Specific Oligonucleotide Research Articles

Abstract 4149: Knock-down of Neuropilin-1 by locked nucleic acid antisense oligonucleotides facilitates cancer immune control

Abstract Immune checkpoint inhibitors have become an important tool for the treatment of cancer. However, the majority of patients does not respond to the currently employed antibody-based treatments, indicating a need for additional targets and new treatment modalities. The invention of 3rd generation chemistries like locked nucleic acids (LNAs) has allowed the development of highly specific and efficacious antisense oligonucleotides (ASOs) that allow target knockdown in vivo without the need for complex delivery formulations. The membrane-bound protein neuropilin-1 (NRP1) was initially identified as a factor involved in cell migration, -survival, and neoangiogenesis making it an attractive target for cancer therapy. It was subsequently shown that it has additional immune-mediated pro-tumorigenic roles. It can suppress anti-tumor immune activity via regulation of trafficking of tumor-associated macrophages, phenotypic stability of Tregs, and exhaustion of effector T cells. Considering that these effects are mediated by multiple domains of NRP1, there is a conceptual advantage to down-regulate the expression of the whole protein over the functional or steric blockade of individual domains. We used our in-house Oligofyer™ bioinformatics system to design both human NRP1 specific LNAplus™ ASOs, as well as murine surrogates, which both achieved target knock-down of more than 85% in vitro. Furthermore, the murine surrogate down regulated NRP1 in tumor mouse models. Systemic application of mouse-specific ASOs without additional carriers or adjuvants has led to a strong delay of tumor growth or complete eradication in syngeneic mouse models. This effect was almost completely abrogated in immune-compromised NSG mice. Furthermore, re-challenge of immune-competent mice after tumor eradication did not result in new tumor growth, suggesting induction of a long-lasting immunity by LNAplus™ ASO-mediated knockdown of NRP1. Taken together, these encouraging results indicate that the ASO-mediated down-regulation of NRP1 has the potential to become a promising treatment option for patients in need. We are currently performing further experiments to fully elucidate the mechanisms that underlie the observed anti-tumor efficacy. Citation Format: Andre Maaske, Nicole Kirchhammer, Julia Festag, Laura Fernandez Rodriguez, Mélanie Buchi, Monika Schell, Stefanie Raith, Sven Michel, Richard Klar, Alfred Zippelius, Frank Jaschinski. Knock-down of Neuropilin-1 by locked nucleic acid antisense oligonucleotides facilitates cancer immune control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4149.

Application of eDNA for monitoring freshwater bivalve Nodularia nipponensis and its glochidium larvae

AbstractHabitat loss, the introduction of invasive species, and climate change due to human activity have threatened many freshwater unionid bivalves worldwide. Unionid bivalves represent important members of freshwater ecosystems, providing bitterling fish with spawning grounds and contributing to water clarification via the filtration of suspended solids. This study examined an environmental DNA (eDNA) approach for monitoring the unionid bivalve Nodularia nipponensis in a lake environment. We developed oligonucleotide primers for the specific detection of mitochondrial DNA from N. nipponensis and evaluated the decay and release rates of eDNA in a laboratory tank experiment under two bivalve densities (49 and 164 individuals m−2) selected to ensure sufficient eDNA yields. The eDNA release rate was weakly and positively correlated with bivalve density but strongly and positively correlated with inorganic nitrogen and phosphorus excretion from the bivalve. In a eutrophic lake, Lake Hachiro, 67% of the samples (16 out of 24) collected from June to July yielded detectable amounts of eDNA and were not detected at all in May and August (0 out of 20). This difference was probably due to the release of N. nipponensis glochidium larvae. During these months, unionid glochidia adhered to bundles of polyvinylidene chloride fibers placed on the lake bottom for 8 h, confirmed by microscopic inspection. We successfully obtained N. nipponensis eDNA from all bundles examined over the months, demonstrating the efficacy of bundle capture in quantifying unionid bivalve eDNA. Our eDNA‐based technique with specific oligonucleotide primers and fiber bundles may be a promising tool for monitoring N. nipponensis in freshwater environments. Fiber bundles are useful for tracing eDNA derived from glochidium larvae, even at low unionid bivalve densities, which may be conducive to conservation efforts.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients’ serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 μg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.

