kdr mutation L1014F, in this study we detected two kdr mutations, L1014H and T929I, that have not previously been reported in the UAE. Also, for the first time in the UAE, a CDC bottle bioassay for deltamethrin resistance was used, which found that 60 min and 4.5 µg/ml were the diagnostic time and dose, respectively. Using this assay, we detected deltamethrin resistance in house flies from two of 16 locations, with a resistance level of 12.5%.ConclusionsUsing DNA sequencing, we confirmed the presence of a known kdr mutation and uncovered two new kdr mutations in house flies from Abu Dhabi. Additionally, we detected deltamethrin resistance in these flies using a CDC bottle bioassay. Further research is recommended to comprehensively identify more kdr mutations in UAE house fly populations and assess their impacts on control strategies.Graphical

Genetic variation of CYP2C9 gene and its correlation with cardiovascular disease risk factors.

The major enzyme that is responsible for Sulfonylureas (SUs) metabolism is hepatic cytochrome P-450 2C9 (CYP2C9). It is encoded by the polymorphic gene CYP2C9, which has many allelic variants, among those the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. People with diabetes mellitus type 2 (T2DM) are more likely to develop cardiovascular disease (CVD), and their risk of dying from it is more than two times higher than that of people without the condition. The purpose of this study was to evaluate the association of genetic variations in the CYP2C9 gene with cardiovascular risk factors by investigating CYP2C9*1, *2, *3, *5, *11, and *13 allelic variants. A total of 226 participants were enrolled in the current case-control study. Allele-specific amplification- PCR (ASA-PCR) was used to determine the allele of different variations and the results were confirmed by sequencing. The findings of this study showed the presence of the CYP2C9*2 allele in the T2DM group does not differ from its percentage in the control group. Also, CYP2C9*3 allele frequencies identified by Hardy-Weinberg equilibrium (HWE) analysis law were not significant, p = 0.6593 and 0.5828 in T2DM and control groups. There is no statistically significant difference between the control and diabetes groups involving the distribution of CYP2C9 alleles and CYP2C9*5, *11, and *13 polymorphisms were absent in the Iraqi population. No carrier for the CYP2C9*3 homozygous state was found in both groups. According to these results T2DM patients with the CYP2C9*2 and *3 variants have an increased risk of developing hypertension.

Pharmacogenetics: Influence of CYP2C9*2 and *3 alleles polymorphisms on Iraqi type 2 diabetic patients

Abstract Background: Sulfonylureas (SUs) are the most prescribed anti-diabetic drugs. The enzyme responsible for metabolizing of SUs is hepatic cytochrome P4502C9 (CYP2C9). The CYP2C9 gene has numerous allelic variations; among those, the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. The pharmacokinetics of SUs are dramatically impacted by CYP2C9 genotype. Objective: Evaluation of the association of genetic polymorphisms in CYP2C9 gene with the efficacy of glibenclamide (GB), second-generation SUs, by investigating two CYP2C9 allelic variants. Materials and Methods: Blood samples were collected from 113 type 2 diabetes patients. Allele specific amplification-polymerase chain reaction was used to genotype the CYP2C9 gene. Fasting serum glucose, fasting insulin, and glycated hemoglobin (HbA1c) levels were measured as part of the biochemical analysis. Results: The CYP2C9 gene variants were analyzed in a study group. The results exposed that 75 patients carried the wild (CYP2C9*1/*1) genotype, 25 were heterozygote allele (CYP2C9*1/*2) for CYP2C9*2 gene, 4 were homozygous for the variant CYP2C9*2 allele (CYP2C9*2/*2), and 9 were heterozygous for the variant CYP2C9*3 allele (CYP2C9*1/*3). Statistically significant difference was found in mean HbA1c between the mutant and wild alleles group (P = 0.044). The mean HbA1c for those carrying the CYP2C9*2 and*3 alleles (n = 38) was 8.4750 compared to 9.3177 for those carrying the CYP2C9*1 allele (n = 75), which indicate better glycemic control. Conclusion: The accordance of CYP2C9*2 and*3 was found to be associated with severe hypoglycemia (odd ratio [OR] = 2.045). The OR suggests a strong association between CYP2C9*2 and*3 alleles and hypoglycemia. Our findings imply that the diabetic patients with CYP2C9 polymorphism are more likely to suffer hypoglycemia than those with wild type alleles when treated with GB.

