Commercial RNA Extraction Kits Research Articles

Frequency of virus in some Diplodia pinea and Gremmeniella abietina isolates originated from Turkey

Diplodia pinea and Gremmeniella abietina are common pathogens causing shoot blight and dieback of pine all over the world. D. pinea is one of the main causal agents of shoot blight of Calabrian pines in the Mediterranean countries including Turkey. G. abietina has been recently observed on saplings and seedlings of Pinus nigra, which remain under snow cover during winter dormancy in Dedegul Mountain in the Mediterranean Region of Turkey. The presence of viruses in fungi has been known for many years. An accumulating number of cloned and sequenced viral genomes have enabled us to detect virus in increasing number of fungal species in the recent years. D. pinea and G. abietina are known to contain members of the virus families Narnaviridae, Totiviridae and Partitiviridae, which can infect single fungal isolates. Viral dispersal in fungi mainly occurs via anastomosis. Some Diplodia and G. abietina isolates have different characteristics, such as reduced virulence and growth rate, lack of pigmentation, altered colony morphology, and reduction in conidial production due to presence of viral particles. In this study, 18 D. pinea and 6 G. abietina isolates were investigated for the presence of dsRNA. Double-stranded RNA was isolated using a commercial RNA extraction kit and visualized in agarose gel electrophoresis. Isolates containing dsRNA were also investigated for their in vitro growth rate and ability to produce conidia. Three (50%) G. abietina and ten (56%) D. pinea isolates contained dsRNA that had an approximate molecular size of 1.6 kb. Key words: Scleroderris canker, calabrian pine, dsRNA.

蛍光RT-multiplex PCR法を用いた食中毒起因微生物の包括的検出

We have developed a reverse transcription (RT)-multiplex PCR assay with fluorescent dye-labeled primers for detection of 12 pathogens, including 9 bacteria and 3 viruses, that are highly associated with food-borne gastroenteritis outbreaks. This assay has a great advantage of comprehensive detection of bacterial DNAs and viral RNAs. PCR products of pathogens are easily discriminated by fluorescent color and fragment size visualized under ultraviolet light without staining, after electrophoresis. Simultaneous extraction of bacterial DNAs and viral RNAs were achieved using a commercial viral RNA extraction kit with some modification. After RT reaction, multiplex PCR was carried out with 3 primer sets (A, B, and C) labeled with 3 to 4 different colors of fluorescent dyes. Primer set A was designed for diarrheagenic Escherichia coli. Primer set B was for Salmonella spp., Campylobacter jejuni and C. coli, and Vibrio parahaemolyticus. Primer set C was for Clostridium perfringens, Norovirus, Sapovirus, and Astrovirus. Upon analysis of 45 food-borne gastroenteritis outbreaks and 15 sporadic cases, this assay had nearly corresponding sensitivity and specificity compared with conventional methods, such as bacterial cultivation and monoplex PCR. This assay can be completed within 6 to 7 hours and is considered effective for rapid screening of food-borne bacteria and viruses.

Comparison of methods for improved RNA extraction from blood for early detection of Classical swine fever virus by real-time reverse transcription polymerase chain reaction

Classical swine fever (CSF) is a highly contagious disease of pigs. Early detection of the Classical swine fever virus (CSFV) in infected animals and routine surveillance is important for a rapid response and control of an outbreak of CSF. The current study investigated whole blood as a clinical specimen for the detection of CSFV by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in experimentally infected pigs. The virus was detectable in pre-clinical animals in whole blood and in different fractions of blood, including white blood cells, red blood cells (RBC), and serum. Based on an in-vitro binding assay, CSFV is retained in the RBC fraction. Naturally occurring PCR inhibitors of whole blood were shown to inhibit detection, and several commercial RNA extraction kits failed to remove these inhibitors. The commercial blood RNA extraction protocols were modified to achieve optimized single tube and high-throughput 96-well plate RNA extraction that efficiently removed PCR inhibitors from whole blood and enhanced detection of CSFV in experimentally inoculated pigs. This enabled detection 1-2 days earlier than observed using unmodified RNA extraction protocols. The results show effective use of whole blood as a clinical specimen for diagnosis and surveillance of CSF in pre-clinical animals.

