Deoxyribonucleic Acid Extraction Research Articles

High-efficient separation of deoxyribonucleic acid from pathogenic bacteria by hedgehog-inspired magnetic nanoparticles microextraction

Efficient separation of deoxyribonucleic acid (DNA) through magnetic nanoparticles (MN) is a widely used biotechnology. Hedgehog-inspired MNs (HMN) possess a high-surface-area due to the distinct burr-like structure of hedgehog, but there is no report about the usage of HMN for DNA extraction. Herein, to improve the selection of MN and illustrate the performance of HMN for DNA separation, HMN and silica-coated Fe3O4 nanoparticles (Fe3O4@SiO2) were fabricated and compared for the high-efficient separation of pathogenic bacteria of DNA. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are typical Gram-negative and Gram-positive bacteria and are selected as model pathogenic bacteria. To enhance the extraction efficiency of two kinds of MNs, various parameters, including pretreatment, lysis, binding and elution conditions, have been optimized in detail. In most separation experiments, the DNA yield of HMN was higher than that of Fe3O4@SiO2. Therefore, a HMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated and used to detect pathogenic bacteria in real samples. Interestingly, the HMN-based MSPE combined qPCR strategy exhibited high sensitivity with a limit of detection of 2.0 × 101 CFU mL−1 for E. coli and 4.0 × 101 CFU mL−1 for S. aureus in orange juice, and 2.8 × 102 CFU mL−1 for E. coli and 1.1 × 102 CFU mL−1 for S. aureus in milk, respectively. The performance of the proposed strategy was significantly better than that of commercial kit. This work could prove that the novel HMN could be applicable for the efficient separation of DNA from complex biological samples.

Prospects for use of genetic tagging technology in animal husbandry

The efficiency of DNA extraction with using different extraction methods: from the cartilage tissue of the outer ear and the leukocyte fraction of whole blood of cattle, followed by an assessment of the possible prospects for the use of “genetic tagging” technology in animal husbandry has been compared in the work. It was established that according to the results of the research the highest concentration of DNA (146,26±13,180 ng/μl at p < 0,001) was obtained in samples from ear tissues when using the method of precipitation with reagents compared to isolating DNA from the leukocyte fraction of whole blood using highly eff ective methods extraction of genomic deoxyribonucleic acid on magnetic particles (22,80±3,138 ng/μl) and on columns (39,46±8,941 ng/μl). It was found when assessing DNA contamination with polypeptides that despite the fact that when using diff erent extraction methods, the ratio of optical densities at the corresponding absorption wavelengths A260/A280 was close to the optimal ~1,8. The smallest scatter of measurement values was obtained in DNA samples from earmarks tissue 1,89±0,008. In a subsequent comparison of available technologies for taking ear pluck tissue samples used for genetic research (population genetic characterization, genomic analysis, etc.), the FlexoPlusGeno “genetic tagging” system turned out to be the most preferable. In this case, the resulting ear pluck tissue sample is correlated with corresponding ear visual or RFID tags, which creates a “link” of the biomaterial to a specific animal with the ability to track it throughout its life. The technology under consideration is applicable for collecting and storing samples of cartilage tissue obtained by ear plucking from cattle, small ruminants, pigs and deer.

Gut Microbiota in Patients with Prediabetes.

Prediabetes is characterized by abnormal glycemic levels below the type 2 diabetes threshold, and effective control of blood glucose may prevent the progression to type 2 diabetes. While the association between the gut microbiota, glucose metabolism, and insulin resistance in diabetic patients has been established in previous studies, there is a lack of research regarding these aspects in prediabetic patients in Asia. We aim to investigate the composition of the gut microbiota in prediabetic patients and their differences compared to healthy individuals. In total, 57 prediabetic patients and 60 healthy adult individuals aged 18 to 65 years old were included in this study. Biochemistry data, fecal samples, and 3 days of food records were collected. Deoxyribonucleic acid extraction and next-generation sequencing via 16S ribosomal ribonucleic acid metagenomic sequencing were conducted to analyze the relationship between the gut microbiota and dietary habits. Prediabetic patients showed a lower microbial diversity than healthy individuals, with 9 bacterial genera being less abundant and 14 others more abundant. Prediabetic patients who consumed a low-carbohydrate (LC) diet exhibited higher diversity in the gut microbiota than those who consumed a high-carbohydrate diet. A higher abundance of Coprococcus was observed in the prediabetic patients on an LC diet. Compared to healthy individuals, the gut microbiota of prediabetic patients was significantly different, and adopting an LC diet with high dietary fiber consumption may positively impact the gut microbiota. Future studies should aim to understand the relationship between the gut microbiota and glycemic control in the Asian population.