Analysis of EGFR Gene Mutations in Lung Adenocarcinoma in Karamay, Xinjiang

Abstract Objective To investigate the mutation types and mutation rate of the epidermal growth factor receptor (EGFR) gene in patients with lung adenocarcinoma and the clinical features of lung adenocarcinoma with EGFR gene mutations in Karamay, Xinjiang, China. Methods Paraffin-embedded tissue samples of adenocarcinoma patients were collected in the Karamay Central Hospital from March 2016 to June 2019, and mutations in exon 18–21 of the EGFR gene were detected by the allele-specific amplification polymerase chain reaction (Amplification Refractory Mutation System–PCR) method. The relationships between the mutation types, mutation incidence, and clinical features were analyzed. Results Of the 170 patients with lung adenocarcinoma, 83 had EGFR mutations. The total mutation rate of EGFR in patients with lung adenocarcinoma was 48.8%, which included mutations in exons 18 (1.2% [2/170]), 19 (19.4% [33/170]), 20 (2.4% [4/170]), and 21 (20.6% [35/170]). Intriguingly, there was a case with 9 mutations in exons 20 and 21. The mutations in exon 19 of EGFR resulted in the deletion of codons 746 to 750. The main mutation in exon 21 was L858R (91.4% [32/35]). There was no significant difference in exons 19 and 21 mutation rates (P &gt; 0.05). The mutation rate of EGFR in female patients was significantly higher than that in male patients (P &lt; 0.05) but had no correlation with the age, smoking status, and clinical stage of patients with non–small cell lung cancer (P &gt; 0.05). The EGFR mutation rate may be related to the degree of tumor differentiation. Conclusion Among patients with lung adenocarcinoma in Kelamayi (city in Xinjiang), EGFR mutations were more frequently detected in female patients, and the main sites of mutations were exons 19 and 21.

BRAFV600E, BANCR, miR-203a-3p and miR-204-3p in Risk Stratification of PTC Patients.

In order to enhance the risk stratification of papillary thyroid carcinoma (PTC) patients, we assessed the presence of the most common mutation in PTC (BRAFV600E) with the expression profiles of long non-coding RNA activated by BRAFV600E (BANCR) and microRNAs, which share complementarity with BANCR (miR-203a-3p and miR-204-3p), and thereafter correlated it with several clinicopathological features of PTC. BRAFV600E was detected by mutant allele-specific PCR amplification. BANCR and miRs levels were determined by quantitative RT-PCR. Bioinformatic analysis was applied to determine the miRs' targets. The expression profile of miR-203a-3p/204-3p in PTC was not affected by BRAFV600E. In the BRAFV600E-positive PTC, high expression of miR-203a-3p correlated with extrathyroidal invasion (Ei), but the patients with both high miR-203a-3p and upregulated BANCR were not at risk of Ei. In the BRAFV600E-negative PTC, low expression of miR-204-3p correlated with Ei, intraglandular dissemination and pT status (p < 0.05), and the mutual presence of low miR-204-3p and upregulated BANCR increased the occurrence of Ei. Bioinformatic analysis predicted complementary binding between miR-203a-3p/204-3p and BANCR. The co-occurrence of tested factors might influence the spreading of PTC. These findings partially describe the complicated network of interactions that may occur during the development of PTC aggressiveness, potentially providing a new approach for high-risk PTC patient selection.

The Relationship Between Odontogenic Cyst and P53 Codon 72 And P53 Codon 175 Variants in Turkish Patients

Objective: Odontogenic cysts that cause bone destruction can exhibit various types of metaplasia. Inherited genetic variants in codons 72 and 175, the hotspot codons of p53, known as the guardian of the genome, can cause a wide variety of cancers. We aimed to investigate the effects of the p53 codon 72 and p53 codon 175 variants on odontogenic cyst formation. Methods: This research encompassed 71 individuals with odontogenic cysts and 90 without any conditions as a control group. After DNA was extracting, the p53 codon 72 was detected using PCR techniques, while p53 codon 175 was identified through allele-specific amplification-PCR. Results: The presence of the p53 codon 72 GG genotype and its G allele was less frequent in the group with odontogenic cysts compared to the healthy participants. Conversely, the C allele was found more often in the cyst-afflicted group. For the p53 codon 175, the AA genotype and A allele were more common in the affected group, while the G allele was more predominant in the control group. Conclusion: The p53 codon 175 AA genotype and A allele, p53 codon 72 C allele, and p53 codon 72/codon 175 CCAA combined genotype may be associated with odontagenic cyst formation. Individuals with this allele and genotype can be considered at risk for odontagenic cyst formation.

