Nucleic Acid Extraction Methods Research Articles

Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method.
 Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method.
 Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk

Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal powders. The improved method presented in this study could be used as an alternative to successfully extract PCR quality DNA from such plant materials. Further, we present a set of robust matK primers which could be used as plant barcoding resource in future studies.

Factors affecting stability and infectivity of SARS-CoV-2

BackgroundIn late 2019, a novel human coronavirus – severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) – emerged in Wuhan, China. This virus has caused a global pandemic involving more than 200 countries. SARS-CoV-2 is highly adapted to humans and readily transmits from person-to-person.AimTo investigate the infectivity of SARS-CoV-2 under various environmental and pH conditions. The efficacies of various laboratory virus inactivation methods and home disinfectants against SARS-CoV-2 were investigated.MethodsThe residual virus in dried form or in solution was titrated on to Vero E6 cells on days 0, 1, 3, 5 and 7 after incubation at different temperatures. Viral viability was determined after treatment with various disinfectants and pH solutions at room temperature (20–25oC).FindingsSARS-CoV-2 was able to retain viability for 3–5 days in dried form or 7 days in solution at room temperature. SARS-CoV-2 could be detected under a wide range of pH conditions from pH 4 to pH 11 for several days, and for 1–2 days in stool at room temperature but lost 5 logs of infectivity. A variety of commonly used disinfectants and laboratory inactivation procedures were found to reduce viral viability effectively.ConclusionThis study demonstrated the stability of SARS-CoV-2 on environmental surfaces, and raises the possibility of faecal–oral transmission. Commonly used fixatives, nucleic acid extraction methods and heat inactivation were found to reduce viral infectivity significantly, which could ensure hospital and laboratory safety during the SARS-CoV-2 pandemic.

First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.

Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Comparative evaluation of nucleic acid detection kits and nucleic acid extraction methods for SARS-CoV-2 using clinical samples

Objective: To compare and analyze 7 new coronavirus nucleic acid detection kits and 5 nucleic acid extraction methods Methods: After extracting nucleic acids from 44 positive coronavirus clinical samples, 7 SARS-CoV-2 nucleic acid detection kits were used for RT-PCR amplification experiments to compare the positive rate and Ct value;33 new coronavirus positive clinical samples were selected to compare the acid extraction methods Five different nucleic acid extraction methods were used to extract the samples, and then RT-PCR amplification experiments were performed to compare the positive rate and Ct value Results: The brand A nucleic acid extraction kit had the highest positive rate and the lowest rate of missed detection;comparison of nucleic acid extraction methods showed that the manual column extraction method had the highest positive rate, followed by the magnetic bead extraction method, and the one-step extraction method had the highest missed detection rate Conclusion: The detection capabilities of the SARS-CoV-2 detection kits are uneven, so evaluation work needs to be done before the selection of the kit The manual column extraction method showed best extraction efficiency but took a long time Because of the possible combination with the automatic nucleic acid extraction instrument, the magnetic bead extraction method had a high extraction efficacy, which might be suitable for use in the ex- traction of large batches of samples Although the one-step extraction method was easily operable, the method had a high missed detection rate, so this method was not recommended for clinical use

Novel rapid identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by real-time RT-PCR using BD Max Open System in Taiwan.

Coronavirus disease 2019 has become a worldwide pandemic. By April 7, 2020, approximately 1,279,722 confirmed cases were reported worldwide including those in Asia, European Region, African Region and Region of the Americas. Rapid and accurate detection of Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is critical for patient care and implementing public health measures to control the spread of infection. In this study, we developed and validated a rapid total nucleic acid extraction method based on real‐time RT-PCR for reliable, high‐throughput identification of SARS-CoV-2 using the BD MAX platform. For clinical validation, 300 throat swab and 100 sputum clinical samples were examined by both the BD MAX platform and in-house real-time RT-PCR methods, which showed 100% concordant results. This BD MAX protocol is fully automated and the turnaround time from sample to results is approximately 2.5 h for 24 samples compared to 4.8 h by in-house real-time RT-PCR. Our developed BD MAX RT-PCR assay can accurately identify SARS-CoV-2 infection and shorten the turnaround time to increase the effectiveness of control and prevention measures for this emerging infectious disease.

Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation.

Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device’s steel wool matrix for subsequent processing steps. We demonstrate the method’s utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5–200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10–17 and 70–96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.

