RESUMEN
Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.
Asunto(s)
Adenovirus Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Infecciones del Sistema Respiratorio , Humanos , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Técnicas de Diagnóstico Molecular/métodos , Infecciones por Adenovirus Humanos/diagnóstico , Infecciones por Adenovirus Humanos/virología , Sensibilidad y Especificidad , ADN Viral/genética , ADN Viral/análisis , Reacción en Cadena de la Polimerasa Multiplex/métodosRESUMEN
Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.
Asunto(s)
Ácido 4-Aminobenzoico , Amidas , Oligonucleótidos , Fosforamidas , Ácidos Fosfóricos , Oligonucleótidos/genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas p21(ras) , Fosfatos , Nucleótidos , Azoles , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Pathogenic variants (PVs) in BRCA1 and BRCA2 genes are essential biomarkers of an increased breast and ovarian cancer risk and tumor sensitivity to poly ADP ribose polymerase inhibitors. In Russia, eight PVs were thought to be the most common, among which BRCA1 c.5266dup is the most frequently identified one. METHODS: We show the distribution of BRCA1/2 PVs identified with quantitative PCR and targeted next-generation sequencing in 1399 ovarian cancer patients recruited into the study from 72 Russian regions in 2015-2021. RESULTS: The most abundant PVs were c.5266dup (41.0%), c.4035del (7.0%), c.1961del (6.3%), c.181 T > G (5.2%), c.3756_3759del (1.8%), c.3700_3704del (1.5%), and c.68_69del (1.5%), all found in BRCA1 and known to be recurrent in Russia. Several other frequent PVs were identified: c.5152 + 1G > T (1.2%), c.1687C > T (1.0%), c.4689C > G (0.9%), c.1510del (0.6%), c.2285_2286del (0.6%) in the BRCA1 gene; and c.5286 T > G (1.2%), c.2808_2811del (0.8%), c.3847_3848del (0.8%), c.658_659del (0.7%), c.7879A > T (0.6%), in the BRCA2 gene. For the most common PV in the BRCA2 gene c.5286 T > G, we suggested that it arose about 700 years ago and is a new founder mutation. CONCLUSION: This study extends our knowledge about the BRCA1 and BRCA2 pathogenic variants variability.
Asunto(s)
Neoplasias de la Mama , Neoplasias Ováricas , Humanos , Femenino , Genes BRCA2 , Predisposición Genética a la Enfermedad , Neoplasias de la Mama/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Mutación de Línea Germinal , Neoplasias Ováricas/epidemiología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Federación de Rusia/epidemiología , Células GerminativasRESUMEN
Detecting copy number variations (CNVs) and alterations (CNAs) in the BRCA1 and BRCA2 genes is essential for testing patients for targeted therapy applicability. However, the available bioinformatics tools were initially designed for identifying CNVs/CNAs in whole-genome or -exome (WES) NGS data or targeted NGS data without adaptation to the BRCA1/2 genes. Most of these tools were tested on sample cohorts of limited size, with their use restricted to specific library preparation kits or sequencing platforms. We developed BRACNAC, a new tool for detecting CNVs and CNAs in the BRCA1 and BRCA2 genes in NGS data of different origin. The underlying mechanism of this tool involves various coverage normalization steps complemented by CNV probability evaluation. We estimated the sensitivity and specificity of our tool to be 100% and 94%, respectively, with an area under the curve (AUC) of 94%. The estimation was performed using the NGS data obtained from 213 ovarian and prostate cancer samples tested with in-house and commercially available library preparation kits and additionally using multiplex ligation-dependent probe amplification (MLPA) (12 CNV-positive samples). Using freely available WES and targeted NGS data from other research groups, we demonstrated that BRACNAC could also be used for these two types of data, with an AUC of up to 99.9%. In addition, we determined the limitations of the tool in terms of the minimum number of samples per NGS run (≥20 samples) and the minimum expected percentage of CNV-negative samples (≥80%). We expect that our findings will improve the efficacy of BRCA1/2 diagnostics. BRACNAC is freely available at the GitHub server.
Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias Ováricas , Neoplasias de la Próstata , Femenino , Humanos , Masculino , Proteína BRCA1/genética , Proteína BRCA2/genética , Genes BRCA2 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Ováricas/genética , Neoplasias Ováricas/diagnóstico , Neoplasias de la Próstata/genéticaRESUMEN
Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Codón , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Exones , Genes Bacterianos , Genes Virales , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Hibridación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Virus/genéticaRESUMEN
Mutations in the BRCA1 and BRCA2 genes are known risk factors and drivers of breast and ovarian cancers. So far, few studies have been focused on understanding the differences in transcriptome and functional landscapes associated with the disease (breast vs. ovarian cancers), gene (BRCA1 vs. BRCA2), and mutation type (germline vs. somatic). In this study, we were aimed at systemic evaluation of the association of BRCA1 and BRCA2 germline and somatic mutations with gene expression, disease clinical features, outcome, and treatment. We performed BRCA1/2 mutation centered RNA-seq data analysis of breast and ovarian cancers from the TCGA repository using transcriptome and phenotype "portrayal" with multi-layer self-organizing maps and functional annotation. The results revealed considerable differences in BRCA1- and BRCA2-dependent transcriptome landscapes in the studied cancers. Furthermore, our data indicated that somatic and germline mutations for both genes are characterized by deregulation of different biological functions and differential associations with phenotype characteristics and poly(ADP-ribose) polymerase (PARP)-inhibitor gene signatures. Overall, this study demonstrates considerable variation in transcriptomic landscapes of breast and ovarian cancers associated with the affected gene (BRCA1 vs. BRCA2), as well as the mutation type (somatic vs. germline). These results warrant further investigations with larger groups of mutation carriers aimed at refining the understanding of molecular mechanisms of breast and ovarian cancers.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Mutación , Neoplasias Ováricas/genética , Transcriptoma , Adulto , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: N-acetyltransferase 2 plays a crucial role in the metabolism of a wide range of xenobiotics, including many drugs, carcinogens, and other chemicals in the human environment. The article presents for the first time data on the frequency of two important "slow" variants of NAT2 gene (NAT2*5, rs1801280 and NAT2*7, rs1799931), which significantly affect the rate of xenobiotics acetylation, among representatives of indigenous populations of Forest and Tundra Nenets in Northern Siberia. The aim of this study was to identify the frequencies of these variants and compare them with frequencies in other ethnic populations. RESULTS: NAT2*5 (T341C) genotyping revealed frequencies of 28,0% and 38,6% for Tundra and Forest Nenets, respectively. The frequencies of NAT2*7 (G857A) variant were 9,8% and 8,2% for Tundra and Forest Nenets, respectively. Polymorphic variants frequencies for Nenets are intermediate between those in populations of Europeans and Asians. These results can probably be explained by the presence of both European and Asian components in Nenets gene pools. CONCLUSIONS: The results of this study expand the knowledge of NAT2 polymorphism in world populations. These data may also help assess the genetic predisposition of Nenets to multifactorial diseases associated with polymorphism in the NAT2 gene and, in general, contribute to the development of personalized medicine in reference to native people of Siberia.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Genética de Población , Etnicidad/genética , Frecuencia de los Genes , Humanos , SiberiaRESUMEN
The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.
