Multiplex Allele-specific PCR Research Articles

Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

The Impact of CYP2C19 Genotype on the Platelet Reactivity Index (PRI) among Chronic Coronary Syndromes (CCS) Patients Undergoing Percutaneous Coronary Intervention (PCI): Affectability of Rapid Genetic Testing.

In the Asian population, the presence of the CYP2C19 loss-of-function (LOF) allele is a known genetic variation. This allele is associated with a reduced capacity to metabolize clopidogrel into its active forms through the CYP2C19 enzyme, resulting in diminished platelet inhibition and an elevated risk of recurrent cardiovascular events. Regulatory authorities have recommended an alternative P2Y12 inhibitor, ticagrelor, for individuals carrying the LOF allele. Consequently, this study seeks to assess the impact of the CYP2C19 genotype on the Platelet reactivity index (PRI) using a rapid genetic testing approach in Asian patients with chronic coronary syndromes (CCS) who undergo percutaneous coronary intervention (PCI). This prospective study employed a parallel design, single-center design, and randomized approach. Genotyping for the CYP2C19*2 and *3 polymorphisms was conducted using the Nested Allele-Specific Multiplex PCR (NASM-PCR) technique. Patients meeting the inclusion criteria underwent genotyping for CYP2C19 polymorphisms. Following PCI, patients were randomly assigned to receive either ticagrelor or clopidogrel. PRI assessments were performed four hours after loading dose administration. The trial was registered with ClinicalTrials.gov under the identifier NCT05516784. Among the 94 patients recruited for the study, 40 (42.55%) were identified as carriers of the LOF allele for CYP2C19*2 and *3 (*1/*2, *2/*2, *1/*3). Out of the 84 patients evaluated for PRI (44 receiving clopidogrel and 40 receiving ticagrelor), 21 (47.7%) of the clopidogrel group and 39 (97.5%) of the ticagrelor group exhibited a favorable response to antiplatelet therapy (PRI < 50). Patients treated with ticagrelor demonstrated superior antiplatelet responses compared to those receiving clopidogrel, regardless of LOF carrier status (P = 0.005 and < 0.001 for non-LOF and LOF carriers, respectively). NASM-PCR as a rapid genetic test holds promise for personalizing antiplatelet therapy in Asian CCS patients.

Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy.

Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.

Assessment of Cdx2 polymorphism in Iranian women with polycystic ovary syndrome

BackgroundWomen’s fertility is affected by polycystic ovarian syndrome (PCOS) as an endocrine disorder with characteristic symptoms such as insulin resistance, polycystic ovaries, menstrual irregularities, and obesity. In polycystic ovarian syndrome, the vitamin D endocrine system is regulated by the vitamin D receptor (VDR) associated with type II diabetes, endocrine dysfunctions, and insulin resistance. Therefore, the current paper deals with the investigation of the connection between Cdx2 VDR gene polymorphism and the biochemical factors in obese PCOS women.Material and methodsIn the current case–control study, 40 obese women without PCOS and 38 obese women with PCOS were enrolled in May–September 2016. Insulin, IGF1, FBS, and HOMA-IR were examined for the participants along with the allelic and genotypic frequency of Cdx2 polymorphism G/A (rs11568820) from Isfahan Fertility and Infertility Center, Iran. The ASM-PCR (multiplex allele-specific PCR) technique was utilized in this regard.ResultsThe age of PCOS women was less (P < 0.001) than the controls. In PCOS women, insulin, FBS, and HOMA-IR serum levels were higher than in the control women (all P values 0.05). For GG, AG, AA,A, and G Cdx2(A/G) genotypic/allelic frequencies were 84.2%, 15.8%, 0%, 7.9%, and 92.1% in cases and 87.5%, 12.5%, 0%, 6.3%, and 93.8% in controls, respectively. HOMA-IR (P = 0.047 and P = 0.033, respectively) and insulin than those with the AG genotype were in PCOS women with the GG Cdx2 genotype. The highest IGF-1 mean value (P = 0.020) was found for the AG genotype in PCOS. In our study, a significant relation was found only between PCOS and FBS, in terms of a logistic regression analysis of Cdx2 and parameters.ConclusionIn the present study, it was indicated that the GG genotype in PCOS subjects was associated with the IGF-1, HOMA-IR, and insulin. Similarly, no association was found between obese PCOS patients and Cdx2 in the 1a promoter area of the VDR gene in our study.

