One-Step Reverse-Transcription PCR on a High-Throughput Micro-Fluidic Device

ASME 2009 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2009-206623 ◽

2009 ◽

Author(s):

Paul Fleming ◽

Tara Dalton

Keyword(s):

High Throughput ◽

Reverse Transcription ◽

Continuous Flow ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Cell Lysates ◽

Thermal Region ◽

One Step ◽

The One

One step reverse-transcription polymerase chain reaction (RT-PCR) assays are an attractive option for further automating gene detection assays. One-step assays can reduce hands–on-time and the risk of sample crossover and contamination. The one-step chemistries are showing increasing use in virus detection and have been reported, in some cases, to be more appropriate than their two-step counterparts [1, 2]. Previous work presented by the Stokes Institute research group outlined a micro fluidic based continuous flow instrument which performed high throughput qPCR in nanolitre sized droplets [3]. This instrument had advantages over commercially available instruments in that it could process far more than the traditional 96 or 384 reaction setup in a single run and the reaction volume was reduced from 20–50 μl down to 30–100 nl sized droplets. Combining one-step chemistry with the technology offered by the devices being developed would lead to a high-throughput RNA-to-signal system capable of reverse transcribing and performing PCR on thousands of nanolitre sized reactions every day. It is envisaged that this technology will also lead to gene expression from single cells contained in nanolitre sized droplets. In this paper, a study was conducted in which an extra thermal region, manufactured from aluminium, was added to the existing continuous flow instruments. This region was maintained at a temperature suitable for reverse transcription, which was 48°C for the one-step kit tested. The thermal region was also a suitable length to maintain the sample at the required temperature for 15 minutes. Using a commercially available one step RT-PCR kit (TaqMan® RNA-to-CT™ 1-Step Kit, 4392653), the device was evaluated for its potential to perform one-step RT-PCR in continuously flowing nanolitre sized droplets. Electrophoresis gels were initially used in assessing specific amplification before an end-point detection method was utilized. RNA was extracted from the leukemic REH cell line with the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the gene of interest. To investigate the possibility of further reducing sample preparation and facilitating further automation, amplification from cell lysates without nucleic acid extraction was carried out on the device. Cell lysates were prepared using the cell lysis buffer from the TaqMan® Gene Expression Cells-to-CT™ Kit (Cat #AM1728). It was found that the device was successful in one-step RT-PCR from extracted RNA samples and samples from cell lysates without nucleic acid extraction.

Download Full-text

Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Download Full-text

Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4618-4627.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4618-4627 ◽

Cited By ~ 40

Author(s):

Carola Burtscher ◽

Stefan Wuertz

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Nucleic Acid ◽

Pathogenic Bacteria ◽

Organic Waste ◽

Detection Methods ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

One Step

ABSTRACT A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37°C and 24 h in Rappaport Vassiliadis medium at 43°C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples that tested positive when classical isolation procedures were followed. The study shows that selective two-step enrichment is useful when very low numbers of bacterial pathogens must be detected in organic waste materials, such as biosolids. There were no false-positive results derived from DNA of dead cells in the waste sample, suggesting that it is not necessary to perform RT-PCR analyses when PCR is combined with selective enrichment. Large numbers of added nontarget bacteria did not affect detection of Salmonella spp., L. monocytogenes, and Y. enterocolitica but increased the detection limit of Staphylococcus aureus from <10 to 104 CFU/g of organic waste. Overall, the detection methods developed using seeded organic waste samples from one waste treatment facility (WTF) needed to be modified for satisfactory detection of pathogens in samples from other WTFs, emphasizing the need for extensive field testing of laboratory-derived PCR protocols. A survey of 13 WTFs in Germany revealed that all facilities complied with the German Biowaste Ordinance, which mandates that the end product after anaerobic digestion or aerobic composting be free of Salmonella. In addition, all biosolids were free of L. monocytogenes, Staphylococcus aureus, and Y. enterocolitica, as evidenced by both classical and PCR-based detection methods.

Download Full-text

Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates

Clinical Chemistry ◽

10.1093/clinchem/44.11.2256 ◽

1998 ◽

Vol 44 (11) ◽

pp. 2256-2263 ◽

Cited By ~ 13

Author(s):

Yohei Hamaguchi ◽

Yoshimasa Aso ◽

Hiroshi Shimada ◽

Masato Mitsuhashi

Keyword(s):

Reverse Transcription ◽

Basic Research ◽

Rt Pcr ◽

Molecular Toxicology ◽

Cell Lysates ◽

Reverse Transcription Pcr ◽

Immobilized Oligonucleotides ◽

One Step ◽

Primer Sets ◽

Research Drug

Abstract To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured ∼40–55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5′ end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.