Asunto(s)
Mycoplasma pneumoniae/genética , Neumonía por Mycoplasma/epidemiología , Neumonía por Mycoplasma/microbiología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Historia del Siglo XXI , Humanos , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Moscú/epidemiología , Tipificación de Secuencias Multilocus , Mycoplasma pneumoniae/clasificación , Mycoplasma pneumoniae/efectos de los fármacos , Neumonía por Mycoplasma/historia , Polimorfismo de Nucleótido Simple , Vigilancia en Salud Pública , ARN Ribosómico 23S/genéticaRESUMEN
Nowadays the advent of innovative high-throughput sequencing allows obtaining high-quality microbiome profiling. However, PCR-based tests are still considered the "golden standard" for many clinical applications. Here, we designed a qPCR-based platform with fluorescent-labeled oligonucleotide probes for assessing human gut microbiome composition. The system allows conducting qualitative and semiquantitative analysis for 12 prokaryotic taxa that are prevalent in the human gut and associated with diseases, diet, age and other factors. The platform was validated by comparing microbiome profile data obtained with two different methods - the platform and high-throughput 16S rRNA sequencing - across 42 stool samples. The test can form the basis for precise and cost-efficient microbiome assay for large-scale surveys including clinical trials with interventions related to diet and disease risks.
Asunto(s)
Microbioma Gastrointestinal/genética , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Cartilla de ADN/metabolismo , Sondas de ADN/metabolismo , Heces/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Ribosómico 16S/genéticaRESUMEN
BACKGROUND: The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes. Novel bioinformatic methods are thus urgently needed for multi-omics data analysis to reveal causative biomarkers with a potential driver role in early stages of cancer. METHODS: We have developed a method for finding potential causal relationships between epigenetic changes (DNA methylations) in gene regulatory regions that affect transcription factor binding sites (TFBS) and gene expression changes. This method also considers the topology of the involved signal transduction pathways and searches for positive feedback loops that may cause the carcinogenic aberrations in gene expression. We call this method "Walking pathways", since it searches for potential rewiring mechanisms in cancer pathways due to dynamic changes in the DNA methylation status of important gene regulatory regions ("epigenomic walking"). RESULTS: In this paper, we analysed an extensive collection of full genome gene-expression data (RNA-seq) and DNA methylation data of genomic CpG islands (using Illumina methylation arrays) generated from a sample of tumor and normal gut epithelial tissues of 300 patients with colorectal cancer (at different stages of the disease) (data generated in the EU-supported SysCol project). Identification of potential epigenetic biomarkers of DNA methylation was performed using the fully automatic multi-omics analysis web service "My Genome Enhancer" (MGE) (my-genome-enhancer.com). MGE uses the database on gene regulation TRANSFAC®, the signal transduction pathways database TRANSPATH®, and software that employs AI (artificial intelligence) methods for the analysis of cancer-specific enhancers. CONCLUSIONS: The identified biomarkers underwent experimental testing on an independent set of blood samples from patients with colorectal cancer. As a result, using advanced methods of statistics and machine learning, a minimum set of 6 biomarkers was selected, which together achieve the best cancer detection potential. The markers include hypermethylated positions in regulatory regions of the following genes: CALCA, ENO1, MYC, PDX1, TCF7, ZNF43.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Metilación de ADN/genética , Retroalimentación Fisiológica , Transducción de Señal/genética , Sitios de Unión/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Islas de CpG/genética , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene. METHODS: In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status. RESULTS: DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1. CONCLUSIONS: We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.