Molecular Identification of invA gene from Salmonella species isolated from Human Sources in Southern Taraba, North-East Nigeria

Place and Duration of the Study: The study was conducted in Wukari, Donga, Ibi, and Takum Local Government Areas in Southern Taraba State, North-East Nigeria. These areas were chosen due to their high population of farmers and traders, making them suitable locations for investigating foodborne pathogens. The experimental research spanned a period of 7 months, during which 200 blood and stool samples were collected from food vendors in the selected areas. Methodology: Sample collection involved the collection of 200 blood and stool samples from food vendors in Wukari, Donga, Ibi, and Takum Local Government Areas. Isolation and confirmation of Salmonella species were carried out through cultural and biochemical analyses, with reference strains used for quality control. Deoxyribonucleic Acid (DNA) extraction was performed using the boiling technique, followed by Polymerase Chain Reaction (PCR) amplification of the invA gene. Electrophoresis on an agarose gel was used to visualize the presence of the invA gene in the isolates. Results: The results of the study revealed a high prevalence of Salmonella species among food vendors in Southern Taraba State, North-East Nigeria. Analysis of the invA gene showed its presence in all isolates, indicating the widespread distribution of virulent strains in the study area. The findings underscore the importance of PCR-based methods for the detection of Salmonella and suggest the need for comprehensive surveillance and control measures to prevent foodborne illnesses. Conclusion: The study demonstrates the significant prevalence of Salmonella species among food vendors in Southern Taraba State, North-East Nigeria. The detection of the invA gene in all isolates highlights the potential virulence of these strains and emphasizes the importance of effective surveillance and control strategies to mitigate the risk of foodborne diseases. Further research is warranted to elucidate the genetic diversity and antimicrobial resistance genes associated with Salmonella strains in the region, facilitating the development of targeted interventions for public health protection.

Polymerase chain reaction-based identification of various serotypes of Streptococcus mutans in adults with and without dental caries.

Dental caries is a multistep process which initiates the development of plaque' defined as a structured biofilm containing microbial communities. Teeth provide unique surfaces for bacterial colonization. Serotypes of Streptococcus mutans implicate the development of dental caries. The aim of the study was to determine the prevalence and association of serotypes of S. mutans in groups with and without dental caries. One hundred and fifty adults aged between 18 and 35 years were included in the study. Supragingival plaque samples were collected, followed by deoxyribonucleic acid extraction. Polymerase chain reaction was performed to identify S. mutans and its serotypes. Proportions of S. mutans and its serotypes were correlated with caries-active (CA) and caries-free (CF) groups. CA group showed 66.7% positivity for S. mutans and CF group showed only 42.7% of positivity. Serotype C showed a higher proportion followed by E' F, and K in the CA group, whereas in the CF group, higher proportion was observed with K followed by C' E, and F. 70.8% cases showed single serotype in the CA group and 83.3% in CF group. Multiple serotypes were seen in 29.2% in the CA group and 16.7% in the CF group. The study clearly established variation in proportions of S. mutans and its serotypes between CA and CF groups. Positive correlation was observed in the CA group for S. mutans and its serotypes.

Genetic variant in TNF-α gene and its plasma level in relation to obstructive sleep apnoea in the Pakistani population.

To assess the link between tumour necrosis factor-alpha -308 guanine/adenine polymorphism and tumour necrosis factor-alpha plasma levels in relation to obstructive sleep apnoea. The cross-sectional study was conducted from December 2018 to March 2021 at the sleep clinic of Dow University Hospital, Karachi, on obstructive sleep apnoea patients and healthy controls. Epworth Sleep Scale score was used to determine daytime sleepiness, while full-night polysomnography was carried out for obstructive sleep apnoea confirmation and categorisation according to severity. Blood sample collection was followed by deoxyribonucleic acid extraction and plasma tumour necrosis factor-alpha measurement using enzyme-linked immunosorbent assay. Genotype distribution and allelic frequency were assessed. Data was analysed using SPSS 20. Out of the 225 subjects, with a mean age of 47.68±9.88 years, 132 (58.7%) were males, and 93 (41.3%) were females. Among them, 150 (66.7%) were patients, and 75 (33.3%) were controls. Heterozygous tumour necrosis factor-alpha -308 guanine/adenine genotypes were significantly higher among the patients (p<0.05). Minor allele - 308 adenine showed an association with obstructive sleep apnoea, its severity, higher tumour necrosis factor-alpha levels, neck circumference, excessive daytime sleepiness and the presence of hypertension (p<0.05). Tumour necrosis factor-alpha -308 adenine allele and higher tumour necrosis factor-alpha levels were found to be linked with obstructive sleep apnoea. The polymorphism also showed an association with hypertension in obstructive sleep apnoea patients.

