Abstract：Influenza in birds is caused by infection with viruses of the family Orthomyxoviridae placed in the genus influenza virus A. Many species of birds have been shown to be susceptible to influenza A viruses. Influenza A viruses are classified into subtypes on the basis of their haemagglutinin (HA) and neuraminidase (NA) antigens. Traditional diagnostic methods of the subtype H5 influenza are laborious, time-consuming or insensitive, such as virus isolation, haemagglutination, neuraminidase inhibition and RT-PCR. So, more sensitive and convenient methods are in demand. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers the advantage of omitting thermocycling, enabling RNA amplification at constant temperature, and is used most widely among the isothermal amplification technologies. In order to develop a detection method of subtype H5 Avian influenza virus (AIV) with RT-LAMP, 3 pairs of primers specifically directing to 8 recognition sites on the hemagglutinin (HA) gene of H5-AIV were designed, and then mixed into the other ingredients as recommended in the instruction of the RT-LAMP kits with or without freeze-dried protecting agents. The recombinant plasmid with the whole genome of subtype H5N1-AIV was extracted, purified and diluted to make each reaction system of RT-LAMP and RT-PCR contain 106, 105, 104, 103, 102, 101 or 100 copies. The sensitivities of the RT-LAMP before and after lyophilization, and the difference on sensitivity between RT-LAMP and RT-PCR by turbidity monitoring and agarose gel electrophoresis analysis, respectively, were determined and compared. The specificities of RT-LAMP and RT-PCR with subtypes H1~H15 AIV, Newcastle disease virus(NDV) and Infectious bronchitis virus (IBV) were tested. The feasibility of the visualization of RT-LAMP was tested by adding the color developing solution (mixture of SYBR Green I and hydroxynaphthol blue) into the isothermal amplification systems after reaction for 1 h at 63 ℃. Finally, totally 326 clinical samples with the established RT-LAMP and RT-PCR, and the percentages of the positive samples were compared between the 2 methods. The results of sensitivity tests showed that, at least as 102 copies of the recombinant plasmids with the target gene could be detected by RT-LAMP after reaction for 1 h at 63 ℃, with a sensitivity of about 10-fold higher than that of the RT-PCR. Moreover, the sensitivity of RT-LAMP won't be interfered by lyophilization with 5% trehalose, 1.25% mannitol and 1.25% bovine serum albumin, or by the color developing agent added when the amplification reaction finished. The specificity tests indicated that all the virus except subtype H5 AIV showed negative results both by RT-LAMP and RT-PCR. Among 326 clinically collected, 38 and 31 swabs were confirmed as positive by RT-LAMP and RT-PCR, respectively. All the 31 positive swabs by RT-PCR were also positive by RT-LAMP, and 7 negative swabs by RT-PCR were detected as positive by RT-LAMP. So, the sensitivity of RT-LAMP was preferable to the RT-PCR assay. The above results suggested that the RT-LAMP had higher sensitivity and good specificity, and could be used as a point-of-care (POC) test product since it is convenient for storage and very easy for the veterinarians to use in the farms.
[1]邓国华, 马文军, 于康震.禽流感病毒核蛋白基因在重组杆状病毒中的表达.[J].中国预防兽医学报(J), 1999, 21(2):81-84[2]侯轩, 付萍, 张海燕等.牛支原体的环介导等温扩增快速检测技术研究.[J].农业生物技术学报(J), 2012, 20(2):218-224[3]黄书林, 付萍, 李佳禾等.环介导等温扩增技术快速检测猪细小病毒的试验.[J].中国兽医杂志(J), 2011, 47(10):3-6[4]蒋菲, 黄书林, 徐威等.H9亚型禽流感病毒环介导等温扩增检测方法的建立与荧光显色剂的应用.[J].农业生物技术学报（J）, 2011, 19(1):191-196[5]张侃, 蒋菲, 张跃伟等.羟基萘酚蓝在伪狂犬病病毒环介导等温扩增检测方法中的应用.[J].农业生物技术学报（J）, 2011, 19(6):1075-1080[6]Fouchier R.A.M., Munster V., Wallensten A. et al.Characterization of a novel Influenza A virus hemagglutinin subtype (H16) obtained from blackheaded gulls.[J].Journal of Virology, 2005, 79(2):2814-2822[7]He F, Soejoedono RD, Murtini S et al.Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus.[J].BMC Microbiology, 2010, 30(10):330-[8]Jia he Li, F Chris Minion, Andrew C Petersen et al. .Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.[J].World Journal Microbiology Bioanalytical Chemistry, 2013, 29(4):607-616[9]Notomi T, Okayama H., Masubuchi H. et al.Loop-mediated isothermal amplification of DNA.[J].Nucleic Acids Research, 2000, 28:E63-[10]Pérez L J, Díaz de Arce H, Cilloni F et al.An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.[J].Molecular and Cellular Probes, 2012, 26(3):137-145[11]Thongtira A, Payungporn S, Keawcharoen J et al.The single step multiplex reverse transcription polymerase chain reaction assay for dectecting H5 and H7 avian influenza A viruses.[J].The Tohoku Journal of Experimental Medicine, 2007, 211(1):75-79[12]World Health Organization Expert Committee .A revision of the system of nomenclature for influenza viruses: A WHO Memorandum.[J].Bulletin of the World Health Organization, 1980, 58(4):585-591[13]Yuewei Zhang, Ping Fu, Jiahe Li et al.Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China.[J].African Journal of Biotechnology, 2011, 10(61):13278-13283