Asunto(s)
Variaciones en el Número de Copia de ADN , Genes BRCA1 , Pruebas Genéticas/métodos , Reacción en Cadena de la Polimerasa Multiplex , Proteína BRCA1/genética , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Pruebas Diagnósticas de Rutina , Femenino , Reordenamiento Génico , Humanos , Neoplasias Ováricas/sangre , Neoplasias Ováricas/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Brain arteriovenous malformations (BAVMs) are formed by hypertrophied arterial vessels (afferents, feeders), a large number of arteriovenous shunts which become tangled to form a body (nidus) of malformation, which then expands draining proximal veins. The aim of this study was a replication of single nucleotide polymorphism (SNP) rs11672433 association with BAVM development with the subsequent meta-analysis of published data. METHODS: A total of 252 Russian patients with brain BAVMs and 480 control subjects were included in the present study. Genotyping was performed using real-time polymerase chain reaction with competitive hydrolysis probes. RESULTS: In our case-control study, we found no significant association with brain arteriovenous malformation for the SNP rs11672433 of ANGPTL4 gene (odds ratio .82, 95% confidence interval = .57-1.17 P value = .27) as well as in meta-analysis (odds ratio 1.18, 95% confidence interval = .81-1.73, P value = .39). CONCLUSIONS: Our data showed that SNP rs11672433 was not associated with the BAVM Russian population and the following meta-analysis did not detect an association in total. Thus, in spite of the fact that ANGPTL4 (protein) participates in the angiogenesis regulation processes, we consider that SNP rs11672433, a high-frequency locus in the ANGPTL4 gene, does not influence the predisposition to BAVM or its effect is too small to be detected in the present size sample set.
Asunto(s)
Proteína 4 Similar a la Angiopoyetina/genética , Malformaciones Arteriovenosas Intracraneales/genética , Polimorfismo de Nucleótido Simple , Adulto , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Malformaciones Arteriovenosas Intracraneales/diagnóstico , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Fenotipo , Factores de Riesgo , Federación de Rusia , Adulto JovenRESUMEN
OBJECTIVES: The objective of this study was to study the association of polymorphisms MTHFR C677T (rs1801133) and MTR A2756G (rs1805087) with the risk of varicose veins in ethnical Russians. METHODS: We genotyped 475 patients with varicose veins, 168 individual without chronic venous disease, and the population-based group of 896 subjects. Association was studied using logistic regression analysis adopting co-dominant, additive, recessive, and dominant models of inheritance. RESULTS: None of the polymorphisms showed a statistically significant association with the risk of varicose veins. CONCLUSIONS: Our results provide evidence that the studied polymorphisms do not contribute to genetic susceptibility to varicose veins in ethnical Russians.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo de Nucleótido Simple , Várices/genética , Etnicidad , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Patrón de Herencia/genética , Modelos Logísticos , Federación de RusiaRESUMEN
Telomere length and telomerase activity have been hypothesized to play a role in cancer development. The aim of our study was to investigate the association of allelic variants of three functional polymorphisms rs2853669, rs2736100, and rs7726159 in the telomerase reverse transcriptase (TERT) gene with the risk of the breast cancer and prostate cancer in Russian population. Six hundred sixty women with breast cancer, 372 men with prostate cancer, and corresponding control groups of 523 women and 363 men were included in the present case-control study. We observed an association of allele rs2853669 C with increased risk of prostate cancer (co-dominant model TC vs. TT OR = 1.65, P = 0.002; additive model OR = 1.42, P = 0.005; dominant model: OR = 1.64, P = 0.001) and allele rs7726159 A with reduced risk of this malignancy (Ño-dominant model: AA vs. CC OR = 0.42, P = 0.002; additive model: OR = 0.69, P = 0.002; dominant model: OR = 0.67, P = 0.01; recessive model: OR = 0.48, P = 0.005). None of the studied polymorphisms showed an association with the risk of breast cancer. Our results provide evidence that the TERT gene variability modulate prostate cancer predisposition in ethnical Russians.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias de la Próstata/genética , Telomerasa/genética , Anciano , Anciano de 80 o más Años , Alelos , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/patología , Factores de Riesgo , Federación de Rusia , Telómero/genéticaRESUMEN
