RT-PCR Based Detection of Rupestris stem pitting associated virus Within Field-Grown Grapevines Throughout the Year

The presence of Rupestris stem pitting associated virus (RSPaV) can go unnoticed since symptoms appear only if additional viruses are present. Detection by reverse transcription-polymerase chain reaction (RT-PCR) is possible; however, this assay could be unreliable if the tissue that is being tested has detection-interfering compounds, or if the virus has a low titer. This paper reports on (i) use of a recently developed extraction method and internal control to determine which tissues from field-grown grapevines yield extracts that are reliable for virus detection by RT-PCR, and (ii) a survey for RSPaV of different tissues from the Vitis vinifera varieties Riesling, Chardonnay, Cabernet Franc, Merlot, Sauvignon Blanc, Pinot Noir, and Gamay, as well as from the rootstocks 3309 and Riparia, which were harvested in Ontario, Canada, at different times of the year. Amplifiable extracts were obtained from virtually all bud, shoot tip, seed, and cane samples tested. Detectable amounts of RSPaV were generally found in all tissues of infected plants except young buds collected in the summer. A combination of three single buds from dormant canes, less time-consuming than the preparation of cane shavings, was a reliable source for RSPaV detection.

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Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48726.14977 ◽

2021 ◽

Author(s):

Rajeev Kumar Jain ◽

Nagaraj Perumal ◽

Rakesh Shrivastava ◽

Kamlesh Kumar Ahirwar ◽

Jaya Lalwani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

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Updating the Quarantine Status of Prunus Infecting Viruses in Australia

Viruses ◽

10.3390/v12020246 ◽

2020 ◽

Vol 12 (2) ◽

pp. 246 ◽

Cited By ~ 1

Author(s):

Wycliff M. Kinoti ◽

Narelle Nancarrow ◽

Alison Dann ◽

Brendan C. Rodoni ◽

Fiona E. Constable

Keyword(s):

Polymerase Chain Reaction ◽

High Throughput Sequencing ◽

Mottle Virus ◽

Rt Pcr ◽

Chain Reaction ◽

Virus Family ◽

First Report ◽

Hop Stunt Viroid ◽

Polymerase Chain ◽

Stem Pitting

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

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First Report of Rupestris stem pitting associated virus in Argentina

Plant Disease ◽

10.1094/pdis.2002.86.8.921b ◽

2002 ◽

Vol 86 (8) ◽

pp. 921-921 ◽

Cited By ~ 2

Author(s):

C. G. Tarnowski ◽

P. A. Worlock ◽

S. Ulanovsky ◽

S. Gómez Talquenca

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Polymerase Chain Reaction Method ◽

Vitis Vinifera L ◽

Chain Reaction ◽

First Report ◽

Cornell University ◽

Polymerase Chain ◽

Rupestris Stem Pitting ◽

Stem Pitting

Rupestris stem pitting associated virus (RSPaV), a component of the rugose wood complex (RWC), is a worldwide graft transmissible disease of grapevines (Vitis vinifera L.). RSPaV has a single-stranded 8,726-nt RNA genome, belongs to the genus Foveavirus, and is often associated with Rupestris stem pitting (RSP) disease (2). In 1995, a grapevine sanitary selection program was implemented in Mendoza to investigate this and other grapevine viral diseases. RSP can be diagnosed when V. rupestris cv. St. George is used as a woody indicator for biological indexing. Chip-bud inoculated St. George plants developed a row of small pits and grooves on the wood cylinder below the graft or around and below the inoculated point (1,2). After three seasons in the field, 15 accessions with RSP wood markings were observed. Mature leaves and bark shavings were extracted, partially purified, and analyzed by a onestep reverse transcription polymerase chain reaction method. The expected 339-bp band was found in only six of the positively indexed samples using the specific 13/14 primer pair (2). Other viruses associated with RWC have been detected in Argentina, but to our knowledge, this is the first report of RSPaV. References: (1) A. C. Goheen. Page 53 in: Compendium of Grape Diseases, R. C. Pearson and A. C. Goheen, eds. American Phytopatological Society, St. Paul, MN, 1988. (2) B. Meng. Rupestris stem pitting: Insights on etiology and development of reverse transcription-polymerase chain reaction and immunoassays for diagnosis. Ph.D. Diss. Cornell University, Ithaca, NY, 1999.

