Viral Nucleic Acid Isolation Research Articles

Prevalence and quantitative assessment of foodborne viruses on the imported mussels in Polish market

The aim of this study was an assessment of the prevalence of human enteric viruses such as norovirus (NoV) and hepatitis A and E virus (HAV, HEV) in mussels sold on the Polish market. In addition, the presence of human (hAdV) and swine (pAdV) adenoviruses as indicators of faecal contamination of shellfish was also studied. In total, 246 shellfish samples representing 41 batches of blue and Mediterranean mussels were purchased from Polish seafood wholesalers. Shellfish were processed according to the method in ISO 15216-2:2019 followed by isolation of viral nucleic acids using NucliSens®Magnetic Extraction Reagents (bioMérieux). For virus detection several duplex real-time PCR protocols with incorporated internal amplification controls (IAC) were used. The presence of human enteric viruses was shown in 42 shellfish samples (17.1%). The most frequently detected were NoV (41 samples; 16.7%) followed by HEV (1 sample; 0.4%). None of the samples was positive for HAV, hAdV or pAdV. 15 shellfish batches (36.6%) were virus positive. By the use of Bayesian inference, a true seasonal and country related virus prevalence in shellfish was estimated. NoV GI was more frequently detected in Dutch mussels (18.9%; 95% CI: 6.7%–34.0%), while NoV GII was more frequent in Danish mussels (17.1%; 95% CI: 5.8%–32.5%). However significant differences in virus prevalence in individual and shellfish batches between producing countries were not shown. A seasonal virus distribution pattern in shellfish samples was observed. Lower level of NoV contamination was found in mussels harvested during spring-summer (0.0%–1.3%, 95% CI: 0.1%–4.8%) compared to the fall-winter season (13.0%–20.7%, 95% CI: 6.1%–31.2%). For shellfish batches, a similar seasonal correlation was only observed for NoV GII. The monitoring of marketed shellfish would be an essential tool allowing a reduction of the number of viral food-borne outbreaks.

A fast, low-cost, robust and high-throughput method for viral nucleic acid isolation based on NAxtra magnetic nanoparticles

The year of 2020 was profoundly marked by a global pandemic caused by a strain of coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19). To control disease spread, a key strategy adopted by many countries was the regular testing of individuals for infection. This led to the rapid development of diagnostic testing technologies. In Norway, within a week, our group developed a test kit to quickly isolate viral RNA and safely detect SARS-CoV-2 infection with sensitivity comparable to available kits. Herein, the procedure employed for the detection of SARS-CoV-2 in swab samples from patients using the NTNU-COVID-19 test kit is described in detail. This procedure, based on NAxtra magnetic nanoparticles and an optimized nucleic acid extraction procedure, is robust, reliable, and straightforward, providing high-quality nucleic acids within 14 min. The NAxtra protocol is adaptable and was further validated for extraction of DNA and RNA from other types of viruses. A comparison of the protocol on different liquid handling systems is also presented. Due to the simplicity and low cost of this method, implementation of this technology to diagnose virus infections on a clinical setting would benefit health care systems, promoting sustainability.