Sensitive and specific clinically diagnosis of SARS-CoV-2 employing a novel biosensor based on boron nitride quantum dots/flower-like gold nanostructures signal amplification

The sudden increase of the COVID-19 outbreak and its continued growth with mutations in various forms has created a global health crisis as well as devastating social and economic effects over the past two years. In this study, a screen-printed carbon electrode reinforced with boron nitride quantum dots/flower-like gold nanostructures (BNQDs/FGNs/SPCE) and functionalized by highly specific antisense DNA oligonucleotide presents an alternative and promising solution for targeting SARS-CoV-2 RNA without nucleic acid amplification. The platform was tested on 120 SARS-CoV-2 RNA isolated from real clinical samples (60 positive and 60 negative confirmed by conventional RT-PCR method). Based on obtained quantitative results and statistical analysis (box-diagram, cutoff value, receiver operating characteristic curve, and t-test), the biosensor revealed a significant difference between the two positive and negative groups with 100% sensitivity and 100% specificity. To evaluate the quantitation capacity and detection limit of the biosensor for clinical trials, the detection performance of the biosensor for continuously diluted RNA isolated from SARS-CoV-2-confirmed patients was compared to those obtained by RT-PCR, demonstrating that the detection limit of the biosensor is lower than or comparable to that of RT-PCR. The ssDNA/BNQDs/FGNs/SPCE showed negligible cross-reactivity with RNA fragments isolated from Influenza A (IAV) clinical samples and also remained stable for up to 14 days. In conclusion, the fabricated biosensor may serve as a promising tool for point-of-care applications.

Highly sensitive fluorescence-based mercury(II) DNA sensor enhanced by silver(I) activation

Mercury exposure constitutes an acute risk to human health and the environment. Driven by the requirement to monitor trace-level mercury, we report a highly sensitive mercury(II) DNA sensor enhanced by silver(I) activation, followed by mercury(II)-specific oligonucleotides (MSO) molecular configuration switch and mercury(II)-modulated FRET. Activating the MSO strands with silver(I), mismatched cytosine–silver(I)–cytosine bridges induce individual MSO strands to fold readily in response to mercury(II) resulting in enhanced fluorescence signal. The structural switches were studied by 2D 1H–1H NOESY and TOCSY NMR spectroscopy, and 260 nm absorbance. The signal decreases with increasing mercury(II) concentration from 100 pM to 0.1 mM. The approach affords outstanding mercury(II) selectivity over other environmentally associated metals. Furthermore, the methodology was deployed for detection of mercury(II) in spiked pond waters with 97.9–100.6% recovery. The simple and feasible format has great potential for developing a cost effective and useful tool for environmental monitoring.

Analysis of loss of heterozygosity at HLA loci in a patient with leukemia

To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

Species-specific PCR-based marker for rapid detection of Aspidiotus rigidus Reyne (Hemiptera: Diaspididae)

• A marker was designed from InDels of Aspidiotus rigidus 28 s rDNA sequences. • Species-specific delineation of A. rigidus works in singleplex and multiplex PCR. • A. rigidus multiplex-based detection still works with parasitized samples. The Philippine coconut production has been greatly affected by the recent devastating infestation of Aspidiotus spp. However, identification of the outbreak species, Aspidotus rigidus , has been a challenge using morphological approaches. Molecular identification via PCR sequencing of insect barcoding genes has been implemented, but the overall process is time-consuming and costly. Thus, we developed and optimized a species-specific PCR-based molecular marker for rapid, efficient and cost-effective molecular identification of A. rigidus . The molecular marker was designed based on the sequences of the partial 28S ribosomal RNA gene from species of Aspidiotus that feed on coconut in the Philippines, A. rigidus, A. destructor and A. excisus . Multiple alignment of nucleotide sequences revealed a conserved 16-bp insertion-deletion (InDel) site common to all A. rigidus specimens identified from which the A. rigidus- specific oligonucleotide (RIG1) primer targeting an approximately 570 bp fragment size was designed. Results showed that the species-specific DNA marker technology consistently delineated laboratory-reared and field-collected A. rigidus samples from A. destructor and A. excisus . The protocol offers a rapid and reliable method for the early detection of A. rigidus infestation in high-risk areas planted with coconut in the country.

Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools.

