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Human leukocyte antigen (HLA) genotyping has been widely used in establishing the database of the donors for hematopoietic stem cell, HLA matching selection between the donor and recipient, establishing the database of platelet donors with known HLA genotype, diagnosis of HLA association with diseases, genetics study and other scientific research. With the increasing number of HLA alleles and the development of HLA genotyping technology, there are some controversy in the definition of resolution for HLA genotyping, the description of the results for HLA genotyping between donor and recipient and the determination of HLA matching level. In order to improve the normalization of the definition and interpretation of HLA genotyping resolution, this expert consensus is formulated by many experts from the fields of HLA genotyping in the laboratory and clinical transplantation according to the relevant domestic and foreign literature and clinical practice. The definition of the resolution of HLA genotyping, the methods of HLA genotyping, the description of the results between donor and recipient and HLA matching determination are summarized, which will further standardize HLA genotyping technology and ensure the accuracy of the results for HLA genotyping.

Abstract Background Amplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis. Methods The new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR. Results NGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada. Conclusions This new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten. Graphical Abstract

Background: Amplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis. Methods: The new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR. Results: NGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada. Conclusions: This new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten. [ABSTRACT FROM AUTHOR]

The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective. [ABSTRACT FROM AUTHOR]

Taste perception is an important factor in determining people's food intake. Understanding the relationship between taste sensitivity and food consumption behavior is one of the most innovative strategies for solving health problems caused by diet, and measuring the taste sensitivity of human subjects is the key to establish this relationship. In this paper, from the aspects of working principle, operation process and application of taste sensitivity determination, the methods of measuring human taste sensitivity at home and abroad are summarized, including psychophysical method, biopotential method, bacterial papilla counting method and genotyping method. Psychophysical measurement method is simple to operate and easy to implement, but subjective and error are large; biopotential measurement method reduces subjective errors, but the subjects need to remain relatively static during the measurement process, otherwise it will affect the stability of the electrode; fungal papilla counting method and genotyping method need to be carried out in conjunction with other methods. In addition, the genotyping method requires high technical content and cost. Through the analysis and evaluation of the above methods, it provides a theoretical basis and guidance for selecting different taste sensitivity determination methods.

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. [ABSTRACT FROM AUTHOR]

The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has been reported worldwide and novel SARS-CoV-2 variants continue to emerge. A novel SARS-CoV-2 strain, the Delta variant (B.1.617.2), is spreading worldwide. The Delta variant has reportedly high infectivity and immune evasion potency. In June 2021, the World Health Organization categorized it as a variant of concern (VOC). Therefore, it is vital to develop tests that can exclusively identify the Delta variant. Here, we developed a rapid screening assay to detect characteristic mutations observed in the Delta variant using high-resolution melting (HRM) analysis. In this assay, we determined L452R and T478K, among which T478K is an identifier of the Delta variant since L452R is seen in other strains (Kappa and Epsilon variants). Additionally, nested PCR-based HRM analysis, which involved RT-PCR (1st PCR) and HRM analysis (2nd PCR), was developed to improve the specificity and sensitivity. Our method discriminat- ed between the L452R mutant and wild-type L452. In addition, HRM analysis distinguished the T478K mutant from the wild-type T478. Seven clinical samples containing the Delta variant were successfully identified as L452R/T478K mutants. These results indicate that this HRM-based genotyping method can identify the Delta variant. This simple method should contribute to rapid identification of the Delta vari- ant and the prevention of infection spread. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus causing coronavirus disease 2019 (COVID-19), has been expanding globally since late 2019. SARS-CoV-2, an RNA virus, has a genome sequence that can easily undergo mutation. Several mutated SARS-CoV-2 strains, including those with higher infectivity than others, have been reported. To reduce SARS-CoV-2 transmission, it is crucial to trace its infection sources. Here, we developed a simple, easy-to-use genotyping method to identify SARS-CoV-2 variants using a high-resolution melting (HRM) analysis. We investigated five mutation sites, A23403G , G25563T , G26144T , T28144C , and G28882A , which are known strain determinants according to GISAID clades (L, S, V, G, GH, and GR). We first employed synthetic DNA fragments containing the five characteristic sites for HRM analysis. All sequences clearly differentiated wild-type from mutant viruses. We then confirmed that RNA fragments were suitable for HRM analysis following reverse transcription. Human saliva did not negatively affect the HRM analysis, which supports the absence of a matrix effect. Our results indicate that this HRM-based genotyping method can identify SARS-CoV-2 variants. This novel assay platform potentially paves the way for accurate and rapid identification of SARS-CoV-2 infection sources. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus (SARS-CoV) was discovered as a novel pathogen in the 2002-2003 SARS epidemic. The emergence and disappearance of this pathogen have brought questions regarding its source and evolution. Within the genome sequences of 281 SARS-CoVs, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and SARS-related CoVs (SARSr-CoVs), a ~430 bp genomic region (from 27 701 bp to 28 131 bp in AY390556.1) with regular variations was investigated. This ~430 bp region overlaps with the ORF8 gene and is prone to deletions and nucleotide substitutions. Its complexity suggested the need for a new genotyping method for coronaviruses related to SARS-similar coronaviruses (SARS-CoV, SARSr-CoV, and SARS-CoV-2). Bat SARSr-CoV presented 3 genotypes, of which type 0 is only seen in bat SARSr-CoV, type I is present in SARS in the early phase, and type II is found in all SARS-CoV-2. This genotyping also shows potential usage in distinguishing the SARS-similar coronaviruses from different hosts and geographic areas. This genomic region has important implications for predicting the epidemic trend and studying the evolution of coronavirus. [ABSTRACT FROM AUTHOR]

