RNA Mini Kit Research Articles

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples

BackgroundNumerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.ResultsIn this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis.ConclusionsThe evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Performance of a commercial assay for detecting and quantifying HEV RNA in faeces

BackgroundDetecting hepatitis E virus (HEV) RNA in faeces is useful for diagnosing and monitoring HEV infections, particularly in immunocompromised patients requiring ribavirin therapy. ObjectivesThis study evaluated the performance of the Altona RealStar HEV RNA kit for detecting and quantifying HEV in faeces. Study designRNA was extracted from 94 stool samples by two methods: QIAamp Viral RNA Mini kit and MagNA Pure 96 automate. The Altona results were compared to a reference laboratory-developed accredited ISO15189 RT-PCR assay. ResultsThe Altona and reference assays detect HEV RNA in 77/93 (82.8%) and 83/93 (89.2%) of the QIAamp extracted samples, respectively, after exclusion of invalid result; they detected HEV RNA in 67/92 (72.8%) and 66/92 (71.7%) of the MagNA Pure extracted samples, respectively, which emphasizes the importance of the RNA extraction method. The HEV RNA concentrations obtained with Altona RT-PCR and the reference RT-PCR were well correlated whatever the extraction method, and Bland Altman analyses indicated that the Altona values were higher than the reference assay values. The Altona values for QIAamp-extracted and MagNA Pure-extracted HEV RNA were very similar. ConclusionsThe Altona RealStar assay is suitable for quantifying HEV RNA in the faeces and monitoring HEV RNA shedding during ribavirin therapy. Extraction is critical for detecting faecal HEV with high performance RT-PCR assays.

Efficiency evaluation of the process control virus “Mengovirus” in real time RT-PCR viral detection in the bivalve mollusc Donax sp

There are many problems associated with extracting viral genetic material from contaminated samples of bivalve molluscs, specifically because the hepatopancreas has many PCR inhibitors. For this reason, nucleic acid extraction methods must consider a process control virus (PCV) that may help to measure extraction efficiency. In the market, there are many commercial kits to extract nucleic acid from RNA viruses, as well as others to perform one-step real time RT-PCR, but most of them have not been evaluated for bivalve molluscs. For this reason, the aim of this study was to evaluate the extraction efficiency of the PCV (Mengovirus), it was performed using 3 different RNA extraction kits and 2 one-step real time RT-PCR kits. 10 μL of Mengovirus at a concentration of 1.6 × 104 viral particles/μL was added to 29 samples of hepatopancreas of Donax sp. Sample processing was performed according to the ISO/TS 15216-2: 2013 standard. RNA was extracted from each sample with the kits: (1) BioMerieux NucliSens®system (BioMérieux SA, France), (2) PureLink™ RNA Mini Kit (Ambion-Life Technologies™, USA) and (3) Hugh Pure RNA Tissue Kit (Roche SA, Germany). Once RNA was extracted, one-step real time RT-PCR was conducted by using the following kits: (A) Ultrasense One-step qRT-PCR Kit (Invitrogen, USA) according to ISO/TS 15216-2:2013, and (B) Mengovirus@ceeramTools™ Kit (Ceeram, France) according to the manufacturer's specifications. The extraction efficiency of PCV when using the extraction kits 1, 2 and 3 combined with real time RT-PCR kit A were: 10.82, 1.90 and 0.64, respectively; and when using real time RT-PCR kit B were: 7.34, 0.97 and 0.47, respectively. It is concluded that the BioMerieux NucliSens®system RNA extraction kit was the most efficient and that the Ultrasense One-step qRT-PCR Kit performed better than the Mengovirus@ceeramTools™ RT-PCR kit.

Serotyping of Dengue Viruses Circulating During 2014–2015 in Assam, India

Dengue has become endemic in India with outbreaks caused by all the four serotypes occurring almost every year. Dengue cases have been increasing alarmingly in Assam also. This study aimed to identify the dengue serotypes circulating in Assam. Clinically suspected dengue fever cases were included in the study. Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Nested reverse transcriptase polymerase chain reaction was done for serotyping. The frequency of dengue was 25.23% with a peak during the period from September (22.56%) to October (26.22%). Two serotypes were detected, DEN-1 (72.7%) and DEN-2 (12.1%) and dual infection in 15.1%.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus.

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

AimTo characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples.BackgroundDNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide.MethodsQIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array.ResultsIrrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA.ConclusionHigh quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes.