Identification of specific codon 201 mutation of the DCC Gene in the colonoscopic specimen of colorectal cancer.

To identify the mutation in codon 201 of the deleted in colorectal cancer gene in colorectal cancer, and to correlate that mutation to the histopathological grading of colorectal cancer. The cross-sectional study was conducted from February 2019 to February 2021 after approval from the ethics review board of the Dow University of Health Sciences, Karachi, and comprised biopsy-proven colorectal cancer patients regardless of age and gender. After histopathological reporting, formalin-fixed paraffin-embedded tissue blocks of colorectal cancer were used for deoxyribonucleic acid extraction, followed by polymerase chain reaction optimisation and deoxyribonucleic acid Sanger sequencing for mutational analysis. Data was analysed using SPSS 25. Of the 100 biopsy specimens assessed, 45(45%) were selected. Of them, 13(29%) samples failed to show any band on gel electrophoresis. The remaining 32(71%) samples were used for Sanger sequencing. Of these, 1(3%) sample did not sequence, while 31(97%) showed sequencing. All the sequenced samples identified a mutation in codon 201 of exon 3 in the deleted in colorectal cancer gene; 30(97%) showed homozygosity, and 1(3%) showed heterozygosity. No significant association of point mutation was noted with various demographic and clinicopathological parameters (p>0.05). The deleted in colorectal cancer gene's missense mutation in codon 201 was frequently observed in colorectal cancer patients.

Metagenomic analysis and biodiversity of bacteria in traditional fermented fish or Budu from West Sumatera, Indonesia.

This research aims to investigate the microbial diversity of Budu prepared from fresh and frozen fish from the Pariaman and Pasaman districts in West Sumatra Province, Indonesia, as well as provide basic information about Budu quality. To obtain the bacterial microbial composition, deoxyribonucleic acid extraction was carried out using amplicon-sequencing of the 16S-rRNA gene in the V3-V4 region from two types of Budu and carried out in duplicate. Budu prepared with fresh (Pariaman) or frozen (Pasaman) fish was dominated by Firmicutes (78.455%-92.37%) and Proteobacteria (6.477%-7.23%) phyla. The total microbial species in Budu from Pariaman were higher (227 species) than in Pasaman (153 species). The bacterial species found are Lentibacillus kimchi (1.878%-2.21%), Staphylococcus cohnii (0.597%-0.70%), Peptostreptococcus russeli (0.00%-0.002%), Clostridium disporicum (0.073%-0.09%), Clostridium novyi (0.00%-0.01%), Nioella sediminis (0.00%-0.001%), and Shewanella baltica (0.00%-0.003%). Lentibacillus kimchi, S. cohnii, and C. disporicum are found in both Budu. Nioella sediminis and S. baltica are found in Budu Pariaman. Peptostreptococcus russeli and C. novyi were found in Budu Pasaman. Metagenomic analysis of Budu from different fish, Pariaman (fresh fish) and Pasaman (frozen fish) showed that the biodiversity of bacteria was barely different. Both Budu found lactic acid bacteria from the Enterococcaceae family, genus Vagococcus, and pathogenic bacteria, such as S. cohnii, P. russeli, C. disporicum, and S. baltica. The discovery of various species of pathogenic bacteria indicates that development is still needed in the Budu production process to improve Budu quality.

Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk

Abstract. Fatmawati M, Suwanti LT, Mufasirin, Subekti DT, Ekawasti F, Lastuti NDR, Lamid M, Suprihati E, Effendi MH, Al-Arif A. 2023. Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk. Biodiversitas 24: 5905-5911. Dairy goats (Capra aegagrus hircus) are intermediate hosts that transmit Toxoplasma gondii to humans through goat milk consumption. T. gondii can invade mammary glands and secrete into milk. The method of detecting gene of the T. gondii in milk is through a Polymerase Chain Reaction (PCR) test based on specific genes. The objective of this study was to compare the efficacy methods of the T. gondii molecular assay from the extraction, master mix for PCR assay, and primer that is possible to amplify the gene of T. gondii in goat milk for PCR assay. The research method for purification used flotation using concentrated sugar. This research compared the extraction kit from Kit Extraction 1 (K1), Kit Extraction 2 (K2), and Kit Extraction 3 (K3) with the extraction method listed in the brochure. Meanwhile, for the PCR assay, three types of master mix were used, including Master Mix 1 (M1), Master Mix 2 (M2), and Master Mix 3 (M3). The specific genes used included primers B1, ROP, GRA 1, GRA 7, BAG 1, and P30. Tris borate EDTA (TBE) or Phosphate Buffer Saline Solution (PBS) as buffer milk samples gave the same PCR test results. The results show that the principle of a flotation method test using PBS can be used to prepare the milk sample. The research showed that extraction kit 1 (K1) and extraction kit 2 (K2) gave positive results for the positive control. However, in terms of price, K2 is cheaper than K1. So, to optimize DNA extraction, K2 can be used. The master mix that can be used for PCR testing of milk samples is M1 because M1 gives positive results for positive controls and negative results for negative controls. Primers for optimal results used GRA 7 and B1 with denaturation temperature of 94°C, annealing at 56°C, and an extension of 73°C 33. This research concluded that the type of DNA extraction kit affects the PCR test results for detecting T. gondii in goat milk. It is necessary to optimize the type of master mix to be used in PCR testing and primers capable of amplifying the T. gondii gene.

Integrated Three-Dimensional Microdevice with a Modified Surface for Enhanced DNA Separation from Biological Samples.

Functional interfaces and devices for rapid adsorption and immobilization of nucleic acids (NAs) are significant for relevant bioengineering applications. Herein, a microdevice with poly(acrylic acid) (PAA) photosensitive resin was integrated by three-dimensional (3D) printing, named DPAA for short. Precise microscale structures and abundant surface carboxyl functional groups were fabricated for fast and high-throughput deoxyribonucleic acid (DNA) separation. Surface modification was then done using polydopamine (PDA) and poly(ethylene glycol) (PEG) to obtain modified poly(acrylic acid) (PAA)-based devices DPDA-PAA and DPEG-PAA rich in amino and hydroxyl groups, respectively. The fabricated device DPAA possessed superior printing accuracy (40-50 μm). Functionalization of amino and hydroxyl was successful, and the modified devices DPDA-PAA and DPEG-PAA maintained a high thermal stability like DPAA. Surface potential analysis and molecular dynamics simulation indicated that the affinity for DNA was in the order of DPDA-PAA > DPEG-PAA > DPAA. Further DNA separation experiments confirmed the high throughput and high selectivity of DNA separation performance, consistent with the predicted affinity results. DPDA-PAA showed relatively the highest DNA extraction yield, while DPEG-PAA was the worst. An acidic binding system is more favorable for DNA separation and recovery. DPDA-PAA showed significantly better DNA extraction performance than DPAA in a weakly acidic environment (pH 5.0-7.0), and the average DNA yield of the first elution was 2.16 times that of DPAA. This work validates the possibility of modification on integrated 3D microdevices to improve their DNA separation efficiency effectively. It also provides a new direction for the rational design and functionalization of bioengineering separators based on nonmagnetic methods. It may pave a new path for the highly efficient polymerase chain reaction diagnosis.

The quality and quantity of compounds affected by viral inactivation methods in dried blood spots

Abstract Objectives The aim is to evaluate the effect of viral inactivation methods on the quality and quantity of compounds in dried blood spots (DBS). Methods Three effective and common inactivation methods were selected via the literature search, including: heating at 56 °C for 30 min, irradiation with UVC for 30 min, and surface wetting with 70 % ethanol. The concentration and clinical predicting significance of hormones, amino acids, and acylcarnitines from DBS were assessed, and the quality and quantity of extracted deoxyribonucleic acid (DNA) from DBS were evaluated. Results Compared to control, we found that there was no significant difference on hormones concentration in the DBS treated by heating at 56 °C for 30 min (thyroid stimulating hormone p=0.36, 17-hydroxyprogesterone p=0.52). And heating at 56 °C for 30 min had a minimal changed coefficient of variation on the concentration of amino acids and acylcarnitines. All three inactivation methods slightly changed the yield of DNA extraction, but did not affect the quality of the DNA. Importantly, the three inactivation methods wouldn’t change the clinical predicting significance of above-compounds mostly, especially heating at 56 °C for 30 min. Conclusions Considering the minimal effect on the quality and quantity of various compounds, the contaminated DBS could be pretreated by the three inactivation methods, as temporary emergency inactivation methods, especially heating at 56 °C for 30 min.

