Mejoramiento de un método no comercial para la extracción de ADN de moscas de interés médico-legal

La obtención de ADN de moscas de interés médico-legal es de relevancia para una variedad de aplicaciones. Aunque existen métodos de extracción comerciales de ADN, su uso rutinario es limitado, en algunos escenarios. En este contexto, el uso de métodos no comerciales constituye una alternativa; sin embargo, su optimización es clave para mejorar el flujo de trabajo y los resultados. Este trabajo evaluó el impacto de variaciones a un método de precipitación salina sobre la concentración y la pureza del ADN recuperado. No se encontraron diferencias significativas en la concentración de ADN extraído entre los diferentes tiempos de incubación, probados durante la fase de extracción, mientras que el incremento en el volumen de etanol absoluto, en la fase de precipitación de ADN, mejoró significativamente la concentración de ADN obtenido. Las modificaciones propuestas reducen el tiempo de ejecución y la concentración de ADN obtenido comparado con el protocolo original.

Obtaining DNA from flies of medico-legal interest is relevant for a variety of applications. Although commercial extraction methods offer optimal DNA, their routine use is limited in some settings. In this context, the use of non-commercial methods constitutes an alternative in laboratories with limited resources however, its optimization is key to improving the workflow and the results. This work evaluated the impact of variations to a saline precipitation method on the concentration and purity of the recovered DNA. No significant differences were found in the concentration of extracted DNA between the different incubation times tested during the extraction phase. In contrast, the increased volume of absolute ethanol in the DNA precipitation phase significantly improved the concentration of DNA obtained. The proposed modifications reduce the runtime and DNA concentration obtained compared with the original protocol.

Species identification of Ligustrum lucidum / 中国中药杂志

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom / 法医学杂志

OBJECTIVES@#To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.@*METHODS@#The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.@*RESULTS@#When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05).@*CONCLUSIONS@#The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index

ABSTRACT Introduction: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. Methods: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. Results: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. Conclusions: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications in neonatal screening and diagnostic confirmation of lysosomal disorders

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since their introduction in the neonatal screening of phenylketonuria almost 50 years ago. The range of its application has been extended to modern approaches, such as next-generation sequencing (NGS) for molecular genetic testing. This study aimed to evaluate the use of a standardized organic method for DNA extraction from DBS samples in the diagnostic setting.Methods: The clinical applicability of the method was tested using 3 samples collected from a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from 3 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and method performance was assessed by standard polymerase chain reaction, restriction length polymorphism, Sanger sequencing, and targeted NGS.Results: Results were compared with the ones obtained from DNA samples extracted following the internally validated in-house extraction protocol that used 6 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to having a lower cost per sample

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Research Progress on Touch DNA on Cartridge Cases in Forensic Field / 法医学杂志

Cartridge cases are crucial physical evidence in gun-related crimes. The successful identification of the touch DNA on cartridge cases can help to screen the suspects and reconstruct the gun-related crime scene. With the improvement of DNA extraction methods and the sensitivity of amplification kit, forensic examiners are expected to obtain more valuable information by testing the touch DNA on cartridge cases. In practical cases, the touch DNA detection on cartridge cases often encounters with low DNA content degradation, mixing and the gunshot residual interference, which brings more challenges to DNA examination and identification. This article reviews forensic research of touch DNA on the cartridge cases from the aspects of factors affecting touch DNA on cartridge cases, advances in the extraction and amplification methods, and the practical applications in order to provide reference for forensic identification of touch DNA on the cartridge cases in real cases.