We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project, which analyzes complete viral genomes from more than 3000 HIV-1-infected Europeans with high-throughput deep sequencing techniques. The goal of the BEEHIVE project is to determine the contribution of viral genetics to virulence. Recently we performed a pilot experiment with 125 patient plasma samples to identify the method that is most suitable for isolation of HIV-1 viral RNA for subsequent long-amplicon deep sequencing. We reported that manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) provides superior results over robotically extracted RNA. The latter approach used the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics), the QIAcube robotic system (Qiagen), or the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular). Here we present a detailed protocol for the labor-intensive manual extraction method that yielded the best results.

First Report of Maize yellow mosaic virus Infecting Maize in Brazil

Maize (Zea mays) is a staple crop for human consumption and widely used as a livestock feed worldwide. Therefore, potential new diseases affecting this crop are of major concern. In May 2013, yellow mosaic and dwarfing symptoms were observed in several maize plants in a field located in the rural area of Casa Branca (21° 46′ 26″ S, 47° 5′ 9″ W), Sao Paulo state (Southeastern Brazil). Symptomatic leaves were sampled, and total RNA was isolated using Trizol™ reagent and further purified with PureLink™ RNA Mini Kit spin columns (Invitrogen, USA). A library was prepared with an Illumina TruSeq Stranded Total RNA sample preparation kit with Ribo-Zero Plant and sequenced on an Illumina Hiseq2500 platform at Macrogen Inc, South Korea. De novo assembly using Abyss 1.9 with K-mer = 64 yieldeda contig of 1183 nt with high similarities (92-93%) to several isolates sequences of Maize yellow mosaic virus (MaYMV), a putative new member of the genus Polerovirus, family Luteoviridae, recently reported in China (Chen et a...

Clinical Significance of AFP, HGF and Micro RNA-122 Levels in Patients with Liver Cirrhosis and Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and is advanced by severe viral hepatitis B or C (HBV or HCV). HCC is frequently diagnosed late requiring costly surgical resection or transplantation. Alpha fetoprotein (AFP) measurements, together with ultrasound are the most non-invasive methods for detection of HCC. Also, serum levels of hepatocyte growth factor (HGF) vary in liver diseases, reflecting hepatic damage and hepatocellular dysfunction. The discovery of miRNAs circulating in the peripheral blood has opened new directions of research to identify new non-invasive markers for diagnosis of diseases . Objectives: To assess the serum level of HGF and AFP in patients with liver cirrhosis with HCV infection and those of HCC .Also, to evaluate the expression levels of circulating plasma miRNA-122 in Egyptian patients. Methodology: Anti-HCV and HCV RNA were detected by an enzyme immunoassay and the viral RNA mini kit respectively among patients with cirrhosis and HCC. Also, serum AFP and HGF concentration was measured by sandwich ELISA for patients with HCC, patients with cirrhosis and normal controls. Correlation between HGF levels AFP and biochemical (transaminases, prothrombin activity, albumin, bilirubin), or virological (hepatitis C virus load) parameters was analyzed. . Expression level of miRNA-122 in plasma using RT-PCR were determined. Results: The serum AFP level was significantly elevated in cirrhotic and HCC patients in comparison to the control, with the highest level in the HCC group. On the other hand, the HGF level was significantly higher in HCC group than patients with cirrhosis. For HGF, the sensitivity and specificity were better than AFP (95% and 80% respectively). MiRNA-122 showed significant elevation in patients with cirrhosis groups and HCC group (P = 0.001) in comparison to the control group. Receiver operator characteristic (ROC) curve analysis for miRNA-122 yielded 73% sensitivity and 96 % specificity for the differentiation of HCC patients from non-HCC at a cutoff 0.165. Conclusion: HGF was better than AFP as noninvasive biomarkers for the detection of HCC in HCV cirrhotic patients. As its specificity was 96 %. MiRNA-122 can be used as novel non-invasive biomarker for monitoring HCV related disease progression.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Comparative Analysis of Methods of Molecular Detection of Avian Influenza Virus

