Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.
RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus.
Jiuhai
J
Wang
Peng
P
Lu
Jieni
J
Yan
Yufan
Y
Zhang
Lanye
L
Huang
Zeeshan
Z
Ali
Zhiyang
Z
Li
Nongyue
N
He
D001483:Base Sequence; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018189:Immunomagnetic Separation / Q000295:instrumentation; D008163:Luminescent Measurements / Q000295:instrumentation*; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000295:instrumentation*; D019562:Viral Load
D058185:Magnetite Nanoparticles; D012367:RNA, Viral