Application of Automatic Nucleic Acid Extractor Combined with Vacuum Concentrator in Forensic Science / 法医学杂志

Objective To explore the application value of automatic nucleic acid extractor combined with vacuum concentrator in forensic DNA extraction. Methods Gradient samples of human peripheral venous blood were collected at 40, 80, 120, 160, 200, 240, 280 and 320 fold dilution. The samples of each gradient were treated with no inhibitor, black oil, rust, fruit acid, tin foil and indigo, respectively. The automatic nucleic acid extractor was used for DNA purification and extraction of the above samples. The extracted DNA eluent （6 μL） was taken for amplification directly, and the rest was concentrated by vacuum concentrator. DNA was amplified and examined using the Investigator 26plex QS kit before and after concentration. Results Only gradient samples treated with fruit acid obtained complete STR typing results at 40 fold dilution. The other 5 methods obtained complete STR typing results at 40-160 fold dilution. The results of STR typing after DNA concentration showed that the average peak height and detection rates of gene loci both increased to a certain extent, but the effect was not obvious. Conclusion The automatic nucleic acid extractor has an efficient inhibitor removal ability and high extracting efficiency of DNA. The vacuum concentrator can concentrate DNA samples to a certain extent. Combining the automatic nucleic acid extractor with the vacuum concentrator can improve the examination success rate of forensic materials.

Molecular Detection of Medically Important Candida species from Droppings of Pigeons (Columbiformes) and Captive Birds (Passeriformes and Psittaciformes)

Abstract Passeriformes and Psittaciformes birds and pigeons (Columba livia) are known to be reservoirs of microorganisms, and their stool allows fungi development. Since accumulated avian excreta can interfere with public health, this study aimed to perform a molecular screening of medically important Candida species in pigeon droppings in public places and birds raised in captivity. Excreta collected from captive birds (3 residences) and pigeons (4 districts) were inoculated on Sabouraud dextrose agar with chloramphenicol for Gram staining and subculture on Hicrome® Candida. Three DNA extraction methods were performed for comparison (commercial kit, in-house and by boiling) and PCR to screen 6 clinically important Candida species among the isolates. The correlation between phenotypic and molecular methods was calculated by kappa/K. Only 6 C. parapsilosis (20%) were identified from captive birds' feces among 30 isolates (80% not identified), while pigeons' feces harbored a greater diversity, with the 6 pathogenic species confirmed among 41 isolates: C. albicans (31.70%/13), C. krusei (14.63%/6), C. tropicalis (14.63%/6), C. parapsilosis (17.10%/7), C. glabrata (14.63%/6) and C. guilliermondii (7.31%/3); 100% correlation between tested methods (K = 1) for the first 3 species. Boiling DNA extraction method was fast and efficient to obtain viable DNA from Candida spp. for PCR. Our results indicate that pigeon droppings harbor more potentially pathogenic species than birds in residential captivity, which probably have non-albicans Candida less frequently isolated in infectious processes. The greater availability of nutrients may have contributed to a diversity of Candida spp. in feces from public environments.

Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

DNA extraction methods for molecular detection of Eimeria spp. in cattle and sheep / Métodos de extração de DNA para detecção molecular de Eimeria spp. em bovinos e ovinos

Molecular detection of Eimeria species in fecal samples can be useful for experimental and diagnostic purposes. However, the parasite quantity presence in feces and the oocyst wall are an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the oocysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of six protocols for DNA extraction from Eimeria spp. present in bovine and sheep. Twenty pools of fecal samples from cattle (10 pools) and sheep (10 pools) were distributed to six DNA extraction protocols: commercial kit, commercial kit with modification, DNAzol, cetyl-trimethyl ammonium bromide (CTAB), glass beads and commercial kit for fecal samples. Fecal samples were submitted to DNA extraction and PCR. Among the protocols tested, CTAB was determined to be most suitable for DNA extraction from oocysts (90% of DNA detection by PCR); DNAzol and CTAB resulted in higher DNA detection from bovine samples (80%). CTAB and commercial kit with modification improved PCR detection of Eimeria spp. in sheep samples, with positive amplification of DNA in all tested samples.(AU)