Evaluation of different RNA-extraction kits for sensitive detection of Hepatitis A virus in strawberry samples

The efficiency of different commercial RNA extraction kits for the detection of hepatitis A virus (HAV) from seeded strawberry samples was assessed by standard RT-PCR and real time RT-PCR (RT-qPCR). The best results with standard RT-PCR were achieved with Aurum™ Total RNA extraction kit (BioRad), obtaining a detection limit of 5–6.25 pfu/mg of tissue. A slightly lower sensitivity was rendered by the RNeasy ® Plant mini kit (Qiagen) (10–12.5 pfu/mg tissue), while the Total Quick RNA Cells and Tissues kit version mini (Talent) rendered a detection limit of 50–100 pfu/mg of tissue. The other tested commercial kits showed worse detection limits (>500 pfu/mg). With RT-qPCR and ten fold diluted RNA all the kits showed an increase of sensitivity, detecting the kits from Qiagen, Talent and BioRad down to 0.05 pfu/mg of strawberry homogenate. These findings indicate that the use of Aurum™ Total RNA extraction kit, with standard RT-PCR technique or RT-qPCR, can not only be labor and time saving but also helpful to improve the sensitivity for the HAV detection from fruits and to facilitate the standardization of detection methods among laboratories.

Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells

BackgroundThe isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix.FindingsWe evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR.ConclusionsWe showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.

Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR)

RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1–0.2ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

BackgroundRNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis.FindingsA protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds.ConclusionsThis is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.

Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil

Nucleic acid extraction is a critical step in the detection of an unknown biological agent. However, success can vary depending on the isolation and identification methods chosen and the difficulty of extraction from environmental matrices. In this work, bacteriophage MS2 RNA was extracted from three soil matrices, sand, clay, and loam, using five commercially available kits: the PowerSoil™ Total RNA Isolation, E.Z.N.A. ® Soil RNA, FastRNA ® Pro Soil-Direct, FastRNA ® Pro Soil-Indirect, and IT 1-2-3 Platinum Path™ kits. Success of the isolation was determined using an MS2-specific RT-PCR assay. The reproducibility and sensitivity of each method in the hands of both experienced and novice users were assessed and compared. Cost, operator time, and storage conditions were also considered in the evaluation. The RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A. ® Soil RNA kit for sand, the IT 1-2-3 Platinum Path™ Sample Purification kit for clay, and the FastRNA ® Pro Soil-Indirect kit for loam. However, if time and storage conditions are important considerations, the IT 1-2-3 Platinum Path™ kit may be appropriate for use with all soils since the kit has the shortest processing time and fewest temperature requirements.

Removal of Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Inhibitors Associated with Cloacal Swab Samples and Tissues for Improved Diagnosis of Avian Influenza Virus by RT-PCR

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion MagMAX, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5-3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.

Gene expression analysis of osteoblasts seeded in coral scaffold

Coral matrix of Porites sp. has the suitable properties for bone cell growth. This study was aimed to study the gene expression levels of osteoblast specific genetic markers; RUNX2, osteopontin, alkaline phosphatase and osteocalcin from osteoblasts seeded in coral scaffold, which are important in determining the feasibility of osteoblasts. Human osteoblasts were inoculated onto the processed coral in Dulbecco's Minimum Essential Medium. The cells were trypsinized on day 1, 7, 14, 18, and 21 and added with RNALater for preservation of RNA in cells. The RNA was extracted using commercial RNA extraction kit and the respective genes were amplified using RT-PCR kit and analyzed qualitatively on 1.5% agarose gel. The expressions were evaluated with the Integrated Density Value based on the intensity of band for different periods of cell harvest. Increased expressions of the RUNX2, osteopontin, alkaline phosphatase and osteocalcin genes in the present study proved that coral is a favorable carrier for osteogenetically competent cells to attach and remain viable.

An integrated microfluidic system using magnetic beads for virus detection

An integrated system capable of sample pretreatment using antibody-conjugated magnetic beads and one-step reverse transcriptase–polymerase chain reaction (RT-PCR) on a microfluidic system was developed to accelerate the detection of RNA viruses such as dengue virus or enterovirus 71. The targeted virus in the sample was first captured by the specific antibody-conjugated magnetic beads, which were manipulated by micro-electromagnets made of micro-electro-mechanical systems technology. The RNA of the targeted virus then underwent thermal lysis and was reverse-transcripted to cDNA using a μRT-PCR module. The sensitivity to detect dengue virus is around 10–100 PFU, which is equivalent to the commercial RNA extraction kit and a large-scale RT-PCR machine. This microsystem can specifically detect 4 serotypes of dengue virus, as well as enterovirus 71. The specificity was warranted by both antibody and primer. The microfluidic system allows automatic process of sample including mixing, incubation, and reaction. The antibody-conjugated magnetic beads offer sample pretreatment of purification and concentration. The integration of antibody-conjugated magnetic beads into the microfluidic system is promising for fast molecular diagnosis of microorganisms.

Improvement of RT-PCR Sensitivity for Fruit Tree Viruses by Small-scale dsRNA Extraction and Sodium Sulfite

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

Bacterial DNA decontamination for reverse transcription polymerase chain reaction (RT-PCR)

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 μg of nucleic acid using 10 U of RNase-free DNase I in 10-μl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.