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A comparison of the efficiency of five
different commercial DNA
extractionkits for extraction of DNA from faecal
samplesby
Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and
Mark P. Nicol .
A comparison of the efficiency of five
different commercial DNA
extractionkits for extraction of DNA from faecal
samplesby
Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and
Mark P. Nicol .
BY
MARIA CAROLINA RAVE AGUIRRE
MARIA ISABEL PALACIO
MOLECULAR BIOLOGY
MEDICINE STUDENTS 3nd SEMESTER
TEACHER LINA MARIA MARTíNEZ
SÁNCHEZ
AUGUST 23, 2013
MEDELLÍN - COLOMBIA
BY
MARIA CAROLINA RAVE AGUIRRE
MARIA ISABEL PALACIO
MOLECULAR BIOLOGY
MEDICINE STUDENTS 3nd SEMESTER
TEACHER LINA MARIA MARTíNEZ
SÁNCHEZ
AUGUST 23, 2013
MEDELLÍN - COLOMBIA
ARTICULO ORIGINAL
INTRODUCTION
Variations in the composition of the gut microbiots between individuals are recognized for the development of independent analytic techniques.
These techniques have contributed to many studies for investigating the human gut microbiots and its role in health and disease of the people.
For this objetive is necesary obtain enough amounts of high quality genomic DNA.
Extraction of Deoxyribonucleic acid (DNA) from faeces is very complicated because many substances may be co-extracted, resulting in inhibitory effects of past applications.
Also in extracting DNA from faeces is possible has incomplete lysis cells and shearing of DNA which may also affect the process.
Extracted genomic DNA is used for represent all microbial communities present in the sample of faeces.
The human gastrointestinal tract (GIT) is colonized with germs which have diferent cell walls structures and compositions, making some species more difficult to lyse than others, like Bifidobacteria and Lactobacillus.
GENETIC MATERIAL
DNA EXTRACTION
FENOL CLOROFORMO •Peptides and proteins are extracted from the organic phase with phenol and then used the proteinase K•very toxic the agents used•you can lose many shows whether degrades too CHELEX•DNA has single strand •reagents are not toxic•You don’t lost a lot of sample
MICROBIOTS
Known like: The normal microbiots, normal microbial flora or human microbiome
Meaning: is the set of microorganisms that are found frequently in different parts of the human body in healthy people.It is in a symbiotic relationship with the host, because are advantages for the host and the microorganism.
It is the community of living microorganisms resident in a particular ecological niche
Classification of Human microbiots
Indigenous microbiots: Microorganisms that colonize the host for a long time, may participate in the physiological functions and have evolved along with the species
Allochthonous microbiots: Microorganisms that can be found in any habitat and system, usually do not contribute to the physiology of the host and are present transiently or latently
Also it´s classified by the time in the host:
Latent Microbiots: Microorganisms that have the host for most of my life, have no major fluctuations in population and often have symbiotic with the host activity.
Transient Microbiots: It is continuous fluctuations in the population and not essential for the survival of the host. can colonize in changes as habitat change, age, season, use of antibiotics, etc.
The term “flora" is a mistake
The term “flora" is a mistake
in this case refers to the host like a
carrier
in this case refers to the host like a
carrier
This microorganisms are called opportunistic
pathogens when they cause diseases.
This microorganisms are called opportunistic
pathogens when they cause diseases.
The most common are: Staphylococcus aureus, Escherichia
coli and Candida albicans
The most common are: Staphylococcus aureus, Escherichia
coli and Candida albicans
STOOL
MICROORGANISMS PRESENT IN THE STOOL
Bacteroides speciesBifidobacteriesEubacteriesColiformesEnterococo feacalisCandidaE. coli
DNA extraction
DNA extraction
MICROBIOTSMICROBIOTS
STOOLSTOOL
OBJECTIVES
Compare the relative efficacy of extracting bacterial genomic DNA from human faecal samples using five commercial DNA extraction kits.
Evaluate the DNA extraction kits based on their ability to efficiently lyse bacterial cells, cause minimal DNA shearing, produce reproducible results and ensure broad-range representation of bacterial diversity.
MATERIALES Y MÉTODOS
SUJETOS Y RECOLECCIÓN DE MUESTRAS
cuatro niños menores de dos años y cuatro adultos, después de obtener el consentimiento de los individuos sanos o de sus tutores. Después de la recolección, la muestras se transportaron en hielo y se almacenaron a 70 °C antes de su procesamiento.
EXTRACCIÓN DE ADN A PARTIR DE MUESTRAS FECALES
Extracción del ADN utilizando inicialmente
dos cantidades de materia fecal: 100 mg y
200 mg
Extracción del ADN utilizando inicialmente
dos cantidades de materia fecal: 100 mg y
200 mg
La lisis celular mecánica se realizó en BEAD-BEATING por todos los kits a 50 Hz
durante 5 minutos usando la TissueLyser LT™
La lisis celular mecánica se realizó en BEAD-BEATING por todos los kits a 50 Hz
durante 5 minutos usando la TissueLyser LT™
TABLA 1.
Extracción automatica utilizandoQIAsymphony® Virus/BacteriaMidi Kit (kit QS)Fuera del tablero de lisis se incorporo : 750 μl de buffer de ZR Fecal DNA MiniPrep™ kit (kit Z) combinada con la lisis celular mecánica.
