Rapid Detection of Norovirus from Fresh Lettuce Using Immunomagnetic Separation and a Quantum Dots Assay

2013 ◽  
Vol 76 (4) ◽  
pp. 707-711 ◽  
Author(s):  
HEE-MIN LEE ◽  
JOSEPH KWON ◽  
JONG-SOON CHOI ◽  
KYEONG-HWAN LEE ◽  
SUNG YANG ◽  
...  
Keyword(s):  
Quantum Dots ◽  
High Speed ◽  
Virus Detection ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Pcr Analysis ◽  
Virus Concentration ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Centrifugation Step

Current molecular methods that include PCR have been used to detect norovirus in many food samples. However, the protocols require removing PCR inhibitors and incorporate time-consuming concentration steps to separate virus from analyte for rapid and sensitive detection of norovirus. We developed an immunomagnetic separation (IMS) and a quantum dots (QDs) assay to detect norovirus eluted from fresh lettuce with Tris buffer containing 1% beef extract (pH 9.5). IMS facilitated viral precipitation with a 10-min incubation, whereas virus concentration using polyethylene glycol (PEG) requires more than 3 h and an additional high-speed centrifugation step to precipitate virus before reverse transcription PCR (RT-PCR) analysis. The fluorescence intensity of QDs was detected qualitatively on norovirus dilutions of 10−1 to 10−3 in a stool suspension (100 RT-PCR units/ml). The results suggest that a fluorescence assay based on IMS and QDs is valid for detecting norovirus qualitatively according to fluorescent signal intensity within the same virus detection limit produced by IMS–RT-PCR and PEG–RT-PCR.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Reverse Transcription-PCR Analysis of the Regulation of the Manganese Peroxidase Gene Family

1998 ◽  
Vol 64 (2) ◽  
pp. 569-574 ◽  
Author(s):  
Jessica M. Gettemy ◽  
Biao Ma ◽  
Margaret Alic ◽  
Michael H. Gold
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
Manganese Peroxidase ◽  
Transcript Level ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Total Rna ◽  
Nitrogen Levels ◽  
Reverse Transcription Pcr ◽  
Low Levels

ABSTRACT Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequencedP. chrysosporium mnp genes, i.e., mnp1,mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 μM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of themnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures,mnp1 was the major expressed mnp gene.

scholarly journals Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

2020 ◽  
Author(s):  
Karina Helena Morais Cardozo ◽  
Adriana Lebkuchen ◽  
Guilherme Goncalves Okai ◽  
Rodrigo Andrade Schuch ◽  
Luciana Godoy Viana ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Low Cost ◽  
Large Population ◽  
Virus Detection ◽  
Standard Test ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fast Processing

Abstract The current outbreak of severe acute respiratory syndrome associated with coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

scholarly journals Expression of Smooth Muscle Calponin in Synovial Sarcoma

Sarcoma ◽  
1999 ◽  
Vol 3 (2) ◽  
pp. 107-113 ◽  
Author(s):  
Hidefumi Ono ◽  
Hideki Yoshikawa ◽  
Takafumi Ueda ◽  
Hisako Yamamura ◽  
Ikuo Kudawara ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Synovial Sarcoma ◽  
Gene Product ◽  
Actin Binding ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Sarcoma Cell ◽  
Reverse Transcription Pcr ◽  
Synovial Sarcomas ◽  
Sarcoma Cells

Purpose.Histogenesis of synovial sarcoma remains controversial and reliable molecular markers for diagnosis are necessary. Expression of basic calponin, a smooth muscle differentiation-specific actin-binding protein, was studied in synovial sarcoma.Subjects and Methods.The basic calponin gene and the gene product were analyzed by reverse transcription PCR analysis (RT-PCR) and immunohistochemistry in 14 synovial sarcomas and a human synovial sarcoma cell line (HS-SY-II).Results and Discussion.Immunoreactivity for basic calponin was detected in the cytoplasm of 6 synovial sarcomas (43% positive). In the basic calponin-positive tumors and the HS-SY-II cells, expression for smooth muscle-specific genes, including basic calponin and SM22α , was detected by RT-PCR, suggesting a lineage relationship between synovial sarcoma cells and smooth muscle-like mesenchymal cells.Conclusions.A subset of synovial sarcomas expressing the basic calponin gene and the gene product were identified. The basic calponin may have potential utility as a novel molecular marker identifying certain synovial sarcomas.