Prevalence of SARS-CoV-2 Variants in COVID-19 Positive Patients

Objective: Of the existing variants, alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and omicron (B.1.1.529) were defined as variants of concern (VOCs) by World Health Organization. We aimed to retrospectively assess the distribution of VOCs in patients with positive COVID-19 real-time polymerase chain reaction (RT-PCR) test. Methods: Between April 2021 and December 31, 2021, 4260 patients whose clinical samples were sent with a COVID-19 PCR test request were included in the study. Of the patients whose samples were evaluated, 2173 (51%) were male, and 2087 (49%) were female. Viral nucleic acid isolation was performed by treating nasopharyngeal swabs, bronchoalveolar lavage, and tracheal aspirate samples with viral nucleic acid buffer (vNAT). SARS-CoV-2 variants were detected by Bio-Speedy® SARS-CoV-2 Variant Plus kit (Bioeksen AR-GE Technologies, Turkey), which targets variant-specific genome regions only found in B.1.1.7, B.1.351, and P.1 as well as ORF1ab and N gene regions. We used Bio-Speedy® SARS-CoV-2 Emerging Plus kit and CFX96 DX real-time PCR system to detect SARS-Cov-2 variants; N D3L mutation was targeted to detect the alpha variant, SL452R mutation to detect the delta variant, and S E484K mutation, which is widespread, to detect the gamma and the mu variants. Results: SARS-CoV-2 RT-PCR was positive in 773 (18.14%) of 4260 samples, and different SARS-CoV-2 variants were detected in 316 (40.88%) of positive samples. 407 (52.65%) of the patients with real-time polymerase chain reaction (RT-PCR) positive test were women, and the mean age was 41.7 years. While no variant type was detected in 457 (59.12%) of the patients, the alpha variant was detected in 156 (20.18%) and the delta variants in 137 (17.72%) patients. Conclusion: In our study, we found that the alpha variant was the dominant type in April, May, and June, and the delta variant became dominant as of July until the end of the year. Decreased rates of delta variants towards the end of 2021 and an increase in the other variants suggest the omicron variant, widely spread globally. Therefore, we believe that the omicron variant has been our country’s dominant type since November 2021. In conclusion, molecular surveillance studies that are planned in the light of epidemiological data and assessment of the frequency and genomic analyzes of regional and global variants are required.

SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab.

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

Investigation of Torque Teno Virus (TTV) DNA as an immunological and virological marker in pediatric hematopoietic stem cell transplantation (HSCT) patients

BackgroundHigh viral loads are observed in Torque Teno Virus (TTV) infection after hematopoietic stem cell transplantation (HSCT). We aimed to analyze the kinetics of plasma TTV-DNA load in pediatric patients who received immunosuppressive therapy and developed infection complications in the first 100 days after HSCT. MethodsAs a patient group; 113 plasma samples taken from 33 pediatric HSCT recipients at a time interval after transplantation and as a control group; 38 plasma samples from 38 children without known chronic disease were included in the study. Viral nucleic acid isolation was performed by using the NucliSENS easyMAG (bioMerieux, France) system. A laboratory designed quantitative polymerase chain reaction process was performed on 7300 Real-Time PCR system (Applied Biosystems, CA, USA) with the amplification mixture containing primer and probe sequences for the UTR gene region. ResultsTTV-DNA was detected in all patient's samples and the median viral load was calculated as 7.67 Log10 copies/mL (range: 2.84–9.59). In the control group, the TTV-DNA median viral load was calculated as 5.51 Log10 copies/mL (range: 2.50–7.04), except for one negative sample. A significant difference was observed between the control group and the patient group in terms of TTV viral load levels. In nine patients, a median 2.15 Log10 copies/mL viral load increase was observed at 31–60 days post-transplant compared to the pre-transplant period. ConclusionTTV-DNA levels should be closely monitored to understand the immune status of the first 100 days after transplantation and the effects of treatment regimens on patients with HSCT.

Tick‐borne viruses: Current trends in large‐scale viral surveillance

AbstractTicks are ectoparasites that transmit pathogens, such as tick‐borne viruses, to their hosts. Tick‐borne viruses are diverse: they can be categorized into two orders, nine families, and at least 12 genera. Almost 25% of these viruses are infectious to humans and some are a serious threat to public health. The global rise in tick‐borne virus diseases has been linked to climate change which has reduced tick mortality in the winter and extended their active period. The spread of tick‐borne viral diseases to humans has received significant interest due to the increased threat to human life; epidemiological monitoring of tick‐borne viruses using molecular, immunological, and environmental methods is now a priority. Nevertheless, many tick‐borne diseases remain undiagnosed, which poses a challenge to public administration and health care officials. This review discusses three major tick‐borne RNA viruses that cause serious infection in humans: severe fever with thrombocytopenia syndrome (SFTS) virus, tick‐borne encephalitis (TBE), and Crimean–Congo hemorrhagic fever (CCHF) virus. Specifically, we discuss the epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants related to these viruses. Furthermore, we review the current surveillance of these tick‐borne viruses with a specific focus on diagnostic approaches that employ molecular interventions such as viral nucleic acid isolation, PCR‐based diagnostics, and high‐throughput sequencing technologies.

