Anchored Pan Dengue RT‐PCR and Fast Sanger sequencing for detection of Dengue RNA in human serum

A large number of human infections are caused by different dengue virus strains, mainly in the tropical and subtropical parts of the world, but also outside the endemic regions. RT‐PCR methods are used widely for detection of dengue virus RNA in acute‐phase serum samples; however, new sequence variation can inhibit these methods. An assay was developed integrating an anchored Pan Dengue RT‐PCR with a new Fast Sanger sequencing protocol. For broad detection and identification of dengue virus RNA, including new strains of all serotypes, the conserved 3′ genome end was targeted for highly specific cDNA synthesis. A combination of degenerated primers was used for second strand synthesis, followed by tag primed amplification. The mixture of generated amplicons was identified directly by the Fast Sanger sequencing from the anchored 3′ genome end. Evaluating the assay on human serum RNA spiked with viral RNA representing the four dengue serotypes demonstrated a detection limit of 44–124 copies viral RNA per reaction for a two‐step format of the anchored Pan Dengue RT‐PCR and 100–500 copies for a one‐step protocol, respectively. The different serotypes were clearly identified from the generated sequences. Further, the 5‐hr procedure was evaluated and compared to standard real‐time RT‐PCR protocols on acute‐phase serum samples from patients with confirmed dengue infections. This assay demonstrates a strategy for virus detection, which combines nucleic acid amplification adapted for dengue virus RNA with direct and rapid sequencing. It provides a tolerance for new sequence variation and the strategy should be applicable for other RNA viruses. J. Med. Virol. 82:1701–1710, 2010. © 2010 Wiley‐Liss, Inc.     

Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: evidence for false-priming and improvement by tagged RT-PCR

Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.     

Strategies for reliable diagnosis of hepatitis C infection: the need for a serological confirmatory assay.

The aim of the study was to examine whether the diagnosis of Hepatitis C (HCV) infection can be obtained reliably without using an immunoblot-based confirmation assay. 1,708 EIA-reactive serum samples were examined retrospectively for (i) optical density value in the screening assay, (ii) reactivity in an immunoblot assay, and (iii) result by RT PCR. In 1,394 (81.0%) samples positive results were obtained by both the HCV EIA and the confirmation assay. OD-values > or = 2.2 were observed in 1026 of these samples, but covered the range from 0.4 to 2.1 in the other 368 samples. The combination of HCV EIA reactivity and indeterminate immunoblot assay was observed in 134 (7.8%) serum samples. HCV RNA was detected in 58 cases by PCR. The OD-values of these 58 samples ranged from 0.4 to >2.2. Especially reactivity against the core recombinant protein was indicative of PCR positivity. The reactivity by the HCV EIA could not be confirmed by immunoblot assay or PCR in 180 (10.5%) sera. These false reactive sera showed OD values by EIA from 0.3 to 2.1. It is concluded that no threshold values can be defined which would allow differentiation between positive, indeterminate, and false reactive result by HCV EIA without producing an unacceptably high number of false negative diagnoses. Not using immunoblot-based confirmation would result in many additional PCR examinations. Therefore, confirmation of reactive HCV EIA results by a serological confirmatory assay must remain an essential part of the diagnostic procedure.     

EB病毒转化人外周血单个核细胞作为HCV复制模型的初步研究

目的　复制丙型肝炎病毒（ＨＣＶ） 感染的细胞模型。方法　用ＥＢ病毒感染其ＨＣＶ 阳性患者的外周血单个核细胞并使其转化，获得ＨＣＶ的外周永生化Ｂ细胞株。以后，每隔１ 个月应用套式逆转录聚合酶链反应（ＲＴＰＣＲ） 检测１ 次培养细胞和上清中的ＨＣＶＲＮＡ。结果　ＨＣＶ可以在传代细胞中持续存在１ 年，培养细胞中的ＨＣＶ负链和培养上清中的ＨＣＶ 则呈间断阳性。结论　ＨＣＶ 可以在该细胞株中较长时间存在、复制和分泌