Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates
Shelly BolotinAnne RobertsonAlireza EshaghiCedric De LimaErnesto LombosEddie Chong-KingLaura BurtonTony MazzulliSteven J. Drews
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Objective To reconstruct the initiative procedure of HIV-1 reverse transcription in vitro and establish a methodology of assessing activity of HIV-1 reverse transcriptase (RT) with real-time PCR Methods The tRNALys-3 gene was amplified from genome in healthy individuals through polymerase chain reaction (PCR), and then T7 transcription promoter was added in 5'-terminal of the tRNALys-3. The tRNA[Lys-3 cRNA product was obtained by applying T7 RNA polymerase through a transcription reaction. The 5'-LTR-PBS DNA was also obtained by transcription reaction from the HIV-1 infectious clone and inserted into pGEM-T easy vectors. 5'-LTR-PBS cRNA was obtained by applying SP6 RNA polymerase whose combining site was located in pGEM-T easy vectors. Then the two RNA samples was catalyzed by two kinds of standard reverse transcriptases (SuperScript Ⅲ and HIV-1 standard reverse transcriptase, respectively) and the cDNA was synthesised. The relative activity of RT was determined with the real-time PCR. Results The tRNALys-3 primer and the SP6-5'-LTR-PBS RNA were procured accurately, whose length were 93bp and 872 bp, respectively. After the following serial dilution of Super Script Ⅲ and HIV-1 standard reverse transeriptase:1 : 10, 1: 100, 1:1 000, 1:10 000, each step of reverse transcription process worked successfully. Real-time PCR results showed that Ct values of the two groups were 13.9, 18. 3, 20. 9, 24. 9 and 20. 4, 25. 5, 28. 7, 32. 5 respectively. Conclusion A novel real-time PCR method is developed to assay the RT activity directly through reconstructing the initiation of HIV reproduction, which may be helpful for clinical management, screening of new antiretroviral drugs, and drug resistance test.
Key words:
HIV-1; HIV reverse transcriptase; Polymerase chain reaction
T7 RNA polymerase
Primer (cosmetics)
Transcription
Primer binding site
RNA-Directed DNA Polymerase
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AbstractSince the polymerase chain reaction (PCR) for DNA amplification was first introduced in 1985 (1), the combination of reverse transcription with subsequent PCR amplification of the cDNA (RT-PCR) has been an increasingly utilized technique to analyze gene expression (2,3). In order for this procedure to be reasonably quantitative, however, appropriate controls must be applied to all steps, including the quantitation of the original RNA, the reverse transcription, and the PCR itself. Several investigators have published methods on quantitative RT-PCR that involve varying cDNA input into the PCR (3–7), varying cycle number (3,4,8,9), or the use of a competitive template as an internal standard (10,11). However, only a few of the competitive PCR methods take into account the efficency of the reverse transcription phase of RT-PCR (4,7,11), which may vary from 10-50% (12,13). In the former methods, it would also be necessary to amplify another control gene in parallel (e.g., actin) to control for RNA input and reverse transcription.KeywordsPolymerase Chain ReactionPolymerase Chain Reaction ProductPolymerase Chain Reaction ReactionCompetitive Polymerase Chain ReactionInput cDNAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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RNA polymerase II
Primer dimer
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clone (Java method)
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Background: The human genome contains a large number of repetitive elements including endogenous retroviruses and long interspersed nuclear elements (LINE). Repetitive elements have been shown to be activated and influence gene expression in Hodgkin lymphoma (HL). Some repetitive elements contain open reading frames that encode a reverse transcriptase. We analyzed RT activity and expression of reverse transcriptase sequences in HL cells. Methods: RNA seq analysis was used for the identification of expressed putative reverse transcriptases in HL cells. Reverse transcriptase activity in HL cells was assessed using phage MS2 RNA as template for reverse transcription and amplification by quantitative polymerase chain reaction. Sensitivity of HL cells for the non-nucleoside reverse transcriptase inhibitor efavirenz was analyzed by flow cytometry. LINE-1 reverse transcriptase sequences were amplified from HL cell line L-428 by reverse transcription-polymerase chain reaction, cloned into vector pGEM-T Easy and sequenced by Sanger sequencing. Results: HL cells showed high reverse transcriptase activity in comparison to normal blood cells. In addition, efavirenz killed HL cells in a dose dependent manner. RNA seq analysis suggested that HL cells express sequences corresponding to LINE-1 reverse transcriptase. By RT-PCR using LINE-1 reverse transcriptase specific primers, several transcripts containing open reading frames with predicted coding capacity for reverse transcriptase molecules were identified. Conclusions: HL cells express LINE-1 reverse transcriptase sequences that might be responsible for the observed reverse transcriptase activity of these cells. LINE-1 reverse transcriptase might be interesting targets for future therapeutic developments. Our work is supported by Mitteldeutsche Kinderkrebsforschung.
RNA-Directed DNA Polymerase
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