With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications including gene synthesis, CRISPR-based gene editing, and DNA data storage. The selective retrieval of specific oligos from these complex pools traditionally uses polymerase chain reaction (PCR). Designing a large number of primers to use in PCR presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool is orders of magnitude larger than other applications. Although a nested primer address system was recently developed to increase the number of accessible files for DNA storage, it requires more complicated lab protocols and more expensive reagents to achieve high specificity, as well as more DNA address space. Here, we present a new combinatorial PCR method that has none of those drawbacks and outperforms in retrieval specificity. In experiments, we accessed three files that each comprised 1% of a DNA prototype database that contained 81 different files and enriched them to over 99.9% using our combinatorial primer method. Our method provides a viable path for scaling up DNA data storage systems and has broader utility whenever one must access a specific target oligo and can design their own primer regions.

Molecular fish sexing on Kohaku Koi (Cyprinus carpio) based on ArS.9-15 gene amplification by PCR method

This study aims to sex determine on Kohaku Koi fish rapidly, precisely, accurately, and as early as possible based on the amplification of the ArS.9-15 gene using the PCR method. This study began with the maintenance of 10 Kohaku Koi fish, obtained from Koi fish farmers in the Sleman area in Yogyakarta. The 10 Kohaku Koi were adapted in a filtered aquarium with O2 aeration and fed ad libitum by fish pellets for 1-3 days. Then, a collection of 0.5 ml peripheral blood samples per fish from the caudalis vein was performed under anesthesia conditions using special anesthetic preparation for Koi fish, namely Koi Anesthesia mixed with fresh water. Those samples were then collected in tubes containing the Ca-EDTA anticoagulant. The DNA then was extracted from peripheral blood samples and used as a template for PCR amplification by specific oligonucleotide primer pairs to amplify the ArS.9-15 gene. PCR products in the form of DNA fragments were visualized with 1.5% agarose gel electrophoresis and SybrSafe staining using Transilluminator-UV in a dark room. The DNA fragments were analyzed descriptively to sex determination between male and female Kohaku Koi. Electrophoresis results of PCR products showed two DNA bands of 850 bp and 1,100 bp in male Kohaku Koi, while in female Kohaku Koi showed only a band DNA in size of 850 bp. The results of the molecular fish sexing of the 10 Kohaku Koi showed that 70% are female and 30% are male.

POS-324 ASSOCIATION OF VOLTAGE-DEPENDENT CALCIUM CHANNEL POLYMORPHISMS 1.3 WITH THE DEVELOPMENT OF DIABETIC NEPHROPATHY, DIABETIC RETINOPATHY AND DIABETIC NEUROPATHY

One of the main characteristics of type 2 diabetes (T2D) is the lack of insulin secretion, this process is regulated by several proteins, within these are voltage-gated calcium channels, mainly CaV1.3 channels, whose single nucleotide polymorphisms (SNPs) have been associated in other populations with the presence of microvascular complications such as nephropathy, retinopathy and neuropathy in patients with T2D. Objective: To determine the association of the polymorphisms of the gene that codes for CaV1.3 with the risk of developing nephropathy, retinopathy and diabetic neuropathy. A case-control, comparative, observational, ambispective and cross-sectional study was carried out, in which men and women between 30 and 90 years of age participated, including patients with T2D according to the guidelines of the American Association. of Diabetes, with and without microvascular complications (nephropathy, retinopathy and neuropathy), treated in the outpatient clinic of the Internal Medicine Service, General Hospital “Dr. Manuel Gea González ”, from March to August 2020, from whom peripheral blood samples were taken for the extraction of genomic DNA by the phenol / chloroform method and subsequent genotyping, with the iPLEX Assay system that requires the design of specific oligonucleotides , amplification by polymerase chain reaction and finally detection of SNPs by mass spectrometry. A total of 200 patients participated in this study, 125 (62.5%) men and 75 (37.5%) women, within which 100 cases were divided into two groups (with a diagnosis of type 2 diabetes and the presence of nephropathy, retinopathy and neuropathy) and 100 controls (with a diagnosis of type 2 diabetes and absence of microvascular complications). A total of 4 polymorphisms of the CACNA1D gene were studied. Wild alleles were the most frequent in all groups. Within the polymorphisms analyzed in the binary logistic regression, rs9841978 GA (OR, 6.2; CI95%: 1.15-13.48, p = 0.03) and rs312486 CC (OR, 6.6; CI95%: 4.60-9.45, p = 0.002) with a Wald significance level of 4.51 and 9.51 respectively. In the multinomial logistic regression model, in diabetic retinopathy the variables were age (OR, 1.12; 95% CI: 1.03-1.22, p = 0.01), HT (OR, 5.92; 95% CI: 4.55-7.11, p = 0.002), rs9841978 GA (OR, 9.72; 95% CI: 3.62-10.72, p = 0.002) and rs312486 CC (OR, 1.09; 95% CI: 1.0004-1.19, p = 0.003). In diabetic nephropathy and neuropathy, the variable was the polymorphism rs9841978 GA, with OR, 12.55; CI95%: 4.80- 32.80, p = 0.03 and OR, 6.18; 95% CI: 2.90-13.16, p = 0.03, respectively. Our study demonstrated the association of well-established genetic variants of the polymorphisms of the CACNA1D gene with the presence of microvascular complications in patients with type 2 diabetes, showing that these SNPs, specifically rs9841978 GA and rs312486 CC, may represent important markers in the development of these complications of the disease, mainly nephropathy and retinopathy, and they are a predisposing factor due to the high frequency and strength of association observed in our study population, representing this the first study in Latin America. This suggests that our population is highly susceptible to developing not only T2D but also complications such as nephropathy and retinopathy that in the medium or long term could represent the cause of death in this sector when these SNPs are present.