Evaluation of a commercial exogenous internal process control for diagnostic RNA virus metagenomics from different animal clinical samples

Metagenomic next generation sequencing (mNGS) is increasingly recognized as an important complementary tool to targeted human and animal infectious disease diagnostics. It is, however, sensitive to biases and errors that are currently not systematically evaluated by the implementation of quality controls (QC) for the diagnostic use of mNGS. We evaluated a commercial reagent (Mengovirus extraction control kit, CeraamTools, bioMérieux) as an exogenous internal control for mNGS. It validates the integrity of reagents and workflow, the efficient isolation of viral nucleic acids and the absence of inhibitors in individual samples (verified using a specific qRT-PCR). Moreover, it validates the efficient generation of viral sequence data in individual samples (verified by normalized mengoviral read counts in the metagenomic analysis). We show that when using a completely random metagenomics workflow: (1) Mengovirus RNA can be reproducibly detected in different animal sample types (swine feces and sera, wild bird cloacal swabs), except for tissue samples (swine lung); (2) the Mengovirus control kit does not contain other contaminating viruses that may affect metagenomic experiments (using a cutoff of minimum 1 Kraken classified read per million (RPM)); (3) the addition of 2.17 × 106Mengovirus copies/mL of sample does not affect the virome composition of pig fecal samples or wild bird cloacal swab samples; (4) Mengovirus Cq values (using as cutoff the upper limit of the 99 % confidence interval of Cq values for a given sample matrix) allow the identification of samples with poor viral RNA extraction or high inhibitor load; (5) Mengovirus normalized read counts (cutoff RPM > 1) allow the identification of samples where the viral sequences are outcompeted by host or bacterial target sequences in the random metagenomic workflow. The implementation of two QC testing points, a first one after RNA extraction (Mengoviral qRT-PCR) and a second one after metagenomic data analysis provide valuable information for the validation of individual samples and results. Their implementation in addition to external controls validating runs or experiments should be carefully considered for a given sample type and workflow.

Current trends in large‐scale viral surveillance methods in mosquitoes

AbstractVector‐borne and zoonotic infectious diseases are serious public health concerns that affect approximately half of the world's population. In particular, arthropod‐borne viruses (arboviruses) have contributed to more mortality and morbidity worldwide with the emergence of dengue, chikungunya, yellow fever, and Zika virus diseases. The infections have scaled up due to urbanization, globalization, and international mobility. Traditionally, the spread of mosquito‐borne viral diseases to humans was considered a low health priority concern. However, their categorization as emerging infectious diseases and public health emergencies of international concern has heightened the attention given by the government, academia, research, and industry for the development of timely, cost‐efficient, and sustainable solutions. The urgency has increased in the wake of global climate change. The focus on effective interventions includes epidemiological monitoring, vector control measures, molecular diagnostics, vaccines, and environmental determinants. In this review, we discuss the etiology and predisposition of mosquito‐borne viruses that are detrimental to public health and economically damaging when disseminated as epidemics. We focus on the large‐scale virus surveillance methods with special reference to innovations and interventions in molecular detection science and technologies that include viral nucleic acid isolation, polymerase chain reaction (PCR)‐based diagnostics, and high‐throughput sequencing technologies. In addition, we discuss the development of a viral RNA extraction and PCR‐based diagnostic kit (Invirustech) that can extract viral RNA from mosquitoes with verified applications in PCR‐based molecular diagnostics of Pan‐flavivirus.

