scholarly journals The use of Armored RNA as a multi-purpose internal control for RT-PCR

2008 ◽  
Vol 150 (1-2) ◽  
pp. 73-76 ◽  
Author(s):  
Jeffery Stevenson ◽  
Weston Hymas ◽  
David Hillyard
Keyword(s):  
Internal Control ◽  
Rt Pcr ◽  
Armored Rna

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

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.

scholarly journals Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

2010 ◽  
Vol 169 (2) ◽  
pp. 375-379 ◽  
Author(s):  
Karen Miyuki Asano ◽  
Sibele Pinheiro de Souza ◽  
Iracema Nunes de Barros ◽  
Giselle Razera Ayres ◽  
Sheila Oliveira Souza Silva ◽  
...  
Keyword(s):  
Internal Control ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Bovine Coronavirus ◽  
Group A Rotavirus ◽  
Group A ◽  
Exogenous Internal Control

scholarly journals Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control

Plant Disease ◽  
2006 ◽  
Vol 90 (2) ◽  
pp. 185-189 ◽  
Author(s):  
ZhiYou Du ◽  
JiShuang Chen ◽  
Chuji Hiruki
Keyword(s):  
Potato Virus ◽  
Internal Control ◽  
18S Rrna ◽  
Degradation Kinetics ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Potato Viruses ◽  
Das Elisa

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

Lineage-specific defect in gene expression in human platelet phospholipase C-β2 deficiency

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 905-911 ◽  
Author(s):  
Guang Fen Mao ◽  
Vijender R. Vaidyula ◽  
Satya P. Kunapuli ◽  
A. Koneti Rao
Keyword(s):  
Gene Expression ◽  
Phospholipase C ◽  
Internal Control ◽  
Healthy Subjects ◽  
Leukotriene B4 ◽  
Calcium Mobilization ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Specific Expression ◽  
Platelet Factor

Abstract Phospholipase C (PLC)–β2 plays a major role in platelet activation. Previous studies have described a unique patient with impaired receptor-mediated platelet aggregation, secretion, calcium mobilization, and phospholipase C (PLC) activation associated with a selective decrease in platelet PLC-β2 isozyme. To identify the mechanisms leading to the defect, platelet RNA from the patient and healthy subjects was subjected to reverse transcription–polymerase chain reaction (RT-PCR) and the products sequenced. The PLC-β2 cDNA sequence in the patient showed no abnormalities. Platelet PLC-β2 and β-actin (internal control) mRNA levels were assessed by RT-PCR; the ratio of PLC-β2 to β-actin mRNA levels was 0.80 to 0.95 in 4 healthy subjects and 0.28 in the patient. PLC-β2 mRNA levels were similarly reduced compared with GPIIb and Gαq mRNA levels. PLC-γ2 and platelet factor 4 mRNA levels were normal. Calcium mobilization was studied in neutrophils upon activation with formyl-Met-Leu-Phe (fMLP), adenosine diphosphate (ADP), platelet-activating factor (PAF), interleukin-8 (IL-8), C5a, and leukotriene B4 (LTB4), and it was normal. Neutrophil elastase secretion upon activation with fMLP, ADP, PAF, IL-8, C5a, and LTB4 was normal, as were neutrophil PLC-β2 mRNA and PLC-β2 on immunoblotting. Thus, responses to activation, PLC-β2 protein, and PLC-β2 mRNA are decreased in patient platelets but not in neutrophils, providing evidence for a hitherto undescribed lineage (platelet)–specific defect in PLC-β2 gene expression. These studies provide a physiologically relevant model to delineate regulation of PLC-β2 gene and its tissue-specific expression.

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