Antisense Oligonucleotide Technologies to Combat Obesity and Fatty Liver Disease.

Synthetic oligonucleotide technologies are DNA or RNA based molecular compounds that are utilized to disrupt gene transcription or translation in target tissues or cells. Optimally, oligonucleotides are 10–30 base pairs in length, and mediate target gene suppression through directed sequence homology with messenger RNA (mRNA), leading to mRNA degradation. Examples of specific oligonucleotide technologies include antisense oligonucleotides (ASO), short hairpin RNAs (shRNA), and small interfering RNAs (siRNA). In vitro and in vivo studies that model obesity related disorders have demonstrated that oligonucleotide technologies can be implemented to improve the metabolism of cells and tissues, exemplified by improvements in fat utilization and hepatic insulin signaling, respectively. Oligonucleotide therapy has also been associated with reductions in lipid accumulation in both the liver and adipose tissue in models of diet-induced obesity. Recent advances in oligonucleotide technologies include the addition of chemical modifications such as N-acetylgalactosamine (GalNAc) conjugates that have been successful at achieving affinity for the liver, in turn improving specificity, and thus reducing off target effects. However, some challenges are still yet to be overcome relating to hepatic injury and off-target effects that have been reported with some compounds, including ASOs. In summary, oligonucleotide-based therapies are an effective tool to elucidate mechanistic insights into metabolic pathways and provide an attractive avenue for translational research into the clinic.

Safe and Effective Cynomolgus Monkey GLP-Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders.

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13.

Investigation of many bacterial and viral infections circulating in pigeons showing nervous symptoms

Pigeon’s flocks have shown several neurological symptoms including circling, torticollis, tremors, paralysis, which caused suspicion for viral or bacterial natural infections. Pigeon paramyxovirus type-1 (PPMV-1) is a notifiable disease-causing high morbidity and mortality with severe nervous symptoms. Clinical represented tissue specimens were collected from 50 infected pigeon flocks in eight governorates. All samples were examined bacteriologically (isolation, identification and serotyping) for E. coli, Salmonella spp., S. aureus and pseudomonas aeruginosa. Antimicrobial susceptibility test (AST) was accomplished for all isolates using a disk-diffusion test. For viral identification, RT-PCR specific oligonucleotide primers were used for distinguishing of Avian influenza virus, PPMV-1 and PPMV-3. Neurological manifestations were observed in pigeon’s flocks mainly in winter and autumn. The mortality rate in eight governorates was about 50% in 10 flocks and other houses mortality rate was ranged from 10 to 20%. Post mortem examination have shown hemorrhagic enteritis, soft and friable brain tissues and/or hemorrhages. The percentage of isolated bacteria E. coli, Salmonella spp., S. aureus and Pseudomonas aeruginosa were 75%, 75%, 50% and 18.75%; respectively. The antibiotic resistance pattern for bacterial isolates showed resist to ampicillin, amoxicillin- clavulinic acid, teteracyclin, ceftriaxone, doxycycline, sulfamethoxazole-trimethoprim and ceftazidine with different result for each type of bacteria, while Salmonella spp., isolates showed only a highly intermediate result for ciprofloxacin. Eight samples are positive with 16% to PPMV-1. Also, sample No.5,6,9 was co-infected with different types of bacterial isolates in addition to NDV. In conclusion, we reported several neurological symptoms in pigeon’s flocks mainly of bacterial infections (E. coli, Salmonella spp., S. aureus and Pseudomonas aeruginosa).