Association of angiotensin-converting enzyme I/D polymorphism and apolipoprotein B with cardiometabolic abnormalities among young adults: a pilot study from Delhi

BackgroundAngiotensin-converting enzyme (ACE) gene polymorphism and elevated apolipoprotein B (apoB) are important risk factors for several cardiometabolic abnormalities. However, much less attention has been given to the relationship between these risk factors and cardiometabolic abnormalities among young adults. Considering this gap, the present study explored the association of ACE I/D polymorphism and apoB with cardiometabolic abnormalities among young adults of Delhi, India.MethodsThis cross-sectional study was conducted among young adults (aged 18–30) of either sex residing in Delhi, India. A total of 330 individuals were invited to participate in the study, and data on the socio-demographic variables were collected using a pre-tested interview schedule. Somatometric and physiological measurements were obtained using standard protocols. However, blood sample collection and biochemical and genetic analyses could successfully be performed for 178 individuals. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were estimated using Erba XL-640 biochemical analyzer. LDL and TG values were used to calculate apoB levels. Genotyping for ACE I/D polymorphism was performed by allele-specific PCR amplification followed by electrophoresis. Statistical analysis was done using SPSS v.20.ResultsACE I/D polymorphism was not found to be associated with hypertension, obesity, and abnormal FBG, TG, and HDL levels. However, DD and ID genotypes and D allele, with II as the reference genotype, significantly reduced the risk for high TC (OR, p value = 0.14, 0.01*; 0.29, 0.04*; 0.22, < 0.01*, respectively) and high LDL (OR, p value = 0.17, 0.03*; 0.20, 0.03*; 0.19, < 0.01*, respectively). Except for abnormal FBG, the prevalence of all the studied cardiometabolic abnormalities was significantly higher in the 4th quartile of apoB when compared to other quartiles. Linear regression model revealed a significant positive association of apoB levels with diastolic blood pressure, studied obesity parameters, TC, TG, and LDL levels.ConclusionThe D allele of ACE I/D polymorphism was not associated with most of the studied cardiometabolic abnormalities in the present study. Further, the association of high apoB with cardiometabolic abnormalities hints toward the importance of apoB in the early diagnosis of CVDs.

Characterization of a pollen-part self-compatible apple (Malus × domestica Borkh.) mutant induced by γ-ray mutagenesis

Self-compatibility (SC) in a naturally self-incompatible fruit tree species is a highly interesting trait for breeding objectives and a valuable tool for investigating the mechanism of the gametophytic self-incompatibility (GSI) system. This study focused on apple (Malus × domestica Borkh.), carried out γ-ray mutagenesis, and succeeded in producing a self-compatible mutant. The reciprocal pollination test showed that a pollen factor is involved in its SC. Results on S-RNase genotyping by allele-specific PCR amplification of the progenies of the mutant revealed that SC in the mutant was not caused by a nonfunctional pollen S factor in either one of the S-haplotypes but presumably by an S-chromosomal alteration (i.e., reciprocal translocation of an S-haplotype or a partial S-chromosomal duplication). Amplicon sequencing of S-haplotypes of progeny individuals suggested that the most probable S-chromosomal alteration in the mutant could be a translocation of an S-haplotype to a nonhomologous chromosome; such S-chromosomal alteration made it possible for the mutant to produce S-heteroallelic pollen grains that overcome self-incompatibility by competitive interaction between the two different S-factors in a pollen grain. These results supported the idea of a “collaborative non-self-recognition system by multiple factors” based on the GSI system in apple, similar to those in Pyrus of Rosaceae and Solanaceae. Implications for breeding self-compatible apple using the mutant induced in this study are briefly discussed.

Modified gold nanoparticle colorimetric probe-based biosensor coupled with allele-specific PCR for rapid detection of G944C mutation associated with isoniazid resistance

The emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) makes tuberculosis (TB) difficult to control and treat. Therefore, rapid and reliable detection of MDR-TB within the community plays a significant role. The aim of this study was to investigate the use of modified gold nanoparticle (AuNP) colorimetric probe-based biosensor coupled with allele-specific PCR for the rapid detection of G944C mutation associated with isoniazid resistance in MTB. Based on the citrate reduction method, the AuNPs were synthesized and functionalized using thiol-modified oligonucleotides (AuNP-biosensor). The AuNP-biosensor method was compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy. The gold standard was defined as a positive result in sequencing.The AuNP-biosensor had 100% sensitivity and specificity for detection of G944C mutation. After allele-specific PCR amplification the results were carried out for <15 min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 309 and 100%, respectively. The method designed in this study can identify the desired mutation with high efficiency after initial amplification, either from the sputum sample or from isolated bacteria. By designing a panel of this method, different MTB resistance-associated mutations could be detected more rapidly.

Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods.

The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.