ВЛИЯНИЕ ОДНОНУКЛЕОТИДНЫХ ПОЛИМОРФИЗМОВ ГЕНОВ IL1β, EDN1 И NOS3 НА ИНДИВИДУАЛЬНЫЙ ГЕНЕТИЧЕСКИЙ ПРОФИЛЬ ПАЦИЕНТОВ С ИШЕМИЧЕСКОЙ БОЛЕЗНЬЮ СЕРДЦА В РЕСПУБЛИКЕ КРЫМ

Abstracts. Introduction. Coronary heart disease prevalence rate grows, which determines the importance of researchers’ inquiries aiming to improve the prevention, diagnosis, and treatment of this pathology. Aim. This study was aimed at identifying the associations of polymorphisms of genes interleukin 1-beta, endothelin-1, and endothelial nitric oxide synthase in patients with coronary heart disease. Materials and Methods. We studied polymorphic markers of endothelin-1 (rs5370), endothelial nitric oxide synthase (rs2070244), and interleukin 1-beta genes (rs1143634 and rs16944) in patients with ischemic heart disease (n=229) and in controls (n=56) by multiplex allele-specific polymerase chain reaction with electrophoretic detection. Odds ratio (OR) was used to state the association of genotypes with the disease; 95% confidence interval limits were calculated to assess the significance of OR. Results and Discussion. Analysis of the T-511C polymorphism in the interleukin 1-beta gene demonstrated a trend towards an increased risk of coronary heart disease associated with the TT genotype in the study group (Odds ratio=2.2, 95%. Confidence interval: 1.3-3.6). At the same time, the CC genotype can be associated with a protective effect (Odds Ratio=0.3, 95%. Confidence Interval: 0.2-0.6). When analyzing the C3953T polymorphism in the interleukin 1-beta gene, the TT genotype showed a significantly increased disease development risk within the pathology under study (Odds Ratio=6.4, 95%. Confidence Interval: 1.7-23.4). Analysis of the polymorphic marker C786T in the NOS3 gene showed a correlation between the TT genotype and susceptibility to coronary heart disease (Odds ratio=2.07, 95%. Confidence interval: 1.3-3.2), while the CC genotype showed a protective effect (Odds ratio=0.3, 95%. Confidence interval: 0.2-0.5). A similar result was obtained in an experiment with the Lys198Asn polymorphism in the endothelin-1 gene. TT genotype showed an association with the increased risk of coronary heart disease (Odds ratio=6.5, 95%. Confidence Interval: 3.4-12.4). Odds ratio analysis of the effect of heterozygotes showed no risk of coronary heart disease (Odds ratio=0.3, 95%. Confidence Interval: 0.1-0.5). Conclusions. This study allowed us to find an association between the polymorphic markers of genes endothelin-1 (rs5370), eNOS (rs2070244), and interleukin-1β (rs114634 and rs16944) and the coronary heart disease risk in the population, which may be useful for both stratification and selection of further therapeutic strategies.

Multiplex allele-specific polymerase chain reaction-based prevalence study of canine trapped neutrophil syndrome in Thailand.

Trapped neutrophil syndrome (TNS) is an autosomal recessive genetic disorder found in Border Collies and is characterized by peripheral neutropenia and myeloid hyperplasia of bone marrow. The underlying cause of TNS is associated with a 4-base pair deletion mutation in the vacuolar protein sorting 13 homolog B (VPS13B) gene. In this study, we proposed and validated a novel multiplex allele specific-polymerase chain reaction (MAS-PCR) technique to assess the prevalence of TNS using VPS13B genotypes of Border Collies and Thai Ridgebacks in Thailand. We assessed the prevalence of TNS in 100 Border Collies and 30 Thai Ridgebacks using MAS-PCR-based allelic discrimination technique of the VPS13B gene. We then confirmed the VPS13B genotypes by direct DNA sequencing. A total of 130 samples were successfully genotyped using MAS-PCR assays. Of the two dog breeds examined, the VPS13B mutation was present in Border Collies, whereas Thai Ridgebacks were unaffected by this mutation. In Border Collies, 96% of dogs tested had an intact VPS13B genotype, whereas the remaining individuals had a heterozygous mutation genotype, with prevalence and mutated VPS13B allele frequencies of 4% and 2%, respectively. Using a novel MAS-PCR assay targeting the VPS13B gene, this study demonstrates for the first time that carriers of TNS exist in Border Collies in Thailand. This assay is a reliable and cost-effective tool for diagnosing TNS based on VPS13B genotypes and is suitable for routine clinical practice.