BACKGROUND: The spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis compromises effective control of tuberculosis (TB) in Siberia. Early identification of drug-resistant isolates is, therefore, crucial for effective treatment of this disease. The aim of this study was to conduct drug susceptibility testing and identify mutations in drug resistance genes in clinical isolates of M. tuberculosis from some TB patients presenting for treatment in Siberia. METHODS: Thirty randomly selected clinical isolates of M. tuberculosis were obtained from the Novosibirsk Research Institute of Tuberculosis, Russia. Isolates were screened for drug resistance and characterized by variable number of tandem repeats (VNTR)-typing using 15 standard and four additional loci. Deligotyping on multiple large sequences was performed using 10 loci. RESULTS: Twenty-nine of the isolates were assigned XDR status. Twenty-eight isolates belonged to the M. tuberculosis Beijing family, from which 11 isolates were considered the M11 type (39%), two the M2 type (7%), and one the M33 type (3%). Seventeen isolates (60.7%) from this family exhibited unique genetic patterns. The remaining two isolates belonged to the Latino-American Mediterranean family. Gene sequences (rpoB, katG, rrs, rpsL, tlyA, gidB, gyrA, gyrB) were analyzed to identify mutations that confer resistance to rifampicin, isoniazid, amikacin, kanamycin, capreomycin, and ofloxacin. The most common mutations among the XDR isolates were S531L in RpoB, S315T in KatG, various codon 94 mutations in gyrA, A90V in GyrA, K43R in RpsL, and 1401 A â G in rrs; these confer resistance to rifampicin, isoniazid, ofloxacin, streptomycin and kanamycin/capreomycin, respectively. There was high congruence between the two typing methods (VNTR typing and deligotyping) and RD105, RD149, RD152, RD181, and RD207 regions of difference were absent from the 28 Beijing family isolates. CONCLUSIONS: Deligotyping can be used for rapid and reliable screening of M. tuberculosis isolates, followed by more in-depth genotyping. Identification of Beijing family isolates with extensive drug resistance confirms that such strains have epidemiological importance in Siberia. Rapid detection of mutations that lead to drug resistance should facilitate selection of effective drug therapies, and the development of early prevention strategies to combat this infection.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Tuberculosis Extensivamente Resistente a Drogas/sangre , Tuberculosis Extensivamente Resistente a Drogas/diagnóstico , Mycobacterium tuberculosis/aislamiento & purificación , Adulto , Amicacina/farmacología , Antituberculosos/farmacología , Capreomicina/farmacología , Estudios Transversales , Tuberculosis Extensivamente Resistente a Drogas/epidemiología , Femenino , Genotipo , Humanos , Isoniazida/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Repeticiones de Minisatélite , Mutación , Ofloxacino/farmacología , Rifampin/farmacología , Siberia/epidemiología , Tuberculosis/genética , Adulto JovenRESUMEN
LAMP (Loop-mediated isothermal amplification) is a popular method for the molecular diagnostics of numerous pathogens, specifically useful for point-of-care testing. However, the efficacy and sensitivity of LAMP still need to be maximised for the best performance in clinical settings. Adding a novel fourth primer pair is a promising way to accelerate the LAMP speed. Here, we report PI primers that are part of inner primers and can be used in LAMP without a specific design. PI primers were tested in quantitative LAMP detecting SARS-CoV-2 and MS2. The new primers have increased the speed and sensitivity of quantitative LAMP, RT-LAMP, and duplex LAMP with artificial templates and RNA samples from nasal swabs. Adding PI primers could become a valuable option for LAMP optimisation, especially when a desirable LAMP target is a highly variable DNA sequence with a few conservative sites for primers.
RESUMEN
BACKGROUND AND AIMS: DiGeorge syndrome (DGS) is a genetic disorder manifesting in polymorphic symptoms related to developmental abnormalities of various organs including thymus. DGS is caused by microdeletions in the 22q11.2 region between several low copy repeats (LCR) occurring in approximately 1 in 4000 live births. Diagnosis of DGS relies on phenotypic examination, qPCR, ultrasound, FISH, MLPA and NGS which can be relatively inaccurate, time-consuming, and costly. MATERIALS AND METHODS: A novel multiplex droplet digital PCR (ddPCR) assay was designed, optimized and validated for detection and mapping 22q11.2 microdeletions by simultaneous amplification of three targets - TUPLE1, ZNF74, D22S936 - within the deletion areas and one reference target - RPP30 - as an internal control. RESULTS: The assay reliable identified microdeletions when the template concentration was >32 copies per reaction and successfully detected LCR22A-B, LCR22A-C, LCR22A-D, and LCR22B-C deletions in clinical samples from 153 patients with signs of immunodeficiency. In patients with the microdeletions, flow cytometry detected a significant increase in B-cell and natural killer cell counts and percentages, while T-cell percentages and T-cell receptor excision circle (TREC) numbers decreased. CONCLUSION: The designed ddPCR assay is suitable for diagnosing DGS using whole blood and blood spots.