Germplasm collections of major crops include thousands of accessions whose agronomic evaluation is prevented by high costs, while their molecular characterization is getting increasingly affordable. This study aimed to assess the consistency between alfalfa (Medicago sativa L.) landrace diversity measures relative to adaptation pattern, eleven morphophysiological traits, and four genotyping strategies using SSR or genotyping-by-sequencing (GBS) SNP markers recorded on either five individual plants or three bulks of 100 plants each per accession. We focused on eleven landraces from Northern Italy featuring large genotype × environment interaction for biomass yield across environments with contrasting level of summer drought. We recorded 34 SSR and 237 SNP polymorphic markers for individual plants, and 41 SSR and 1274 SNP markers for bulked plants. Landrace diversity based on SNP data from bulked plants was the only molecular diversity measure associated with both adaptive diversity as expressed by pattern analysis (r = 0.34) and drought stress at collecting sites (r = 0.50). No molecular diversity measure was associated with overall morphophysiological diversity. Molecular diversity was highly consistent only between SSR and SNP data from bulked plants (r = 0.67). SSR-based diversity from individual plants exhibited marked inconsistency with other molecular diversity measures but was useful for genetic structure assessment, indicating much smaller among-population variation relative to within-population variation (7.5% in terms of variance ratio; 7.8% as global FST). Moderate inferences on germplasm adaptive diversity may be achieved by molecular characterization that maximizes the sampling of genomic areas and genotypes within accession through GBS applied to bulked plants. [ABSTRACT FROM AUTHOR]

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.O objetivo deste trabalho foi padronizar um método semi‑automatizado para genotipagem de soja, baseado na metodologia de iniciadores com sequências de cauda universal (PSCU), e compará‑lo ao método de genotipagem convencional de eletroforese em gel de poliacrilamida. Trinta cultivares de soja foram caracterizadas genotipicamente por ambos os métodos, com o uso de 13 locos microssatélites. Para o método PSCU, o número de alelos (NA) foi de 50 (2-7 por marcador) e o conteúdo de informação polimórfica (PIC) variou de 0,40 a 0,74. Para o método convencional, o NA foi de 38 (2-5 por marcador) e o PIC variou de 0,39 a 0,67. As matrizes de dissimilaridade genética obtidas pelos dois métodos apresentaram alta correlação entre si (0,8026), e os grupos formados foram coerentes com dados fenotípicos utilizados para o registro varietal. Os 13 marcadores permitiram a distinção de todas as cultivares analisadas. O baixo custo do método PSCU, associado a sua alta acurácia, torna‑o ideal para a caracterização de cultivares de soja e a determinação de pureza genética.