EXCEPTO QIAamp® DNA Stool
Mini Kit (kit QA), por
recomendaciones del fabricante
EXCEPTO QIAamp® DNA Stool
Mini Kit (kit QA), por
recomendaciones del fabricante
El lisado se centrifugó a 10000 rpm durante 1 minEl lisado se centrifugó a 10000 rpm durante 1 min
300 μl del sobrenadante resultante fueron usados para la extraccion del DNA con
ayuda del QIAsymphony® SP instrument (Qiagen, Hombrechtikon, Switzerland)
300 μl del sobrenadante resultante fueron usados para la extraccion del DNA con
ayuda del QIAsymphony® SP instrument (Qiagen, Hombrechtikon, Switzerland)
Para el kit de Z, se agrego un paso adicional centrifugación a 14000 rpm durante 1 minPara el kit de Z, se agrego un paso adicional centrifugación a 14000 rpm durante 1 min
Muestras fecales, en Buffer ASL, para el kit de control de calidad mediante el TissueLyser LT ™ (Qiagen, Fritsch GmbH, Idar-Oberstein, Alemania) a 50 Hz durante 1
min
Muestras fecales, en Buffer ASL, para el kit de control de calidad mediante el TissueLyser LT ™ (Qiagen, Fritsch GmbH, Idar-Oberstein, Alemania) a 50 Hz durante 1
min
Calefacción opcional del lisado fecales a 95 °C en lugar de 75 °CCalefacción opcional del lisado fecales a 95 °C en lugar de 75 °C
El ADN extraído se trató con 3,0 g de RNasa A (Sigma-Aldrich, Carlsbad, Estados Unidos de
América)
El ADN extraído se trató con 3,0 g de RNasa A (Sigma-Aldrich, Carlsbad, Estados Unidos de
América)
Todos los DNA extraidos fueron eluidos en 50 μl de agua destilada, excepto el kit QS, donde el volumen de elución minimo que sugiere
el proveedor es 60 μl usando Buffer AVE.
Todos los DNA extraidos fueron eluidos en 50 μl de agua destilada, excepto el kit QS, donde el volumen de elución minimo que sugiere
el proveedor es 60 μl usando Buffer AVE.
LA CALIDAD Y CANTIDAD DE ADN EXTRAÍDO DE LA MUESTRA
FECALEl rendimiento de ADN (ng) y pureza (relación de absorbancia a 260/280) de ADN genómico extraído se determinó espectrofotométricamente utilizando el Nano gota ® ND-1000 (Nanodrop Technologies Inc., Wilmington, Estados Unidos de América)
.
Integridad del ADN genómico se determinó por visualización de 200 ng de ADN en gel de agarosa al 1% (w / v) que contiene 0,25 g / l de bromuro de etidio (EtBr), se ejecutan en 1 x buffer Tris-EDTA a 100 Voltios
Integridad del ADN genómico se determinó por visualización de 200 ng de ADN en gel de agarosa al 1% (w / v) que contiene 0,25 g / l de bromuro de etidio (EtBr), se ejecutan en 1 x buffer Tris-EDTA a 100 Voltios
EVALUACIÓN DE LOS MÉTODOS DE EXTRACCIÓN DE ADN POR
PCR EN TIEMPO REALGENERACIÓN DE UNA CURVA ESTÁNDAR CON EL ADN
GENÓMICO
Cepas: Bacteroides fragilis 638R y Bifidobacterium longum spp. longum JCM 1217
se cultivaron en agar Brucella en un dióxido de carbono (CO2) en condiciones atmosféricas (85% de nitrógeno, 10% de CO2 y 5%
de hidrógeno) a 37 ° C. Escherichia coli ATCC (American Type Culture Collection) 25922
se cultivó aeróbicamente en agar Mueller-Hinton a 37 ° C.
EVALUACIÓN DE LA EFICIENCIA DE EXTRACCIÓN DE ADN MEDIANTE
TÉCNICAS DE TOMA DE HUELLAS DACTILARES
ANÁLISIS DGGE DE 16S RDNA AMPLIFICADOS
Análisis DGGE de 16S rDNA amplificadosDos rondas de PCR se realizaron enfocandose en el gen 16S de rDNA bacteriano
T-RFLP DEL 16S RDNA AMPLIFICADOS
RESULTADOS
•QA comparado con U y P, > mediana de rendimiento de DNA.•QS muchísimo mayor que U•QA < puro que P y Z (100mg), tambn < Z y U
•qPCR diana de las tres bacterias que se encuentran comúnmente en las heces no mostraron diferencias consistentes en la ampliación de los DNA de estas bacterias• Como se ha demostrado mediante pruebas de qPCR utilizando DNA extraído por los kits
DISCUSSION
REFERENCE WHAT THEY SAID AGREE OR DESAGREE
De Vos and De Vos; Fujimura et al.
“Over the past decade, the influence of the human gut microbiota on health and desease has been the focus of numerous studies”
Agree
Maukonen et al.; Penders et al.
“with faecal samples being widely used”
Agree
Hayashi et al. “Even though faeces may only partly represent the complexity of the GIT microbiota”
Agree
Dave et al. “it is sampled non-invasively and easy to acquire at minimal cost”
Agree
CONCLUSIONS
The gut microbiot is different in each person, even in the same person, so it is important to identify various strains to knowledge deseases.
It is important use RNAse to purify the samples. The gastrointestinal tract have a variety of
microorganism living in symbiosis. We have many analysis techniques for DNA and all use
different principles, but all of them get to the same result.
Mapas conceptuales
Maria Isabel Palacio
Maria Carolina Rave Aguirre
BIBLIOGRAPHY
MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 7 ed. Medellín: UPB. Fac. de Medicina, 2013. 275-290 p.
Shantelle Claassen, Elloise du Toit ,,Mamadou Kaba, Clinton Moodley ,Heather J. Zar and Mark P. Nicol “A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples”. 14 Mayo de 2013.
Bibliografía