Development of a sensitive and user-friendly allele-specific PCR assay for adulteration detection of spices

Spice adulteration not only seriously interferes with their flavoring functions but also leads to life-threatening poisoning for consumers. To overcome the limitations of traditional methods in spice adulteration detection, a multiplex allele-specific PCR system was developed for molecular discrimination of four commonly used spices, Foeniculum vulgare Mill., Zanthoxylum bungeanum Maxim., Illicium verum Hook.f., and Syzygium aromaticum (L.) Merr. & L.M.Perry, from their corresponding adulterants based on chloroplast SNP markers. The developed assay, eliminating the obstacles of DNA sequencing and false negative results, can detect 0.1% of spice adulteration down to 0.01 ng level of genomic DNA with absolute allelic specificity and favorable efficiency. Based on the results, a standard operating procedure for using multiplex allele-specific PCR for spice adulteration detection was established. Therefore, the present study provided a simple, reliable, and sensitive molecular method for adulteration detection of spices.

Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of SDHIs, resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and 8 resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern Ⅲ (BosVHR, FluoSS, PenLR, CarS); pattern Ⅳ (BosLR, FluoLR, PenS, CarR); pattern Ⅴ (BosMR, FluoLR, PenS, CarS); pattern Ⅵ (BosMR, FluoLR, PenLR, CarS); pattern Ⅶ (BosVHR, FluoHR, PenHR, CarS); and pattern Ⅷ (BosLR, FluoLR, PenLR, CarS). We successfully established 9 AS-PCR assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µL, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µL. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

Detection of katG, inhA and ahpC gene mutation in clinical isolates of isoniazid-resistant Mycobacterium tuberculosis in Makassar City, South Sulawesi, Indonesia

BackgroundTuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). The world is currently facing challenges due to the spread of anti-tuberculosis drug-resistant of M. tuberculosis. Isoniazid-resistant (INH), is one of the first-line anti-tuberculosis agents that has a high resistance case. This study used Multiplex allele-specific Polymerase Chain Reaction (MAS-PCR) to detect the most common mutations associated with isoniazid resistance on inhA, katG, and ahpC gene. MethodsThis study used samples from clinical isolates of M. tuberculosis which had been tested for their antibiotic sensitivity of first-line anti-tuberculosis drugs. The DNA extraction process was carried out using the boiling method and then amplified with specific primers for inhA, katG, and ahpC genes using the MAS-PCR method. The results are then read on the electrophoretic gel with an interpretation of the mutation gene when the target gene DNA bands were absent according to the allele-specific fragments target. ResultsA total of 200 isolates were tested in this study consisting of isoniazid-resistant and susceptible with the largest distribution of Multi-Drug Resistant (MDR) isolates with a total of 146 isolates (73%). The most significant gene mutation was on the ahpC gene in 61 isolates (30,5%) and the combination mutation of the katG + ahpC gene in 52 isolates (26%) with sensitivity and specificity of the test reaching 87% and 42% for the detection of INH-resistant. ConclusionMutation on the ahpC gene has the highest percentage in this study. AhpC gene can be considered one of the essential genes to be tested for the cause of isoniazid-resistant. Using MAS-PCR for detecting gene mutation in isoniazid-resistant was simple and easy, it has the potential to be widely used as a rapid screening molecular test.

Method Development of Nested Allele-Specific Multiplex Polymerase Chain Reaction (NASM-PCR) for the Irritable Bowel Syndrome (IBS)-related Gene Polymorphisms