Selection of Aptamer for N-Methyl Mesoporphyrin IX to Develop Porphyrin Metalation DNAzyme.

Mesoporphyrin IX (MPIX) contains a planar macrocycle center that can interact with various divalent metal ions through the exposed binding sites, leading to the metalation of MPIX. The DNA aptamers for porphyrin molecules usually display different catalytic functions (termed deoxyribozymes or DNAzymes), which can accelerate such chemical reactions. Inspired by this, an affinity chromatography selection approach was designed for identifying a porphyrin metalation DNAzyme. In our experiment, N-methyl mesoporphyrin IX (NMM), an analog of MPIX, is used as the target molecule, owing to its stable and high fluorescence enhancement after combining with specific oligonucleotides. Our results showed that the selected aptamer Nm1 is capable of binding to NMM with a low micromolar dissociation constant (0.75±0.08μM) and displays a catalytic activity for MPIX metalation with 3.3-fold rate enhancement. The protocol for isolation of such a porphyrin metalation DNAzyme is described in detail here.

Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

ABSTRACT Emerging evidence showed that lncRNAs play important roles in a wide range of biological processes of fungi such as Saccharomyces cerevisiae. However, systemic identification of lncRNAs in non-model fungi is a challenging task as the efficiency of rRNA removal has been proved to be affected by mismatches of universal rRNA-targeting probes of commercial kits, which forces deeper sequencing depth and increases costs. Here, we developed a low-cost and simple rRNA depletion method (rProbe) that could efficiently remove more than 99% rRNA in both yeast and mycelium samples of Talaromyces marneffei. The efficiency and robustness of rProbe were demonstrated to outperform the Illumina Ribo-Zero kit. Using rProbe RNA-seq, we identified 115 differentially expressed lncRNAs and constructed lncRNA-mRNA co-expression network related to dimorphic switch of T. marneffei. Our rRNA removal method has the potential to be a useful tool to explore non-coding transcriptomes of non-model fungi by adjusting rRNA probe sequences species specifically.

ПОРІВНЯННЯ СИСТЕМ GENEXPERT MTB/RIF І GENOTYPE ЗІ СТРИПАМИ MTBDRPLUS ДЛЯ ВИЯВЛЕННЯ МУТАЦІЙ, ПОВ’ЯЗАНИХ З РЕЗИСТЕНТНІСТЮ M. TUBERCULOSIS ДО РИФАМПІЦИНУ ПРИ ТУБЕРКУЛЬОЗІ

