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

Abstract

ABSTRACT. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.

Evaluation of the Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) as a Screening Method for the Detection of Influenza Viruses in the Fecal Materials of Water Birds

“…In addition, processing of multiple samples was available using 96-well plates for viral RNA extraction and then for RT-LAMP. Compared to previous reports [3,12,24], we clearly indicated RT-LAMP as a promising tool with a high throughput capability before virus isolation using embryonated chicken eggs. We consider that using this assay as a part of the isolation procedure for influenza viruses will facilitate surveillance and timely control of avian influenza.…”

“…Practically, HA subtypes and genetic background of the M gene of influenza viruses evaluated in this study were various, suggesting that the primer set used in this study is able to detect the M gene of multiple influenza viruses. In addition, by using loop primers (FluAFL and FluABL), viral gene is amplified efficiently and the amplification time is shortened to 35 min compared to previous reports [3,12,24].…”

“…The loop-mediated isothermal amplification (LAMP) is one of the molecular methods to amplify the genome under isothermal conditions with high specificity, efficiency, and rapidity [20]. This method has been used for rapid detection of influenza virus genes [3,11,12,24]. In the present study, we assessed sensitivity and specificity of the reverse transcription (RT)-LAMP of influenza virus detection from fecal materials of ducks.…”

Loop-Mediated Isothermal Amplification as a Simple Molecular Method for the Detection of Derzsy’s Disease Virus

“…The DNA sensitivity reaction was 0.1 TCID 50 of strain 24/03 GPV for gel electrophoresis and observation of the fluorescence after addition of SYBR Green or Gel Red TM dyes. In comparison, the sensitivity of LAMP specific for AIV reached the limit of 1,000 EID 50 (9). The high sensitivity of LAMP was also confirmed by Song et al (10), who estimated its detection limit as 1.45 pg of DHV-1 RNA.…”

Development and Evaluation of a Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Enterovirus 71

“…This novel detection method works on the principle of a strand displacement reaction with the specific stem-loop structures, and the whole amplification reaction takes place continuously under isothermal conditions. Previously, LAMP methods for detection of different viruses have been developed, including West Nile virus (27), dengue virus serotypes 1 to 4 (26), Japanese encephalitis virus (20,28), avian H5 and H7 influenza viruses (29,32), the 2009 pandemic H1N1 influenza virus (15,21), Marburg virus (16), Ebola virus (17), foot-and-mouth disease virus (25), and herpes simplex virus 1 (30). All these assays showed high specificity, efficiency, and sensitivity that were similar to or higher than PCR assays.…”