ObjectiveThe performance of comparative analysis of sensitivity and resultsof detection of avian influenza virus by real time polymerase chainreaction (PCR-RT) and loop-mediated isothermal amplification of thenucleic acids (LAMP) was the main goal of the study.IntroductionAs part of this surveillance study for Avian Influenza both activeand passive surveillance samples were tested using PCR and alsoutilized to validate the LAMP method. Active surveillance samplesinclude pathological material and tracheal and cloacal swabs fromill poultry, which were subsequently assessed for avian influenzaduring diagnosis, and birds collected by hunters. Passive surveillanceincluded environmental samples such as sand and bird faeces.Active surveillance samples were taken mostly from poultry farmsacross Ukraine, where infected birds are required to be diagnosedby State Scientific Research Institute of Laboratory Diagnosticsand Veterinary Sanitary Expertise (SSRILDVSE) by Ukraine Law.Passive surveillance samples were taken primarily during the annualbird migration season. Development of simple, sensitive, and cheapmethods for diagnostics of avian influenza is a very important taskfor practical veterinary medicine. LAMP is one of such methods.The technique is based on isothermal amplification of nucleic acids.It does not require special conditions and equipment (PCR cyclers),therefore it is cheaper in comparison with PCR. Accurate diagnosisis necessary for determining the risk associated with avian influenzain Ukraine and along the Dnipro River during the migratory season.MethodsFor the research, we used PCR-RT commercial kit Bird-Flu-PCR(Ukrzoovetprompostach, Ukraine), LAMP (the protocol has beenoptimized and patented by SSRILDVSE), QIAamp®Viral RNA MiniKit. For the study, we used pathological and biological materials frombirds, which were sent to the SSRILDVSE from all regions of Ukraineaccording to the 2013–2014 State monitoring plan.Set up of the real time PCR reactions and parameters ofamplifications are indicated in the instruction to the kit.The following protocol was used to set up the RT- LAMP: 2.5μL10 X Thermopol buffer, 1 mmol/L betaine, 5 mmol/L MgSO4,1.4 mmol/L - BNTP, 12.5μmol/L SYBR GREEN, 0.5 mmol/LMnCL2, up to 25μL Nuclease-free water, 8 U Bsm DNA polymerase,0.1μM/1 of F3, 0.1μM/1 of B3, 0.8μM/1 of FIP, 0.8μM/1 of BIP,0.4μM/1 of LF, 0.4 of LB, 2μL cDNA.During our work, we used the following optimal temperature andtime for the amplification – 59°C and 60 minutes.The sensitivity of diagnostic kit Bird-Flu-PCR and RT- LAMP wasdetermined by testing cDNA of the reference strain of AIV H5N1,which was provided to us by NSC Institute for Experimental andClinical Veterinary Medicine (Kharkiv, Ukraine). For the standard,we employed concentration in the range of 10.0-0.01 ng/sample.ResultsTable 1.This table shows the reproducibility results obtained by bothmethods. However, taken into account absence of highly pathogenicavian influenza virus circulating in Ukraine during the studied period,it was not possible to confirm these results with protocols of positivesamples.Table 2.It has been established that the sensitivity of PCR-RT kit Bird-Flu-PCR is 0.01 ng/sample for gene M and 0.1 ng/sample for subtypeH5N1.Fig. 1. Visual detection of LAMP products with differentconcentrations of cDNA of avian influenza virus (ng per sample):1 – 10; 2 – 5; 3 – 1.0; 4 – 0.1; 5–7 – 0.01; 8–9 – 0.1; 10 – negative.We have examined the LAMP results using electrophoresis forthe confirmation of visual detection and correct interpretation of theresults (Fig. 2).Fig.2. Electrophoresis results for LAMP products. M –molecular weight marker; 1 – 10.0; 2 – 5.0; 3 – 1.0; 4 – 0.1; 5–7– 0.01; 8 - negative control.It has been established that the sensitivity of LAMP is0.1 ng/sample. Slightly lower sensitivity of LAMP in comparisonto PCR-RT can be explained by visual detection of the products ofthe LAMP reaction.Conclusions1. Sensitivity of both methods is high.2. LAMP is a perspective screening method for the diagnosis ofviral infectious diseases supported by confirmation of positive resultsby PCR-RT.