COMPARISON OF GENEXPERT MTB/RIF AND GENOTYPE SYSTEMS WITH MTBDRPLUS STRIPS FOR DETECTION OF MUTATIONS, ASSOCIATED WITH M. TUBERCULOSIS RESISTANCE TO RIFAMPICIN IN TUBERCULOSIS A. A. Zhurilo, A. I. Barbova, Yu. A. Cherednik, P. S. Trofi mova, S. V. Mironchenko, O. V. Pavlova, A. V. Chernov, L. M. Sladkova Summary The aim was to analyze the level of compliance of two molecular genetic methods GeneXpert MTB/RIF and GenoTypeDRplus in determining the drug resistance of M. tuberculosis to rifampicin when detecting mutations in the RRDR region associated with drug resistance. Object and methods. We studied strains of M. tuberculosis with resistance to rifampicin, which was detected by any of the studied molecular genetic methods. 96 sputum samples were taken. Sputum smears were examined for the presence of acid-fast bacteria by microscopy after staining with the Ziehl-Neelsen method. The material was inoculated into Middlebrook 7H9 broth and on Lowenstein-Jensen medium. The liquid medium was incubated in the BACTEC MGIT system. An immunochromatographic test was used to identify the strains. The drug susceptibility test of M. tuberculosis to rifampicin was performed using the BACTEC MGIT system. The GeneXpert MTB/RIF test was performed according to the manufacturer’s instructions. The GenoTypeDRplus assay was performed on decontaminated and concentrated sputum samples. The process was carried out in three stages: DNA extraction from the processed sputum sample; amplification of the RRDR region by PCR; hybridization of the PCR product to specific oligonucleotide probes immobilized on the test strip. For sequencing, M. tuberculosis DNA isolation was performed using the QIAamp® DNA Mini Kit. The DNA concentration was measured on a Denovix Quantus spectrophotometer. Targeted panel amplification was performed using the Deeplex Myc-TB kit. Amplicon purification was performed using Agencourt AMPure XP magnetic beads. Quantitative analysis of the purified amplification products was performed using a Qubit fluorometer. The M. tuberculosis DNA library was prepared for sample sequencing using the Nextera XT DNA library preparation kit. The library used 5.0 μl of input DNA at a concentration of 0.2 ng/μl. Sequencing was performed on MiSeq equipment with the library normalization and denaturation protocol. Results and discussion. The GeneXpert MTB/RIF and MTBDRplus systems target the same 81 bp rifampicin resistance domain. (RRDR) subunits of bacterial RNA polymerase (rpoB) for mutation detection using DNA probes, i.e. there is a correspondence of probes to each other and an expected similarity of probe binding. We analyzed all sputum samples using GeneXpert MTB/RIF and GenoType MTBDRplus and phenotypic BACCTEC MGIT methods. The level of agreement between two molecular genetic methods for the detection of rifampicin-associated mutations in the RRDR region has been established. The RRDR 81bp region of the rpoB gene of mismatched cases was studied by sequencing. GeneXpert MTB/RIF and GenoType DRplus matched the phenotypic method in 92.7% and 89.6% of cases of M. tuberculosis resistance, respectively. Complete agreement between the results of GeneXpert MTB/RIF and GenoTypeMTBDRplus was observed in 92.7% of cases. GeneXpert MTB/RIF and GenoType DRplus showed a similar pattern of binding failure of wild type probes (WT-probes) when scanning the 81 bp region (RRDRdomain), which leads to stability diagnostics through probe failure software. Sequencing of the RRDR region of “mismatched” strains showed that GeneXpert probes detected seven “mismatched” cases correctly, and GenoTypeDRplus was erroneous in all cases. GeneXpert has demonstrated greater accuracy in R-resistance detection for mismatched isolates compared to GenoTypeDRplus. GeneXpert MTB/RIF has a number of other benefits over GenoTypeDRplus. GeneXpert MTB/RIF is relatively easier to implement, biosafety requirements are minimal, study times are shorter, and the study process is more automated, resulting in less human error and more reproducible results. Given these facts and the results of GeneXpert MTB/RIF found in the study, it is recommended that GeneXpert MTB/RIF be used to detect MDR-TB. Conclusions. Sequencing of the 81bp RRDR region of mismatched M. tuberculosis strains showed that GeneXpert MTB/RIF performed more accurately than GenoTypeDRplus in detecting mutations associated with rifampicin resistance. GeneXpert MTB/RIF is relatively easier to perform, biosafety requirements are minimal, time to study is shorter, and the study process is more automated, resulting in less human error and greater reproducibility of results, so it is reasonable to use it for the detection of multidrug-resistant tuberculosis. The Deeplex Myc-TB analytical solution delivers a wealth of insightful information from antimycobacterial drug resistance markers and speeds up data analysis with easy-to-use software. Key words: tuberculosis, Mycobacterium tuberculosis, drug resistance, GeneXpert MTB/RIF and GenoTypeDRplus molecular genetic systems, BACTEC MGIT phenotypic system, sequencing. Ukr. Pulmonol. J

Lack of association of the KIR and HLA class I ligands with ZIKV infection in south and southeast of Brazil.

BACKGROUND Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes.OBJECTIVES This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility.METHODS KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique.FINDINGS No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed.MAIN CONCLUSIONS KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

Development of a novel molecular assay based on conventional PCR liquid hybridization and ELISA (NAT-ELISA) for the detection of HIV, HCV, and HBV in blood donors

