Establish and optimization of real-time fluorescent reverse transcription loop-mediated isothermal amplification for detection of avian influenza H5 hemagglutinin gene.
- Author:
Yun LIU
1
;
Jin-ming TANG
2
;
Hong TAO
2
;
Jie SUN
3
;
Ti-kang LU
2
;
Li-shan LIAO
2
;
Jian-li LIU
2
;
Shao-ling ZENG
2
;
Chen-fu CAO
2
;
Cai-hong ZHANG
2
;
Zhou-xi RUAN
2
;
Jian-qiang LV
2
;
Jun-xing YANG
2
;
Qun-yi HUA
2
;
Zheng-li CHEN
4
;
Zhi-feng QIN
2
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Chickens; Hemagglutinin Glycoproteins, Influenza Virus; genetics; Influenza A Virus, H5N1 Subtype; genetics; isolation & purification; Influenza in Birds; diagnosis; virology; Poultry Diseases; diagnosis; virology; Reverse Transcriptase Polymerase Chain Reaction; instrumentation; methods; Sensitivity and Specificity
- From: Chinese Journal of Virology 2013;29(5):488-494
- CountryChina
- Language:Chinese
- Abstract: H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.