Methods of virus detection in various work environment

Viruses are harmful biological agents, which can pose a risk for workers from different occupational groups. Information regarding the occurrence of viruses in occupational environment is still scarce, primarily due to analytical difficulty of their routine detection. Hygiene control applied for microbiological contamination are not effective in case of viruses, so the occupational risk for workers exposed to biological agents is still underestimated. Such a state of affairs fundamentally precludes a proper management of occupational safety. The effectiveness of virus detection in the environmental samples depends on methods of sampling and transport, and isolation and identification method. The aim of this paper is an attempt to critically review the literature of the subject, determination of the best and the most effective method of virus detection and identification in the occupational environment, which could be used as a routine methodology for assessing the risk of viruses in various workplaces. The article discusses methods of viral samples collection, viral nucleic acid isolation, and, their detection based on viral activity, molecular biology and immunoenzymatic assays.

A Method for Isolation of the Virome from Plasma Samples.

Virome studies are of special interest nowadays. Understanding viral communities in different body compartments will help guide future personalized treatments and to discern between homeostasis and disease. High-throughput sequencing technologies allow us to detect all the nucleic acids present in a sample, including viral ones, by random sequencing. One of the major challenges in virome studies is the correct isolation of the viral nucleic acids from a specific sample. This can be done during the extraction steps (e.g., enrichment of viral capsids), or during the bioinformatic analysis (e.g., removing all human and bacterial sequences). Furthermore, it is an important remark that the treatment of the sample will strongly influence the results. Samples will be treated differently if the ultimate goal is the study of all replicating and encapsidated viruses, including both RNA and DNA ones, if we are only focused on DNA ones, or if we want to analyze all the possible viral nucleic acids in the specific sample, even if the genome is degraded. Here, we present a technique that allows for isolation of viral nucleic acids from plasma samples.

Hemodiyaliz hastalarında gizli Hepatit B enfeksiyonunun polimeraz zincir reaksiyonu ile araştırılması

Aim: It is known as the occult Hepatitis B that Hepatitis B virus DNA (HBV DNA) exists in serum very little if Hepatitis B virus surface antigen in serum is negative. The studies about occult Hepatitis B infection prevalence are limited in patients who undergo hemodialysis implements for along time. HBV existence is able to continue in plasma and liver tissue of some patients whose HBsAg test is negative. This condition causes many diagnostic problems. It is very crucial the description of people who have this clinical risk of HBV transmission in the community, especially in dialysis units. This study is aimed to determine the prevalence of occult Hepatitis B in dialysis patients. Material and Methods: 160 HBsAg-negative patients and 28 predialysis total of 188 patients were included in this study. 5 ml venous blood samples taken from the patients into sterile vacutainer tubes contain anticoagulant. Blood tubes to separate serum 800-1600 x g for 20 min. centrifuged. The separated serum was collected into polypropylene tubes and was stored at -20 oC until the applied test procedure. For the detection of HBV DNA; HBV Isolation Device (Magnesia 16 Geneworks Anatolia, Turkey) and HBV DNA isolation kit (16/201 viral nucleic acid isolation kit) was used and isolated, then serum were tagged on the elution tube. Real-Time PCR Device (Montanide 483 Geneworks Anatolia, Turkey) and Real-time PCR kit (Bosphore Quantification Kit V2) was used in PCR process. Real Time PCR kit, which is used in the study, was detected whole HBV genotypes between A and H of the Hepatit B virus DNA in human serum or plasma sample. Analytical sensitivity of the kit was 10 IU / mL linear range 1x101-1x109 IU/ml. Hepatitis B virus markers (HBsAg, anti-HBcAg, anti-HBs) ) were studied using commercial ELISA kits (Abbott ARCHITECT Qualitative Reagan Kit) and ELISA device (Abbott ARCHITECT i2000 SR), CRP (SIEMENS cardiophase to hsCRP the kit and SIEMENS BN II device. Likewise, aspartate aminotransferase (AST), alanine aminotransferase (ALT), (aspartate aminotransferase A / Alanine aminotransferase Roche Cobas INTEGRA / cobas c system) were studied using with test kits Roche Cobas 501 devices. Results: In 11.3% of hemodialysis patients (18/160) HBV DNA positivity (occult Hepatitis B infection) were detected. Predialysis patients who are in the control group was not observed HBV DNA positivity (0/28). Anti-HBs positivity in 142 hemodialysis patients (88.7%), while 14 predialysis patients (50%) were found (p <0.05). There was no statistically significant difference between the other parameters. Conclusions : Dialysis centers are areas of risk for transmission of HBV. HBV DNA screening of all patients undergoing dialysis must be performed. In addition; suggested to vaccination of susceptible patients, GHB has been diagnosed patients isolating dialyzer, the diagnosis of HBV patients receiving a specific service area. Key Words: HBV, occult Hepatitis B, hemodialysis