Cytochrome P450 2B6 6 distribution among patients living with HIV in Burkina Faso

The function of cytochrome P450 (CYP) enzymes, one of the major catalysts in drug metabolism, is significantly influenced by genetic polymorphisms leading to substantial inter-individual variability in drug response and/ or adverse reactions activity. The aim of this study was to determine the frequency of the CYP2B6 516G&gt;T allele in patients living with HIV receiving treatment at the Medical Center of Camp General Aboubacar Sangoule Lamizana for whom the treatment was changed after the initiation of treatment because of the appearance of side effects. 155 HIV-positive patients were enrolled. Genomic Deoxyribonucleic acid (DNA) extraction from blood was performed using the GenJET® Genomic DNA according to the manufacturer’s instructions. Genotyping for the cytochrome P450 variant alleles was performed using predesigned primers. The amplification was carried out by Polymerase Chain Reaction (PCR), while the differentiation between the alleles was carried out after digestion with restriction enzymes by electrophoresis. The results obtained showed. Among these patients, 61 changed treatments during their follow-up. The majority of the 61 patients, 32 patients, received an initial treatment regimen based on the AZT (3'-Azido-3'-Deoxythymidine)/3TC (Lamivudine)/ NVP(Nevirapine) combination, followed by 18 patients for the TDF (tenofovir)/3TC (lamivudine)/ EFV(Efavirenz) regimens. Currently, the majority of patients, 48 patients, have benefited from a therapeutic regimen based on the TDF (tenofovir)/3TC (lamivudine) /DTG(Dolutegravir) combination, followed by 11 patients for the TDF (tenofovir)/3TC (lamivudine)/EFV(Efavirenz) regimens. The distribution of the major allele CYP2B6*T was 68%, and the minor CYP2B6*G allele was 32%. Heterozygote (GT genotype) and Homozygous mutants (TT genotype) and Heterozygote were 31% and 52,5%. Studies could be continued to set up cytochrome mutation detection kits to anticipate the onset of side effects and adapt treatments.

Comparison of Purity and Concentration Values of Mycobacterium tuberculosis DNA Extraction Result from the Boiling and Spin Column Method

The Polymerase Chain Reaction (PCR) technique is extensively employed in molecular biology to precisely detect Mycobacterium tuberculosis. Prior to conducting PCR, extracting of high-quality genomic Deoxyribonucleic Acid (DNA) is crucial to ensure accurate and reliable results. The primary objective of this study is to conduct a comparative analysis of the purity and concentration of M. tuberculosis DNA acquired through the utilization of the boiling method and the spin column extraction methods. A descriptive comparative research design was employed, utilizing a sample of 16 sputum specimens that had previously been confirmed as positive for M. tuberculosis through Acid-Fast Bacteria (AFB) examination and Molecular Rapid Test (MRT). The extraction of DNA was carried out using the boiling method and the spin column method. Subsequently, the concentration and purity of the extracted DNA were assessed using the NanoDrop Spectrophotometer, and the results were compared. The obtained yield of M. tuberculosis DNA isolates through the boiling method ranged from 9.6 ng/µL to 1258.7 ng/µL, with an average purity value of 1.23. Conversely, for the spin column method, the concentration of M. tuberculosis DNA isolates ranged from 8.7 ng/µL to 207.8 ng/µL, with an average purity value of 1.83. In conclusion, there is a significant difference between the purity and concentration of M. tuberculosis DNA extraction results using the boiling method and spin column methods.

