scholarly journals Comparison of DNA extraction methods for non-marine molluscs: Is modified CTAB DNA extraction method more efficient than DNA extraction kits?

2019 ◽  
Author(s):  
Sudeshna Chakraborty ◽  
Anwesha Saha ◽  
N.A. Aravind
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
High Molecular Weight Dna ◽  
Long Term Storage ◽  
Dna Extraction Method ◽  
Successful Amplification ◽  
Dna Extraction Methods ◽  
Sequence Quality

AbstractIsolation of high molecular weight DNA from gastropod molluscs and its subsequent PCR amplification is considered difficult due to excessive mucopolysaccharides secretion which co-precipitate with DNA and obstruct successful amplification. In an attempt to address this issue, we describe a modified CTAB DNA extraction method that proved to work significantly better with a number of freshwater and terrestrial gastropod taxa. We compared the performance of this method with Qiagen® DNeasy Blood and Tissue Kit. Reproducibility of amplification was verified using a set of taxon-specific primers wherein, modified CTAB extracted DNA could be replicated at least four out of five times but kit extracted DNA could not be replicated. Additionally, sequence quality was significantly better with CTAB extracted DNA. This could be attributed to the removal of polyphenolic compounds by polyvinyl pyrrolidone (PVP) which is the only difference between conventional and modified CTAB DNA extraction methods for animals. The genomic DNA isolated using modified CTAB protocol was of high quality (A260/280 ≥ 1.80) and could be used for downstream reactions even after long term storage (more than two years).

scholarly journals Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

2020 ◽  
Vol 5 (2) ◽  
pp. 95 ◽  
Author(s):  
Rajashree Chowdhury ◽  
Prakash Ghosh ◽  
Md. Anik Ashfaq Khan ◽  
Faria Hossain ◽  
Khaledul Faisal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Recombinase Polymerase Amplification ◽  
National Guidelines ◽  
Diagnostic Efficacy ◽  
Rapid Extraction ◽  
Kala Azar ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

scholarly journals Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

2019 ◽  
Author(s):  
Q.R. Ducarmon ◽  
B.V.H. Hornung ◽  
A.R. Geelen ◽  
E.J. Kuijper ◽  
R.D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACTWhen studying the microbiome using next generation sequencing, DNA extraction method, sequencing procedures and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing, and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with maximum three-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for estimating richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs and variation induced by DNA extraction methods was lower than inter-subject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we demonstrate the importance of including negative controls when analyzing low bacterial biomass samples.IMPORTANCEMethod choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls, and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiome studies. Our methods were not only applied to commonly studied samples for microbiota analysis, e.g. feces, but also for more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g. urine and tumor biopsies) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiome studies, especially when low bacterial biomass is expected.

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

scholarly journals Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Q. R. Ducarmon ◽  
B. V. H. Hornung ◽  
A. R. Geelen ◽  
E. J. Kuijper ◽  
R. D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples. IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

scholarly journals Comparative investigation of DNA extraction methods in black gram (Vigna mungo (L.)

2019 ◽  
Author(s):  
M. . Prakash ◽  
B. . Priyadharshini ◽  
M. . Vignesh ◽  
R. . Anandan
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Large Scale ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Comparative Investigation ◽  
Black Gram ◽  
Dna Extraction Method ◽  
Ctab Method ◽  
Dna Extraction Methods

Isolation of intact, double stranded, pure and non- contaminated genomic DNA is prerequisite for large scale genotyping analysis including DNA-banks. Three methods of DNA isolation (Dellaporta, CTAB and Hi-PurAg DNA isolation kits) from 25 black gram genotypes were compared in terms of the yield, purity, integrity, and stability of extracted DNA. Purity and quantification of isolated DNA samples was confirmed by using the UV nano-spectrophotometer at OD260/280 and the same is confirmed based by agarose gel electrophoresis. The CTAB method showed the best results followed by Hi-PurAg and Dellaporta method. The CTAB DNA extraction method was found to be the most efficient DNA extraction method, capable of providing high quality, pure and stable DNA and could be used for various molecular related works. All the 25 black gram genotypes for this research gave good yield of DNA from the established modified CTAB protocol.