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Detecção e caracterização biológica e molecular de Rupestris stem-pitting associated virus e seu efeito na fotossíntese de videiras

Fitopatologia Brasileira ◽

10.1590/s0100-41582004000200016 ◽

2004 ◽

Vol 29 (2) ◽

pp. 209-214 ◽

Cited By ~ 3

Author(s):

Thor V.M. Fajardo ◽

Marcelo Eiras ◽

Henrique P. Santos ◽

Osmar Nickel ◽

Gilmar B. Kuhn

Keyword(s):

Rio Grande ◽

Rio Grande Do Sul ◽

Rt Pcr ◽

Cabernet Franc ◽

Rupestris Stem Pitting ◽

Stem Pitting ◽

Vitis Spp

Rupestris stem pitting associated virus (RSPaV) é o agente causal das "caneluras do tronco de Rupestris" da videira (Vitis spp.). Neste trabalho, um isolado de RSPaV, encontrado em videiras cv. Cabernet Franc no Rio Grande do Sul, foi estudado. O vírus foi detectado biologicamente, por enxertia em videira indicadora cv. Rupestris du Lot, em 26,2% das amostras avaliadas. A seqüência parcial do gene da replicase do RSPaV, isolado sul-brasileiro, com 831 nucleotídeos amplificados por RT-PCR e 276 aminoácidos deduzidos, apresentou maior identidade de nucleotídeos (98,1%) e aminoácidos deduzidos (99,6%), com dois isolados norte-americanos. O RSPaV estudado apresentou baixa homologia (37-41%) com outros vírus do gênero Foveavirus. A maioria das mudas de videira cv. C. Franc infetadas com RSPaV apresentou diminuição no potencial fotossintético (2,68 a 5,12 vezes) e aumento na taxa respiratória no escuro quando comparadas a mudas sadias, salientando os impactos que esse vírus pode proporcionar no potencial produtivo de videiras.

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Development and evaluation of a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control

Journal of Virological Methods ◽

10.1016/j.jviromet.2003.10.009 ◽

2004 ◽

Vol 116 (1) ◽

pp. 45-54 ◽

Cited By ~ 15

Author(s):

Sidhartha Hazari ◽

Subrat Kumar Acharya ◽

Subrat Kumar Panda

Keyword(s):

Polymerase Chain Reaction ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Internal Control ◽

Reverse Transcription ◽

Rt Pcr ◽

Hepatitis C Virus Rna ◽

Chain Reaction ◽

Virus Rna ◽

Polymerase Chain

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Real-time reverse transcription-polymerase chain reaction for detection of SYT-SSX translocation in synovial sarcoma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.9553 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 9553-9553

Author(s):

J. Liu ◽

K. Qu ◽

C. Chai ◽

H. Li ◽

A. Sferruzza ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Synovial Sarcoma ◽

Internal Control ◽

Reverse Transcription ◽

Gel Electrophoresis ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Polymerase Chain

9553 Background: Synovial sarcoma is the most common non-rhabdomyosarcomatous soft tissue sarcoma in children and adolescents. A specific translocation, t(X;18), induces fusion of the SYT gene on chromosome 18 to an SSX gene on chromosome X. The resulting fusion gene consists of at least 2 subtypes with different breakpoints: SYT-SSX1(X;18)(p11.23;q11.2) and SYT-SSX2 (X;18)(p11.21;q11.2). Because t(X;18) transcripts occur in >90% of synovial sarcoma subtypes, this marker may be useful for diagnosis. We evaluated the accuracy of a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of the primary SYT-SSX fusion transcript types in formalin-fixed, paraffin-embedded (FFPE) tissues and frozen tissues. Methods: 17 tumors (7 synovial sarcomas, 4 Ewing’s sarcomas, 5 rhabdomyosarcomas, 1 small round blue-cell tumor), 4 normal tissues, and 4 control samples were tested for SYT-SSX translocations using real-time RT-PCR. Results were compared to those obtained with gel electrophoresis detection of amplified transcripts; discrepant results were confirmed by sequencing. Results: Concordance between real time RT-PCR and gel electrophoresis was 100% (25/25) for internal control genes and SYT-SSX1, and 92% (23/25) for SYT-SSX2. Of the 2 samples with discordant SYT-SSX2 results, 1 was positive by real-time RT-PCR but not gel electrophoresis and 1 was positive by electrophoresis but not real-time RT-PCR; in both cases, DNA sequencing confirmed the real-time RT-PCR results. The minimum percentage of tumor to normal cells required for detection of SYT-SSX fusion transcripts by real-time RT-PCR was 6.25%. Conclusions: This real-time RT-PCR assay appears to provide greater accuracy than gel electrophoresis for identification of SYT-SSX translocation and fusion types. No significant financial relationships to disclose.