Insecticide Resistance and Mechanisms of Culex pipiens Populations in the Mediterranean and Aegean Regions of Turkey During 2017-2018.

Culex pipiens has a significant public health importance since it is an important vector of West Nile virus and Rift Valley fever virus. We, therefore, aimed to determine the insecticide resistance level in Cx. pipiens populations in the Aegean and Mediterranean regions of Turkey. Bioassays have been carried out against Dichlorodiphenyltrichloroethane (DDT) (4%), Malathion (5%), Fenitrothion (1%), Propoxur (0.1%), Bendiocarb (0.1%), Permethrin (0.75%) and Deltamethrin (0.05%). Biochemical analyses have been performed to detect non-specific esterase, mixed function oxidase, glutathione-s-transferase and acetylcholinesterase levels. A knockdown resistance (kdr) (L1014F) and Acetylcholinesterase (Ace-1) (G119S) mutations have been detected by using allele-specific primers and a polymerase chain reaction (PCR) amplification of specific alleles (PASA) diagnostic test was performed for detection of F290V mutation. Bioassay results showed that all Cx. pipiens populations were resistant to DDT, Malathion, Fenitrothion, Bendiocarb, Propoxur and some of the populations have started to gain Permethrin and Deltamethrin resistance. Biochemical analyses results revealed that altered glutathione-s-transferases, P450 monooxygenases, esterase levels might be responsible for DDT, organophosphate, carbamate and pyrethroid resistance in Cx. pipiens populations. Results showed mild to high frequency of L1014F, low frequency of F290V but no Ace-1 G119S mutation within the populations. Additionally, acetylcholinesterase insensitivity was not significantly high within the most of these populations. Overall results may help to fulfil the lacking information in the literature regarding insecticide resistance status and underlying mechanism of Culex pipiens populations of the Mediterranean and Aegean region of Turkey by using all bioassays, molecular tests and biochemical assays.

Frequency of Pyrethroid Insecticide Resistance kdr Gene and Its Associated Enzyme Modulation in Housefly, Musca domestica L. Populations From Jhang, Pakistan

Housefly, Musca domestica, is considered responsible for transmitting a wide variety of human and veterinary diseases. Mostly, insecticides are being used for their control and more commonly, pyrethroid insecticides worldwide. However, resistance has been reported against various pyrethroid insecticides. Houseflies become resistant by two major mechanisms, i.e., target site insensitivity through knockdown resistance gene mutation (kdr) and enzyme detoxification. Thus, the current study was designed to monitor the frequency of pyrethroid resistance gene kdr in housefly populations of District Jhang. The flies were collected from seven sampling sites and then reared in the lab for molecular and biochemical assays. The amplification of template DNA was performed for knockdown resistance gene through the outer primers kdr1 and kdr4, and the inner primers kdr1 and kdr2 using PASA (PCR Amplification of Specific Alleles) method which specifically amplify the domain-II of kdr gene. Three populations were found homozygous susceptible (+/+; 42.85%), whereas two populations were found genetically homozygous resistant (−/−; 28.57%) which are insensitive to pyrethroid insecticides. Similarly, two populations were found heterozygous (+/−; 28.57%) for kdr suggesting thereby that at least 1/4th homozygous-resistant (−/−) housefly populations with insensitivity to pyrethroids would be produced in the future keeping in view the Mendelian ratio. Biochemical assay showed that homozygous-resistant populations had increased activity of Acetylcholinesterase (AChE), α-Carboxylesterases (α-Carboxyl), β-Carboxylesterase (β-Carboxyl), Alkaline Phosphatase (AkP), and Acidic Phosphatase (AcP) enzymes. In addition, heterozygous populations also showed increased activities of these enzymes. The current results would not only help avoid the indiscriminate load of insecticides onto the environment but also serve as a hallmark for the management of housefly populations in target areas in the future.

Enhancing Cohort PASA Efficiency from Lessons Assimilated by Mutant Genotyping in C. elegans.

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.

