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The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. KEY POINTS: • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps, and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens.

2019 年爆发的 2019 年冠状病毒病（COVID-19）严重损害了世界经济和公共卫生，使人们更加关注呼吸道传染病。然而，传统的定量实时聚合酶链反应（qRT-PCR）核酸检测试剂盒需要 RNA 提取、逆转录、扩增以及大型设备的支持来富集和纯化核酸和精确的温度控制。因此，新型、快速、方便、敏感和特异的检测方法正在紧急开发并进入概念验证测试。在本研究中，我们开发了一种新的核酸检测系统，称为 4 恒温步骤（4TS），它创新性地允许所有检测过程在恒温设备中完成，在 40 分钟内完成提取、扩增、目标切割和检测。该检测方法可以特异性和灵敏地检测五种呼吸道病原体，即 SARS-CoV-2、猫支原体（MF）、猫衣原体（CF）、猫杯状病毒（FCV）和猫疱疹病毒（FHV）。此外，设计和开发了一种具有成本效益和实用的小型反应装置，以保持稳定的反应条件。五种病毒的检测结果表明，该系统的灵敏度大于 94%，特异性为 100%。4TS 系统不需要复杂的设备，操作方便快捷，可在家中或小型诊所立即对疑似传染病原体进行检测。因此，该检测系统具有诊断价值，对进一步降低传染病早期筛查的成本和扩大其应用具有重要意义。