Virulence and Molecular Detection of Cassava Mosaic and Brown Streak Virus Genes on Elite Mutant Cassava Lines

Unavailability of germplasm with Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD) tolerance genes remains the main bottleneck towards management of the two diseases in tropic and sub-tropic regions in Africa. We assessed the pathogenicity of two viruses and established the presence of genes linked to CBSD and CMD among some elite cassava lines using field techniques and PCR methods. The screen-house experiment was arranged in split plot in completely randomized block design replicated thrice. Severity data was done on a scale of 1 - 5 at an interval of 14 days post inoculation. Symptomatic fresh leaves were used in the extraction of genomic deoxyribonucleic acid and amplification done using markers linked to African cassava mosaic virus (ACMV), separated bands scored as present (+) or absent (-) and visualized under UV trans-illuminator. Virulence and pathogenicity results showed higher tolerance to CBSD and CMD under single inoculation. In contrast, dual inoculation synergistically increased severity expression leading defoliation at early stages of growth. DNA amplification found no CBSD and CMD alleles in CAS1. However, some traces of the alleles were amplified on CAS2, CAS3, KME3 and KME4. Mutant cassava lines exhibited low severity compared to parental lines which were genetically different in terms of viral genes detected by the ACMV linked markers.

Usefulness of truenat-derived DNA from extrapulmonary specimens in direct detection of drug resistant tuberculosis by line probe assay

Objectives: Isoniazid (INH) and second-line drug resistance (DR) detection through line probe assay (LPA) takes long in extrapulmonary (EP) specimens because culture growth needs to be obtained to perform deoxyribonucleic acid (DNA) extraction due to the paucibacillary nature of these specimens. Knowing the DR pattern at the earliest is key to success of the treatment. Delay in appropriate tuberculosis (TB) treatment in EP TB patients runs the risk of DR amplification, significant disease damage, and patient loss to follow-up. Here, LPA was attempted on truenat-derived DNA elute from EP specimens, which, in routine, is discarded after the truenat test, to determine drug sensitivity test (DST) for INH and, where necessary, for second-line drugs (Fluoroquinolones, Kanamycin, amikacin, and capreomycin). Material and Methods: Truenat, acid-fast bacilli culture, and fluorescent microscopy were performed on all EP samples that were received at the laboratory during June–September 2022. DNA elute that was left over from 59 truenat Mycobacterium tuberculosis (MTB) positive EP samples were subjected to Genotype MTBDR plus Ver 2.0 assay. Results: MTBDR plus assay (DNA elute) detected MTB and rifampicin (RIF) and INH DST in 47 samples (79.6%) having truenat MTB count of 7.8 × 102 colony-forming unit/milliliter and above. It also detected RIF DST in 65.2% truenat RIF indeterminate samples and DST for both RIF and INH in 60% of culture negative EP specimens. DST results by LPA (DNA elute) completely concorded with standard indirect LPA (on 21 culture isolates from smear-negative specimens). The MTBDRsl yield was however relatively low (11.1%), although second line LPA (SLLPA) was performed only on 9 first-line DR samples. Conclusions: Left-over truenat-derived DNA elute is a significant sample by-product that can significantly speed up and increase the yield of determination of MTB DST in EP samples for RIF and INH, the most critical drugs for TB treatment.

Clinical Significance of Pathogenicity of Somatic Mutations in Oral Leukoplakia: a Prospective Observational Study

Background. The vast majority of malignant neoplasms of the oral mucosa refer to squamous cell carcinomas. The development of squamous cell carcinoma of the oral mucosa is often promoted by previous potentially malignant diseases, with oral leukoplakia dominating among them.Objective. To determine the clinical significance of the pathogenicity of somatic mutations in oral mucosal leukoplakia.Methods. The study material included 24 samples of abnormal epithelium of the oral mucosa from leukoplakia patients. QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) was used for deoxyribonucleic acid (DNA) extraction from the samples. DNA sequencing was performed using IlluminaNextSeq 550 sequencer and TruSight™ Oncology 500 DNA Kit For Use with NextSeq (Illumina, USA). All DNA extractions from biological samples, preparation and sequencing of DNA libraries were performed step-by-step in strict accordance with the guidelines provided with the respective reagent kits. Bioinformatics analysis was carried out using specific software Illumina Base Space (Illumina, USA) and Galaxy Project (The Galaxy Community, a non-profit international project) according to current guidelines. The desired power of the study accounted for 90%. Two Proportions Z test was performed by means of The Sample Size Calculation of Statistica 12 (StatSoft, Inc.) with the set option “one-tailed hypothesis”, because it was initially assumed that pathogenic (oncogenic) genetic variants occur in the tissue of oral leukoplakia much more frequently than in the human reference genome used for sequence alignment.Results. The pathogenic somatic mutations in the TP53, KRAS, APC, NRAs and BRAF genes, identified in this study, alone or in combination, are highly likely (hazard ratio 3000-11000) to be associated with the development of oral mucosal leukoplakia and low-grade epithelial dysplasia. The multiplicity of pathogenic and likely pathogenic genetic variants associated with epithelial dysplasia, as well as the fact that a number of variants do not occur in all patients, suggests that the same histotype of oral mucosal dysplasia may develop under the influence of different mutations.Conclusion. The pathogenic and likely pathogenic variants of the TP53, KRAS, APC, NRAS and BRAF genes, identified in this study, alone or in combination, are highly likely (hazard ratio 3000–11000) to be associated with the development of leukoplakia and low-grade epithelial dysplasia.

