Nucleic Acid Extraction Kit Research Articles

Beyond Diagnostics: The role of a RT-PCR laboratory in a pandemic

PurposeThe gold standard for the detection of SARS-CoV-2 is Real Time Reverse Transcriptase PCR (rRT-PCR). A combination of nasopharyngeal and oropharyngeal swabs improves the sensitivity of detection of SARS-CoV-2 in samples. The first wave of COVID-19 impacted Chennai in May 2020 and lasted till October 2020. The second wave began in March 2021 and lasted till June 2021. The aim of this study is to compare and analyze the COVID-19 waves in 2020 and 2021 based on the data from a tertiary care centre in Chennai, India.Methods & MaterialsA total of 42,374 samples were tested by rRT-PCR (Rotor Gene-Q) between March 2020 and June 2021. RNA was extracted using Zybio nucleic acid extraction kit. The target regions of the RT-PCR kits (ViroQ SARS CoV2, Bag Diagnostics, Germany) were the E-gene and RdRp gene. Quality control is maintained by routine participation in the external quality assurance scheme (EQAS).ResultsIn 2020, 22,905 samples were tested and 3,415 samples tested positive for COVID 19. During the first wave of COVID-19 between May-October 2020, 2989 samples tested positive for COVID 19. Between January and June 2021, 19,469 samples were tested and 2,320 samples were COVID-19 positive. A total of 2,155 samples were positive for COVID-19 in the second wave between March-June 2020. The duration of the first wave was longer with a smaller peak in incidence of COVID 19. The second wave in 2021 had a shorter duration with a more pronounced spike in COVID-19 cases in April 2021.ConclusionThe number of COVID 19 positive cases are similar between the two waves of COVID 19. However, the first wave of COVID-19 took place over a period of nearly six months, while the second wave occurred over three to four months. There is a noticeable surge in COVID-19 incidence observed in April-May 2021. This could indicate the increased infectivity of the virus as observed with the cases doubling rapidly during this time. Documenting the characteristics of the wave in terms of its rapidity of onset of disease and peaks in incidence would offer insight into the progression of COVID-19.

Viral RNA extraction using an automatic nucleic acid extractor with magnetic particles and genetic characterization of bovine viral diarrhea virus in Tokachi Province, Japan, in 2016-2017.

In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016-2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.

Comparative Study of Gut Microbiome signatures in cirrhotics and their healthy donors undergoing Living Donor Liver Transplantation

Background and Aim: There is emerging evidence of gut dysbiosis in end-stage liver disease and its association with poor survival. We intend to study the gut microbiome profile in cirrhotics and compare with their liver donors as healthy individuals, when these patients are proceeding for living donor liver transplantation. Also, to understand the correlation of baseline gut microbiome with severity of liver disease. Methods: Fecal samples were collected from 14 patient donor pairs prior to transplant in this observational study. Genomic DNA was extracted from faecal samples using stool nucleic acid extraction kit and analysed by using 16s rRNA V3-V4 variable region next generation sequencing and metagenomics. Results: The mean age of recipients was 42.7±9.1 years and 37.6±9.7 years in donors. Mean MELD score was 19.4±7.5 and MELD-Na was 23.4±7.3. Predominant etiology was ethanol (50%) followed by MAFLD (21.4%). In phyla distribution, compared to recipients, donors had more Bacteroidetes (48.3%vs.31.9%, p=0.007), less Proteobacteria (7.71%vs.26.57%, p=0.001). At genera level, recipients compared to donors, showed less Prevotella (8.9%vs.29.4%), Lachnospira (4.16%vs.10.1%), more Enterobacteriaceae (20.1%vs.3.5%), p<0.05. Based on MELD score, recipients were categorized into three groups (<15,15-25,>25) and compared with donors. Proteobacteria, Enterobacteriaceae positively correlated and Prevotella, Lachnospira negatively correlated as the MELD score increased (p < 0.05). Based on aetiology, MAFLD>HCV>ethanol groups negatively correlated with Bacteroidetes and positively with Proteobacteria (p<0.05). Interestingly, as events of portal hypertension (SBP, bleed, HE, sepsis) increased, there was more Actinobacteria, Bacteroidetes/Firmicutes ratio, more Lactobacillaceae, Bifidobacteria and reduced Ruminococcus (p<0.05). Conclusions: Compared to donors, there was high abundance of pathogenic bacteria and low abundance of Bacteroides in recipients. This dysbiosis was more evident in increasing MELD scores and with more events of portal hypertension, which may affect prognosis. In conclusion, our study shows that in recipients compared to donors there is significant gut dysbiosis which can have implications on post-transplant outcomes.

Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p&lt;0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020

IntroductionThe Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. MethodsWe diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. ResultsAs a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. ConclusionThe false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

Human Papillomavirus (HPV) Prevalence and Type Distribution in Urban Areas of Malaysia.

Background:Cervical cancer is the third leading cause of death in Malaysia, and Human Papilloma Virus (HPV) is the principal aetiology that is responsible for its development. This study was aimed to determine the prevalence and distribution of HPV types among different age groups, ethnicity, and areas in Malaysia. Materials and Methods:A total of 764 women aged 20-74 years old within the cities of Johor Bahru, Kuala Lumpur, Ipoh, Penang, and Kota Kinabalu underwent both cervical cytological assessment and HPV DNA analysis. Cervical cytology glass slides were prepared using the liquid base technique (Path TEZT TM). HPV DNA was extracted using TANBead® Nucleic Acid Extraction Kit (Taiwan Advanced Nonotech Inc.), then the types were further identified using a DR.HPV Genotyping IVD kit. Results:The prevalence of HPV infection was 14.0% (107/764) with high-risk type at 10.7% (82/764) and low-risk type at 3.27% (25/764). The most common high-risk HPV types were HPV-52, 66, 33, 39, and 58 whereas low-risk HPV types were HPV-6, 40, and 81. The majority of HPV infections (80.37%) were detected in women with normal cytology results. The most prevalent HPV type among Chinese is 33 (n=6) followed by 16, 44, 58, 66 and 68 (n=5). Among Malays, HPV 16 and 51 were the two most prevalent types (n=2). The sensitivity of the HPV DNA test compared to cytology was 100% with a specificity of 88.37%.Conclusion:This study revealed that the most common high-risk HPV type among women living in urban areas in Malaysia is HPV 52, unfortunately which is not the type of infection the current HPV vaccine is covered for protection among females. These findings may contribute beneficial information to health care providers for the appropriate use of HPV vaccine in the prevention of cervical cancer in Malaysia.

Use of guanidine thiocyanate-based nucleic acid extraction buffers to inactivate poliovirus in potentially infectious materials

The efforts of the Global Poliovirus Eradication Initiative (GPEI) have brought about the near elimination of poliovirus worldwide. The World Health Organization has issued guidelines for the safe handling and containment of infectious materials (IM) and potentially infectious materials (PIM) following poliovirus eradication. Inactivation of poliovirus in IM and PIM is needed to prevent inadvertent re-introduction of polioviruses post-eradication. In this study, we investigated the use of guanidine thiocyanate-based nucleic acid extraction buffers from commercially available nucleic acid extraction kits to inactivate poliovirus in cell culture isolates and stool suspensions, two common types of poliovirus IM and PIM, respectively. Incubation with selected nucleic acid extraction buffers or extraction buffers supplemented with ethanol reduced the infectivity of high-titer wild poliovirus type 1 (WPV1), wild poliovirus type 3 (WPV3), Sabin 1 (SL1), and Sabin 3 (SL3) cell culture isolates below the limit of detection in CCID50 assays. Stool suspensions containing WPV1, WPV3, SL1, SL2, or SL3 were also inactivated by the extraction buffers tested. Blind passage of WPV1-spiked stool suspensions confirmed complete inactivation of WPV1 after incubation with extraction buffers. Moreover, treatment with a buffer consisting of 4 M guanidine thiocyanate with 30 % ethanol inactivated a high-titer WPV1 culture isolate and a WPV1-spiked stool suspension. Taken together, these results show that guanidine thiocyanate-based nucleic acid extraction buffers are an effective means of inactivating poliovirus IM and PIM, and thus will be instrumental in ensuring containment compliance and preventing potential re-emergence of contained polioviruses.

Extraction of Viral Nucleic Acids with Carbon Nanotubes Increases SARS-CoV-2 Quantitative Reverse Transcription Polymerase Chain Reaction Detection Sensitivity.

The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using “capture” ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.

Low risk of SARS-CoV-2 in blood transfusion.