Evaluating the efficacy of commercial kits for viral DNA/RNA extraction

Extraction of viral DNA/RNA from environmental samples as part of the analytical procedure in quantifying waterborne viruses, is of great importance. In this study, two commercially available kits were compared to assess their performance, the MO BIO PowerViral Environmental DNA/RNA Isolation kit and the Qiagen QIAamp Viral RNA Mini kit. A performance assessment of extraction kits for detecting and quantifying six human enteric viruses as the commonest waterborne pathogens and one plant virus as an alternative fecal indicator has been carried out using quantitative PCR (qPCR). Water samples were collected from seven sites in Singapore during March and April 2015. In general, a strong association was observed between two different viral DNA/RNA extraction kits and detection frequency of targets (P = 0.017). The Qiagen kit showed higher extraction efficiency than the MoBio kit. However, in terms of quantification, a significant difference was only observed in the occurrences of NoV GI and PMMoV between two different kits (P < 0.05), although the kits showed similar efficiency removing qPCR inhibitors. The Qiagen kit was preferred for routine water quality monitoring.

High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos.

The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

The Prevalence of Hepatitis C Virus Genotypes in Mazandaran Province, Iran

Background: Hepatitis C virus (HCV) infection is a major public health concern and the third most common cause of death from cancer worldwide. This infection is the most common risk factor for the progression of chronic liver disease, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Hepatitis C virus infection is classified into six major genotypes and multiple subtypes. The distribution of HCV genotypes seems to vary in different areas around the globe. Objectives: In this study, we aimed to identify HCV RNA and perform genotyping in anti-HCV-positive patients through polymerase chain reaction (PCR). We compared the results of PCR and ELISA tests among patients referred to several healthcare centers of Mazandaran province, situated in North of Iran, during 2015 - 2016. Methods: A total of 157 patients with anti-HCV-positive specimens were enrolled in this study during 2015-2016. Hepatitis C virus-RNA was extracted from the serum samples, using pure link viral RNA Mini kit. Detection of HCV nucleic acids was performed by using the serum samples from anti-HCV positive specimens. The genotypes of the HCV genome were determined by PCR, using the RNA-PCR kit according to the specified protocol. Also, the PCR products (DNA fragments) from these samples were run on 1.5% agarose gel and visualized on a UV analyzer. All the samples were examined at the virology laboratory of Mazandaran University of Medical Sciences, Sari, Iran. Results: The mean age of the patients was 39 ± 14.3 years (range: 20 - 68 years). In total, 110 (70.06%) and 47 (29.94%) anti-HCV-positive patients were male and female, respectively. Hepatitis C virus–RNA was detected in 84 (53.50%) out of 157 patients with positive HCV antibodies. Based on the findings, the majority of HCV-RNA positive patients were intravenous drug users (46.43%). In this study, the 3a HCV genotype was predominant (59.52%), followed by the 1a/b genotype (40.48%). Conclusions: The present findings indicate that the 3a genotype is the most frequent HCV genotype, followed by 1a/b in the study region. The prevalence and incidence of HCV genotypes seem to vary in different areas, with each genotype showing a different response to interferon therapy. Therefore, it is essential to determine the predominant subtypes of HCV.

Abstracts of the 34th International Congress of the International Society of Blood Transfusion, Dubai, United Arab Emirates, September 3-8, 2016.