Metagenomic characterization of viral communities in corals: mining biological signal from methodological noise.

Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities and requires specialized methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this minireview, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by double-stranded DNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral and other eukaryotes in close association.

Metagenomic characterisation of viral communities in corals: Mining biological signal from methodological noise

Summary Reef-building corals form close associations with organisms from all three domains of life and therefore have many potential viral hosts. Yet, knowledge of viral communities associated with corals is barely explored. This complexity presents a number of challenges in terms of the metagenomic assessments of coral viral communities, and requires specialised methods for purification and amplification of viral nucleic acids, as well as virome annotation. In this mini-review, we conduct a meta-analysis of the limited number of existing coral virome studies, as well as available coral transcriptome and metagenome data, to identify trends and potential complications inherent in different methods. The analysis shows that the method used for viral nucleic acid isolation drastically affects the observed viral assemblage and interpretation of the results. Further, the small number of viral reference genomes available, coupled with short sequence read lengths might cause errors in virus identification. Despite these limitations and potential biases, the data show that viral communities associated with corals are diverse, with double- and single-stranded DNA and RNA viruses. The identified viruses are dominated by dsDNA-tailed bacteriophages, but there are also viruses that infect eukaryote hosts, likely the endosymbiotic dinoflagellates, Symbiodinium spp., host coral, and other eukaryotes in close association.

Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) infections are potentially dangerous complications of transfusion therapy. The aim of this study was to determine the prevalence of HDV markers examined by serological and molecular methods in hepatitis B surface antigen (HBsAg)-reactive sera among blood donors. Samples from 88 HBsAg-reactive blood donors were investigated for total anti-delta antibody (anti-HDV) and HDV-RNA between April 2010 and February 2011. HBsAg screening tests were performed by "microparticle enzyme immunoassay" (MEIA) method using the AxSYM system (Abbott Laboratories, USA), and total anti-delta antibody tests were performed by MEIA method using the Alisei system (Radim, Italy). HDV-RNA was quantified using the polymerase chain reaction (PCR) method. Viral nucleic acid isolation system (Anatolia Geneworks) was used with Bosphore HDV quantification kit. HBsAg reactivity was determined as 1% (124/12.423) among blood donors as a whole. Eighty-eight of these 124 samples were investigated further for HDV. Three (3.4%) of the 88 HBsAg-reactive serum samples were total anti-delta antibody-reactive. Of the 3 anti-HDV-reactive sera, 2 were reactive for HDV-RNA. Therefore, HDV-RNA reactivity was determined as 2.3% (2/88) in HBsAg-reactive donors as a whole. The 2 HDV-RNA-reactive donors were brothers. Investigation of HDV is important because HBV infection is endemic in Turkey. Intrafamilial transmission is important in HDV transmission.

Automated nucleic acid isolation in viral molecular diagnostics: evaluation of the QIAsymphony SP

The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors’ laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories.