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Development of PCR Methods for Enteric Virus Detection in Water

Water Science & Technology ◽

10.2166/wst.1993.0348 ◽

1993 ◽

Vol 27 (3-4) ◽

pp. 211-218 ◽

Cited By ~ 17

Author(s):

K. J. Schwab ◽

R. De Leon ◽

M. D. Sobsey

Keyword(s):

Hepatitis A ◽

Hepatitis A Virus ◽

Detection System ◽

Virus Detection ◽

Enteric Virus ◽

Rt Pcr ◽

Chain Reaction ◽

Sample Concentration ◽

Spin Column ◽

Polymerase Chain

This study developed a reverse transcriptase-polymerase chain reaction (RT-PCR) detection system for enteric viruses in sample concentrates obtained by conventional filter adsorption-elution methods. One liter beef extract (BE)-glycine (G) eluant seeded with poliovirus 1 and hepatitis A virus (HAV) was used as a model system and the eluant further processed for RT-PCR compatibility. Sample concentration and purification procedures consisted of polyethylene glycol (PEG) precipitation, Pro-Cipitate (Affinity Technology, New Brunswick, NJ) precipitation, a second PEG precipitation, spin-column chromatography, and ultrafiltration. Sample volumes are reduced from 1 L to 20-40 µL and purified sufficiently for viral detection by RT-PCR. As little as 3 PFU of poliovirus 1 in an initial 1 L eluate were detected by RT-PCR.

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Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR

Arquivos de Neuro-Psiquiatria ◽

10.1590/0004-282x20160119 ◽

2016 ◽

Vol 74 (10) ◽

pp. 810-815 ◽

Cited By ~ 5

Author(s):

Sérgio Monteiro de Almeida ◽

Sônia Mara Raboni ◽

Meri Bordignon Nogueira ◽

Luine R. Renaud Vidal

Keyword(s):

Polymerase Chain Reaction ◽

Cerebrospinal Fluid ◽

Red Blood Cells ◽

Blood Cells ◽

Virus Detection ◽

Inhibitory Factor ◽

Rt Pcr ◽

Chain Reaction ◽

Negative Results ◽

Polymerase Chain

ABSTRACT The presence of hemoglobin in samples are considered an important inhibitory factor for polymerase chain reaction (PCR). The aim of this study was to examine the influence of red blood cells (RBC)s in cerebrospinal fluid (CSF) as an inhibitory factor to reverse transcription polymerase chain reaction (RT-PCR) for enteroviruses (EV). Forty-four CSF samples from patients showing characteristics of viral meningitis were assessed for EV by RT-PCR. Viral RNA extracted with guanidine isothyocianate buffer and virus detection was performed by in-house nested PCR. Positivity for EV RT-PCR was higher in CSF samples without RBCs than in samples with RBCs: 13(26%) and 36(9.2%), p = 0.001. In the group with positive EV RT-PCR, the mean + SD CSF RBC was 37 ± 183 cell/mm3; the group with negative results had 580 + 2,890 cell/mm3 (p = 0.007). The acceptable upper limit for CSF RBCs that could not influence RT-PCR was 108 cells/mm3. CSF samples with negative results for EV RT-PCR have more erythrocytes.

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Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control

Phytopathology ◽

10.1094/phyto-96-1223 ◽

2006 ◽

Vol 96 (11) ◽

pp. 1223-1229 ◽

Cited By ~ 99

Author(s):

Giorgio Gambino ◽

Ivana Gribaudo

Keyword(s):

Polymerase Chain Reaction ◽

Internal Control ◽

Reverse Transcription ◽

Rna Extraction ◽

Simultaneous Detection ◽

Grapevine Virus ◽

Rt Pcr ◽

Chain Reaction ◽

Arabis Mosaic Virus ◽

Polymerase Chain

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10-3 or 10-4 extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

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