LIDDLE SYNDROME – DETECTION OF NOVEL MUTATION IN A FAMILY WITH HYPERTENSION

Abstract Objective: Liddle syndrome is a rare monogenic form of arterial hypertension with autosomal dominant inheritance. It is caused by mutation of genes coding one of three subunits of epithelial sodium channel (ENaC), which is located in distal nephron. Under physiological conditions, its expression and activity is regulated to maintain fluid and electrolytes homeostasis by reabsorbing Na+. Mutated channel cannot be degraded and therefore, it is accumulated on cell membrane; this results in enhanced sodium reabsorption. The most common clinical presentation of Liddle syndrome is early onset of hypertension, hypokalemia, suppressed plasma renin activity and low plasma aldosterone level. Up to now, 36 mutations causing Liddle syndrome were described. Design and method: Genetic assessment was performed in a Czech family of three generations (n = 14, see Figure) where hypertension and tendency to hypokalemia was present in several members. We used PCR amplification of specific alleles of SCNN1B and SCNN1G genes which was followed by Sanger sequencing. Results: We present a novel mutation causing Liddle syndrome: Tyr604∗ in beta-subunit of epithelial sodium channel. This mutation is supposed to generate a premature stop codon at position 604, resulting in truncated beta-subunit of EnaC. It was found in 7 members of the family: I-1, II-1 and 2, and III-1 to 4. Phenotypes of these subjects were different: from severe hypertension and hypokalemia (subjects I-1 and III-3, manifestation in 10 and 5 years, respectively) to normotension with normal potassium levels (III-1 and 2, now aged 8 and 10 years, respectively); however, all the seven subjects with mutation had hypoaldosteronism (serum level &lt; 0.13 ng/ml). Conclusions: A new mutation causing Liddle syndrome is described. It is manifested with different severity and mild forms that are not manifested in early childhood exist; thus, Liddle syndrome may be more frequent among adult hypertensive subjects than currently supposed. Proper diagnosis is important as the patients respond well to treatment with ENaC blocker amiloride.

Differential expression following ammonia stress and growth-related SNP of ferritin: Applications for establishment of stress-tolerant stocks with high growth performance of Pacific white shrimp Litopenaeus vannamei in Thailand

Effects of ammonia challenge on expression of ferritin (LvFer) transcripts in Pacific white shrimp Litopenaeus vannamei were examined. One family of second generation (G2) of a growth-improved stock was generated and subjected to a non-lethal ammonia stress test (control, 10 and 20 mg/l ammonia-N). The expression level of LvFer transcripts in hemocytes and hepatopancreas of L. vannamei juveniles during a 72 h trial was significantly changed after exposed to 20 mg/l ammonia-N compared to the control (unchanged water). In addition, a lethal dose challenge (32.15 mg/l ammonia-N) was carried out. The expression levels of LvFer transcripts in susceptible (died before 72 h post treatment, hpt) was significantly lower than that of tolerant (survived after 72 hpt) shrimp (P < 0.05). One single nucleotide polymorphism (SNP, T>G 208) was found in the LvFer gene segment. Real-time PCR amplification of specific alleles (real-time PASA) was successfully developed for genotyping of a T>G 208 SNP. The expression level of juveniles in the tested family carrying a T/T 208 genotype was greater than those carrying a T/G 208 genotype. Association analysis was further tested in a genetically improved stock (4 full-sib families). Juveniles with a T/T 208 genotype showed a greater average body weight and total length than those with a T/G 208 genotype. The expression level of LvFer can be further applied for selection of ammonia-tolerant families while its SNP can be used for selection of shrimp families with a better growth performance in our on-going breeding program of L. vannamei.

A Simple PCR Assay for Discrimination of Dengue Vectors from Nsukka LGA, Nigeria

Mosquitoes of Aedes species are vectors of several arboviral diseases which continue to be a major public health problem in Nigeria. This study among other things, morphologically identified Aedes mosquitoes collected from Nsukka LGA and used an allele specific PCR amplification for discrimination of dengue vectors. Larval sampling, BG-sentinel traps and modified human landing catches were used for mosquito sampling in two selected autonomous communities of Nsukka LGA (Nsukka and Obimo). A total of 124 Aedes mosquitoes consisting of five (5) different species were collected from April to June, 2019 in a cross-sectional study that covered 126 households, under 76 distinct geographical coordinates. Larvae was mainly collected from plastic containers 73% (n=224), metallic containers 14% (n=43), earthen pots 9% (n=29) and used car tyres 3% (n=9), reared to adult stage 69.35% (n=86), and all mosquitoes were identified using standard morphological keys. Five (5) Aedes mosquito species were captured; Aedes aegypti 83(66.94%), Aedes albopictus 33(26.61%), Aedes simpsoni (4.48%), Aedes luteocephalus (≤1%) and Aedes vittatus (≤1%). Nsukka autonomous community had higher species diversity than Obimo. Allele specific amplification confirmed dengue vectors, Aedes aegypti and Aedes albopictus species on a 2% agarose gel. Since the most recent re-emergence of arboviral diseases is closely associated with Aedes species, findings of this study therefore, give further evidence about the presence of potential arboviral vectors in Nigeria and describe the role of a simple PCR in discriminating some. Further entomological studies should integrate PCR assays in mosquito vector surveillance.