Feasibility of Qualitative Testing of BCR-ABL and JAK2 V617F in Suspected Myeloproliperative Neoplasm (MPN) Using RT-PCR Reversed Dot Blot Hybridization (RT-PCR RDB)

2019 ◽  
Vol 19 (4) ◽  
pp. 220-227
Author(s):  
Najmiatul Masykura ◽  
Ummu Habibah ◽  
Siti Fatimah Selasih ◽  
Soegiarto Gani ◽  
Cosphiadi Irawan ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Jak2 V617f ◽  
Rt Pcr ◽  
Dot Blot ◽  
Dot Blot Hybridization

scholarly journals Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽  
1998 ◽  
Vol 82 (1) ◽  
pp. 84-88 ◽  
Author(s):  
R. H. Li ◽  
G. C. Wisler ◽  
H.-Y. Liu ◽  
J. E. Duffus
Keyword(s):  
Western Blot ◽  
Blot Hybridization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Plants ◽  
Dot Blot Hybridization ◽  
Field Samples ◽  
Infected Tomato ◽  
Tomato Infectious Chlorosis Virus

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

scholarly journals An Integrated Diagnostic Approach for Citrus Exocortis Viroid

2021 ◽  
Author(s):  
Chih-Hsu Lin ◽  
Ting-Hsuan Hung ◽  
I Hu ◽  
Ta-Hsin Ku ◽  
Chun-Yi Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Severe Disease ◽  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Cultivar ◽  
Dot Blot Hybridization ◽  
Reverse Transcription Pcr ◽  
One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

scholarly journals Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248581
Author(s):  
Clyde S. Manuel ◽  
Cassandra Suther ◽  
Matthew D. Moore ◽  
Lee-Ann Jaykus
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Blot Hybridization ◽  
Molecular Techniques ◽  
Rt Pcr ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
One Step

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.

scholarly journals RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses

2001 ◽  
Vol 26 (2) ◽  
pp. 170-175 ◽  
Author(s):  
MARCELO EIRAS ◽  
RENATO O. RESENDE ◽  
ALEXANDRE A. MISSIAGGIA ◽  
ANTÔNIO C. DE ÁVILA
Keyword(s):  
Blot Hybridization ◽  
Impatiens Necrotic Spot Virus ◽  
Virus Species ◽  
Rt Pcr ◽  
Dot Blot ◽  
Spot Virus ◽  
Dot Blot Hybridization ◽  
Tospovirus Species ◽  
Universal Detection

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.

Utility of RT-PCR-based Dot-blot Hybridization for Detecting and Genotyping Echoviruses

2007 ◽  
Vol 37 (3) ◽  
pp. 153 ◽  
Author(s):  
Ahyoun Kim ◽  
Wooyoung Choi ◽  
Yoonseok Chung ◽  
Kisoon Kim ◽  
Youngmee Jee ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Dot Blot Hybridization

Myogenin and oxidative enzyme gene expression levels are elevated in rat soleus muscles after endurance training

2004 ◽  
Vol 97 (1) ◽  
pp. 277-285 ◽  
Author(s):  
Parco M. Siu ◽  
David A. Donley ◽  
Randall W. Bryner ◽  
Stephen E. Alway
Keyword(s):  
Enzymatic Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Blot Hybridization ◽  
Mitochondrial Enzymes ◽  
Rt Pcr ◽  
Dot Blot ◽  
Expression Levels ◽  
Dot Blot Hybridization ◽  
Mrna Content

The intent of this study was to determine whether endurance exercise training regulates increases in metabolic enzymes, which parallel modulations of myogenin and MyoD in skeletal muscle of rats. Adult Sprague-Dawley rats were endurance trained (TR) 5 days weekly for 8 wk on a motorized treadmill. They were killed 48 h after their last bout of exercise. Sedentary control (Con) rats were killed at the same time as TR animals. Myogenin, MyoD, citrate synthase (CS), cytochrome- c oxidase (COX) subunits II and VI, lactate dehydrogenase (LDH), and myosin light chain mRNA contents were determined in soleus muscles by using RT-PCR. Myogenin mRNA content was also estimated by using dot-blot hybridization. Protein expression levels of myogenin and MyoD were measured by Western blots. CS enzymatic activity was also measured. RT-PCR measurements showed that the mRNA contents of myogenin, CS, COX II, COX VI, and LDH were 25, 20, 17, 16, and 18% greater, respectively, in TR animals compared with Con animals ( P < 0.05). The ratio of myogenin to MyoD mRNA content estimated by RT-PCR in TR animals was 28% higher than that in Con animals ( P < 0.05). Myosin light chain expression was similar in Con and TR muscles. Results from dot-blot hybridization to a riboprobe further confirmed the increase in myogenin mRNA level in TR group. Western blot analysis indicated a 24% greater level of myogenin protein in TR animals compared with Con animals ( P < 0.01). The soleus muscles from TR animals had a 25% greater CS enzymatic activity than the Con animals ( P < 0.01). Moreover, myogenin mRNA and protein contents were positively correlated to CS activity and mRNA contents of CS, COX II, and COX VI ( P < 0.05). These data are consistent with the hypothesis that myogenin is in the pathway for exercise-induced changes in mitochondrial enzymes.

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