Rapid detection of goose astrovirus genotypes 2 using real-timereverse transcription recombinase polymerase amplification
Haiqin LiYujun ZhuChunhe WanZhangzhang WangLei LiuMeifang TanFanfan ZhangYanbing ZengJiangnan HuangChengcheng WuYu HuangZhaofeng KangXiaoqiao Guo
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Abstract Background Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. Results The real-time RT-RPA reaction was carried out at a constant temperature of 39°C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/µL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. Conclusions The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.Keywords:
Recombinase Polymerase Amplification
Recombinase Polymerase Amplification
Clostridium perfringens
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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.
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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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재조합효소-중합효소 증폭법(RPA)은 등온에서 매우 빠른 반응속도를 보이는 유전자 증폭법으로 근래 여러 병원체의 검출법에 폭넓게 적용되고 있다. 본 연구는 kSBV의 빠른 검색을 위하여 kSBV 특이 실시간 재조합효소-중합효소 증폭법을 새로이 개발하였으며, 특이 cDNA로부터 2분 46초만에 kSBV특이 유전자의 존재를 확인할 수 있었다. 또한, 이를 바탕으로 목적 유전자의 정량이 가능한, 보편적 응용이 가능한 정량 실시간 재조합효소-중합효소 증폭법(quantitative real-time RPA)을 제안하였으며, 이 정량법이 현장시료에서 유용함을 입증하였다. 본 실험법은 꿀벌 질병체의 정량 검출 뿐 아니라, 일반 병원체의 정량 검출에서도 응용되기를 기대한다.
Recombinase Polymerase Amplification
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Organoid
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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.
Transcription
RNA polymerase II
Primer dimer
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목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균 율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용 하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription poly-merase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행 받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA) 를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체 를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외 하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지 는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법 은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학 자료 수집에 큰 도움이 될 것으로 기대한다.
Carriage
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Reverse engineering
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Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.
Recombinase Polymerase Amplification
Coronavirus
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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
RNase H
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