PurposeDevelopment of a simple, affordable, and sensitive method based on gene amplification by PCR followed by liquid hybridization for detection of HIV-1 & 2, HCV and HBV in plasma samples (NAT-ELISA) for detection of these viruses particularly in their window period. MethodsViral nucleic acid extracted from WHO International Standards for HIV1, HIV2, HCV and HBV and 199 blood donor plasma samples were amplified by reverse transcription -PCR with forward and biotinylated reverse primers designed from the conserved regions of p-24 gene of HIV-1 and HIV-2, 5’ UTR of HCV and middle part of S gene of HBV respectively. The biotinylated amplicons were immobilized on streptavidin coated ELISA plates, denatured chemically, and were hybridized with digoxigenin labelled specific oligonucleotide probes of HIV-1, HIV2, HCV and HBV. Hybridization signals were measured colorimetrically by indirect ELISA using anti-digoxigenin HRP conjugate and TMB substrate at 450 ​nm. Analytical Sensitivity or lower limit of detection (LOD) of this assay was determined with WHO standards. ResultsPCR amplified products of WHO standards were 321bp of HIV-1, 291bp of HIV- 2, 229bp of HBV, and 105bp of HCV. The LOD95 and LOD99.7 endpoints of HIV1, HIV2, HCV and HBV were 13, 6, 15 and 11 IU/ml and 15, 7, 17 and 12 IU/ml respectively. Diagnostic sensitivity and specificity of NAT-ELISA assay were calculated on 199 samples and was 97.4% and 99.4% respectively. ConclusionsThe results suggest that this assay is highly suitable for the blood banks which do not have facilities for exorbitantly expensive NAT test for the detection of these viruses during their window period particularly in the blood donors who test negative by antibody/antigen screening tests.

CATALASE CRS-262T GENE POLYMORPHISM AND CHANGES IN VENTILATION LUNG CAPACITY IN CHILDREN-RESIDENTS OF RADIOACTIVELY CONTAMINATED TERRITORIES.

to determine the association of catalase С-262Т gene polymorphism with the presence of bronchial hyper-reactivity in children living in radioactively contaminated territories. There were examined school-age children-residents of radioactively contaminated territories (RCT), who did not have clinical signs of respiratory pathology. Catalase (CAT) С-262Т gene deletion polymorphism was studied in the molecular genetic laboratory of the State Institution «Reference Center for Molecular Diagnostic of Public Health Ministry of Ukraine». Determination of the polymorphic variant by the catalase С-262Т gene was performed by Polymerase Chain Reaction (PCR) using specific oligonucleotide primers, followed by Restriction Fragment Length Polymorphism (RFLP) analysis. The CAT С-262Т gene polymorphism in children living in RCT was compared with that in the reference group of practically healthy individuals. Ventilation lung capacity was performed by computer spirometry according to the analysis of the loop «the flow-volume». A pharmacological inhalation test with a bronchodilator that acts on β2-adrenergic receptors of the lungs was used to detect early changes in the ventilatory capacity of the lungs - bronchial hyperreactivity. Comparative analysis showed that in the presence of bronchial hyperreactivity in children living in RCT, the CT genotype was more common than in children without bronchial hyperreactivity, and the frequency of the CC genotype was correspondingly reduced. There was a trend towards a decrease in the frequency of the TT genotype. An analysis of the frequency distribution of allelic variants of the CAT С-262Т gene polymorphism in children living in the RCT revealed a tendency to increase in the frequency of the T-allele and according to the decrease in the frequency of C-allele in the presence of bronchial hyperreactivity.Сonclusions. Thus, among children living in RCT, CT-homozygotes of CAT С-262Т gene polymorphism had bronchial hyperreactivity probably more often than CC-heterozygotes. In the presence of bronchial hyperreactivity, there was a trend towards an increase in the frequency of the T-allele and, accordingly, a decrease in the frequency of the C-allele.

Unknown Mutation Detection via Restriction Hybridization Method Instead of Using Next Generation Sequencing Method

In biology mutation is a change in the nucleotide sequences of the DNA of an organism Mainly there are three types of mutation: point mutation, deletion and insertions. Once the mutation has been defined allele specific oligonucleotide hybridization, amplification, heteroduplex formation method referred to as a diagnostic method some advance technique like CRISPR cas9 system is using for selected mutagenesis. Using restriction method system we can detect a mutation. Let’s say you have a DNA sample with fluorescent labeled from patient and you want to make sure that gene you are interested is in healthy gene. We can design different short fragment sequences to scan through DNA or find specific gene or mutation. The sequences scan the DNA if the sequences does not find targeted gene it does not bind to it its means that no fluorescence color appears under UV-light each different short fragment sequences is label with different colors. If the different short fragments sequence does not bind to the DNA or specific gene or area this means that there will be no color appear under UV light this part or gene will be separated from the DNA by using Restriction enzyme to do a Sanger sequencing gel electrophoresis. Result of the Sanger sequencing will provide the information about sequence of unknown part or gene of the DNA this method is easier and cost economic method instead of Next generation sequencing method [1-7].