Polymorphisms in the genes of G-protein subunit beta 3 (GNB3); rs5443, tryptophan hydroxylase 1 (TPH1); rs211105 and rs4537731, tryptophan hydroxylase 2 (TPH2); rs4570625 and sodium voltage-gated channel alpha subunit 5 (SCN5A); rs1805124, have known to cause the abnormalities in the gastrointestinal tract that are implicated to irritable bowel syndrome (IBS) predisposition. Upfront genetic polymorphism genotyping in IBS-related gene polymorphisms will help to intervene and guide the decision-making in the management of IBS patients. This study aimed to develop a genotyping method to detect the respective polymorphisms using nested allele-specific multiplex polymerase chain reaction (NASM-PCR). A combination of nested and allele-specific multiplex PCR method was developed to determine the five single nucleotide polymorphisms (SNPs) mentioned. Annealing temperature, annealing and extension times, and the concentrations of MgCl2, primers, and DNA samples were optimized in the PCR. Sanger sequencing was performed to validate the genotyping results. NASM-PCR for IBS-related gene polymorphisms were successfully developed. DNA bands which correspond to the genes and SNPs have shown 100% homologous with the gene database. The developed method of NASM-PCR was reproducible and specific to be used for determining the respective polymorphisms of IBS. Notably, the described method can easily be integrated into other laboratories for population study or clinical use.

Performance of a Commercial Multiplex Allele-Specific Polymerase Chain Reaction Kit to Genotype African-Type Glucose-6-Phosphate Dehydrogenase Deficiency.

8-Aminoquinoline antimalarial drugs (primaquine, tafenoquine) are required for complete cure of Plasmodium vivax malaria, but they are contraindicated in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In the absence of spectrophotometry, which is a gold standard for measuring G6PD activity, G6PD genotyping is one of the alternatives to establish a database and distribution map of G6PD enzyme deficiency in Mauritania, which has become a new epicenter of P. vivax malaria in West Africa. The aim of our study was to assess the performance of multiplex allele-specific polymerase chain reaction (PCR) (African-type Diaplex C™ G6PD kit) against PCR-restriction fragment length polymorphism and sequencing. Of 146 mutations associated with G6PD A- genotypes in 177 blood samples from Mauritanian patients, all but two samples were identified correctly using multiplex allele-specific PCR (100% sensitivity and 99% specificity; "almost perfect agreement" between allele-specific PCR and PCR-restriction fragment length polymorphism/sequencing, with a kappa coefficient of 0.977). Despite a suboptimal PCR protocol for dried blood spots and the inability of the commercial assay to predict unequivocally the G6PD enzyme level in heterozygous females, the African-type Diaplex C™ G6PD genotyping kit seemed to be a valuable screening tool for male subjects and for research purposes in resource-limited countries where spectrophotometer and DNA sequencing are not available.

Platelet reactivity index after treatment of clopidogrel versus ticagrelor based on CYP2C19 genotypes among patients undergoing percutaneous coronary intervention: results of a randomized study

Abstract Funding Acknowledgements Type of funding sources: None. Background Although clopidogrel is the basic drug for antiplatelet therapy in patients with coronary artery disease (CAD). However, loss of function (LOF) allelic variants located within the CYP2C19 gene may reduce the production of clopidogrel active metabolites and weaken the antiplatelet effect, increasing platelet rates reactivity and associated with increased rates of adverse events after percutaneous coronary intervention (PCI). Thus, drug regulatory authorities have warned of clopidogrel's reduced efficacy and suggested using alternative treatments (such as ticagrelor). However, in clinical practice, the strategy of genotype-guided selection for antiplatelet therapy has been limited by the lack of access to immediately available results. Purpose We sought to evaluate the effect of point-of-care genetic testing for CYP2C19 genotypes on the antiplatelet choice of therapy (i.e., clopidogrel vs ticagrelor), using platelet reactivity index (PRI) as a proxy of clinical outcome Method This study was a prospective, randomized, parallel design, an open-label investigation conducted among Malaysian patients with stable coronary artery disease (SCAD) undergoing PCI. Patients underwent rapid point-of-care genetic testing using the nested allele-specific multiplex PCR assay, which defines CYP2C19 genetic status within 3 hours, allowing patients to be genotyped on the same day before PCI. Patients who were carriers of at least one +LOF or -LOF (*2 or *3) allele was randomized to receive either clopidogrel [600mg loading dose (LD) - 75mg/day maintenance dose (MD)] or ticagrelor (180mg LD - 90mg MD). The induced platelet reactivity index (PRI) was assessed by Vasodilator-Associated Stimulated Phosphoprotein (VASP) Assay to whole blood at one standardized time point after 4 hours of LD. All patients were treated with aspirin. The primary endpoint of the study was the non-inferiority in platelet reactivity, measured as PRI, at 4 hours of clopidogrel vs ticagrelor in +LOF or -LOF allele carriers Result In 1638 SCAD patients, 94 underwent PCI and were genotyped and randomized to receive either clopidogrel (n=44) or ticagrelor (n=50). A total of 42 (44.68%) were carriers of at least one +LOF. PRI levels at 4 hours for clopidogrel vs ticagrelor 44 and 40, respectively (mean difference = 23.08; 95% CI: 17 to 30; p=&amp;lt;0.001); this will meet the primary endpoint of superiority in a non-inferiority. Ticagrelor significantly reduced PRI more than clopidogrel. The cross-tabulation of the response classification showed that ticagrelor is superior to clopidogrel based on the -LOF and +LOF (P= 0.005 and &amp;lt;0.001, respectively) Conclusion A rapid point-of-care genetic test using a nested allele-specific multiplex PCR assay can be used to personalize antiplatelet therapy and provides timely results for patients undergoing PCI. In addition, the results showed that ticagrelor was superior to clopidogrel in the level of platelet inhibition in both groups (+LOF or -LOF)

Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Clinical Isolates from Sputum Samples by High-Resolution Melting Analysis.

The effective management of multidrug-resistant tuberculosis (MDR-TB) and the need for rapid and accurate screening of rifampin (RIF) and isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) isolates are the most fundamental and difficult challenges facing the global TB control. The present study aimed to compare the diagnostic accuracy of high-resolution melting-curve analysis (HRMA) in comparison to multiplex allele-specific PCR (MAS-PCR) and xpert MTB/RIF as well as the conventional drug-susceptibility test (DST) and gene sequencing for the detection of INH and RIF resistance in the Mtb isolates. In the present study, a total of 431 Mtb isolates including 11 MDR (%2.55), 7 INH resistance (%1.62), two RIF resistance (%0.46), and 411 sensitive isolates were phenotypically confirmed. HRMA assay identified katG gene mutations and the mabA-inhA promoter region in 15 of 18 INH-resistant samples and rpoB gene mutations were successfully evaluated in 11 out of 13 RIF-resistant samples. The sensitivity and specificity of the HRMA method were 83.3% and 98.8% for INH and 84.6% and 99% for RIF, respectively. The most common mutation in RIF-resistance-determining region (RRDR) occurred at codon 531 (TCG → TTG)(84.6%) and then at codon 513 (CAA → GTA)(7.6%) and 526 (CAC → TAC) (7.6%), which resulted in the amino-acid changes. Also, 88.8% of INH-resistant samples had mutations in the katG gene and the mabA-inhA promoter region, of which the highest mutation occurred at codon 315 (AGC → ACC) of the katG gene. In conclusion, all these results indicated that the sensitivity and specificity of the HRM method were increased when the katG gene and the mabA-inhA promoter region were used as a target.

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Molecular survey of pyrethroid and fipronil resistance in isolates of Rhipicephalus microplus in the north of Uruguay

The resistance of Rhipicephalus microplus to acaricides is a serious control problem, so its early diagnosis by a molecular technique is important. This study aims to develop a multiplex allele–specific polymerase chain reaction (PCR) for single-nucleotide polymorphisms (SNPs) in the para-sodium channel gene and in the GABA-Cl gene, associated with pyrethroids (cypermethrin and flumethrin) and fipronil resistance, respectively. We used 22 tick field isolates from farms with tick control problems (sampling convenience). These farms are located in departments of northern Uruguay. Three mutations in the sodium channel gene (Domain II S4–5: C190A and G215T; domain III S6: T2134A) and one in the GABA-Cl gene (A286S/L: CG856CC/TG) were studied. Mutations G215T and T213A were not detected. In all field isolates, the resistant allele (R) for C190A mutation (knockdown resistance, kdr) was detected, mainly in heterozygous individuals (SR) (11.1% to 86.7%). The highest incidence of the kdr mutant allele occurred in the Tacuarembó tick field isolates, where on 7 out of 10 farms >30% of individuals were SR and on one farm > 30% of individuals were RR. The next highest was Artigas (half of farms had>30% SR individuals and a quarter had >30% RR individuals). The resistance to dieldrin locus (rdl) mutation (CG856CC/TG) was absent in five field isolates. The highest incidenceof the mutant allele was observed in ticks from farms in Rivera (all farms had SR in >30% of individuals and two farms had RR in >12.5 and >16.7% of individuals) followed by farms in Tacuarembó (3 of 10 farms had >30% SR and 2 with >30% RR). Less than half of the farms had rdl in homozygous individuals. No significant association was observed between phenotypic bioassays and the rdl resistance allele. Several field isolates were phenotypically susceptible to the presence of the rdl allele. Several causes are possible (bioassay sensitivity, discriminating concentration). Individuals with simultaneous kdr and rdl mutations were present in 17 field isolates, and their frequency varied between 0.06% and 60%. Genotypic analysis shows that tick resistance to both acaricides, especially pyrethroids, is a serious problem. It is important to monitor the resistance using molecular techniques to plan efficient control measures. This is the first report describing kdr and rdl detection in R. microplus in Uruguay.

