H5、H7和H9是重要的禽流感病毒(AIV)亚型,会造成巨大的经济损失,并对全球公共卫生构成潜在威胁。在这项研究中,开发了一种新的三重荧光逆转录环介导的等温扩增(TLAMP)检测方法,其中传统的LAMP技术与探针结合进行检测.通过这种创新的方法,可以同时识别和区分AIV的H5,H7和H9亚型,从而为预防和控制工作提供关键的技术支持。基于每种亚型的血凝素基因的保守区域设计了三个引物组和复合探针。探针在其3'末端用不同的荧光团标记,在扩增过程中分离以释放荧光信号。基于TLAMP产品的颜色解释检测结果。然后,优化了反应条件,并将三个引物组和探针组合在同一反应系统中,导致TLAMP检测分析用于AIV亚型的鉴别诊断。用体外转录的RNA进行的灵敏度测试表明,TLAMP测定的检测限对于H5为每个反应205个拷贝,对于H7为360个拷贝,对于H9为545个拷贝。TLAMP分析显示出优异的特异性,与相关的禽类病毒没有交叉反应,与先前发表的定量聚合酶链反应(qPCR)测定具有100%的一致性。因此,由于它的简单性,快速性,灵敏度,和特异性,该TLAMP检测方法适用于流行病学调查,是检测和区分临床样本中AIV的H5,H7和H9亚型的有价值的工具.
H5, H7, and H9 are pivotal avian influenza virus (AIV) subtypes that cause substantial economic losses and pose potential threats to public health worldwide. In this study, a novel triplex fluorescence reverse transcription-loop-mediated isothermal amplification (TLAMP) assay was developed in which traditional LAMP techniques were combined with probes for detection. Through this innovative approach, H5, H7, and H9 subtypes of AIV can be simultaneously identified and differentiated, thereby offering crucial technical support for prevention and control efforts. Three primer sets and composite probes were designed based on conserved regions of the haemagglutinin gene for each subtype. The probes were labelled with distinct fluorophores at their 3\' ends, which were detached to release the fluorescence signal during the amplification process. The detection results were interpreted based on the colour of the TLAMP products. Then, the reaction conditions were optimized, and three primer sets and probes were combined in the same reaction system, resulting in a TLAMP detection assay for the differential diagnosis of AIV subtypes. Sensitivity testing with in vitro-transcribed RNA revealed that the detection limit of the TLAMP assay was 205 copies per reaction for H5, 360 copies for H7, and 545 copies for H9. The TLAMP assay demonstrated excellent specificity, no cross-reactivity with related avian viruses, and 100% consistency with a previously published quantitative polymerase chain reaction (qPCR) assay. Therefore, due to its simplicity, rapidity, sensitivity, and specificity, this TLAMP assay is suitable for epidemiological investigations and is a valuable tool for detecting and distinguishing H5, H7, and H9 subtypes of AIV in clinical samples.