A detecção molecular de espécies de Eimeria em amostras fecais pode ser útil para fins experimentais e de diagnóstico. No entanto, a quantidade de parasitas nas fezes e a parede do oocisto são um obstáculo nos protocolos de extração de DNA. Portanto, uma amostragem adequada e a ruptura efetiva dos oocistos são essenciais para melhorar a precisão da detecção de DNA por PCR. Os objetivos deste estudo foram avaliar seis protocolos para extração de DNA de Eimeria spp. em amostras de bovinos e ovinos. Foram distribuídos 20 grupos de amostras fecais de bovinos (10 grupos) e ovinos (10 grupos) em seis protocolos de extração de DNA: kit comercial, kit comercial com modificação, DNAzol, brometo de cetil-trimetil amônio (CTAB), pérolas de vidro e kit comercial para amostras fecais. As amostras fecais foram submetidas à extração de DNA e PCR. Entre os protocolos testados, CTAB foi considerado o mais adequado para extração de DNA de oocistos (90% de detecção de DNA por PCR); DNAzol e CTAB resultaram em maior detecção de DNA em amostras de bovinos (80%). CTAB e kit comercial com modificação melhoraram a detecção por PCR de Eimeria spp. em amostras de ovinos, amplificação positiva de DNA em todas as amostras testadas.(AU)

Application of Two Kinds of Automatic Nucleic Acid Purifiers / 法医学杂志

Objective To investigate the effect of automatic nucleic acid purifiers QIAsymphony SP and QIAcube in the DNA extraction of samples of trace amount or mixed with inhibitors. Methods Different kinds of purification methods using QIAsymphony SP and QIAcube were applied to extract swabs which contained 30, 100, 150 and 300 cells and other samples which contained six types of inhibitors-heme, humic acid, lard, soil, rust and grease. PCR amplification and STR typing were performed on the extracted DNA templates to compare extracting efficiency and inhibitor removal ability of four different purification methods. Results Different purification methods showed similar extraction effects, 70.83%-100.00% of loci could be detected by amplification of DNA extracted from samples with 30, 100 and 300 cells, and the six types of inhibitors could be removed well. Conclusion The two automatic nucleic acid purifiers have a good inhibitor removal effect. For swabs with only 30 cells, after DNA extraction and amplification, the locus detection rate of samples can still be high, which can meet the requirements of local DNA laboratory work, and realize the standardization construction of the laboratory.

Integrando Universidade e Escola: Relato de experiência da Oficina "Brincando de Geneticista: descobrindo o DNA" / Integrating University and school: Experience report of the Workshop "Playing Geneticist: discovering DNA" / Integración de la universidad y la escuela: informe de experiencia del taller "Jugando Genetista: descubriendo el ADN"

Objetivo: relatar uma experiência didática envolvendo diálogo entre a Universidade e a Escola Pública, por meio da aplicação da oficina pedagógica denominada "Brincando de Geneticista: descobrindo o DNA". Método: A oficina direcionada para alunos da Educação Básica foi realizada na Universidade Estadual de Feira de Santana, durante a Semana Nacional de Ciência e Tecnologia do ano de 2019. A atividade pedagógica foi fundamentada na extração de material genético. Foram utilizados materiais de baixo custo e fácil acesso. Resultados: 80 estudantes Ensino Fundamental II participaram da oficina. Os alunos foram divididos em pequenos grupos, possibilitando maior interação entre estudantes e professor. A oficina de caráter participativo possibilitou que os estudantes pudessem compreender a importância da genética para a vida, além de estimular a interação dos escolares com a Universidade. Ademais, foi observado que muitos escolares apresentam dificuldades para transpor e contextualizar assuntos relacionados a conteúdos celulares e moleculares. Conclusão: No presente trabalho, o uso dessa abordagem didática ampliou a receptividade dos alunos aos conteúdos trabalhados, facilitou o diálogo aluno-professor e se mostrou uma ótima ferramenta de interface entre universidade e escola básica.