Hepatitis E (HEV) has become the topic in transfusion medicine a few years ago since its occurence is increasing in developed countries. Therefore there is a risk of viral transmission by blood. Blood donors in Croatia are not routinely screened for HEV and there are no data regarding HEV seroprevalence in the general population. We also had no trace-back for HEV. Aims were to estimate the seroprevalence of HEV among voluntary blood donors (VBD) in the Croatian Institute of Transfusion Medicine (CITM). In October and November 2014 a total of 1036 serum samples of VBD were collected. All donors had previously completed the medical questionnaire to verify that they fulfilled the criteria for blood donation and had given informed consent. The study was approved by the Ethics Committee of CITM. There were 913 (88, 1%) males and 123 (11, 9%) females. All of the testing was done according to the producer’s instructions. Samples were primarily tested for total HEV antibodies using a commercial enzyme immunoassay, HEV Ab, Dia.Pro Srl, Milan, Italy (performed on Gemini analyzer). All reactive samples were tested for HEV IgG/IgM by comercial immunoassays, HEV IgG/IgM, Dia.Pro Srl, Milan, Italy (performed on Gemini analyzer). All HEV IgM reactive samples were confirmed by a comercial immunoblot assay, recomLine HEV IgM, Mikrogen, Neuried, Germany (performed on Dynablot Plus analyzer). IgM-positive samples were further tested for the presence of HEV RNA. Viral RNA extraction (QIAamp viral RNA Mini Kit®, Qiagen, Hilden, Germany) was performed from 140 µl of each sera sample. A real time RT-PCR protocol (Jothikumar et al., 2006) for detecting a highly conserved fragment within ORF3 was carried out. The amplification was done in a Rotor-Gene Q machine (Qiagen, Hilden, Germany) by the use of commercially available kits (Rotor-Gene Probe RT-PCR kit, Qiagen, Hilden, Germany) according to the producer’s instructions. 223/1036 (21, 5%) samples were reactive for total HEV antibodies. 210/223 were reactive for HEV IgG and overall anti-HEV IgG seroprevalence was 20, 3%. 45/223 samples were anti-HEV IgM reactive, 18/45 (1, 7%) were confirmed by immunoblot HEV IgM test and HEV RNA was not detected in any of those 18 samples. There is a significant association between age (less and more then 40) and a higher seroprevalence (P<0, 001). The difference between males and females was not significant (P=0, 807). Conclusion: We observed a high HEV IgG seroprevalence of 20, 3% among voluntary blood donors in comparison to the results of previous Croatian data (used Anti-hepatitis E virus ELISA test, Euroimun, Lubeck, Germany) for different population groups: alcohol abusers 8, 9%, war veterans 8, 6%, injecting drug users 6, 1% and healthcare professionals 2, 7%. Results suggest that most autochthonous HEV infections are either asymptomatic or unrecognized. The HEV seroprevalence studies done in developed countries show significant differences, from 0, 3% in Greece to 52, 5% in southwest France, depending on different HEV antibodies detection assays “Wenzel et al, The Journal of Infectious Diseases, 2013”. So, further study will be conducted that compare 3 different anti-HEV IgG/IgM tests.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes.Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests.

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection.

Gene Expression Profiling of Prion‐Infected Brains: a Novel Disease Signature for Neurodegeneration in Humans

ObjectivesIn a previous study (Barbisin et al. 2014), we identified a five‐gene signature able to distinguish BSE‐infected macaques (Macaca fascicularis) from healthy ones: in brief, hemoglobin beta (HBB) and alpha2 (HBA2) were down‐regulated in infected monkeys, whereas transthyretin (TTR), apolipoprotein C1 (APOC1) and serpin peptidase inhibitor clade A (SERPINA3) were up‐regulated. The aim of the present work is to investigate whether the gene dysregulation pattern observed in macaques is also a specific gene signature for Creutzfeldt– Jakob disease (CJD) in humans compared to both healthy subjects and other neurodegenerative diseases.MethodsWe collected about 120 samples from frontal cortex of frozen brain tissue, obtained from prion‐infected patients (variant (v)CJD, sporadic (s)CJD, iatrogenic (i)CJD), patients with non‐CJD neurodegenerative disorders (Alzheimer's Disease, Parkinson's Disease, tauopathies) and age‐matched controls). RNA was extracted using TRIzol with PureLink® RNA Mini Kit and on‐column DNase I digestion. Quantity, purity and integrity were checked and only samples with a RIN≥5 were included in the study. Reverse transcription was carried out using Superscript III and qPCR was performed for the selected transcripts, with ACTB, RPL19, GAPDH and B2M as reference genes. In addition, erythrocyte markers expression analysis was performed in order to exclude any relevant blood contamination.ResultsGene expression trend observed in macaques was confirmed in humans, with similar FC values, for four genes. HBA1/2 was down‐regulated in sCJD cases and also in patients affected by non‐CJD neurodegenerative diseases, while APOC1, TTR and SERPINA3 were up‐regulated in CJD patients, but not in patients affected by non‐CJD neurodegenerative diseases. IHC and WB analyses on related proteins are ongoing. We also found a consistent up‐regulation of SERPINA3, in both RML‐infected ScGT1 and ScN2a cell lines.ConclusionsBesides providing important hints on some prion‐specific alterations in CJD, these results suggest that various pathways, such as oxygen and iron homeostasis (HBB, HBA2) and in particular cholesterol and lipids metabolism (APOC1, SERPINA3, TTR) have to be taken into consideration to identifying potential biomarkers and drug targets for prion diseases and other neurodegenerative disorders.Support or Funding InformationFIRB Accordi di programma 2011 “genomica funzionale delle malattie neurodegenerative” (Prot. RBAP11FRE9_001)

IDENTIFIKASI MOLEKULER BROAD BEAN WILT VIRUS 2 (BBWV2) DAN CYMBIDIUM MOSAIC VIRUS (CYMMV) ASAL TANAMAN NILAM (POGOSTEMON CABLIN BENTH.)

