Detection of avian influenza virus-H5N1 by multiplex reverse transcription and multiple cross displacement amplification combined with lateral flow biosensor technology
10.3760/cma.j.cn114452-20240307-00117
- VernacularTitle:多重逆转录交叉置换扩增结合测流生物传感器技术检测H5N1型禽流感病毒
- Author:
Pengfei WANG
1
;
Fan ZHAO
;
Linlin YAN
;
Jing WANG
;
Ruifeng YANG
;
Shoukui HU
Author Information
1. 航天中心医院临床检验科，北京 100049
- Keywords:
Influenza A virus, H5N1 subtype;
Reverse transcription;
Thermostatic amplification;
Lateral flow biosensor;
Diagnostic method
- From:
Chinese Journal of Laboratory Medicine
2024;47(11):1326-1331
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish of a newmethod:for the rapid detection of H5N1-Avian Influenza virus by combining reverse transcription (RT), multiple cross displacement amplification (MCDA) and nanoparticle-based lateral flow biosensor (LFB).Methods:MCDA primers were designed based on gene sequences specific to Hemagglutinin (HA) and Neuraminidase (NA) in H5N1 avian influenza virus. The target genes HA and NA were amplified through reverse transcription and MCDA in one reaction system. Results were displayed by LFB. The assay was named as H5N1-mRT-MCDA-LFB. The reaction conditions of the H5N1-RT-MCDA-LFB method were optimized, and the sensitivity and specificity were also assessed.Results:The H5N1-RT-MCDA-LFB assay could achieve good amplification effect at a constant temperature of 65 ℃ for 40 minutes. The method had a lower limit of detection of 100 fg per reaction with 100-fold higher sensitivity than that of the RT-qPCR (lower limit of detection 10 pg per reaction). The assay was negative in detecting 28 common viruses, mycoplasmas, chlamydias, bacterias and funguses, except for H5N1. In addition, the H5N1-RT-MCDA-LFB method showed better validation in simulated clinical samples with a lower limit of detection at 1×10 2 copies/ml. Conclusion:The H5N1-RT-MCDA-LFB assay is a valuable molecular diagnostic technique for detecting H5N1 avian influenza virus due to its simplicity, rapidity, sensitivity and specificity.