Download Full-text

One-Step Release of DNA Rings From Polyoma Virions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009991x ◽

1968 ◽

Vol 26 ◽

pp. 34-35

Author(s):

C. Vasquez ◽

A. K. Kleinschmidt

Keyword(s):

Nucleic Acid ◽

Room Temperature ◽

Sodium Perchlorate ◽

Length Distribution ◽

Uranyl Acetate ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

One Step ◽

Protein Monolayer ◽

The One

A tightly packed regularly coiled DNA ring is assumed to be present inside the polyoma virion. Applying the “one-step” technique of nucleic acid extraction from virions while being adsorbed to a protein monolayer, we studied the configurational changes of the DNA.Intact polyoma virions were mixed with DFP-trypsin (100μg/ml) and spread onto a subphase of a mixture of urea and sodium perchlorate (6M and 2M, or 3M and 1M respectively), at pH 8.2 and room temperature. After spreading the film, aliquots were transferred to grids at different periods of time, and the DNA stained by acetonic uranyl acetate.The length distribution of the DNA molecules measured from electron micrographs is shown in Fig. 1. The number of twists present at different times after spreading were also counted. At 12 minutes, with 3M urea and 1M sodium perchlorate in the subphase, the DNA was found to be 85% supertwisted (Fig. 2); 12% was opened to rings, and 3% existed as linear filaments.

Download Full-text

NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications

Plant Methods ◽

10.1186/1746-4811-6-3 ◽

2010 ◽

Vol 6 (1) ◽

pp. 3 ◽

Cited By ~ 24

Author(s):

Yellamaraju Sreelakshmi ◽

Soni Gupta ◽

Reddaiah Bodanapu ◽

Vineeta Chauhan ◽

Mickey Hanjabam ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Acid Extraction ◽

Reverse Genetic ◽

Nucleic Acid Extraction ◽

Simplified Procedure

Download Full-text

Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.579432 ◽

2020 ◽

Vol 7 ◽

Author(s):

Jun Ji ◽

Qinxi Chen ◽

Zhengli Yu ◽

Xin Xu ◽

Xinhao Mu ◽

...

Keyword(s):

Reverse Transcription ◽

Cost Effective ◽

Pcr Primers ◽

Probit Regression ◽

Conventional Pcr ◽

Specific Detection ◽

Isothermal Method ◽

Naked Eye ◽

One Step ◽

The One

In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/μL at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.

Download Full-text

Detection of Cherry leaf roll virus and Strawberry latent ring spot virus by one-step RT-PCR

Plant Protection Science ◽

10.17221/3/2009-pps ◽

2009 ◽

Vol 45 (No. 4) ◽

pp. 140-143 ◽

Cited By ~ 2

Author(s):

S. Kumari

Keyword(s):

Leaf Roll ◽

Rt Pcr ◽

Spot Virus ◽

Cherry Leaf Roll Virus ◽

One Step ◽

Polymerase Chain ◽

The One ◽

Ring Spot ◽

Screen House ◽

Xiphinema Diversicaudatum

A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed and used for the detection of Cherry leaf roll virus (CLRV) and Strawberry latent ring spot virus (SLRSV). The protocol was used to test infected screen house plants and also plants from orchards and vineyards where the vector (Xiphinema diversicaudatum) of SLRSV was detected from the soil. The one-step RT-PCR protocol is rapid and sensitive and has the potential to be used for the diagnosis of CLRV and SLRSV in routine diagnostic laboratories.

Download Full-text

Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870902100513 ◽

2009 ◽

Vol 21 (5) ◽

pp. 679-683 ◽

Cited By ~ 4

Author(s):

Pamela J. Ferro ◽

Jason Osterstock ◽

Bo Norby ◽

Geoffrey T. Fosgate ◽

Blanca Lupiani

Keyword(s):

Polymerase Chain Reaction ◽

Avian Influenza ◽

Real Time ◽

High Throughput ◽

Reverse Transcription ◽

Virus Isolation ◽

Rt Pcr ◽

Chain Reaction ◽

Response Planning ◽

Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

Download Full-text