scholarly journals Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

2007 ◽  
Vol 189 (22) ◽  
pp. 8120-8129 ◽  
Author(s):  
Rosa Alduina ◽  
Luca Lo Piccolo ◽  
Davide D'Alia ◽  
Clelia Ferraro ◽  
Nina Gunnarsson ◽  
...  
Keyword(s):  
Response Regulator ◽  
Sequence Similarity ◽  
Streptomyces Griseus ◽  
Phosphate Limitation ◽  
Pcr Analysis ◽  
Binding Motif ◽  
Glycopeptide Antibiotics ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Key Steps

ABSTRACT The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His6-tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

Optimisation of the PEG reconcentration procedure for virus detection by cell culture or genomic amplification

1997 ◽  
Vol 35 (11-12) ◽  
pp. 455-459 ◽  
Author(s):  
Ph. Vilaginès ◽  
A. Suarez ◽  
B. Sarrette ◽  
R. Vilaginès
Keyword(s):  
Cell Culture ◽  
Virus Detection ◽  
Second Step ◽  
Virus Concentration ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Field Samples ◽  
Group A ◽  
Virus Recovery ◽  
Higher Sensitivity

A double reconcentration procedure was developed for virus detection in tapwater concentrates obtained by conventional adsorption-elution techniques suitable for cell inoculation as well as for genomic amplification. Using 7.5% PEG 6000 and 2.5% NaCl, a 15min contact time under agitation at room temperature followed by centrifugation (first step: 3,500xg, 90min, 4°C; second step 10,000xg, 20min, 4°C) were the conditions to obtain overall average virus recovery efficiencies of 71% for poliovirus from 900ml eluates and 88, 83 and 75% for poliovirus, coxsackie B2 and rotavirus respectively (400ml eluates). Direct extraction of viral RNA from the first PEG pellet with TrizolTM was efficient for RT-PCR assays without any further treatment. Primer pairs were selected to amplify rotavirus group A and poliovirus in seeded tapwater concentrated by adsorption elution through glass wool. A positive signal was obtained for theoretic virus concentration of 1 PFU. Analysis of field samples (1001) by cell culture and genomic amplification resulted in a higher sensitivity with the latter.

scholarly journals Comparison of a Reporter Assay and Immunomagnetic Separation Real-Time Reverse Transcription-PCR for the Detection of Enteroviruses in Seeded Environmental Water Samples

2007 ◽  
Vol 73 (7) ◽  
pp. 2338-2340 ◽  
Author(s):  
Yu-Chen Hwang ◽  
Oymon M. Leong ◽  
Wilfred Chen ◽  
Marylynn V. Yates
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Plaque Assay ◽  
Virus Detection ◽  
Resonance Energy ◽  
Environmental Water ◽  
Immunomagnetic Separation ◽  
Cell System ◽  
Separation Procedure ◽  
Reverse Transcription Pcr

ABSTRACT Two newly developed protocols for infective virus detection were compared to the plaque assay. An immunomagnetic separation procedure coupled with real-time reverse transcription-PCR of viral nucleic acids was developed to identify intact enteroviral particles, and a reporter cell system responding to viral replication based on fluorescent resonance energy transfer for detection of infectious enteroviruses was tested. Both new procedures detected infective viruses in environmental samples at the same level as the plaque assay.

scholarly journals Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

2014 ◽  
Vol 81 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Hiroaki Furumai
Keyword(s):  
Humic Acid ◽  
River Water ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Water Samples ◽  
Virus Detection ◽  
Virus Concentration ◽  
Organic Substances ◽  
Rt Pcr ◽  
River Water Samples

ABSTRACTReverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances.

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