Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis E Virus

Abstract

The one-step single-tube betaine-free reverse transcription (RT)-loop-mediated isothermal amplification assay was developed for rapid diagnosis of hepatitis E virus. This assay amplified the target gene in less than 45 min (even as short as 20 min) under isothermal conditions at 63°C, and the sensitivity of this assay was 100-fold greater than that of RT-PCR. This assay demonstrated a detection limit of 0.045 fg (nine copies/reaction).

Standardization of Hepatitis E Virus (HEV) Nucleic Acid Amplification Technique-Based Assays: an Initial Study To Evaluate a Panel of HEV Strains and Investigate Laboratory Performance

“…NAT-based assays have been developed for the detection of HEV RNA to confirm active HEV infection. These assays include conventional reverse transcriptase PCR (RT-PCR) as well as realtime RT-PCR and reverse transcription-loop-mediated isothermal amplification (10) for the detection of HEV RNA in serum and plasma or in fecal samples. While consensus assays have been developed for the detection of HEV genotypes 1 to 4 (5,8), no studies have been performed so far to compare the abilities of laboratories to detect HEV RNA.…”

Development of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Detection of Avian Influenza Viruses in Field Specimens

“…The method is applicable to amplification of RNA templates by combination with a reverse transcription reaction (RT-LAMP), and this approach has been applied successfully to the detection of numerous viruses, such as West Nile virus [15], viral hemorrhagic septicemia virus (VHS) [19], Spring viremia of carp virus [18], respiratory syncytial virus (RSV) [20], H3 swine influenza virus [7], infectious bursal diseases virus [25], hepatitis E virus [11] and Newcastle disease virus [12]. Moreover, RT-LAMP has been used for diagnosis of infection by influenza viruses, including H5 and H9 subtypes [1,10].…”

Methylation-Specific Loop-Mediated Isothermal Amplification for Detecting Hypermethylated DNA in Simplex and Multiplex Formats

“…LAMP is a DNA-amplification technique that possesses a number of advantageous features for routine molecular diagnostics applications: (a) It is isothermal and therefore requires no thermal cycler; (b) it is rapid, because no temperature ramping is needed and the reaction thresholds are typically reached in Ͻ30 -40 min; and (c) its detection limit and quantitative performance parallel that of the PCR (29,30 ). A distinct advantage of LAMP over PCR-based amplification is its high specificity for the intended DNA target, which is conferred by its unique amplification mechanism and the sequence-specific binding of multiple primers as a requirement for amplification.…”