Advances in Fully Automated Nucleic Acid Extraction System Based on Magnetic Nanobeads

Using magnetic nanobeads (MNBs) to extract nucleic acids is an efficient, inexpensive, easy automation, high throughput and good repeatability, instead of the traditional nucleic acid extraction (NAE) methods. Advances in fully automated MNBs-based nucleic acid extraction systems (MNAES) can push the frontiers of point-of-care testing (POCT) devices towards low-cost, automation, and enhanced accuracy molecular-level diagnostics. So, this paper introduces the pipettingbased MNAES with position of magnetic separation kit or tip, and based on magnetic bar MNAES with blending manner is shock or rotation. Further, advantages and disadvantages of various MNAES are compared. We envisage that the future trend in molecular diagnosis and monitoring will be facilitation, intelligent, miniaturization, and high throughput MNAES with sample-in-answer-out capability.

A novel approach for tuberculosis diagnosis using exosomal DNA and droplet digital PCR

ObjectivesThe rapid diagnosis of tuberculosis (TB) is important for patient treatment and infection control. Current molecular diagnostic techniques for TB have insufficient sensitivity to detect samples with low bacterial loads. The sensitivity of molecular testing depends on not only the performance of the assay technique but also the nucleic acid extraction method. Here, we present a novel approach using exosomal DNA (exoDNA) and droplet digital PCR (ddPCR) platforms to detect Mycobacterium tuberculosis DNA in clinical samples. MethodsThe ddPCR platform targeting IS6110 was evaluated in parallel using total DNA and exoDNA. The clinical performance of ddPCR method was assessed with 190 respiratory samples from patients with suspected pulmonary TB. ResultsCompared with mycobacterial culture, sensitivity and specificity of ddPCR were 61.5% (95% CI 44.6–76.6%) and 98.0% (95% CI 94.3–99.6%) using total DNA, and 76.9% (95% CI 60.7–88.9%) and 98.0% (95% CI 94.3–99.6%) using exoDNA, respectively. Among 15 culture-positive specimens with low concentrations of target molecules (2~99 positive droplets with exoDNA), only 53.3% (8/15), 46.7% (7/15), and 26.7% (4/15) of cases were detected using ddPCR with total DNA, real-time PCR with exoDNA, and real-time PCR with total DNA, respectively. DiscussionOur platform using ddPCR and exoDNA has the potential to provide sensitive and accurate methodology for TB diagnosis.

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 of spread of infection in medical and hospitality sectors. However, methodological limitations have restricted the analysis of microbial diversity on fabrics. Hence, the study aimed to develop a robust method for extraction of DNA from different types 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 types of fabric so as to come up with recommendation for fabrics bearing minimal microbial load for medical and hospitality sectors.

Detection of Cryptosporidium Recovered from Large-Volume Water Samples Using Dead-End Ultrafiltration.

The procedure described here provides instructions for detection of Cryptosporidium recovered from large-volume water samples. Water samples are collected by dead-end ultrafiltration in the field and ultrafilters are processed in a laboratory. Microbes recovered from the filters are further concentrated and subjected to Cryptosporidium isolation or nucleic acid extraction methods for the detection of Cryptosporidium oocysts or Cryptosporidium DNA.

Abstract 4213: Stool-derived eukaryotic RNA (seRNA) assay for noninvasive detection of colorectal cancer and high-risk adenomas

Abstract Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. The high mortality rate for CRC is largely attributable to the frequency of late-stage diagnoses, caused by low patient compliance with screening guidelines. A novel nucleic acid extraction method that isolates stool-derived eukaryotic RNA (seRNA) has permitted development of a colorectal cancer stool diagnostic test (CRC-SDT) that uses a fecal immunochemical test (FIT) and 11 seRNA transcripts to serve as a reliable and noninvasive screening alternative to lower mortality associated with CRC. Stool samples were obtained from 190 individuals prior to undergoing a screening colonoscopy. Patients were diagnosed as either having colorectal cancer, having high-risk adenomas (high-grade dysplasia, villous growth pattern, or >1.0cm in size), or healthy (low-risk adenomas, benign polyps, and/or no findings on a colonoscopy). FITs were obtained for each sample. Samples that had a positive FIT were considered positive for the CRC-SDT. Samples that had a negative FIT underwent seRNA isolation, library preparation (Illumina TruSeq Targeted RNA) and subsequent sequencing (Illumina NextSeq 550). A random forest model was built using 11 transcripts that were differentially expressed (log2 fold-change > 1; ANOVA p < 0.05) between diseased patients (CRC and high-risk adenomas) relative to the healthy cohort. Internal 10-fold cross-validation was performed for the training set and a receiver operating characteristic (ROC) area under the curve (AUC) determined model accuracy for cancer and high-risk adenomas relative to the healthy cohort. Model predictions for an independent hold-out test set were used to determine model accuracy for colorectal cancer and high-risk adenomas. Internal cross-validation for 154 samples attained a ROC AUC of 0.82. The trained model was employed on the independent hold-out test set of 36 samples and model predictions were compared to colonoscopy results. The model attained a 100% sensitivity for CRC (n = 7), a 60% sensitivity for high-risk adenomas (n = 5), and an 88% combined specificity for the healthy cohort (n = 24). CRC-SDT uses 12 biomarkers (FIT and expression from 11 seRNA transcripts) to accurately detect CRC and high-risk adenomas. Use of seRNA biomarkers dramatically improves detection of high-risk adenomas relative to existing noninvasive screening methods for CRC. Citation Format: Erica K. Barnell, Yiming Kang, Katie M. Campbell, Kimberly R. Kruse, Andrew R. Barnell, Elizabeth M. Wurtzler. Stool-derived eukaryotic RNA (seRNA) assay for noninvasive detection of colorectal cancer and high-risk adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4213.