СОМАТИЧЕСКИЕ МУТАЦИИ ПРИ ЛЕЙКОПЛАКИИ СЛИЗИСТОЙ ОБОЛОЧКИ РОТОВОЙ ПОЛОСТИ

Introduction. Genetic basis for the development of leukoplakia of the oral mucosa is not well understood. Early prediction of oral mucosa cancer development is an important public health problem, which results in the importance of studying the pathogenesis of precancerous diseases. Malignant neoplasms of oral mucosa are mainly the cases of squamous cell carcinoma which commonly develops as the outcome of previous potentially malignant diseases, the leading of which is oral mucosa leukoplakia. Aim. The goal of study is to determine pathogenic somatic mutations in patients with oral mucosa leukoplakia and grade 1 epithelial dysplasia. Material and methods. We have studied 24 samples of altered epithelium of patients with oral mucosa leukoplakia. The QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) was used to isolate deoxyribonucleic acid from the samples. Sequencing was performed with Illumina NextSeq 550 sequencer and TruSight™ Oncology 500 DNA Kit (Illumina, USA). All operations for deoxyribonucleic acid extraction, sampling and sequencing were performed step by step in strict accordance with the instructions supplied with the reagent kits. Bioinformatic analysis was performed using Illumina BaseSpace and Galaxy Project software in accordance with current guidelines. Results and discussion. Pathogenic and probably pathogenic somatic mutations identified during this study in TP53, KRAS, APC, NRAS and BRAF genes, both singly and in combination, are reliably (risk ratios 3000-11000) associated with the development of leukoplakia of the oral mucosa with epithelial dysplasia of 1st degree. The multiplicity of genetic variants associated with epithelial dysplasia, as well as the fact that a number of variants do not occur in all patients simultaneously, suggests that the same histotype of oral mucosa dysplasia may develop under the influence of various mutations. Conclusion. Pathogenic and probably pathogenic variants of TP53, KRAS, APC, NRAS and BRAF genes, both singly and in combinations, are reliably associated with the development of leukoplakia of the oral mucosa with epithelial dysplasia of 1st degree (risk ratios 3000-11000).

Washing solution of tissue biopsy compared with paraffin-embedded tissue for NSCLC gene test.

e21104 Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death, whose targeted therapy is precisely based on gene type. Gene diagnosis is as important as pathological diagnosis and driver gene mutations should be detected as soon as possible. There are generally a few sources to obtain patients’ deoxyribonucleic acid (DNA), such as paraffin tissue of tumor, cytological specimens from bronchoalveolar fluid or pleural effusion, free DNA from blood, hydrothorax, ascite, or cerebrospinal fluid. Our research aims to find out whether washing solution of lung mass biopsy in double distilled water is feasible for detecting gene mutation, and whether its gene type is consistent with that from Next Generation Sequencing (NGS) of paraffin tissue. Methods: 25 clinically diagnosed NSCLC patients from Department of Oncology, Second Xiangya Hospital, Central South University were collected. We used double distilled water to wash the CT-guided percutaneous pulmonary mass biopsy, which was processed to extract DNA. DNA samples were tested for epidermal growth factor receptor (EGFR) gene mutation by amplification refractory mutation system (ARMS)-PCR. 6 of them as well as all 25 paraffin tissue samples were tested for large panel gene mutations by NGS. The gene type consistency between washing solution and paraffin tissue was analyzed by GraphPad Prism V9.4.1. Results: 16 of 25 washing solution DNA samples tested by ARMS-PCR were EGFR mutation positive, including 9 cases of EGFR exon19 deletion, 5 cases of EGFR exon21 L858R, and 2 cases of EGFR exon21 L858R and T790M. Other 9 of 25 samples were EGFR mutation negative. 17 of 25 paraffin tissue samples tested by NGS were EGFR mutation positive, including 9 cases of EGFR exon19 deletion, 7 cases of EGFR exon21 L858R, and 1 case of EGFR exon21 L858R with T790M. There was one washing solution sample showed no EGFR mutation while its tissue sample showed EGFR exon21 L858R. Compared to NGS on paraffin tissue, the sensibility of ARMS-PCR on washing solution was 94.1%. The specificity was 100%, positive predictive value was 100%, negative predictive value was 88.9%, accuracy rate was 96.0%, misdiagnosis rate was 0, and omission diagnostic rate was 5.9%. EGFR types of 6 washing solution samples tested by NGS were exactly the same as that by ARMS-PCR and that of paraffin tissue samples by NGS. The time to obtain gene result was significantly different. ARMS-PCR of washing solution took 1 day, NGS of washing solution took around 7 days, while pathological examination and NGS of paraffin-embedded tissue took around 14 days. Conclusions: The washing solution of CT-guided percutaneous lung mass biopsy in double distilled water is feasible to obtain EGFR type rapidly and efficiently for NSCLC. It is the fastest method to get patients’ gene type at present. Thus, the washing solution of biopsy could be a new DNA source for NSCLC gene test and making decision on targeted therapy ahead of time.