Objective: to report a didactic experience involving dialogue between the University and the Public School, through the application of the pedagogical workshop called "Playing Geneticist: discovering the DNA". Method: The workshop directed to students of Basic Education was held at the State University of Feira de Santana, during the National Week of Science and Technology of the year 2019. The pedagogical activity was based on the extraction of genetic material. Low-cost and easy-to-access materials were used. Results: 80 elementary school students participated in the workshop. The students were divided into small groups (12 to 15 students), allowing greater interaction between students and the teacher. The participatory workshop enabled students to understand the importance of genetics for life, in addition to stimulating the interaction of students with the University. Furthermore, it was observed that many students have difficulties in transposing and contextualizing issues related to cellular and molecular content. Conclusion: In the present work, the use of this didactic approach increased the students' receptivity to the contents worked on, facilitated the student-teacher dialogue and proved to be a great interface tool between university and basic school.

Objetivo: reportar una experiencia didáctica que involucra el diálogo entre la Universidad y la Escuela Pública, a través de la aplicación del taller pedagógico llamado "Jugando Genetista: descubriendo el ADN". Método: El taller dirigido a estudiantes de Educación Básica se realizó en la Universidad Estatal de Feira de Santana, durante la Semana Nacional de Ciencia y Tecnología del año 2019. La actividad pedagógica se basó en la extracción de material genético. Se utilizaron materiales de bajo costo y de fácil acceso. Resultados: 80 estudiantes de primaria participaron en el taller. Los alumnos se dividieron en pequeños grupos (de 12 a 15 alumnos), lo que permitió una mayor interacción entre los alumnos y el profesor. El taller participativo permitió a los estudiantes comprender la importancia de la genética para la vida, además de estimular la interacción de los estudiantes con la Universidad. Además, se observó que muchos estudiantes tienen dificultades para transponer y contextualizar cuestiones relacionadas con el contenido celular y molecular. Conclusión: En el presente trabajo, el uso de este enfoque didáctico aumentó la receptividad de los estudiantes a los contenidos trabajados, facilitó el diálogo estudiante-maestro y demostró ser una gran herramienta de interfaz entre la universidad y la escuela básica.

Comparación de métodos de extracción de ADN simples y económicos para el diagnóstico molecular de leptospirosis animal

La leptospirosis bovina es una importante enfermedad zoonótica cuyo diagnóstico molecular está ampliamente divulgado. Sin embargo, no existe un método único de extracción de ADN para leptospiras patógenas a partir de muestras clínicas. En este trabajo se utilizó orina bovina contaminada con cultivo de L. interrogans serovar Pomona Pomona para analizar el mejor método comparando: M1.) resina Chelex-100, M2.) papel FTA Whatman y M3.) hervido de la muestra (protocolo casero). De estas tres técnicas, la primera (M1) presentó la mayor sensibilidad al realizar la PCR de diagnóstico, detectándose hasta 2x102 leptospiras/mL. La metodología aquí planteada resultó tener buen rendimiento para la detección de leptospiras en muestras clínicas animales, aunque es necesario su validación con mayor número y diversidad de muestras.

Bovine leptospirosis is an important zoonotic disease whose molecular diagnosis is widely reported. However, there is not a unique method of extraction of DNA for pathogenic leptospires using clinical samples. In this study, bovine urine was contaminated with pure culture of L. interrogans serovar Pomona Pomona in order to compare three of them: M1.) Chelex-100 resin, M2.) FTA Whatman paper and M3.) boiling of the sample (in-house protocol), being the first one the most sensitive when used in diagnostic PCR, detecting up to 2x102 leptospiras/mL. The methodology proposed in this study turned out to have good performance for the detection of leptospires in animal clinical samples, although it should be applied to a greater number of samples and in different stages of the pathology.