Development of a reverse transcription-loop- mediated isothermal amplification (LAMP) assay for the rapid detection of onion yellow dwarf virus

Onion yellow dwarf virus (OYDV) is one of the most important viral pathogens of onion. In particular, on ‘Rossa di Tropea’ onion, granted with Protected Geographical Indication (PGI) trademarks, this pathogen represents the most limiting biotic stress in terms of spread, severity of symptoms and damage, and its detection is necessary to preserve high quality standards and avoid yield losses. A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed for detection of OYDV. The specificity, sensitivity, repeatability and reproducibility of the assay were validated according to EPPO standard PM7/98 (2). Diagnostic specificity, diagnostic sensitivity and diagnostic accuracy were determined in both leaf and bulb tissues. To enhance the feasibility of a LAMP-based method for field diagnosis, several nucleic acid extraction methods were compared to simplify sample preparation. The results showed the reliability of the method for OYDV detection, with a limit of detection (LOD) comparable to real time reverse transcription polymerase chain reaction (RT-qPCR). The ease of sample preparation, and the more than acceptable LOD, indicated that the RT-LAMP assay could be used in plant pathology laboratories with limited facilities and resources, as well as directly in the field. This work was carried out in the frame of “SI.ORTO” project.

An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance

BackgroundMutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance.MethodsMethods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification.ResultsThe optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use.ConclusionsThe optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.

Rapid detection of Schistosoma japonicum-infected snails with recombinase-aided isothermal amplification assay

To develop a florescent recombinase-aided amplification (RAA) assay for rapid detection of Schistosoma japonicum-infected Oncomelania snails and explore the optimal method for treatment of snail samples. Snail samples were divided into 3 groups, and each group consisted of 7 subgroups. There were 50 uninfected snails mixed with 1, 2, 3, 4, 5 and 10 infected snails in the 6 subgroups, respectively, and the remaining subgroup contained 100 uninfected snails mixed with 1 infected snails. DNA was extracted from snails in the three groups using a genomic DNA extraction kit following snail crushing and snail shells removal, crude nucleic acid extraction assay following snail crushing and snail shells removal, and crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, and subjected to florescent RAA and PCR as says. The detection results were compared between the two assays. A florescent RAA assay was developed, which completed the detection of S. japonicum-infected snails at 39 ℃ within 30 min. Following DNA extraction from mass snail samples with a genomic DNA extraction kit following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was one infected snail mixed in 50 uninfected snails. Following DNA extraction using crude nucleic acid extraction method following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was 3 infected snails mixed in 50 uninfected snails. Following DNA extraction with a crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, the lowest detection limit of the florescent RAA assay was 10 infected snails mixed in 50 uninfected snails, while the lowest detection limit of PCR assay was 10 infected snails mixed in 50 uninfected snails. A fluorescent RAA assay that is rapid to detect S. japonicum-infected snails in mass snail samples is successfully developed, which is fast, sensitive and easy to perform. Crude nucleic acid extraction following snail crushing and snail shells removal is the optimal method for the treatment of snail samples.

Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples

Influenza A virus is a negative-sense RNA virus with a segmented genome consisting of eight RNA segments. Avian influenza A virus (AIV) primarily infects avian hosts and sporadically infects mammals, which can lead to adaptation to new species. Next-generation sequencing (NGS) of emerging AIV genomes extracted from respiratory samples collected on sequential days from animal models and clinical patients enables analysis of the emergence of evolutionary variants within the virus population over time. However, obtaining codon complete AIV genome at a sufficient coverage depth for nucleotide variant calling remains a challenge, especially from post-inoculation respiratory samples collected at late time points that have low viral titers. In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage depth at each time point sampled. The study revealed that DNase treatment was critical to the amplification of influenza genome segments and the downstream success of codon complete NGS from nasal wash samples. The findings from this study can be applied to improve NGS of influenza and other RNA viruses that infect the respiratory tract and are collected from respiratory samples.