Acinetobacter baumannii is clustered with other phenotypically similar species into what has commonly become known as the ACB complex: A. calcoaceticus, A. pittii and, A. nosocomialis. The ecology and pathology of most of these species are not well understood, mainly because current specific phenotypic techniques have, to date, been insufficient. This has inhibited both the precise identification of, as well as the ability to discriminate between, these clinically important and closely related Acinetobacter strains. However, new genotypic methods have greatly enhanced our capacity to identify the ACB complex. This has resulted in the implementation of more rational infection control programs. Several genotypic identification methods are explored in this study, including non-polymerase chain reaction (PCR)-based and PCR-based methods. These methods include ribotyping, pulsed-field gel electrophoresis, 16S rRNA identification, multilocus sequence typing, single locus sequence typing, restriction fragment length polymorphism analysis, restriction analysis of 16S-23S rRNA intergenic spacer sequences, rapid amplification of polymorphic DNA, and repetitive extragenic palindromic PCR; however, there is no current single ideal genotyping method. Each one has its own advantages and disadvantages. With this in mind we reviewed current and new genotyping methods used to characterize the Acinetobacter species. [ABSTRACT FROM AUTHOR]

Today, the genomic revolution in epidemiology, medicine and population based genetic association studies are results of on-going refocusing efforts toward development of inexpensive and accurate techniques for SNP genotyping. Despite this considerable gain, high throughput and routinely applicable newer SNP detection techniques are still needed. Therefore, aim of this study was to develop and validate a simple, rapid and inexpensive restriction enzyme based method for genotyping of corticotrophin-releasing hormone receptor1 ( CRHR1; rs1396862: C>T) gene variant. This polymorphism has been investigated in a variety of psychiatric and association studies of asthma. A total of 250 healthy volunteers were recruited from same ethnicity and their blood DNA samples were employed for genotyping. Primers were designed using Batch primer3 Software. Specificity and functionality of primers were tested with BLAST database and UCSC In-silco PCR respectively. The lake of a PstI recognition site was seen with T allele. The allele frequencies for rs1396862: C>T were 0.88 (C allele) and 0.12 (T allele). We get 100 % concordant genotyping results for sequencing and PCR-RFLP. This newer genotyping approach lowers the cost and increased the speed. It is particularly useful for small basic research studies of complex genetic disorder. [ABSTRACT FROM AUTHOR]

Abstract: This review gives an overview on ovine and caprine defects/disorders, disease predispositions, production traits and coat colours for which causal gene variants are known. Most phenotypes are inherited autosomal-recessive or dominant and in the majority are caused by single nucleotide substitutions or deletions. Causative sequence variants mainly were identified by sequencing candidate genes in the past, and recently also by whole genome analysis using the ovine 50k SNP chip. While PCR-fragment length polymorphism analyses were developed for the majority of causative sequence variants, other low- to medium-throughput PCR-based methods as PCR-single strand conformation analysis and allele-specific PCR were also established frequently. For processing large sample numbers, high-throughput methods as MALDI-ToF MS or real-time PCR are available for some gene variants. Further progress in development of ovine and caprine genome sequences and SNP chips will be beneficial for the discovery of additional causative variants in these two species. [Copyright &y& Elsevier]

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5′ end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 ( PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele. [ABSTRACT FROM AUTHOR]

Abstract: Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by , corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture. [Copyright &y& Elsevier]

Abstract: Objective: The growth hormone (GH) receptor (GHR) exon 3 deleted/full-length (d3/fl) polymorphism has been suggested to affect GH sensitivity. The conventional genotyping method used for this polymorphism (multiplex PCR with fragment detection by gel electrophoresis) is laborious and requires large amount of DNA template. This has restricted analysis of this polymorphism to small cohorts. Our aim was to evaluate the accuracy of using a tagging single nucleotide polymorphism (tagSNP) as a marker for the d3/fl polymorphism. Design: The d3/fl polymorphism was analyzed using TaqMan SNP genotyping of the tagSNP rs6873545 in 183 patients with adult GH deficiency (GHD). The results were compared to d3/fl genotypes determined by the conventional method. Results: Genotyping success rate for the tagSNP was 100%. Frequency of the d3-allele was 24.0% (d3/d3 7.7%, d3/fl 32.2% and fl/fl 60.1%) and the results from the two different methods were identical. Moreover, three samples previously undetermined when genotyped using the conventional method were successfully analyzed using the tagSNP. Conclusion: The GHR d3/fl polymorphism can be studied by TaqMan SNP genotyping. Use of the tagSNP facilitates investigations of the effects of the d3/fl polymorphism in large cohorts. [Copyright &y& Elsevier]