scholarly journals Evaluation of rapid extraction methods coupled with Recombinase polymerase amplification assay for point-of-need diagnosis of Post-kala-azar-dermal leishmaniasis

2019 ◽  
Author(s):  
Rajashree Chowdhury ◽  
prakash ghosh ◽  
Md. Anik Ashfaq Khan ◽  
Faria Hossain ◽  
Khaledul Faisal ◽  
...  
Keyword(s):  
Skin Biopsy ◽  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Recombinase Polymerase Amplification ◽  
Diagnostic Efficacy ◽  
Rapid Extraction ◽  
Kala Azar ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Introduction Post kala-azar dermal leishmaniasis (PKDL) usually develops as sequelae of visceral leishmaniasis (VL) and can manifest in multiple dermatological forms. Since PKDL patients harbor Leishmania donovani parasites and can potentially trigger inter-epidemic transmission of the disease, the success of kala-azar elimination programme could be jeopardized by these cases. Although several molecular methods with promising diagnostic efficacy have been developed to detect PKDL cases, albeit complicated and expensive DNA extraction methods limit their application in resource poor settings. To address this, in comparison to a reference DNA extraction method (Qiagen), we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay.Methods Thirty suspected PKDL cases were enrolled after diagnosis by clinical examination and a positive rk39 strip test. DNA was extracted from three skin biopsy samples using either a spin column-based method (Qiagen) or one of two rapid DNA extraction methods, (Boil & Spin (B&S) and SpeedXtract (SE)). RPA and qPCR were subsequently performed with the extracted samples to detect L. donovani DNA.Results Using DNA extracted by Qiagen method, the qPCR and RPA assays exhibited sensitivities of 86.7% and 93.3% respectively. In contrast, the sensitivity of RPA assay dropped to 76.7% and 63.3%, respectively, when the B&S and SE rapid extraction methods were performed. Despite this compromised sensitivity, B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k =0.831) techniques. Moreover, SE-RPA showed good agreement with Q-qPCR (k = 0.755), Q-RPA (k =0.692) and B&S-RPA (k =0.635) assays. As expected, with all of the three DNA extraction methods, both qPCR and RPA assay showed absolute specificity.Conclusions This study finding substantiates the superior diagnostic efficacy of Qiagen DNA extraction method over B&S and SE method in detecting LD DNA through RPA assay from skin biopsy of PKDL patients. To apply these rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

scholarly journals Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens

2020 ◽  
Vol 8 (11) ◽  
pp. 1768
Author(s):  
Céline Nourrisson ◽  
Julie Brunet ◽  
Pierre Flori ◽  
Maxime Moniot ◽  
Virginie Bonnin ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Fecal Microbiota Transplantation ◽  
Parasite Load ◽  
Extraction Methods ◽  
Fecal Microbiota ◽  
Automated Method ◽  
Dna Extraction Method ◽  
Blastocystis Sp ◽  
Dna Extraction Methods

Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation.

scholarly journals DNA Extraction from Bronchial Aspirates for Molecular Cytology: Which Method to Take?

2003 ◽  
Vol 25 (2) ◽  
pp. 83-88 ◽  
Author(s):  
Hans Jürgen Grote ◽  
Viola Schmiemann ◽  
Mario Sarbia ◽  
Alfred Böcking
Keyword(s):  
Success Rate ◽  
Dna Extraction ◽  
Extraction Method ◽  
Globin Gene ◽  
Extraction Methods ◽  
High Quality ◽  
High Molecular Weight Dna ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Dna Extraction Method

Objective: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods.Methods: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated.Results: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of β‐globin gene fragments (268, 536, and 989 bp) was 100%. Conclusions: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR‐based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.

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