BackgroundThe novel coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), continues to remain a global challenge. There is emerging evidence of SARS-CoV-2 virus found in the blood of patients from China and some developed countries. However, there is inadequate data reported in Ghana and other parts of Africa, where blood transfusion service heavily relies on voluntary and replacement blood donors. This study aimed to investigate whether plasma of infected individuals could pose significant transfusion transmitted risk of COVID-19 in Ghanaian populations.MethodsThis cross-sectional retrospective study was conducted at the Kumasi Centre for Collaborative Research into Tropical Medicine (KCCR), KNUST, Ghana. Study subjects comprised contacts of COVID-19 individuals, those with classical symptoms of COVID-19 and individuals who had recovered based on the new Ghana discharge criteria. Whole blood, sputum or deep coughed saliva samples were collected and transported to KCCR for SARS-CoV-2 testing. Viral nucleic acid was extracted from sputum/nasopharyngeal samples using Da An Gene column based kit and from plasma using LBP nucleic acid extraction kit. Real-Time PCR was performed specifically targeting the ORF1ab and Nucleocapsid (N) genomic regions of the virus.ResultsA total of 97 individuals were recruited into the study, with more than half being males (58; 59.7%). The mean age of all subjects was 33 years (SD = 7.7) with minimum being 22 years and maximum 56 years. Majority (76; 78.4%) of all the subjects were asymptomatic, and among the few symptomatic subjects, cough (10; 10.3%) was the most predominant symptom. Of the 97 sputum samples tested, 79 (81.4%) were positive for SARS-CoV-2. We identified SARS-CoV-2 viral RNA in the plasma of 1 (1.03%) subject who had clinically recovered.ConclusionThis study reports the identification of SARS-CoV-2 viral RNA in a convalescent individual in Ghana. Due to the low prevalence observed and the marginal cycling thresholds associated, the risk of transfusion transmission of SARS-CoV-2 is negligible. Well-powered studies and advanced diagnostics to determine infectious viremia is recommended to further evaluate the potential risk of hematogenous transmission among recovered patients.

Evaluation of sample pooling for screening of SARS CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed. To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2. We evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200μL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay. We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency. The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don't advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.

Direct Detection of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Without RNA Purification

Coronavirus disease 2019 (COVID-19) is an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has spread globally. Shortage of nucleic acid extraction kits may delay the speed of diagnosis. In order to address this need, we propose a simple method for direct RNA extraction from nasopharyngeal swab samples without RNA purification. Here, we describe a reverse transcription loop-mediated isothermal amplification technique with 95.4% sensitivity (95% CI: 84.2-99.4%) and 100% specificity (95% CI: 78-100%). Combined with a simple RNA extraction step, this optimized RT-LAMP method is able to amplify SARS-CoV-2 directly from clinical nasopharyngeal swab samples in viral transport media in 50 minutes. This method provides an opportunity to facilitate SARS-CoV-2 detection especially in resource limited settings where nucleic acid extraction kits are few.Funding: This study was supported by Prototype Research Grant Scheme (PRGS), PR001-2020B (PRGS/2/2020/SKK09/UM/02/1) from the Ministry of Education, Malaysia.Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.Ethics Approval Statement: Ethical approvals for this study was obtained from UMMC 85 Medical Ethics Committee (202041-8418) and Medical Research Ethics Committee 86 (MREC) Ministry of Health Malaysia (NMRR-20-2344-56994).

Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100–1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

Adventure of the CRISPR Coronavirus Disease 2019 Diagnostic.

Adventure of the CRISPR Coronavirus Disease 2019 Diagnostic.

Identification of Somatic Mutations in Thirty-year-old Serum Cell-free DNA From Patients With Breast Cancer: A Feasibility Study

IntroductionThe aim of this study was to assess the feasibility of cell-free DNA (cfDNA) extraction and circulating tumor DNA sequencing in 30-year-old serum samples. Materials and MethodsWe evaluated serum samples from 52 patients with breast cancer, which were collected between 1983 and 1991, with correlating clinicopathologic data. cfDNA was extracted by using the QIAamp Circulating Nucleic Acid Extraction Kit (Qiagen). Of these 52 cfDNA samples, 10 were randomly selected and sequenced with the Oncomine Breast cfDNA Assay (A31183). In a second step, high-depth targeted sequencing of 15 additional cfDNA samples was performed using a custom Ampliseq Ion Torrent panel targeting breast cancer-related genes. ResultscfDNA extraction was successful in 52 (100%) of 52 patients with a total concentration of 0.2 to 54 ng/uL. A total of 24 cancer-specific mutations were found in 22 (88%) of the 25 samples undergoing sequencing. Of the 52 patients, 32 (62%) had died from breast cancer after a median follow-up of 7.9 years (interquartile range, 3.7-15.5 years). ConclusionThe present study shows that current next generation sequencing technology is sufficiently robust and specific to analyze 30-year-old serum. Therefore, longitudinal studies can be designed with storage of serum samples over many years, thereby obviating the need for timely and continuous cfDNA extraction and sequencing. The samples can be pooled and processed at once with the most modern technology available at the end of the study, when accumulation of events allows correlation of clinical outcomes with adequate power.

Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.

Optimization of a direct real-time quantitative reverse transcription polymerase reaction (DRT-qPCR) assay for the detection of grapevine rupestris stem-pitting associated viruses (GRSPaV) in grapevine

A single step Direct real-time quantitative reverse transcription quantitative polymerase chain reaction (DRT-qPCR) was applied and optimized for the detection of Grapevine rupestris stem-pitting associated virus (GRSPaV) in grapevine. A probe and primer set were designed from conserved genomic regions from eight GRSPaV accessions from GenBank and used with the Direct Plant Extraction Buffer (DiPEB) system in place of total nucleic acid extraction using commercial kits to identify 14 grapevine accessions and field grapevine isolates for the presence of GRSPaV. Optimal detection of GRSPaV was obtained using foliar tissue macerated first in ELISA extraction buffer that was further diluted in DiPEB buffer (1:9 v/v). Middle-aged grape leaves on young canes gave acceptably low Cq (quantification cycle) values, with no significant differences detected between bisected half leaves, refrigerated and frozen leaves, and frozen leaf macerates made in ELISA buffer. Cq values of macerates derived from cambial tissue were comparable to those of leaf tissue macerates. The DRT-qPCR was shown to be a reproducible, specific test capable of detection of as little as 4.59 pg of viral RNA. This is the first study to use this simplified method for the detection of viruses that infect grapevine. Because this system does not require the use of a commercial nucleic acid extraction kit, it provides savings in cost and time allowing higher throughput testing.

Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

Adsorption of DNA by using polydopamine modified magnetic nanoparticles based on solid-phase extraction

A polydopamine magnetic composite (PDA@Fe3O4) was prepared for the extraction of human genomic DNA and characterized by transmission electron microscopy, X-ray diffraction, FT-IR spectrometer, zeta potential and vibrating sample magnetometry. PDA@Fe3O4 based on magnetic solid phase extraction (MSPE) method have highly efficient capture of genomic deoxyribonucleic acid (DNA)and gene fragments ranging from about 100 bp to 200 bp. Compared with commercial beads (Shenggong, China) and spin column nucleic acid extraction kit (Tiangen, China), the PDA coated magnetic nanoparticles display superior genomic DNA extraction capacity (116 mg/g) and yield (90.2%). The isolation protocol used the solutions (composed of PEG and NaCl) with a specific pH for the binding and release of DNA. The procedure can be attributed to the charge switch of amino and hydroxyl groups on surface of the magnetic particle. The extracted DNA with high quality (A260/A280 = 1.82 ± 0.04) can be directly used as template for polymerase chain reaction (PCR) followed by agarose gel electrophoresis. The results showed the new composite to be an ideal adsorbent for separation of DNA which had the advantage of its low cost, high extraction capacity and yield.

Comparison of five viral nucleic acid extraction kits for the efficient extraction of viral DNA and RNA from cell-free samples

Comparison of five viral nucleic acid extraction kits for the efficient extraction of viral DNA and RNA from cell-free samples

A Magnetic Bead-Based DNA Extraction Protocol Suitable for High-Throughput Genotyping in Shrimp Breeding Programs

Due to their convenience, magnetic bead-based nucleic acid extraction kits are commonly used in shrimp genotyping and pathogen screening applications. However, in advanced breeding programs requiring the testing of many thousands of shrimp, their cost can be prohibitive. Various permutations of different Proteinase K digestion, tissue lysis and bead washing buffers as well as magnetic bead types were thus evaluated to devise a high-throughput shrimp DNA extraction (SDE) protocol capable of recovering high-purity DNA using a KingFisher™ Flex Magnetic Particle processor. When genotyped using a MassARRAY® platform (Agena Bioscience) requiring 60-61 genome regions to be co-amplified in a single multiplexed PCR, DNA extracted from shrimp muscle tissue using either the SDE protocol or a commercial kit generated comparable single-nucleotide polymorphism (SNP) call data. The SDE protocol also extracted high-purity DNA from salmon fin clips. It thus offers potential to markedly reduce the costs of large-scale genotyping in shrimp and salmon breeding programs.