MagaZorb: A Simple Tool for Rapid Isolation of Viral Nucleic Acids

Effective isolation of nucleic acids from samples containing viral materials is an essential step for accurate diagnosis of viral infections. The necessity of this critical step before analytical identification and diagnosis of viral infections is paramount to screening programs and to identifying and monitoring epidemics and pandemics. With molecular assays rapidly evolving into routine practice, clinical laboratories face several challenges, including presence of small amounts of viral nucleic acids in abundant levels of genomic DNA and total RNA, processing of various sample types, and carry-over of polymerase chain reaction inhibitors, which could significantly affect polymerase chain reaction and microarray results. MagaZorb nucleic acid isolation technology overcomes these challenges and offers a simple and reliable method for isolation of highquality and high-yield nucleic acids. Although the MagaZorb technology is readily adaptable to automated platforms, it is also well suited to laboratories in remote areas of resource-poor countries, because a simple magnet is the only device required to perform the procedure manually. Performance characteristics and clinical application of the MagaZorb technology are briefly described here.

Easy to use and rapid isolation and detection of a viral nucleic acid by using paramagnetic microparticles and carbon nanotubes-based screen-printed electrodes

A method that is easy to use, rapid, with a low cost of detecting viral nucleic acid in a biological sample represents the essential tool in targeted therapy. In this study, we report the use of paramagnetic microparticles covered by streptavidin and modified by an oligonucleotide probe with a specific viral sequence labeled by biotin to detect human immunodeficiency virus (HIV) and influenza virus subtype H5N1. The viral nucleic acids were primarily detected by adsorptive transfer stripping technique coupled with square wave voltammetry using carbon paste, hanging mercury drop or carbon nanotubes-based screen-printed working electrodes. Detection limits were estimated for both sequences down to picograms per 3 μl. To isolate the viral sequences, paramagnetic microparticles covered with biotin-labeled oligonucleotides were used. We calculated the yield of isolation for H5N1 and/or HIV sequences, which was defined as “isolated concentration of viral nucleic acid sequence”/“given viral nucleic acid sequence” × 100. We estimated the yield for both sequences as 59%. Moreover, we studied the influence of human serum, dsDNA and non-complementary sequence of nucleic acids on isolation of viral nucleic acids. We also used carbon nanotubes-based screen-printed electrodes coupled with micro-flow instrument to detect viral nucleic acids. We were able to isolate and detect nanogram amounts of nucleic acids.

Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA viral load in blood is a clinically validated tool for the management and follow-up of patients infected with this virus. HIV-1 RNA quantification can be performed by several kinds of commercially available polymerase chain reaction (PCR) assays. The sensitivity of these assays is affected by the nucleic acid isolation procedures that are performed before PCR. Especially for the samples containing low numbers of HIV-1 RNA, an effective nucleic acid isolation method is very important to obtain a high nucleic acid output. The purpose of this study was to compare and evaluate the conventional manual extraction method, automated MagNA Pure system, and automated AmpliPrep system for the nucleic acid isolation before TaqMan real-time PCR assay for the quantification of HIV-1 RNA in plasma samples. Plasma samples of 40 patients in which anti-HIV antibodies were found as positive serologically were included in this study. Nucleic acid isolation from these samples was performed by manual High Pure Viral Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany), automated MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics), and automated Cobas AmpliPrep kit (Roche Diagnostics). The nucleic acids obtained by these 3 methods were amplified and detected in the Cobas Taqman 48 (Roche Diagnostics) instrument. Viral RNA copies detected by AmpliPrep system, MagNA Pure system, and manual isolation method were found to be between 40 and 420 000 (average, 100 135), 53 and 1 200 000 (average, 220 554), and 183 and 590 000 (average, 104 761) copies/mL, respectively. The results of isolation methods were evaluated for each sample, and it was found out that the number of HIV-1 RNA obtained by MagNA Pure system was higher than the other 2 methods. All of the 3 methods were found to be reliable in detecting very small amounts of HIV-1 RNA in samples. Manual isolation method is a cheaper alternative, but because the nucleic acid isolation can be performed in a shorter time by automated systems when compared with manual isolation method, these systems can be preferred especially in the laboratories that have high number of samples.