Molecular identification Broad Bean Wilt Virus 2 (BBWV2) and Cymbidium Mosaic Virus (CymMV) from patchouli plant (Pogostemon cablin Benth.). Several viruses have been reported to be associated with mosaic disease on patchouli plant in Indonesia. This study aims to identify the two viruses in patchouli cultivation in West Java by studying the molecular characterization. Mosaic symptomatic leaf samples taken from patchouli cultivation in Manoko (Bandung Barat District, West Java Province). RNA extraction was performed using Xprep Plant RNA mini kit. RNA amplification with RT-PCR technique using primers for the cp gene region of BBWV2 and CymMV. The PCR product was sent to PT. Science Genetics Indonesia to do sequencing, then analyzed nucleotide sequences. Results of RT-PCR were performed successfully obtained DNA bands with size accordance with the predictions of the primer design for BBWV2 and CymMV cp region. Further, based on nucleotide and amino acid sequence analyses, the two virus isolates were confirmed as BBWV2 and CymMV respectively. Phylogenetic analyses revealed that BBWV2 Manoko clustered with BBWV2 from Singapore (original host of Brazilian red-cloak), China (pepper) and South Korea (chili). Whereas, CymMV Manoko become one cluster with CymMV from India (Phaius sp.), Indonesia (Dendrobium), China (vanilla), Thailand (Oncidium), Hawai (Dendrobium) and South Korea Cymbidium).

Identification of Concurrent Infection by Multiple Dengue Virus Serotypes During an Epidemic in 2011 in Pakistan

J Microbiol Exp 2016, 3(3): 00088 DENV-1 and DENV-2 [6]. In 1994 the first lab confirmed cases of DENV-2 was reported from Baluchistan Province [7]. The prevalence of dengue virus serotype 3 in Pakistan was detected during the 2005 dengue virus outbreak in Karachi [8]. The cocirculation of DENV-2 and DENV-3 was identified in 2006 during the outbreak of dengue virus in Karachi [9]. In 2008 the cocirculation of three serotypes DENV-2, DENV-3 and DENV-4 was also documented during the outbreak of dengue virus in Lahore [10]. Recently it is reported that the dominant serotypes of dengue virus circulating in Pakistan from 2007-2009 are DENV2 and DENV-3. The dengue is a major public health problem in Pakistan over the last 20-25 years. In last few years co-circulation of different dengue virus serotypes is identified with mixed infection. From Pakistan only one study in which they reported some cases of mixed infection with DENV-2 and DENV-3.In present study we for the first time reported many cases of mixed infection with DENV-1/DENV-2, DENV-1, DENV-2 and DENV-3 from different geographic areas of Pakistan. The laboratory detection of dengue virus infection is achieved by virus isolation in cell culture, Serologic diagnosis by detection of IgM/IgG antibodies and more sensitive and rapid technique is the Molecular detection. From August to October 2011 a total of 87 serum samples were received at the department of virology National Institute of Health Islamabad Pakistan during an outbreak. These samples were collected from suspected dengue virus cases along the complete history sheet including the date of onset, age; sex etc. All serum samples were stored at -70 until further processing. All serum samples were analyzed for the detection of dengue virus IgM antibodies by using IgM capture ELISA (Panbio Tech Co Brisbane, Australia) according to the instructions provided by the manufacturer. RNA was extracted from 140ul of ELISA Positive serum samples by using QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to manufacturer instructions. In the present study RNA samples were analyzed by the molecular detection by using the real time PCR assay. Real time PCR is a one step assay using Primer pair and Probes specific to dengue serotypes. In single plex reaction mixture 5ul of RNA template was added with 50pmol of each primer and 9pmol of probe in 50ul total iScript one-step RT-PCR kit for Probes. Each reaction tube contains primers and probe for only one dengue serotype. In this assay one RNA sample was tested against four dengue serotypes in four separate reactions tubes [11]. In this technique the use of fluorescent probe enables the detection of PCR reaction product without need of gel electrophoresis. Another advantage of this assay is the ability to determine viral load in the patient sample which is important to understand the severity of dengue infection [12].