Multiplex allele-specific polymerase chain reaction to detect kdr and rdl mutations in the cattle tick Rhipicephalus microplus (Acari: Ixodidae)

The resistance of Rhipicephalus microplus to pyrethroids is widely dispersed worldwide and has been associated with several nucleotide substitutions in its target site, the para-sodium ion channel (Na-channel) gene. The resistance of the tick to fipronil has been increasing in South America, and mutations in the GABA-gated chloride channel (GABA-Cl) have been described in fipronil-resistant tick strains. We developed a multiplex allele-specific PCR (mAS-PCR) to screen for single-nucleotide polymorphisms (SNPs) associated with the resistance to pyrethroids (knockdown resistance or kdr) and fipronil (resistance to dieldrin or rdl) in susceptible tick populations from Uruguay (n = 11) and the Rio Grande do Sul state in Southern Brazil (n = 15). Toxicological in vitro assays with larvae and adults were used to confirm the resistance to cypermethrin, flumethrin, and fipronil. Three SNPs in the Na-channel gene were investigated (C190A, G215 T, and T2134A), and the mAS-PCR included the detection of an SNP (G858 T) coding a non-synonymous mutation in the GABA-Cl gene. C190A was present in all pyrethroid-resistant populations from Uruguay and Brazil, most frequently homozygous. The SNPs G215 T and T2134A were not found. Of the seventeen fipronil-resistant populations, fourteen presented at least one mutant GABA-Cl gene allele, more frequently in heterozygosis. Other mechanisms apart from target site insensitivity may be involved in fipronil resistance since in some resistant populations, the SNP G858 T was not detected. Sixteen (61,5%) of the populations presented individuals with simultaneous mutations in the Na-channel and GABA-Cl genes. This could be a significant problem for the future control of R. microplus. This study shows the wide dispersion of a pyrethroid resistance–associated SNP in high frequency in the region. Fipronil resistance mutations are also dispersed across the region and increasing.

Establishment of multiplex allele-specific blocker PCR for enrichment and detection of 4 common EGFR mutations in non-small cell lung cancer.

BackgroundLung cancer is one of the most severe cancers and the majority of patients miss the best timing for surgery when diagnosed, thus having to rely on radiotherapy, chemotherapy or target therapy. Epidermal growth factor receptor (EGFR) upregulation occurs in a large percentage of patients, who can then benefit from tyrosine kinase inhibitors (TKI). However, the EGFR mutations they carry will vary the effectiveness of TKI. Circulating tumor DNA (ctDNA) contains genetic information from cancer tissue that can be used as a liquid biopsy by non-invasive sampling. This study aimed to provide a solution for minor allele detection from ctDNA.MethodsOur novel method, named multiplex allele-specific blocker PCR (MAB PCR), combines amplification refractory mutation system (ARMS), blocker PCR and fluorescent-labeled probes for better discrimination and higher throughput. MAB PCR was specially designed for low-quality samples such as ctDNA. A sensitive assay based on MAB PCR was developed for enriching and detecting four common EGFR mutations. This assay was optimized and evaluated with manufactured plasmids, and validated with 34 tissue samples and 94 plasma samples.ResultsThe limit of detection of this assay was 102 copies and the detection sensitivity reached 0.1% mutant allele fraction (MAF). The results of clinical sample testing had 100% accordance with sequencing, which proved that this assay was accurate and applicable in clinical settings.ConclusionsThis assay could accomplish low-cost and rapid detection of 4 common EGFR mutations sensitively and accurately, which has huge potential in clinical usage for guiding medication. Furthermore, this design could be used to detect other mutations.