Asunto(s)
Síndrome de DiGeorge , Reacción en Cadena de la Polimerasa Multiplex , Humanos , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Cromosomas Humanos Par 22/genética , Deleción CromosómicaRESUMEN
Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.
Asunto(s)
5-Metilcitosina , Metilación de ADN , Várices , Várices/genética , Várices/metabolismo , Humanos , Masculino , Femenino , Persona de Mediana Edad , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Adulto , Anciano , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sitios GenéticosRESUMEN
OBJECTIVE: Polymorphisms of glutathione S-transferase (GST) genes in mothers may be involved in teratogenesis in their offspring. This study aims to investigate the association of GST genes (T1, M1 and P1) with the risk of having children with congenital malformations (CMs) in residents of the West Siberian region of Russia. METHOD: We studied 235 women with offspring's with CMs, and 273 women with one or more healthy children. Null genotypes of GSTM1 and GSTT1 were identified through multiplex real-time polymerase chain reaction, and GSTP1 gene (Ile105Val) polymorphism was determined through TaqMan-real-time polymerase chain reaction. RESULTS: The study showed that the maternal genotype GSTT1 «0/0¼ is associated with CMs in the offspring (odd ratio (OR) = 3.63, P = 5.18 × 10(-9) ). A significant association of the maternal genotype GSTT1 «0/0¼ with CMs of the cardiovascular system (OR = 5.03, P = 2.93 × 10(-7) ), urinary system (OR = 4.20, P = 3.51 × 10(-6) ) and central nervous system (OR = 4.40, P = 6.69 × 10(-5) ) was found in the child. No association of maternal GSTM1 (del) and GSTP1 (Ile105Val) genetic polymorphisms with CMs of the child was identified. CONCLUSION: Homozygous deletion of the GSTT1 gene in women of the West Siberian region is a risk factor for birth defects in the child.
Asunto(s)
Anomalías Congénitas/genética , Gutatión-S-Transferasa pi/genética , Glutatión Transferasa/genética , Polimorfismo Genético , Adolescente , Adulto , Sustitución de Aminoácidos , Estudios de Casos y Controles , Anomalías Congénitas/epidemiología , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Recién Nacido , Isoleucina/genética , Madres , Embarazo , Factores de Riesgo , Siberia/epidemiología , Valina/genética , Adulto JovenRESUMEN
DNA polymerases are a superfamily of enzymes synthesizing DNA using DNA as a template. They are essential for nucleic acid metabolism and for DNA replication and repair. Modern biotechnology and molecular diagnostics rely heavily on DNA polymerases in analyzing nucleic acids. Among a variety of discovered DNA polymerases, Bst polymerase, a large fragment of DNA polymerase I from Geobacillus stearothermophilus, is one of the most commonly used but is not as well studied as Taq polymerase. The ability of Bst polymerase to displace an upstream DNA strand during synthesis, coupled with its moderate thermal stability, has provided the basis for several isothermal DNA amplification methods, including LAMP, WGA, RCA, and many others. Bst polymerase is one of the key components defining the robustness and analytical characteristics of diagnostic test systems based on isothermal amplification. Here, we present an overview of the biochemical and structural features of Bst polymerase and provide information on its mutated analogs.