Abstract: Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite, and intronic single-nucleotide polymorphisms (SNPs: G7395A, G12390C, T14215C, T35587C, and G35991A) in the AS3MT gene were shown to be related to inter-individual variation in the arsenic metabolism. In the present study, the genotyping for these SNPs was developed using the polymerase chain reaction and restriction fragment length polymorphism technique. Applying this method, the genotype distribution among the Ovambo, Turkish, Mongolian, Korean, and Japanese populations was investigated, and our results were compared with those from other studies. G7395, G12390, T35587, and A35991 were predominant among the five populations in our study. However, a previous study in Argentina, C12390 and G35991 showed the highest allele frequency among the eight populations studied in other studies. The dominant allele of T14215C differed among populations: the T14215 allele was predominant in Argentina, the allele frequency of C14215 was higher than that of T14215 among Turks, Mongolians, Europeans, and American ancestry. In Korea and Japan, similar allele frequencies were observed in T14215 and C14215. Higher allele frequencies were observed in haplotype G7395/G12390/C14215/T35587 with frequencies of 0.40 (Turks), 0.28 (Mongolians), and 0.23 (Koreans). On the other hand, the allele frequency for G7395/G14215/T35587/A35991 was the highest among the Ovambos (0.32), and the frequency for G7395/G12390/C35587/G35991 was the highest among the Japanese (0.27). It is noteworthy that the Japanese haplotype differs from that of the Koreans and Mongolians, which indicates the importance of investigating other intronic polymorphisms in AS3MT, especially in Asians. [Copyright &y& Elsevier]

Abstract: Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n =185), Turkish (n =191), Mongolian (n =233), Korean (n =200), and Japanese (n =370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning. [Copyright &y& Elsevier]

The influence of diet and genetics was investigated in a healthy white person who had distinctly low methylprednisolone clearance. Pharmacokinetic and pharmacodynamic parameter values were similar on 2 occasions during the consumption of a low-carbohydrate diet and a Weight Watchers diet, indicating that the decreased clearance was unlikely attributable to a change in diet composition. Although the subject was found to be homozygous for CYP3A5*3, genetic findings were not significant for a number of other CYP3A4 and CYP3A5 allelic variants. Because of the high prevalence of CYP3A5*3/*3 in whites and because 5 of 7 white control subjects are also homozygous for CYP3A5*3, this genotype cannot fully explain the reduced metabolism of the drug. Other genetic or contributing factors might have been involved. New polymerase chain reaction-based genotyping methods for functionally defective CYP3A5*6, *8, *9, and*10 alleles were developed in this study. These assays will be useful for CYP3A5 genotype analysis in future clinical studies. [ABSTRACT FROM PUBLISHER]

Abstract: As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost. [Copyright &y& Elsevier]

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, the Omicron variant (Pango lineage B.1.1.529), was identified in South Africa in late September 2021. This variant has multiple spike protein deletions and mutations, with 15 amino acid substitutions detected in the receptor-binding domain (RBD). These RBD substitutions are hypothesized to increase infectivity and reduce antibody affinity, which is supported by recent data showing that the Omicron variant spreads faster than the Delta variant (Pango lineage B.1.617.2). Thus, this increase in infectivity should lead to Omicron being the dominant variant and developing screening tests that discriminate between Omicron and Delta variants is urgently needed. In this study, we successfully developed a novel screening assay using high-resolution melting analysis, in which two genotypes at G446/L452 and S477/T478 RBD were determined (G446S/L452 and S477N/T478K for Omicron; G446/L452R and S477/T478K for Delta). Using synthetic DNA fragments, we confirmed both melting point and melting peak shape of the RBD Omicron variant was distinguishable from those of wild-type and the Delta variant. Although this study was conducted without clinical samples, these results suggest that our high-resolution melting (HRM)-based genotyping method can readily identify the Omicron and Delta variants. This simple method should contribute to the rapid identification of SARS-CoV-2 variants and thus prevent potential widespread infection and inflow of the Omicron variant.