Reverse-transcription quantitative PCR directly from cells without RNA
          extraction and without isothermal reverse-transcription: a ‘zero-step’ RT-qPCR
          protocol

Abstract We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.

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Evaluation of a Surface Sampling Method for Recovery of Human Noroviruses Prior to Detection Using Reverse Transcription Quantitative PCR

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-276 ◽

2017 ◽

Vol 80 (2) ◽

pp. 231-236 ◽

Cited By ~ 3

Author(s):

Grace Tung-Thompson ◽

Blanca I. Escudero-Abarca ◽

Janie Outlaw ◽

Arnaud Ganee ◽

Sylvanie Cassard ◽

...

Keyword(s):

Quantitative Pcr ◽

Reverse Transcription ◽

Rna Extraction ◽

Field Tests ◽

Viral Gastroenteritis ◽

Surface Sampling ◽

Human Norovirus ◽

Green Pepper ◽

Human Noroviruses ◽

Reverse Transcription Quantitative Pcr

ABSTRACT Human noroviruses are the most common cause of acute viral gastroenteritis, and the environmental persistence of these viruses contributes to their transmissibility. Environmental sampling is thus an important tool for investigating norovirus outbreaks and for assessing the effectiveness of cleaning and decontamination regimens. The purpose of this study was to evaluate a sampling material (wipes) for their efficacy at recovering human norovirus from hard surfaces and foods. Dilutions of a human norovirus GII.4 stool specimen derived from an outbreak were applied to hard surfaces (stainless steel and ceramic) and the surfaces of representative foods (green pepper, apple, tomato, and cheese). The viruses were recovered at various times postinoculation using the wipes, followed by RNA extraction and reverse transcription quantitative PCR. Recovery efficiency ranged from 74% to almost 100% for all artificially inoculated hard surfaces and for most fresh produce surfaces. Less efficient recovery was observed for cheese. Viral RNA could be recovered from select surfaces for up to 7 days postinoculation, with a <1 log reduction in genome copy number. In field tests, 24 (11%) of 210 environmental samples collected during winter 2012 from restrooms in North Carolina were presumptively positive for human norovirus, and six of these samples were confirmed as GII.4 by sequencing. These wipes may be a valuable tool for investigations of norovirus outbreaks and studies of norovirus prevalence.

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Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR

BIO-PROTOCOL ◽

10.21769/bioprotoc.1695 ◽

2016 ◽

Vol 6 (1) ◽

Author(s):

Fan-Yan Wei ◽

Kazuhito Tomizawa

Keyword(s):

Quantitative Pcr ◽

Reverse Transcription ◽

Transfer Rnas ◽

Mitochondrial Transfer ◽

Reverse Transcription Quantitative Pcr

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Establishment and Application of Rapid Diagnosis for Reverse Transcription-Quantitative PCR of Newly Emerging Goose-Origin Nephrotic Astrovirus in China

mSphere ◽

10.1128/msphere.00380-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 4

Author(s):

Xiaoyuan Yuan ◽

Kai Meng ◽

Yuxia Zhang ◽

Lihong Qi ◽

Wu Ai ◽

...

Keyword(s):

Quantitative Pcr ◽

Clinical Application ◽

Reverse Transcription ◽

Test Sample ◽

Sensitivity Testing ◽

Content Type ◽

Reverse Transcription Quantitative Pcr ◽

Emerging Virus ◽

New Type ◽

And Control

ABSTRACT In 2017, a new type of goose-origin astrovirus (GoAstV) that is completely different from previously identified avian astroviruses (which have only 30.0% to 50.5% homology with GoAstV) has been isolated from diseased geese in China. This disease can cause joint swelling in sick geese, and the anatomy shows a clear precipitation of urate in the kidney. The rate of death and culling can reach more than 30%, revealing the disease’s severe pathogenicity. To quickly and accurately diagnose the newly emerging disease, we established a highly specific reverse transcription-quantitative PCR (RT-qPCR) method of detecting GoAstV. Sensitivity testing showed that the minimum amount of test sample for this method is 52.5 copies/μl. Clinical application confirmed that this method can quickly and effectively detect GoAstV, providing a diagnostic platform for the prevention and control of goose disease. IMPORTANCE Goose-origin astrovirus (GoAstV), as a newly emerging virus in 2017, is different from previously known astroviruses in the genus Avastrovirus. So far, few studies have focused on the novel virus. Considering the infectious development of astrovirus (AstV), we established a reverse transcription-quantitative PCR (RT-qPCR) assay with a strong specificity to quickly and accurately diagnose GoAstV. Confirmed by clinical application, this method can quickly and accurately detect prevalent GoAstV. The assay is thus convenient for clinical operation and is applicable to the monitoring of GoAstV disease.

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