Genetic analysis for mucinous ovarian carcinoma with infiltrative and expansile invasion and mucinous borderline tumor: a retrospective analysis

BackgroundMucinous carcinoma (MC) is a histological subtype of ovarian cancer that has a worse prognosis at advanced stages than the most prevalent histological subtype, high-grade serous carcinomas. Invasive patterns have been recognized as prognostic factors for MCs. MCs with infiltrative invasion were more aggressive than those with expansile invasion. MC with an expansile pattern exhibited behavior similar to mucinous borderline tumors (MBT). However, genomic analysis of invasive patterns is insufficient. This study aimed to compare genetic information between groups with MC and infiltrative invasion (Group A) and those with MC with expansile invasion or MBT (Group B).MethodsTen cases each of MC with infiltrative invasion, MC with expansile invasion, and MBT between 2005 and 2020 were identified. Deoxyribonucleic acid (DNA) extraction from formalin-fixed paraffin-embedded tissues was performed, and cases with DNA fragmentation or the possibility of DNA fragmentation were excluded. Mutant base candidates and tumor mutation burden (TMB) values (mutations/megabase) were calculated.ResultsAfter assessing the quality of purified DNA, seven cases of MC with infiltrative invasion, five cases of MC with expansile invasion, and three cases of MBT were included. More patients in group A experienced recurrence or progression (p < 0.01) and died of disease (p = 0.03). Moreover, the TMB value was statistically higher in group A than in group B (p = 0.049). There were no statistical differences in the incidence of the mutations of KRAS, TP53, and CREBBP. KRAS, TP53, and CREBBP mutations were discovered in 8/15 (53.3%), 6/15 (40.0%), and 5/15 (33.3%) cases, respectively.ConclusionsGenetic analysis revealed that Group A had higher TMB than Group B. Therefore, this result might be useful for future treatment.

Lateral flow biosensor based on LAMP-CRISPR/Cas12a for sensitive and visualized detection of Salmonella spp.

Salmonella is a major pathogen that causes serious foodborne diseases in humans and poses a serious threat to food safety and public health worldwide. Its rapid and accurate diagnosis is essential to prevent bacterial contamination of food. This study aimed to develop a lateral flow biosensor (LFB) based on loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 12a (Cas12a) that is affordable and can aid in visually detecting Salmonella in a food sample. False positives were prevented by synthesizing CRISPR ribonucleic acid (crRNA) with a selected 21-base pair protospacer that did not overlap with the used LAMP primers. A single-stranded deoxyribonucleic acid (DNA) probe labeled with biotin and 6-Carboxyfluorescein (6-FAM) was used for designing the LFB. The duration of these processes was as follows: DNA extraction (20 min), LAMP (60 min), Cas12a cleavage (5 min), and LFB (5 min). The detection limit was 1.22 × 10° CFU/mL in pure culture. The validity of the LAMP-CRISPR/Cas12a based LFB was verified by detecting Salmonella in onions, melon, and salami. Consequently, the developed LAMP-CRISPR/Cas12a based LFB assay show the potential for rapid on-site detection of Salmonella with high specificity, sensitivity, convenience, mobility, low cost, and affordability. • LFB based on LAMP-CRISPR/Cas12a was developed for detection of Salmonella. • The developed method demonstrated suitability for naked-eye detection of Salmonella. • The detection can be completed within 100 min for on-site detection.