A robust method for nucleic acid extraction from fabrics to study bacterial diversity

The importance of studying microbial load on fabrics has been recently realized with reports on fabrics being a source ofspread of infection in medical and hospitality sectors. However, methodological limitations have restricted the analysis ofmicrobial diversity on fabrics. Hence, the study aimed to develop a robust method for extraction of DNA from differenttypes of fabrics. Bacterial community profiles could be successfully generated with DNA extracted from real life samples,together with identification of different bacterial genera on fabrics. The study opens up venues to study effect of environmental factors on microbial load on fabrics. Also, such a technique will aid correlation between microbial load and typesof fabric so as to come up with recommendation for fabrics bearing minimal microbial load for medical and hospitalitysectors.

A crude method of DNA extraction and identification from exfoliated human buccal mucosa cells

Background: DNA analysis has a key role in forensic dentistry. However, techniques of DNA extraction and analysis are far from the reach of majority of medical professionals owing to its expensive set up. Aim: The present study was aimed at formulating a crude method of extracting DNA from human buccal mucosa cells using materials commonly available in the laboratory so that the medical professionals could get more exposure to molecular biology techniques. The objectives were to identify the DNA and to assess its purity. Methods: Buccal mucosa cells from 10 healthy volunteers were taken for DNA extraction following the protocol of cell lysis, purification, and precipitation. DNA was identified using standardized techniques like Diphenylamine test and its purity was assessed using a spectrophotometer. A gel electrophoresis apparatus was also constructed using readily available materials. Results: DNA was extracted from human buccal mucosa cells using a crude method. The standardized tests confirmed the presence of DNA contaminated with proteins. The locally made Gel electrophoresis model exhibited a faint halo around the wells instead of DNA bands. Conclusion: DNA extraction from human buccal mucosa cells was made possible using locally available materials and a crude method, but it was not of high purity.

Urine as a promising sample for Leishmania DNA extraction in the diagnosis of visceral leishmaniasis - a review

ABSTRACT Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.

Development of an Extraction Protocol for Fetal Sexing from the Fourth Week of Gestation by Real Time PCR

Abstract Low number of fetal cells in maternal blood limited the use of fetal materials in diagnostic and clinical applications. This research developed a technology which allowed the extraction of fetal DNA by a non-invasive method that offers no risk to the mother or fetus. A total of 132 pregnant women participated in this inquiry. The DNA extraction was performed employing an in-house method based on guanidine thiocyanate and magnetic bead. For the amplification it was used the Quantifier Y Kit™. The fetal sexing analysis of the 132 pregnant women were 100% in agreement with the ultrasound. Sensitivity and specificity for detection of Y chromosome sequences was possible using Real-Time Polymerase Chain Reaction since the 4th week of gestation. This non-invasive early determination could be employed in fetal gender and also to be extended to detection of genetic diseases in the shortest possible time avoiding invasive methods that puts the fetus at risk.

development and application of dna extraction methods, pcr parameter optimization and rapid detection reagent for fetus cervi / 中国药学杂志

OBJECTIVE: To optimize DNA extraction methods and PCR reaction parameters, and develop an excellent and accurate rapid detection reagent for Fetus Cervi. METHODS: The DNA of Fetus Cervi was extracted by the modified salting method, modified SDS method A and modified SDS method B. Four DNA polymerase were chosen from the market and compared with each other. The annealing temperature and annealing time were optimized by classical control variable method and intersected experiment. A rapid detection reagent of Fetus Cervi was developed and then evaluated. RESULTS: The A260 /A280 ratio of the DNA extracted by modified SDS method B was (1.74 ± 0.05), and the mass concentration was (0.250 ± 0.005) μg•L -1. With high fidelity Taq DNA polymerase, the PCR product concentration could reach (0.185 ± 0.005) μg•L-1. Through these experiments, the annealing temperature was set at 58 ℃ and the annealing time was 30 s. The rapid detection reagent course was established to quickly and accurately identify Fetus Cervi and their artefacts, with one clear and bright band at 563 bp. CONCLUSION: The rapid detection reagent of Fetus Cervi combines the optimal DNA extraction method and the optimal PCR reaction parameters, and can accomplish the identification of Fetus Cervi and its pseudo-products with high accuracy and specificity.