Prospective clinical study using expressed sequencing variants on stool-derived eukaryotic RNA transcripts (seRNA) for colorectal cancer screening.

516 Background: Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. The high mortality rate is largely attributable to the high frequency of late-stage diagnoses, caused by low patient compliance with screening guidelines. A reliable and noninvasive screening alternative is needed for the 40 million noncompliant patients. The development of a novel nucleic acid extraction method to isolate stool-derived eukaryotic RNA (seRNA) permits reliable and noninvasive evaluation of biomarkers derived from the gastrointestinal (GI) epithelium. This method enables sequencing-based tools for the detection of patients with CRC and adenomas. Methods: Stool samples were obtained from 96 individuals prior to undergoing a screening colonoscopy. Fecal immunochemical tests (FITs) were obtained for each sample. RNA isolates underwent custom library preparation, next-generation sequencing, and somatic variant identification. An seRNA assay assessed the probability of CRC risk using results from the FIT, mutational burden of transcripts implicated in precancerous change (APC), and mutational burden of transcripts associated with malignant transformation (KRAS / TP53). Results: When compared to results from a colonoscopy and subsequent biopsy, the seRNA risk assessment attained a 100% sensitivity for CRC, a 71.4% sensitivity for advanced adenomas, and an 88.5% specificity for no neoplastic findings. Conclusions: A single-center, IRB-approved, prospective and blinded clinical study is being conducted in 450 patients to further develop this seRNA assay. Supplemental data will include expression from 408 seRNA transcripts. Preliminary analysis described herein indicates this assay could be the most sensitive noninvasive screening test for the detection of CRC and adenomas. [Table: see text]

New DNA Extraction Method for the Detection of Pneumocystis in Lung Tissue Samples of Colonized Individuals

Background: Pneumocystis jirovecii colonization in patients is associated with a low organism burden, which supports the need to use highly sensitive molecular techniques, such as nested-PCR to determine the presence of the organism. However, few studies have considered the effect of nucleic acid extraction methods on the detection of P. jirovecii. Here, we evaluate how pre-treatment affects microbial detection. Methods: Lung tissue samples from fifteen autopsied infants were processed using two different DNA extraction protocols: 1) the QIAamp DNA Mini kit (Qiagen) (method A) and the QIAamp DNA Mini kit (Qiagen) with additional steps including a pre-treatment, bead-beating and phenol-chloroform steps (method B). Detection of P. jirovecii was performed using the conventional nested-PCR in lung tissue samples. In addition, we estimated the bacterial, fungal and human DNA using qPCR method. Results: Higher DNA yields was obtained with DNA extraction method B compared to method A. P. jirovecii was detected in 10 out of 15 lung tissue samples for both methods at the first or second round of PCR. However, P. jirovecii was detected in 9 out of 10 at the first round with the DNA extraction method B, rather than those processed with DNA extraction method A that were detected in 5 out of 10. Moreover, the faint PCR band intensity observed in 2 out of 6 samples processed with DNA extraction method A was increased with method B. We also observed that DNA extraction method B increased significantly the bacterial and fungal DNA, but not the human DNA, compared to method A. Conclusions: Our findings show that the additional steps (i.e. pre-treatment, bead-beating and Phenol: Chloroform: Isoamyl alcohol steps) supplemented to the QIAamp DNA Mini kit (Qiagen) improves the detection of P. jirovecii in lung tissue samples.

DNA extraction from FFPE tissue samples - a comparison of three procedures.

Aim of the studyOne of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA.Material and methodsSlides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies).ResultsThe results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method.ConclusionsTo our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

An improved method for rapid isolation of DNA and RNA from leaves, flowers and roots of blackgram [Vigna mungo (L.) Hepper] for detection of begomovirus infection and RT-PCR.

Blackgram is an important pulse crop grown for its protein rich seeds. Its tissues are rich in polyphenols which hampered pure nucleic acid extraction and thus use of molecular techniques in breeding programme. In this study, a well established DNA extraction method for legumes, Gem-CTAB method has been modified to isolate PCR compatible DNA from blackgram in a quick and economical way from a very small tissue. The amplification rate of DNA extracted through modified Gem-CTAB method was found to be high compared to Gem-CTAB method for begomovirus specific genes. While for RNA isolation, among different protocols, column based kit method followed by lithium chloride precipitation yielded high quality RNA for leaves compatible with RT-PCR. For flowers and roots, kit method alone or with its modifications yielded pure RNA. The above modified nucleic acid extraction method yielded PCR compatible DNA and RNA from phenolics rich blackgram tissues.